50 results on '"Kristopher Clark"'
Search Results
2. 553 First-in-class inhibitors of ERAP1 alter the immunopeptidome of cancer, driving a differentiated T cell response leading to tumor growth inhibition
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Peter Joyce, Martin Quibell, Jason Shiers, Carmen Tong, Kristopher Clark, Nicola Ternette, Andrew Leishman, Kate Anderton, Jessica Sette, and Wayne Paes
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Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Published
- 2021
- Full Text
- View/download PDF
3. Rapid Cycle Evaluation and Adaptation of an Inpatient Tobacco Treatment Service at a U.S. Safety-Net Hospital
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Hasmeena Kathuria, Nicole Herbst, Bhavna Seth, Kristopher Clark, Eric D. Helm, Michelle Zhang, Charles O’Donnell, Carmel Fitzgerald, Indira Swetha Itchapurapu, Meg Waite, Carolina Wong, Lakshmana Swamy, Jen Olson, Rebecca G. Mishuris, and Renda Soylemez Wiener
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Mental healing ,RZ400-408 ,Psychiatry ,RC435-571 - Abstract
Background To address disparities in smoking rates, our safety-net hospital implemented an inpatient tobacco treatment intervention: an “opt-out” electronic health record (EHR)-based Best Practice Alert + order-set, which triggers consultation to a Tobacco Treatment Consult (TTC) service for all hospitalized patients who smoke cigarettes. We report on development, implementation, and adaptation of the intervention, informed by a pre-implementation needs assessment and two rapid-cycle evaluations guided by the Consolidated Framework for Implementation Research (CFIR) and Expert Recommendations for Implementing Change (ERIC) compilation. Methods We identified stakeholders affected by implementation and conducted a local needs assessment starting 6 months-pre-launch. We then conducted two rapid-cycle evaluations during the first 6 months post-implementation. The CFIR informed survey and interview guide development, data collection, assessment of barriers and facilitators, and selection of ERIC strategies to implement and adapt the intervention. Results Key themes were: (1) Understanding the hospital's priority to improving tobacco performance metrics was critical in gaining leadership buy-in (CFIR Domain: Outer setting; Construct: External Policy and Incentives). (2) CFIR-based rapid-cycle evaluations allowed us to recognize implementation challenges early and select ERIC strategies clustering into 3 broad categories (conducting needs assessment; developing stakeholder relationships; training and educating stakeholders) to make real-time adaptations, creating an acceptable clinical workflow. (3) Minimizing clinician burden allowed the successful implementation of the TTC service. (4) Demonstrating improved 6-month quit rates and tobacco performance metrics were key to sustaining the program. Conclusions Rapid-cycle evaluations to gather pre-implementation and early-implementation data, focusing on modifiable barriers and facilitators, allowed us to develop and refine the intervention to improve acceptability, adoption, and sustainability, enabling us to improve tobacco performance metrics in a short timeline. Future directions include spreading rapid-cycle evaluations to promote implementation of inpatient tobacco treatment programs to other settings and assessing long-term sustainability and return on investment of these programs.
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- 2021
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4. 446 Immunopeptidome changes mediated by a novel ERAP1 inhibitor leads to tumor growth inhibition
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Fergus Poynton, Lesley Young, Peter Joyce, Martin Quibell, Jason Shiers, Carmen Tong, Kristopher Clark, Edd James, Emma Reeves, Alihussein Remtulla, Henry Leonard, Camila de Almeida, Elisa Lori, Nicola Ternette, and Andrew Leishman
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Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Published
- 2020
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- View/download PDF
5. The NEDD8 E3 ligase DCNL5 is phosphorylated by IKK alpha during Toll-like receptor activation.
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Yann Thomas, Daniel C Scott, Yosua Adi Kristariyanto, Jesse Rinehart, Kristopher Clark, Philip Cohen, and Thimo Kurz
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Medicine ,Science - Abstract
The activity of Cullin-RING ubiquitin E3 ligases (CRL) is regulated by NEDD8 modification. DCN-like proteins promote Cullin neddylation as scaffold-like E3s. One DCNL, DCNL5, is highly expressed in immune tissue. Here, we provide evidence that DCNL5 may be involved in innate immunity, as it is a direct substrate of the kinase IKKα during immune signalling. We find that upon activation of Toll-like receptors, DCNL5 gets rapidly and transiently phosphorylated on a specific N-terminal serine residue (S41). This phosphorylation event is specifically mediated by IKKα and not IKKβ. Our data for the first time provides evidence that DCNL proteins are post-translationally modified in an inducible manner. Our findings also provide the first example of a DCNL member as a kinase substrate in a signalling pathway, indicating that the activity of at least some DCNLs may be regulated.
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- 2018
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6. The IkappaB kinase family phosphorylates the Parkinson's disease kinase LRRK2 at Ser935 and Ser910 during Toll-like receptor signaling.
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Nicolas Dzamko, Francisco Inesta-Vaquera, Jiazhen Zhang, Chengsong Xie, Huaibin Cai, Simon Arthur, Li Tan, Hwanguen Choi, Nathanael Gray, Philip Cohen, Patrick Pedrioli, Kristopher Clark, and Dario R Alessi
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Medicine ,Science - Abstract
Mutations in leucine-rich repeat kinase 2 (LRRK2) are strongly associated with late-onset autosomal dominant Parkinson's disease. LRRK2 is highly expressed in immune cells and recent work points towards a link between LRRK2 and innate immunity. Here we demonstrate that stimulation of the Toll-Like Receptor (TLR) pathway by MyD88-dependent agonists in bone marrow-derived macrophages (BMDMs) or RAW264.7 macrophages induces marked phosphorylation of LRRK2 at Ser910 and Ser935, the phosphorylation sites that regulate the binding of 14-3-3 to LRRK2. Phosphorylation of these residues is prevented by knock-out of MyD88 in BMDMs, but not the alternative TLR adaptor protein TRIF. Utilising both pharmacological inhibitors, including a new TAK1 inhibitor, NG25, and genetic models, we provide evidence that both the canonical (IKKα and IKKβ) and IKK-related (IKKε and TBK1) kinases mediate TLR agonist induced phosphorylation of LRRK2 in vivo. Moreover, all four IKK members directly phosphorylate LRRK2 at Ser910 and Ser935 in vitro. Consistent with previous work describing Ser910 and Ser935 as pharmacodynamic biomarkers of LRRK2 activity, we find that the TLR independent basal phosphorylation of LRRK2 at Ser910 and Ser935 is abolished following treatment of macrophages with LRRK2 kinase inhibitors. However, the increased phosphorylation of Ser910 and Ser935 induced by activation of the MyD88 pathway is insensitive to LRRK2 kinase inhibitors. Finally, employing LRRK2-deficient BMDMs, we present data indicating that LRRK2 does not play a major role in regulating the secretion of inflammatory cytokines induced by activation of the MyD88 pathway. Our findings provide the first direct link between LRRK2 and the IKKs that mediate many immune responses. Further work is required to uncover the physiological roles that phosphorylation of LRRK2 by IKKs play in controlling macrophage biology and to determine how phosphorylation of LRRK2 by IKKs impacts upon the use of Ser910 and Ser935 as pharmacodynamic biomarkers.
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- 2012
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7. Massive autophosphorylation of the Ser/Thr-rich domain controls protein kinase activity of TRPM6 and TRPM7.
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Kristopher Clark, Jeroen Middelbeek, Nick A Morrice, Carl G Figdor, Edwin Lasonder, and Frank N van Leeuwen
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Medicine ,Science - Abstract
TRPM6 and TRPM7 are bifunctional proteins expressing a TRP channel fused to an atypical alpha-kinase domain. While the gating properties of TRPM6 and TRPM7 channels have been studied in detail, little is known about the mechanisms regulating kinase activity. Recently, we found that TRPM7 associates with its substrate myosin II via a kinase-dependent mechanism suggesting a role for autophosphorylation in substrate recognition. Here, we demonstrate that the cytosolic C-terminus of TRPM7 undergoes massive autophosphorylation (32+/-4 mol/mol), which strongly increases the rate of substrate phosphorylation. Phosphomapping by mass spectrometry indicates that the majority of autophosphorylation sites (37 out of 46) map to a Ser/Thr-rich region immediately N-terminal of the catalytic domain. Deletion of this region prevents substrate phosphorylation without affecting intrinsic catalytic activity suggesting that the Ser/Thr-rich domain contributes to substrate recognition. Surprisingly, the TRPM6-kinase is regulated by an analogous mechanism despite a lack of sequence conservation with the TRPM7 Ser/Thr-rich domain. In conclusion, our findings support a model where massive autophosphorylation outside the catalytic domain of TRPM6 and TRPM7 may facilitate kinase-substrate interactions leading to enhanced phosphorylation of those substrates.
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- 2008
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8. Implementation of a Phenobarbital-based Pathway for Severe Alcohol Withdrawal: A Mixed-Method Study
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Mari-Lynn Drainoni, Michael H. Ieong, Jaime Murphy, Nicholas A Bosch, Allan J. Walkey, Erika L. Crable, Kristopher Clark, Valerie Grim, and Kimberly A. Ackerbauer
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Pulmonary and Respiratory Medicine ,Care process ,medicine.medical_specialty ,business.industry ,Alcohol ,macromolecular substances ,medicine.disease ,chemistry.chemical_compound ,chemistry ,Alcohol withdrawal syndrome ,Critical illness ,medicine ,Phenobarbital ,Psychiatry ,business ,medicine.drug - Abstract
Rationale: Several institutions have implemented phenobarbital-based pathways for the treatment of alcohol withdrawal syndrome (AWS). However, little is known about the care processes, effectivenes...
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- 2021
9. Bronchial gene expression signature associated with rate of subsequent FEV1 decline in individuals with and at risk of COPD
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Maarten van den Berge, Xiaohui Xiao, Marc E. Lenburg, George T. O'Connor, Stephen Lam, Wim Timens, Katrina Steiling, Pieter S. Hiemstra, Kristopher Clark, Gang Liu, Yuriy O. Alekseyev, Avrum Spira, Alen Faiz, Elizabeth J. Becker, Groningen Research Institute for Asthma and COPD (GRIAC), and Guided Treatment in Optimal Selected Cancer Patients (GUTS)
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0301 basic medicine ,Pulmonary and Respiratory Medicine ,False discovery rate ,COPD ,business.industry ,Respiratory System ,Mucin ,1103 Clinical Sciences ,airway epithelium ,respiratory system ,medicine.disease ,respiratory tract diseases ,respiratory measurement ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,030228 respiratory system ,Immunology ,Gene expression ,Respiratory epithelium ,Medicine ,business ,Airway ,Transcription factor ,Gene - Abstract
BackgroundCOPD is characterised by progressive lung function decline. Leveraging prior work demonstrating bronchial airway COPD-associated gene expression alterations, we sought to determine if there are alterations associated with differences in the rate of FEV1 decline.MethodsWe examined gene expression among ever smokers with and without COPD who at baseline had bronchial brushings profiled by Affymetrix microarrays and had longitudinal lung function measurements (n=134; mean follow-up=6.38±2.48 years). Gene expression profiles associated with the rate of FEV1 decline were identified by linear modelling.ResultsExpression differences in 171 genes were associated with rate of FEV1 decline (false discovery rate 1 decline signature was replicated in an independent dataset of bronchial biopsies from patients with COPD (n=46; p=0.018; mean follow-up=6.76±1.32 years). Genes elevated in individuals with more rapid FEV1 decline are significantly enriched among the genes altered by modulation of XBP1 in two independent datasets (Gene Set Enrichment Analysis (GSEA) pConclusionWe have identified and replicated an airway gene expression signature associated with the rate of FEV1 decline. Aspects of this signature are related to increased expression of XBP1-regulated genes, a transcription factor involved in the unfolded protein response, and genes related to mucin production. Collectively, these data suggest that molecular processes related to the rate of FEV1 decline can be detected in airway epithelium, identify a possible indicator of FEV1 decline and make it possible to detect, in an early phase, ever smokers with and without COPD most at risk of rapid FEV1 decline.
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- 2021
10. 553 First-in-class inhibitors of ERAP1 alter the immunopeptidome of cancer, driving a differentiated T cell response leading to tumor growth inhibition
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Jason Shiers, Kate Anderton, Kristopher Clark, Nicola Ternette, Martin Quibell, Peter Joyce, Carmen Tong, Jessica Sette, Wayne Paes, and Andrew Leishman
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Pharmacology ,Cancer Research ,Class (set theory) ,Immunology ,Cancer ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,Biology ,T cell response ,medicine.disease ,Oncology ,Cancer research ,medicine ,Molecular Medicine ,Immunology and Allergy ,Tumor growth inhibition ,RC254-282 - Abstract
BackgroundClinical data demonstrates increased antigen presentation diversity is an important factor in determining response rates to checkpoint inhibitors.1 In addition to tumor mutational burden, increased HLA heterozygosity and HLA evolutionary diversity are non-overlapping factors which further diversify the immunopeptidome and improve clinical response to checkpoint therapies.2 3 Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an enzyme that trims peptides loaded into classical and nonclassical MHC Class I molecules.4 5 Ablation of mouse ERAAP modifies the immunopeptidome, resulting in improved immunogenicity, generation of CD8+ T cell responses and tumor growth inhibition.6 7 We report the characterisation of ERAP1 inhibitors in syngeneic tumor models and development of biomarkers to enable translation of this mechanism into the clinic.MethodsHuman and mouse cancer cell lines treated with ERAP1 inhibitors were assessed by immunopeptidomics8 to profile peptide repertoire changes. ERAP1 inhibitor with and without checkpoint inhibition were used to treat syngeneic mouse tumor models, followed by analysing effects on the T cell receptor (TCR) repertoire, RNA sequencing profile, immune cell infiltration and tumor growth inhibition.ResultsExtensive analysis of the immunopeptidomes of diverse cancer cell lines robustly show that ERAP1 inhibition modulates the cancer-related antigen repertoire across diverse ERAP1 and HLA genotypes and cancer-type backgrounds. ERAP1 inhibition drives changes in T cell activation and response, leading to increased T cell infiltration into CT26 syngeneic tumors and alteration of the TCR repertoire at early and late timepoints in tumor growth. Consistent peptide length changes in the immunopeptidome, caused by ERAP1 inhibition, is a proof of mechanism biomarker, whilst tumor immunohistochemisty, TCR repertoire analysis and RNA sequencing are potential proof of principle biomarkers that can all be translated into the clinic. Importantly, the antigen and T cell changes we see following ERAP1 inhibition lead to robust tumor growth inhibition in different syngeneic mouse models when combined with anti-PD-1. We are also exploring the potential of ERAP1 inhibitors to enhance tumour immune responses in combination with additional therapies (e.g. chemotherapy and radiotherapy), across different tumor microenvironments.ConclusionsGrey Wolf Therapeutics ERAP1 inhibitors significantly modify the immunopeptidome and combination with anti PD-1 leads to significant TCR repertoire change, T cell infiltration and tumor growth inhibition in syngeneic mouse tumor models. These data provide the foundation from which we will explore the potential of our first-in-class ERAP1 inhibitor development candidate in the clinic, as well as identifying useful biomarkers to demonstrate desired biological activity.ReferencesRizvi N, Hellmann MD, Snyder A, et al. Mutational landscape determines sensitivity to PD-1 blockade in non–small cell lung cancer. Science 2015;348(6230):124–128.Chowell D, uc G T Morris LGT,2 3, Grigg CM, et al Patient HLA class I genotype influences cancer response to checkpoint blockade immunotherapy. Science 2018;359 (6375):582–587.Chowell D, Chirag Krishna, Federica Pierini, et al Evolutionary divergence of HLA class I genotype impacts efficacy of cancer immunotherapy. Nature Medicine 2019;25(11):1715–1720.Shastri N, Nagarajan N, Lind KC, et al. Monitoring peptide processing for MHC class I molecules in the endoplasmic reticulum. Curr Opin Immunol 2014;26:123–127.Mpakali A, Maben Z, Stern LJ, et al. Molecular pathways for antigenic peptide generation by ER aminopeptidase 1. Mol Immunol 2018;13:50–57.James E, Bailey I, Sugiyarto G, et al. Induction of protective antitumor immunity through attenuation of ERAAP function. J Immunol 2013;190(11):5839–5846.Manguso RT, Pope, HW, MD Zimmer, et al In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target. Nature 2017;547(7664):413–418.Purcell AW, Ramarathinam SH, Ternette N. Mass spectrometry–based identification of MHC-bound peptides for immunopeptidomics. Nat Protoc 2019;14(6):1687–1707.
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- 2021
11. Salt-inducible kinase 2 regulates fibrosis during bleomycin-induced lung injury
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Manuel van Gijsel-Bonnello, Nicola J. Darling, Takashi Tanaka, Samuele Di Carmine, Francesco Marchesi, Sarah Thomson, Kristopher Clark, Mariola Kurowska-Stolarska, Henry J. McSorley, Philip Cohen, and J. Simon C. Arthur
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Vascular Endothelial Growth Factor A ,Mice ,Bleomycin ,Animals ,Lung Injury ,Cell Biology ,Protein Serine-Threonine Kinases ,Fibrosis ,Lung ,Molecular Biology ,Biochemistry ,Idiopathic Pulmonary Fibrosis - Abstract
Idiopathic pulmonary fibrosis is a progressive and normally fatal disease with limited treatment options. The tyrosine kinase inhibitor nintedanib has recently been approved for the treatment of idiopathic pulmonary fibrosis, and its effectiveness has been linked to its ability to inhibit a number of receptor tyrosine kinases including the platelet-derived growth factor, vascular endothelial growth factor, and fibroblast growth factor receptors. We show here that nintedanib also inhibits salt-inducible kinase 2 (SIK2), with a similar IC
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- 2022
12. A Veteran Presenting With Chronic Progressive Dyspnea on Exertion
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Kristopher Clark, Bradley A. Maron, Anthony C. Breu, and Ronald H. Goldstein
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medicine.medical_specialty ,Text mining ,business.industry ,MEDLINE ,Physical therapy ,medicine ,Exertion ,business ,Features - Published
- 2020
13. 446 Immunopeptidome changes mediated by a novel ERAP1 inhibitor leads to tumor growth inhibition
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Elisa Lori, Camila de Almeida, Carmen Tong, Alihussein Remtulla, Jason Shiers, Fergus Poynton, Henry Leonard, Edd James, Emma Reeves, Martin Quibell, Andrew Leishman, Nicola Ternette, Kristopher Clark, Lesley Young, and Peter Joyce
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medicine.medical_treatment ,T cell ,Antigen presentation ,Biology ,lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,Major histocompatibility complex ,lcsh:RC254-282 ,medicine.anatomical_structure ,Cancer immunotherapy ,Antigen ,MHC class I ,Cancer research ,medicine ,biology.protein ,Cytotoxic T cell ,Checkpoint Blockade Immunotherapy - Abstract
Background Clinical data demonstrates increased antigen presentation diversity is a key factor in determining response rates to checkpoint inhibitors.1 In addition to tumour mutational burden/microsatellite instability, increased HLA heterozygosity and HLA evolutionary diversity are non-overlapping factors recently identified to further diversify the immunopeptidome and improve clinical response to checkpoint therapies.2 3 Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an enzyme that trims peptides loaded into classical and nonclassical class I MHC molecules.4 5 Ablation of mouse ERAAP modifies the immunopeptidome, resulting in improved immunogenicity, generation of CD8 T cell responses and tumor growth inhibition.6 7 Recently identified selective small molecules potently inhibit ERAP1 across key species and haplotypes.8 We report the further profiling of lead candidate ERAP1 inhibitors in human primary T cell in vitro assays and in vivo tumor models in mice. Methods Human cancer cell lines treated with ERAP1 inhibitors in vitro or in vivo in xenograft mouse models were assessed by immunopeptidomics9 to profile peptide repertoire changes. Novel or upregulated peptides were also tested in human immunogenicity assays. FACS analysis of T cells stimulated with Tyrosinase mRNA transfected human dendritic cells ± ERAP1 inhibition was to assess T cell repertoire changes. ERAP1 inhibitor and anti PD-1 mAb combination was assessed in syngeneic mouse tumor models to investigate tumour growth inhibition and PD end-points (e.g. IHC). Results Analysis of human cervical, lung, colorectal and melanoma cell lines carrying distinct HLA haplotypes demonstrates a consistent and profound effect of ERAP1 inhibition on the immunopeptidome. Novel and upregulated cancer associated antigens identified in association with multiple different HLA-A and B alleles stimulate IFNγ production in primary naive human T cell immunogenicity assays. The impact of ERAP1 inhibition on the T cell repertoire to the melanoma antigen tyrosinase is ongoing. The combination of ERAP1 inhibitor and anti PD-1 mAb led to significant tumor growth inhibition in the CT26 syngeneic mouse tumor model that correlated with increased infiltration of T cells to the tumor. Further PD end-points to be analysed include immune gene array and TCR Vbeta repertoire. Conclusions Grey Wolf ERAP1 inhibitors significantly modify the immunopeptidome both in vitro and in vivo across a broad range of HLA and tumor types. Combination of these inhibitors with anti PD-1 leads to significant T cell infiltration and tumor growth inhibition. Thus, ERAP1 mediated modulation of the immunopeptidome has the potential to drive anti tumor T cell responses and be a transformative immunotherapy. References Rizvi N, Hellmann MD, Snyder A, et al. Mutational landscape determines sensitivity to PD-1 blockade in non–small cell lung cancer. Science. 2015;348(6230):124–128. Chowell D, Morris LGT, Grigg CM, et al. Patient HLA class I genotype influences cancer response to checkpoint blockade immunotherapy. Science 2018;359 (6375):582–587. Chowell D, Krishna C, Pierini F, et al. Evolutionary divergence of HLA class I genotype impacts efficacy of cancer immunotherapy. Nature Medicine 2019;25(11):1715–1720. Shastri N, Nagarajan N, Lind KC, et al. Monitoring peptide processing for MHC class I molecules in the endoplasmic reticulum. Curr Opin Immunol 2014; 26:123–127. Mpakali A, Maben Z, Stern LJ, et al. Molecular pathways for antigenic peptide generation by ER aminopeptidase 1. Mol Immunol 2018; 13:50–57. James E, Bailey I, Sugiyarto G, et al. Induction of protective antitumor immunity through attenuation of ERAAP function. J Immunol 2013;190(11):5839–5846. Manguso RT, Pope HW, Zimmer MD, et al. In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target. Nature 2017;547(7664):413–418. Leonard, H Remtulla A, Poynton F, et al. AACR Annual Meeting 2020. Purcell AW, Ramarathinam SH, Ternette N. Mass spectrometry–based identification of MHC-bound peptides for immunopeptidomics. Nat Protoc 2019;14(6):1687–1707.
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- 2020
14. Authors' response to letter to the editor by Zhiqiang Wu, Jiazhang Wu, and Zhibin Lan
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Kristopher, Clark, Rebekah L, Goldstein, Jaime E, Hart, Merilee, Teylan, Antonio A, Lazzari, David R, Gagnon, Carlos G, Tun, and Eric, Garshick
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Humans ,Longitudinal Studies ,Vitamin D ,Spinal Cord Injuries - Published
- 2020
15. Plasma vitamin D, past chest illness, and risk of future chest illness in chronic spinal cord injury (SCI): a longitudinal observational study
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Rebekah L. Goldstein, Carlos G. Tun, Kristopher Clark, Antonio A. Lazzari, Eric Garshick, Jaime E. Hart, David R. Gagnon, and Merilee Teylan
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Adult ,Lung Diseases ,Male ,medicine.medical_specialty ,Article ,Risk Factors ,Internal medicine ,medicine ,Vitamin D and neurology ,Humans ,Longitudinal Studies ,Vitamin D ,Spinal cord injury ,Veterans Affairs ,Spinal Cord Injuries ,Aged ,business.industry ,Repeated measures design ,General Medicine ,Middle Aged ,medicine.disease ,Prognosis ,United States ,Pneumonia ,United States Department of Veterans Affairs ,Neurology ,Relative risk ,Cohort ,Chronic Disease ,Bronchitis ,Female ,Neurology (clinical) ,business - Abstract
STUDY DESIGN Observational study. OBJECTIVE Assess associations between vitamin D levels and other risk factors on future chest illness in a chronic spinal cord injury (SCI) cohort. SETTING Veterans Affairs Boston and the Boston, MA community. METHODS Between August 2009 and August 2017, 253 participants with chronic SCI were followed over a median of 3.2 years (up to 7.4 years) with two to four visits a median of 1.7 years apart. At each visit, plasma 25-hydroxyvitamin D level was obtained, spirometry performed, and a respiratory questionnaire assessing chest illnesses since last visit was completed. Repeated measures negative binomial regression was used to assess chest illness risk longitudinally. RESULTS At entry, 25% had deficient vitamin D levels (
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- 2020
16. Bronchial gene expression signature associated with rate of subsequent FEV
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Elizabeth J, Becker, Alen, Faiz, Maarten, van den Berge, Wim, Timens, Pieter S, Hiemstra, Kristopher, Clark, Gang, Liu, Xiaohui, Xiao, Yuriy O, Alekseyev, George, O'Connor, Stephen, Lam, Avrum, Spira, Marc E, Lenburg, and Katrina, Steiling
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Pulmonary Disease, Chronic Obstructive ,Forced Expiratory Volume ,Smoking ,Humans ,Bronchi ,Transcriptome ,Respiratory Function Tests - Abstract
COPD is characterised by progressive lung function decline. Leveraging prior work demonstrating bronchial airway COPD-associated gene expression alterations, we sought to determine if there are alterations associated with differences in the rate of FEVWe examined gene expression among ever smokers with and without COPD who at baseline had bronchial brushings profiled by Affymetrix microarrays and had longitudinal lung function measurements (n=134; mean follow-up=6.38±2.48 years). Gene expression profiles associated with the rate of FEVExpression differences in 171 genes were associated with rate of FEVWe have identified and replicated an airway gene expression signature associated with the rate of FEV
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- 2019
17. Abstract 1715: First in class inhibitors of ERAP1 have the potential to be a transformative immunotherapy in oncology
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Kristopher Clark, Jason Shiers, Fergus Poynton, Andrew Leishman, Camila de Almeida, Emma Reeves, Peter Joyce, Henry Leonard, Nicola Ternette, Carmen Tong, Edd James, Martin Quibell, and Elisa Lori
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Cancer Research ,Class (computer programming) ,Psychotherapist ,Transformative learning ,Oncology ,medicine.medical_treatment ,medicine ,Immunotherapy ,Psychology - Abstract
Clinical data demonstrates increased antigen presentation diversity is an important factor in determining response rates to checkpoint inhibitors. In addition to tumor mutational burden, increased HLA heterozygosity and HLA evolutionary diversity are non-overlapping factors which further diversify the immunopeptidome and improve clinical response to checkpoint therapies. Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an enzyme that trims peptides loaded into classical and nonclassical MHC Class I molecules. Human genome-wide association studies have identified single nucleotide polymorphisms within ERAP1 that are associated with immune-related diseases, such as ankylosing spondylitis, providing human genetic validation for ERAP1's role in human disease and antigen presentation. Further, ablation of mouse ERAAP modifies the immunopeptidome, resulting in improved immunogenicity, generation of CD8+ T cell responses and tumor growth inhibition. Grey Wolf Therapeutics have developed highly potent and selective ERAP1 inhibitors. These inhibitors demonstrate significant modulation of the cancer-related antigen repertoire across diverse ERAP1 and HLA genotypes and cancer-type backgrounds, both in vitro and in vivo. These changes in the antigen repertoire drive changes in T cell activation and response, leading to increased T cell infiltration into CT26 syngeneic tumors and T cell receptor (TCR) diversification when combined with anti-PD-1. We have identified immune related markers that are modulated following ERAP1 inhibition in syngeneic tumor models which have the potential to be used as biomarkers. Importantly, ERAP1 inhibitor induced immunopeptidome and T cell changes lead to significant tumor growth inhibition in syngeneic mouse models when combined with anti-PD-1. In parallel, we have demonstrated the ability of ERAP1 inhibitor induced novel cancer associated antigens to stimulate human CD8+ T cell responses. Extensive assessment of the potential of ERAP1 inhibitors to enhance tumor immune responses in combination with additional therapies (e.g. chemotherapy and radiotherapy), across different tumor microenvironments, is ongoing. These data provide the foundation from which we plan to explore the potential of our first-in-class ERAP1 inhibitor development candidate in the clinic. Citation Format: Andrew Leishman, Fergus Poynton, Nicola Ternette, Elisa Lori, Camila de Almeida, Henry Leonard, Emma Reeves, Edd James, Kristopher Clark, Carmen Tong, Jason Shiers, Martin Quibell, Peter Ian Joyce. First in class inhibitors of ERAP1 have the potential to be a transformative immunotherapy in oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1715.
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- 2021
18. Feasibility, Acceptability, and Adoption of an Inpatient Tobacco Treatment Service at a Safety-Net Hospital: A Mixed-Methods Study
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Kristopher Clark, Charles O’Donnell, Hasmeena Kathuria, Renda Soylemez Wiener, Nicole Herbst, Bhavna Seth, Carmel Fitzgerald, Carolina Wong, Katia Oleinik, and Eric D. Helm
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Pulmonary and Respiratory Medicine ,Adult ,Male ,medicine.medical_specialty ,Referral ,Adolescent ,Best practice ,medicine.medical_treatment ,Safety net ,03 medical and health sciences ,Young Adult ,0302 clinical medicine ,Pregnancy ,Intervention (counseling) ,Adaptation, Psychological ,medicine ,Electronic Health Records ,Humans ,030212 general & internal medicine ,Medical prescription ,Aged ,Service (business) ,Aged, 80 and over ,business.industry ,Smoking ,Middle Aged ,Patient Acceptance of Health Care ,Nicotine replacement therapy ,Hospitalization ,030228 respiratory system ,Massachusetts ,Family medicine ,Smoking cessation ,Feasibility Studies ,Female ,Smoking Cessation ,business ,Safety-net Providers - Abstract
Rationale: Hospitalization is an opportunity to engage smokers who may not seek tobacco treatment. Our safety-net hospital developed and implemented an inpatient intervention consisting of an "opt-out" electronic health record-based Best Practice Alert (BPA)+order-set, designed to trigger referral to the Tobacco Treatment Consult (TTC) service (a team staffed by tobacco treatment specialists) for all hospitalized smokers, regardless of motivation to quit.Objectives: We performed a sequential explanatory mixed-methods study to evaluate the feasibility, acceptability, and adoption of the TTC service.Methods: Among all admissions of adult "current smokers" between July 2016 and June 2017, we calculated the percentage of patients whose clinicians accepted the order-set (through a simple "order" click), thus generating the TTC referral. We then determined the extent of clinician follow-through of TTC recommendations for prescribing nicotine replacement therapy among 1,651 consecutive smokers seen by the TTC service. Finally, we conducted qualitative interviews with inpatient clinicians (n = 25) to understand their rationale for adoption or nonuse of the TTC intervention, including perceived usefulness, barriers to adoption, and strategies to improve the utility of the service.Results: Clinicians accepted the TTC order-set for 4,100 out of 6,598 "current smokers" (62.1%) for whom the BPA fired, typically after initially deferring the BPA. Rates of acceptance significantly differed across clinical services (range: 8% [obstetrics-gynecology] to 82.2% [cardiology]; P < 0.00001). A chart review showed that 43.5% (719/1,651) of the patients seen by the TTC service desired outpatient nicotine replacement therapy, but only half of these patients (48.8%; 351/719) received a discharge prescription from the inpatient team. Clinicians expressed that they valued the TTC service, but that BPA fatigue, time constraints, competing priorities, and poor communication with the TTC service were barriers to using the service and following recommendations. Clinicians suggested strategies to address barriers to the use of tobacco treatment interventions during hospitalization and after discharge.Conclusions: Implementing a large-scale "opt-out" tobacco treatment service for hospitalized smokers at a safety-net hospital is feasible and acceptable, but suffers from inconsistent adoption due to a variety of clinician barriers. System-level changes are needed to increase uptake and sustain inpatient tobacco treatment interventions to promote smoking cessation.
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- 2019
19. PSM Peptides of Staphylococcus aureus Activate the p38–CREB Pathway in Dendritic Cells, Thereby Modulating Cytokine Production and T Cell Priming
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Hubert Kalbacher, Katja Schenke-Layland, Manina Günter, Kristopher Clark, Dorothee Kretschmer, Jens Schreiner, Stella E. Autenrieth, Nicole S. Armbruster, Jennifer R. Richardson, Juliane Klenk, Simone Pöschel, and Publica
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0301 basic medicine ,MAPK/ERK pathway ,Staphylococcus aureus ,MAP Kinase Signaling System ,T-Lymphocytes ,medicine.medical_treatment ,T cell ,Bacterial Toxins ,Immunology ,Priming (immunology) ,Biology ,Lymphocyte Activation ,Formyl peptide receptor 2 ,Proinflammatory cytokine ,Mice ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Animals ,Immunology and Allergy ,Cyclic AMP Response Element-Binding Protein ,Immune Evasion ,Dendritic Cells ,Staphylococcal Infections ,Flow Cytometry ,Cell biology ,Mice, Inbred C57BL ,Disease Models, Animal ,TLR2 ,030104 developmental biology ,Cytokine ,medicine.anatomical_structure ,Biochemistry ,Cytokines ,Female ,Signal transduction ,Peptides ,030215 immunology - Abstract
The challenging human pathogen Staphylococcus aureus has highly efficient immune evasion strategies for causing a wide range of diseases, from skin and soft tissue to life-threatening infections. Phenol-soluble modulin (PSM) peptides are major pathogenicity factors of community-associated methicillin-resistant S. aureus strains. In previous work, we demonstrated that PSMs in combination with TLR2 ligand from S. aureus induce tolerogenic dendritic cells (DCs) characterized by the production of high amounts of IL-10, but no proinflammatory cytokines. This in turn promotes the activation of regulatory T cells while impairing Th1 response; however, the signaling pathways modulated by PSMs remain elusive. In this study, we analyzed the effects of PSMs on signaling pathway modulation downstream of TLR2. TLR2 stimulation in combination with PSMα3 led to increased and prolonged phosphorylation of NF-κB, ERK, p38, and CREB in mouse bone marrow–derived DCs compared with single TLR2 activation. Furthermore, inhibition of p38 and downstream MSK1 prevented IL-10 production, which in turn reduced the capacity of DCs to activate regulatory T cells. Interestingly, the modulation of the signaling pathways by PSMs was independent of the known receptor for PSMs, as shown by experiments with DCs lacking the formyl peptide receptor 2. Instead, PSMs penetrate the cell membrane most likely by transient pore formation. Moreover, colocalization of PSMs and p38 was observed near the plasma membrane in the cytosol, indicating a direct interaction. Thus, PSMs from S. aureus directly modulate the signaling pathway p38–CREB in DCs, thereby impairing cytokine production and in consequence T cell priming to increase the tolerance toward the pathogen.
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- 2016
20. Abstract 5551: Potent oral ERAP1 inhibitors modify the immunopeptidome in vivo and are novel immunotherapy agents
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Carmen Tong, Lesley Young, Henry Leonard, Camila de Almeida, Peter Joyce, Michael J. Ford, Kristopher Clark, Jason Shiers, Martin Quibell, Jamie Ware, Nicola Ternette, Fergus Poynton, Edd James, Emma Reeves, Elisa Lori, Alihussein Remtulla, and Patrick McIntyre
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Cancer Research ,biology ,medicine.medical_treatment ,T cell ,Antigen presentation ,Immunotherapy ,Major histocompatibility complex ,Immune checkpoint ,Immune system ,medicine.anatomical_structure ,Oncology ,Antigen ,Cancer research ,medicine ,biology.protein ,Cytotoxic T cell - Abstract
Immune checkpoint therapy has changed the cancer treatment paradigm, however the majority of patients respond poorly to T cell checkpoint blockade alone. Emerging evidence suggests factors that increase the diversity of the immunopeptidome such as high tumor mutation burden and HLA-I evolutionary divergence (HED), improve clinical response to checkpoint blockade providing an impetus to develop strategies that increase the presentation of cancer associated antigens to the immune system. Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an aminopeptidase that trims peptides loaded into classical and nonclassical MHC molecules. Human genome-wide association studies have identified single nucleotide polymorphisms within ERAP1 that are associated with immune-related diseases, such as ankylosing spondylitis, providing human genetic validation for ERAP1's role in human disease and antigen presentation. Furthermore, in mice, genetic ablation, reduction or pharmacological inhibition of ERAP1 changes the immune repertoire, creating novel antigens with improved immunogenicity that lead to the generation of productive CD8 T cell responses and tumor growth inhibition in various syngeneic models. We have generated and profiled highly potent, species cross reactive and selective ERAP1 inhibitors through a suite of in vitro and in vivo assays to develop these as novel immunotherapy agents. In vitro CETSA measurements show potent cellular target engagement that correlates with in vitro SPR and enzyme potency. Using the model antigen SIINFEKL and Class I MHC expression as key pharmacodynamic measurements, we demonstrate Class I modulation in vitro and in vivo in line with published data from ERAP1 knockout mice. These assays are being used to elucidate the kinetics and temporal dynamics of the antigen change to optimise dosing schedules for in vivo studies. Implementation of our immunopeptidomics pipeline has demonstrated treatment with ERAP1 inhibitors both generate novel antigens in vitro and in vivo and lead to profound and consistent increased overall immunopeptidome length in mouse and human cells. To establish the functional consequence of these changes, we developed a human primary T cell immunogenicity platform. The immunogenic potential of these novel cancer associated antigens has been confirmed by the ability to stimulate IFNγ production in naïve T cells and suggests responses to these antigens could reinvigorate anti-tumor responses. Extensive assessment of mouse and human CD8 T cells responses is ongoing, in order to characterise and select Grey Wolf Therapeutics' first lead ERAP1 inhibitor for use as monotherapy or in combination with other immunotherapies such as checkpoint blockade. Citation Format: Jamie Ware, Patrick McIntyre, Kristopher Clark, Carmen Tong, Jason Shiers, Elisa Lori, Camila de Almeida, Emma Reeves, Henry Leonard, Alihussein Remtulla, Michael Ford, Nicola Ternette, Fergus Poynton, Edd James, Lesley Young, Martin Quibell, Peter I. Joyce. Potent oral ERAP1 inhibitors modify the immunopeptidome in vivo and are novel immunotherapy agents [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5551.
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- 2020
21. Authors’ response to letter to the editor by Zhiqiang Wu, Jiazhang Wu, and Zhibin Lan
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Carlos G. Tun, Antonio A. Lazzari, Merilee Teylan, Jaime E. Hart, Eric Garshick, Kristopher Clark, David R. Gagnon, and Rebekah L. Goldstein
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Letter to the editor ,Neurology ,business.industry ,Medicine ,Neurology (clinical) ,General Medicine ,Theology ,business - Published
- 2020
22. Correction: Plasma vitamin D, past chest illness, and risk of future chest illness in chronic spinal cord injury (SCI): a longitudinal observational study
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Kristopher Clark, Rebekah L. Goldstein, Jaime E. Hart, Merilee Teylan, Antonio A. Lazzari, David R. Gagnon, Carlos G. Tun, and Eric Garshick
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Neurology ,Neurology (clinical) ,General Medicine - Published
- 2020
23. The NEDD8 E3 ligase DCNL5 is phosphorylated by IKK alpha during Toll-like receptor activation
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Jesse Rinehart, Kristopher Clark, Daniel C. Scott, Thimo Kurz, Philip Cohen, Yann Thomas, and Yosua Adi Kristariyanto
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0301 basic medicine ,NEDD8 ,Biochemistry ,Mice ,White Blood Cells ,Cell Signaling ,Animal Cells ,Medicine and Health Sciences ,Serine ,Small interfering RNAs ,Membrane Receptor Signaling ,Peptide Synthases ,Phosphorylation ,Post-Translational Modification ,Amino Acids ,Oncogene Proteins ,Toll-like receptor ,Multidisciplinary ,biology ,Kinase ,Chemistry ,Organic Compounds ,Immune Receptor Signaling ,Ubiquitin ligase ,Cell biology ,I-kappa B Kinase ,Precipitation Techniques ,Nucleic acids ,Physical Sciences ,Medicine ,Signal transduction ,Cellular Types ,Cullin ,Signal Transduction ,Research Article ,NEDD8 Protein ,Immune Cells ,Science ,Immunology ,Immunoblotting ,Molecular Probe Techniques ,Research and Analysis Methods ,03 medical and health sciences ,Hydroxyl Amino Acids ,Genetics ,Animals ,Humans ,Immunoprecipitation ,Non-coding RNA ,Molecular Biology Techniques ,Molecular Biology ,Blood Cells ,030102 biochemistry & molecular biology ,Macrophages ,Organic Chemistry ,I-Kappa-B Kinase ,Chemical Compounds ,Biology and Life Sciences ,Proteins ,Cell Biology ,Immunity, Innate ,Gene regulation ,030104 developmental biology ,HEK293 Cells ,RAW 264.7 Cells ,biology.protein ,RNA ,Neddylation ,Gene expression - Abstract
The activity of Cullin-RING ubiquitin E3 ligases (CRL) is regulated by NEDD8 modification. DCN-like proteins promote Cullin neddylation as scaffold-like E3s. One DCNL, DCNL5, is highly expressed in immune tissue. Here, we provide evidence that DCNL5 may be involved in innate immunity, as it is a direct substrate of the kinase IKKα during immune signalling. We find that upon activation of Toll-like receptors, DCNL5 gets rapidly and transiently phosphorylated on a specific N-terminal serine residue (S41). This phosphorylation event is specifically mediated by IKKα and not IKKβ. Our data for the first time provides evidence that DCNL proteins are post-translationally modified in an inducible manner. Our findings also provide the first example of a DCNL member as a kinase substrate in a signalling pathway, indicating that the activity of at least some DCNLs may be regulated.
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- 2018
24. An unexpected twist to the activation of IKKβ: TAK1 primes IKKβ for activation by autophosphorylation
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Jiazhen, Zhang, Kristopher, Clark, Toby, Lawrence, Mark W, Peggie, and Philip, Cohen
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IL-1, interleukin-1 ,TNF, tumour necrosis factor ,linear ubiquitin chain assembly complex (LUBAC) ,M-CSF, macrophage colony-stimulating factor ,Mice ,Serine ,TRAF, TNF receptor-associated factor ,nuclear factor κB (NF-κB) ,PP1γ, protein phosphatase 1γ ,Gene Knock-In Techniques ,Phosphorylation ,IKK, IκB kinase ,Cells, Cultured ,IκB, inhibitor of NF-κB ,Intracellular Signaling Peptides and Proteins ,interleukin-1 (IL-1) ,MAP Kinase Kinase Kinases ,Recombinant Proteins ,BMDM, bone-marrow-derived macrophage ,I-kappa B Kinase ,JNK, c-Jun N-terminal kinase ,LPS, lipopolysaccharide ,HA, haemaglutinnin ,NF-κB, nuclear factor κB ,TLR, Toll-like receptor ,Mice, Transgenic ,Accelerated Publication ,MKK, MAPK kinase ,HEK, human embryonic kidney ,Animals ,Humans ,Protein Interaction Domains and Motifs ,TAK1, transforming growth factor β-activated kinase-1 ,Protein Kinase Inhibitors ,inhibitor of nuclear factor κB kinase (IKK) ,E, embryonic day ,HOIP, HOIL1 [haem-oxidized IRP2 (iron regulatory protein 2) ubiquitin ligase 1]-interacting protein ,TAB, TAK1-binding protein ,NEMO, NF-κB essential modulator ,Ubiquitination ,Embryo, Mammalian ,MEF, mouse embryonic fibroblast ,Enzyme Activation ,HEK293 Cells ,Amino Acid Substitution ,transforming growth factor β-activated kinase-1 (TAK1) ,LUBAC, linear ubiquitin chain assembly complex ,Mutant Proteins ,Protein Processing, Post-Translational ,MAPK, mitogen-activated protein kinase - Abstract
IKKβ {IκB [inhibitor of NF-κB (nuclear factor κB)] kinase β} is required to activate the transcription factor NF-κB, but how IKKβ itself is activated in vivo is still unclear. It was found to require phosphorylation by one or more ‘upstream’ protein kinases in some reports, but by autophosphorylation in others. In the present study, we resolve this contro-versy by demonstrating that the activation of IKKβ induced by IL-1 (interleukin-1) or TNF (tumour necrosis factor) in embryonic fibroblasts, or by ligands that activate Toll-like receptors in macrophages, requires two distinct phosphorylation events: first, the TAK1 [TGFβ (transforming growth factor β)-activated kinase-1]-catalysed phosphorylation of Ser177 and, secondly, the IKKβ-catalysed autophosphorylation of Ser181. The phosphorylation of Ser177 by TAK1 is a priming event required for the subsequent autophosphorylation of Ser181, which enables IKKβ to phosphorylate exogenous substrates. We also provide genetic evidence which indicates that the IL-1-stimulated, LUBAC (linear ubiquitin chain assembly complex)-catalysed formation of linear ubiquitin chains and their interaction with the NEMO (NF-κB essential modulator) component of the canonical IKK complex permits the TAK1-catalysed priming phosphorylation of IKKβ at Ser177 and IKKα at Ser176. These findings may be of general significance for the activation of other protein kinases., We have discovered how a key enzyme that controls the immune system is switched on during infection by bacteria and viruses. Known by the acronym IKKβ, it triggers the production of many proteins that are needed to combat these pathogens.
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- 2014
25. Protein kinase networks that limit TLR signalling
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Kristopher Clark
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Feedback, Physiological ,MAP kinase kinase kinase ,Toll-Like Receptors ,Receptor Cross-Talk ,Biology ,Mitogen-activated protein kinase kinase ,Biochemistry ,Protein kinase R ,Immunity, Innate ,MAP2K7 ,TANK-binding kinase 1 ,Immunology ,Animals ,Cytokines ,Humans ,ASK1 ,Cyclin-dependent kinase 9 ,Phosphorylation ,CHUK ,Protein Kinases ,Protein Processing, Post-Translational ,Signal Transduction - Abstract
TLRs (Toll-like receptors) detect invading micro-organisms which triggers the production of pro-inflammatory mediators needed to combat infection. Although these signalling networks are required to protect the host against invading pathogens, dysregulation of TLR pathways contributes to the development of chronic inflammatory diseases and autoimmune disorders. Molecular mechanisms have therefore evolved to restrict the strength of TLR signalling. In the present review, I highlight recent advances in our understanding of the protein kinase networks required to suppress the innate immune response by negatively regulating TLR signalling and/or promoting the secretion of anti-inflammatory cytokines. I present my discoveries on the key roles of the IKK (inhibitor of nuclear factor κB kinase)-related kinases and the SIKs (salt-inducible kinases) in limiting innate immunity within the greater context of the field.
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- 2014
26. Molecular control of the NEMO family of ubiquitin-binding proteins
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Philip Cohen, Sambit K. Nanda, and Kristopher Clark
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congenital, hereditary, and neonatal diseases and abnormalities ,Cell signaling ,Ubiquitin binding ,macromolecular substances ,Plasma protein binding ,environment and public health ,Ubiquitin ,Humans ,Phosphorylation ,Polyubiquitin ,skin and connective tissue diseases ,Molecular Biology ,Innate immune system ,biology ,Tumor Necrosis Factor-alpha ,Kinase ,NF-kappa B ,Cell Biology ,Immunity, Innate ,I-kappa B Kinase ,Cell biology ,biology.protein ,Signal transduction ,Protein Binding ,Signal Transduction - Abstract
Research over the past decade has revealed how NF-κB essential modulator (NEMO; also known as IKKγ) regulates the IKKα-IKKβ signalling axis in the innate immune system. The discovery that NEMO is a polyubiquitin-binding protein and that the IKK complex is modulated by other protein kinases that are themselves controlled by polyubiquitin chains has provided a deeper molecular understanding of the non-degradative roles of ubiquitylation. New mechanistic insights of NEMO and related polyubiquitin-binding proteins have become a paradigm for how the interplay between phosphorylation and ubiquitylation controls cell signalling networks in health and disease.
- Published
- 2013
27. Inhibition of SIK2 and SIK3 during differentiation enhances the anti-inflammatory phenotype of macrophages
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Nicola J, Darling, Rachel, Toth, J Simon C, Arthur, and Kristopher, Clark
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salt-inducible kinase ,Primary Cell Culture ,HG-9-91-01 ,Gene Expression ,Mice, Transgenic ,macrophage ,Protein Serine-Threonine Kinases ,Mice ,Animals ,Gene Knock-In Techniques ,Transgenes ,Protein Kinase Inhibitors ,Research Articles ,Interleukin-12 Subunit p40 ,Interleukin-6 ,Tumor Necrosis Factor-alpha ,Macrophages ,Phenylurea Compounds ,Cell Differentiation ,Immunity, Innate ,Interleukin-10 ,Mice, Inbred C57BL ,Phenotype ,Pyrimidines ,inflammation ,TNF-α ,Indans ,IL-10 ,Protein Kinases ,Research Article - Abstract
The salt-inducible kinases (SIKs) control a novel molecular switch regulating macrophage polarization. Pharmacological inhibition of the SIKs induces a macrophage phenotype characterized by the secretion of high levels of anti-inflammatory cytokines, including interleukin (IL)-10, and the secretion of very low levels of pro-inflammatory cytokines, such as tumour necrosis factor α. The SIKs, therefore, represent attractive new drug targets for the treatment of macrophage-driven diseases, but which of the three isoforms, SIK1, SIK2 or SIK3, would be appropriate to target remains unknown. To address this question, we developed knock-in (KI) mice for SIK1, SIK2 and SIK3, in which we introduced a mutation that renders the enzymes catalytically inactive. Characterization of primary macrophages from the single and double KI mice established that all three SIK isoforms, and in particular SIK2 and SIK3, contribute to macrophage polarization. Moreover, we discovered that inhibition of SIK2 and SIK3 during macrophage differentiation greatly enhanced the production of IL-10 compared with their inhibition in mature macrophages. Interestingly, macrophages differentiated in the presence of SIK inhibitors, MRT199665 and HG-9-91-01, still produced very large amounts of IL-10, but very low levels of pro-inflammatory cytokines, even after the SIKs had been reactivated by removal of the drugs. Our data highlight an integral role for SIK2 and SIK3 in innate immunity by preventing the differentiation of macrophages into a potent and stable anti-inflammatory phenotype.
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- 2016
28. The TRPM7 interactome defines a cytoskeletal complex linked to neuroblastoma progression
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Edwin Lasonder, Kirsten Vrenken, Jeroen Middelbeek, Kristopher Clark, Frank N. van Leeuwen, Daan Visser, Jan Koster, Kees Jalink, and Oncogenomics
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0301 basic medicine ,Histology ,Cancer development and immune defence Radboud Institute for Molecular Life Sciences [Radboudumc 2] ,TRPM Cation Channels ,Protein Serine-Threonine Kinases ,Biology ,Pathology and Forensic Medicine ,Mice ,Neuroblastoma ,03 medical and health sciences ,Cell Line, Tumor ,Databases, Genetic ,medicine ,Animals ,Humans ,Progenitor cell ,Cytoskeleton ,Cell adhesion ,Cell growth ,C100 ,Neural crest ,Actomyosin ,Cell Biology ,General Medicine ,medicine.disease ,Embryonic stem cell ,C900 ,Neoplasm Proteins ,Cell biology ,Crosstalk (biology) ,030104 developmental biology - Abstract
Item does not contain fulltext Neuroblastoma is the second-most common solid tumor in children and originates from poorly differentiated neural crest-derived progenitors. Although most advanced stage metastatic neuroblastoma patients initially respond to treatment, a therapy resistant pool of poorly differentiated cells frequently arises, leading to refractory disease. A lack of insight into the molecular mechanisms that underlie neuroblastoma progression hampers the development of effective new therapies for these patients. Normal neural crest development and maturation is guided by physical interactions between the cell and its surroundings, in addition to soluble factors such as growth factors. This mechanical crosstalk is mediated by actin-based adhesion structures and cell protrusions that probe the cellular environment to modulate migration, proliferation, survival and differentiation. Whereas such signals preserve cellular quiescence in non-malignant cells, perturbed adhesion signaling promotes de-differentiation, uncontrolled cell proliferation, tissue invasion and therapy resistance. We previously reported that high expression levels of the channel-kinase TRPM7, a protein that maintains the progenitor state of embryonic neural crest cells, are closely associated with progenitor-like features of tumor cells, accompanied by extensive cytoskeletal reorganization and adhesion remodeling. To define mechanisms by which TRPM7 may contribute to neuroblastoma progression, we applied a proteomics approach to identify TRPM7 interacting proteins. We show that TRPM7 is part of a large complex of proteins, many of which function in cytoskeletal organization, cell protrusion formation and adhesion dynamics. Expression of a subset of these TRPM7 interacting proteins strongly correlates with neuroblastoma progression in independent neuroblastoma patient datasets. Thus, TRPM7 is part of a large cytoskeletal complex that may affect the malignant potential of tumor cells by regulating actomyosin dynamics and cell-matrix interactions.
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- 2016
29. Phosphorylation of CRTC3 by the salt-inducible kinases controls the interconversion of classically activated and regulatory macrophages
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Patrick G. A. Pedrioli, Lorna Plater, J. Simon C. Arthur, Mark Peggie, Kasparas Petkevicius, Hwan Geun Choi, Nathanael S. Gray, Jiazhen Zhang, Ed McIver, Kristopher Clark, Kirsty F. MacKenzie, Yosua Adi Kristariyanto, and Philip Cohen
- Subjects
Proteomics ,DNA, Complementary ,Magnetic Resonance Spectroscopy ,Morpholines ,Immunoblotting ,Fluorescent Antibody Technique ,Inflammation ,Protein Serine-Threonine Kinases ,Biology ,CREB ,Polymerase Chain Reaction ,Regulatory macrophages ,Cell Line ,Proinflammatory cytokine ,Mice ,medicine ,Animals ,Phosphorylation ,Transcription factor ,DNA Primers ,Mice, Knockout ,Analysis of Variance ,Multidisciplinary ,Molecular Structure ,Kinase ,Macrophages ,Phenylurea Compounds ,Biological Sciences ,Molecular biology ,Interleukin-10 ,Cell biology ,Interleukin 10 ,Pyrimidines ,Indans ,biology.protein ,Cytokines ,RNA Interference ,medicine.symptom ,Cyclobutanes ,Transcription Factors - Abstract
Macrophages acquire strikingly different properties that enable them to play key roles during the initiation, propagation, and resolution of inflammation. Classically activated (M1) macrophages produce proinflammatory mediators to combat invading pathogens and respond to tissue damage in the host, whereas regulatory macrophages (M2b) produce high levels of anti-inflammatory molecules, such as IL-10, and low levels of proinflammatory cytokines, like IL-12, and are important for the resolution of inflammatory responses. A central problem in this area is to understand how the formation of regulatory macrophages can be promoted at sites of inflammation to prevent and/or alleviate chronic inflammatory and autoimmune diseases. Here, we demonstrate that the salt-inducible kinases (SIKs) restrict the formation of regulatory macrophages and that their inhibition induces striking increases in many of the characteristic markers of regulatory macrophages, greatly stimulating the production of IL-10 and other anti-inflammatory molecules. We show that SIK inhibitors elevate IL-10 production by inducing the dephosphorylation of cAMP response element-binding protein (CREB)-regulated transcriptional coactivator (CRTC) 3, its dissociation from 14-3-3 proteins and its translocation to the nucleus where it enhances a gene transcription program controlled by CREB. Importantly, the effects of SIK inhibitors on IL-10 production are lost in macrophages that express a drug-resistant mutant of SIK2. These findings identify SIKs as a key molecular switch whose inhibition reprograms macrophages to an anti-inflammatory phenotype. The remarkable effects of SIK inhibitors on macrophage function suggest that drugs that target these protein kinases may have therapeutic potential for the treatment of inflammatory and autoimmune diseases.
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- 2012
30. Abstract 4834: CT7001: An orally bioavailable CDK7 inhibitor is a potential therapy for breast, small-cell lung and haematological cancers
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Stuart Thomson, Andrew Leishman, Bo Li, Elaine Sullivan, William M. Gallagher, Ash Bahl, Adam Peall, Raoul Charles Coombes, Edward K. Ainscow, Simak Ali, and Kristopher Clark
- Subjects
Cancer Research ,Programmed cell death ,biology ,Cell growth ,business.industry ,Cell ,Cell cycle ,medicine.disease ,medicine.anatomical_structure ,Oncology ,Cyclin-dependent kinase ,Apoptosis ,Cancer cell ,medicine ,biology.protein ,Cancer research ,Lung cancer ,business - Abstract
CDK7 inhibition has emerged as a promising strategy in a range of cancer indications. CDK7 acts as a regulator of transcription through its role in phosphorylating and activating RNA Polymerase II (polII) as well as a master regulator of the cell cycle through phosphorylation of members of the CDK family. Inhibition of CDK7 in cancer cells has been shown to be effective in blocking transcriptional addiction to a defined cluster of genes (Wang et al 2015) and leads to decreased cell cycling, a decrease in the levels of proto-oncogenes such as c-Myc and induction of apoptosis. Here we describe the properties of a potent, selective and orally bioavailable CDK7 inhibitor CT7001 (formerly known as ICEC0942). Profiling across a pan-cancer cell line panel showed that CT7001 has anti-proliferative activity in a range of cancer types including acute myeloid leukaemia (AML), small cell lung (SCL) and hormone sensitive and triple negative breast cancers (TNBC).Mechanistic investigations revealed that CT7001s activity was associated with both a decrease in cell proliferation and an induction of apoptosis. Inhibition of cell proliferation was commensurate with a decrease in the levels of c-Myc, Mcl-1 and phospho-Pol II as determined by Western Blot analysis.CT7001 was then evaluated in a range of pre-clinical cancer models. CT7001 was shown to lead to cell death in a panel of patient derived models of small-cell lung cancer (SCLC) in 3D spheroid assays. In an in vivo orthotopic-patient derived xenograft model of TNBC in nu/nu mice, orally administered CT7001 monotherapy produced strong and sustained regression of the tumour that persisted during the dosing schedule, and strong suppression was still maintained upon cessation of treatment. At doses that produced regression CT7001 was well tolerated with little effect on body weight ( Citation Format: Edward K. Ainscow, Andrew Leishman, Elaine Sullivan, Bo Li, William Gallagher, Adam Peall, Kristopher Clark, Stuart Thomson, Simak Ali, Raoul C. Coombes, Ash Bahl. CT7001: An orally bioavailable CDK7 inhibitor is a potential therapy for breast, small-cell lung and haematological cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4834.
- Published
- 2018
31. Use of the Pharmacological Inhibitor BX795 to Study the Regulation and Physiological Roles of TBK1 and IκB Kinase ϵ
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Lorna Plater, Philip Cohen, Mark Peggie, and Kristopher Clark
- Subjects
Kinase ,Autophosphorylation ,I-Kappa-B Kinase ,Cell Biology ,IκB kinase ,Biology ,environment and public health ,Biochemistry ,Molecular biology ,enzymes and coenzymes (carbohydrates) ,TANK-binding kinase 1 ,Phosphorylation ,Signal transduction ,Protein kinase A ,Molecular Biology - Abstract
TANK-binding kinase 1 (TBK1) and IκB kinase e (IKKe) regulate the production of Type 1 interferons during bacterial and viral infection, but the lack of useful pharmacological inhibitors has hampered progress in identifying additional physiological roles of these protein kinases and how they are regulated. Here we demonstrate that BX795, a potent and relatively specific inhibitor of TBK1 and IKKe, blocked the phosphorylation, nuclear translocation, and transcriptional activity of interferon regulatory factor 3 and, hence, the production of interferon-β in macrophages stimulated with poly(I:C) or lipopolysaccharide (LPS). In contrast, BX795 had no effect on the canonical NFκB signaling pathway. Although BX795 blocked the autophosphorylation of overexpressed TBK1 and IKKe at Ser-172 and, hence, the autoactivation of these protein kinases, it did not inhibit the phosphorylation of endogenous TBK1 and IKKe at Ser-172 in response to LPS, poly(I:C), interleukin-1α (IL-1α), or tumor necrosis factor α and actually enhanced the LPS, poly(I:C), and IL-1α-stimulated phosphorylation of this residue. These results demonstrate that the phosphorylation of Ser-172 and the activation of TBK1 and IKKe are catalyzed by a distinct protein kinase(s) in vivo and that TBK1 and IKKe control a feedback loop that limits their activation by LPS, poly(I:C) and IL-1α (but not tumor necrosis factor α) to prevent the hyperactivation of these enzymes.
- Published
- 2009
32. Eosinophil degranulation in the allergic lung of mice primarily occurs in the airway lumen
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Paul S. Foster, Joerg Mattes, Klaus I. Matthaei, Nicole Newcombe, Lindsay A. Dent, Nancy A. Lee, Ljubov Simson, James J. Lee, Aulikki Koskinen, and Kristopher Clark
- Subjects
Male ,Pathology ,medicine.medical_specialty ,Eosinophil Peroxidase ,Ovalbumin ,Immunology ,Mice, Transgenic ,Respiratory Mucosa ,Cytoplasmic Granules ,Cell Degranulation ,Allergic inflammation ,Mice ,Bone Marrow ,Eosinophil activation ,Cell Adhesion ,Respiratory Hypersensitivity ,Animals ,Humans ,Immunology and Allergy ,Medicine ,Eosinophil degranulation ,Lung ,Mice, Inbred BALB C ,Eosinophil cationic protein ,biology ,business.industry ,Degranulation ,Immunotherapy, gene therapy and transplantation [UMCN 1.4] ,Cell Biology ,respiratory system ,Eosinophil ,Extracellular Matrix ,respiratory tract diseases ,Eosinophils ,medicine.anatomical_structure ,Peroxidases ,biology.protein ,Major basic protein ,Female ,Interleukin-5 ,business ,Bronchoalveolar Lavage Fluid ,Eosinophil peroxidase - Abstract
Eosinophil degranulation is thought to play a pivotal role in the pathogenesis of allergic disorders. Although mouse models of allergic disorders have been used extensively to identify the contribution of eosinophils to disease, ultrastructural evidence of active granule disassembly has not been reported. In this investigation, we characterized the degree of eosinophil activation in the bone marrow, blood, lung tissue, and airways lumen [bronchoalveolar lavage fluid (BALF)] of ovalbumin-sensitized and aero-challenged wild-type and interleukin-5 transgenic mice. Degranulation was most prominent in and primarily compartmentalized to the airways lumen. Eosinophils released granule proteins by the process of piecemeal degranulation (PMD). Accordingly, recruitment and activation of eosinophils in the lung correlated with the detection of cell-free eosinophil peroxidase in BALF and with the induction of airways hyper-reactivity. As in previous studies with human eosinophils, degranulation of isolated mouse cells did not occur until after adherence to extracellular matrix. However, higher concentrations of exogenous stimuli appear to be required to trigger adherence and degranulation (piecemeal) of mouse eosinophils when compared with values reported for studies of human eosinophils. Thus, mouse eosinophils undergo PMD during allergic inflammation, and in turn, this process may contribute to pathogenesis. However, the degranulation process in the allergic lung of mice is primarily compartmentalized to the airway lumen. Understanding the mechanism of eosinophil degranulation in the airway lumen may provide important insights into how this process occurs in human respiratory diseases.
- Published
- 2004
33. Mice deficient in the Rac activator Tiam1 are resistant to Ras-induced skin tumours
- Author
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Maarten van der Valk, John G. Collard, Angeliki Malliri, Rob A. van der Kammen, Frits Michiels, and Kristopher Clark
- Subjects
Skin Neoplasms ,Genotype ,9,10-Dimethyl-1,2-benzanthracene ,Gene Dosage ,Apoptosis ,GTPase ,Biology ,Metastasis ,Mice ,medicine ,Animals ,Guanine Nucleotide Exchange Factors ,T-Lymphoma Invasion and Metastasis-inducing Protein 1 ,Mice, Knockout ,Multidisciplinary ,Activator (genetics) ,TIAM1 Gene ,Proteins ,Actin cytoskeleton ,medicine.disease ,Immunohistochemistry ,In vitro ,Immunology ,Disease Progression ,ras Proteins ,Cancer research ,Keratins ,Tetradecanoylphorbol Acetate ,Guanine nucleotide exchange factor ,Cell Division - Abstract
Proteins of the Rho family control signalling pathways that regulate the actin cytoskeleton and gene transcription. In vitro studies have implicated Rho-like GTP-hydrolysing enzymes (GTPases) in cell migration, cell-cycle progression, and Ras-induced focus formation, suggesting a role for these GTPases in the formation and progression of tumours in vivo. To study this, we have generated mice lacking the Rac-specific activator Tiam1, a T-lymphoma invasion and metastasis inducing protein. Here we show that such Tiam1(-/-) mice are resistant to the development of Ras-induced skin tumours initiated with 7,12-dimethylbenzanthracene and promoted with 12-O-tetradecanoylphorbol-13-acetate. Moreover, the few tumours produced in Tiam1(-/-) mice grew much slower than did tumours in wild-type mice. Tiam1-deficient primary embryonic fibroblasts were also resistant to Ras(V12)-induced focus formation. Analysis of Tiam1 heterozygotes indicated that both tumour initiation and promotion were dependent on the Tiam1 gene dose. Tiam1 deficiency was associated with increased apoptosis during initiation, and with impeded proliferation during promotion. Although the number of tumours in Tiam1(-/-) mice was small, a greater proportion progressed to malignancy, suggesting that Tiam1 deficiency promotes malignant conversion. Our studies identify the Rac activator Tiam1 as a critical regulator of different aspects of Ras-induced tumour formation.
- Published
- 2002
34. Antiviral potential of chemokines
- Author
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Paul S. Foster, Surendran Mahalingam, Klaus I. Matthaei, and Kristopher Clark
- Subjects
Cytotoxicity, Immunologic ,CCR1 ,Chemokine ,biology ,Cell ,Antiviral Agents ,Models, Biological ,General Biochemistry, Genetics and Molecular Biology ,Chemokine receptor ,medicine.anatomical_structure ,Immune system ,Viral replication ,Virus Diseases ,Immunology ,Leukocytes ,biology.protein ,medicine ,Animals ,Humans ,Cytotoxic T cell ,Receptors, Chemokine ,Chemokines ,Receptor - Abstract
In the past few years, a large number of new chemokines (chemotactic cytokines) and chemokine receptors have been discovered. The growth in knowledge about these molecules has been achieved largely through advances in bioinformatics and the expansion of expression sequence tag (EST) databases. It is now clear that chemokines are crucial in controlling both the development and functioning of leukocytes and that their role is not restricted to cell attraction, as originally assumed. In particular, recent findings provide strong support for the idea that chemokines and their receptors are especially important in the control of viral infection and replication. Thus, specific chemokines are now known to enhance the cytotoxic activity of infected cells, thus inhibiting further virus replication. In addition, some chemokines orchestrate the recruitment of activated leukocytes to foci of infection to aid viral clearance. Viruses, in turn, have evolved various defences against chemokines. These range from the production of proteins that inhibit biological activity of the host chemokine to the hijacking of the chemokine system, whereby certain viruses utilize chemokine receptors for their entry. The latter viral defence can itself be blocked by chemokines. Altogether, these findings illustrate the central role of chemokines in many different phases of the immune response, particularly those aspects involving antiviral defence, a variety and versatility that was not fully appreciated even a few years ago.
- Published
- 2001
35. ZRP-1, a Zyxin-related Protein, Interacts with the Second PDZ Domain of the Cytosolic Protein Tyrosine Phosphatase hPTP1E
- Author
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Denis Banville, Kristopher Clark, Yves Fortin, Kishore K. Murthy, and Shi-Hsiang Shen
- Subjects
Proteasome Endopeptidase Complex ,DNA, Complementary ,Molecular Sequence Data ,Protein domain ,PDZ domain ,Protein Tyrosine Phosphatase, Non-Receptor Type 13 ,Biology ,Biochemistry ,Zyxin ,Cytosol ,pharmaceutical ,Humans ,Amino Acid Sequence ,Molecular Biology ,Adaptor Proteins, Signal Transducing ,LIM domain ,Binding Sites ,Base Sequence ,Sequence Homology, Amino Acid ,Cell Biology ,Protein phosphatase 2 ,LIM Domain Proteins ,Molecular biology ,Cell biology ,Lymphocyte cytosolic protein 2 ,GATAD2B ,ATPases Associated with Diverse Cellular Activities ,Protein Tyrosine Phosphatases ,Carrier Proteins ,LHX3 ,Transcription Factors - Abstract
Protein-protein interactions play an important role in the specificity of cellular signaling cascades. By using the yeast two-hybrid system, a specific interaction was identified between the second PDZ domain of the cytosolic protein tyrosine phosphatase hPTP1E and a novel protein, which was termed ZRP-1 to indicate its sequence similarity to the Zyxin protein family. The mRNA encoding this protein is distributed widely in human tissues and contains an open reading frame of 1428 base pairs, predicting a polypeptide of 476 amino acid residues. The deduced protein displays a prolinerich amino-terminal region and three double zinc finger LIM domains at its carboxyl terminus. The specific interaction of this novel protein with the second PDZ domain of hPTP1E was demonstrated both in vitro, using bacterially expressed proteins, and in vivo, by co-immunoprecipitation studies. Deletion analysis indicated that an intact carboxyl terminus is required for its interaction with the second PDZ domain of hPTP1E in the yeast two-hybrid system and suggested that other sequences, including the LIM domains, also participate in the interaction. The genomic organization of the ZRP-1 coding sequence is identical to that of the lipoma preferred partner gene, another Zyxin-related protein, suggesting that the two genes have evolved from a recent gene duplication event., UI - 99329089
- Published
- 1999
36. PGE2 Induces Macrophage IL-10 Production and a Regulatory-like Phenotype via a Protein Kinase A–SIK–CRTC3 Pathway
- Author
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Kirsty F. MacKenzie, Shaista Naqvi, Pierre C McCarthy, Gesa Nöehren, Patrick G. A. Pedrioli, Geoff J. Barton, Manikhandan Mudaliar, Yosua Adi Kristariyanto, Alan R. Prescott, Rachel Toth, Kristopher Clark, Victoria A. McGuire, J. Simon C. Arthur, Michael J. Pattison, and Mirjam W. M. Van Den Bosch
- Subjects
Transcription, Genetic ,p300-CBP coactivator family ,Immunology ,Protein Serine-Threonine Kinases ,CREB ,Dinoprostone ,Cell Line ,Immune Regulation ,Mice ,Sp3 transcription factor ,Coactivator ,Cyclic AMP ,Immunology and Allergy ,Animals ,RNA, Messenger ,Phosphorylation ,Transcription factor ,biology ,General transcription factor ,Macrophages ,Molecular biology ,Cyclic AMP-Dependent Protein Kinases ,Interleukin-10 ,Protein Transport ,Phenotype ,Transcription Coactivator ,TAF2 ,biology.protein ,Signal Transduction ,Transcription Factors - Abstract
The polarization of macrophages into a regulatory-like phenotype and the production of IL-10 plays an important role in the resolution of inflammation. We show in this study that PGE2, in combination with LPS, is able to promote an anti-inflammatory phenotype in macrophages characterized by high expression of IL-10 and the regulatory markers SPHK1 and LIGHT via a protein kinase A–dependent pathway. Both TLR agonists and PGE2 promote the phosphorylation of the transcription factor CREB on Ser133. However, although CREB regulates IL-10 transcription, the mutation of Ser133 to Ala in the endogenous CREB gene did not prevent the ability of PGE2 to promote IL-10 transcription. Instead, we demonstrate that protein kinase A regulates the phosphorylation of salt-inducible kinase 2 on Ser343, inhibiting its ability to phosphorylate CREB-regulated transcription coactivator 3 in cells. This in turn allows CREB-regulated transcription coactivator 3 to translocate to the nucleus where it serves as a coactivator with the transcription factor CREB to induce IL-10 transcription. In line with this, we find that either genetic or pharmacological inhibition of salt-inducible kinases mimics the effect of PGE2 on IL-10 production.
- Published
- 2013
37. Su1374 Correlation Between Worrisome Features and Pathology of Pancreatic Cyst in a Single Center
- Author
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Rajvinder Sidhu, Andrew Bain, Kristopher Clark, Amanpal Singh, and Sandeep Samuel
- Subjects
Pathology ,medicine.medical_specialty ,Hepatology ,business.industry ,Pancreatic cyst ,Gastroenterology ,Medicine ,business ,Single Center - Published
- 2016
38. Synthesis and structure-activity relationships of a novel series of pyrimidines as potent inhibitors of TBK1/IKKε kinases
- Author
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Ahmad Kamal, Jasveen Chugh, Michelle Newman, William Tsang, Debra L. Taylor, Edward G. McIver, Kristian Birchall, Philip Cohen, Alison Levy, Justin S. Bryans, Kristopher Clark, J. Simon C. Arthur, Joanne Osborne, Stephen John Lewis, Tom Drake, and Ela Smiljanic-Hurley
- Subjects
Cell Membrane Permeability ,Stereochemistry ,Clinical Biochemistry ,Anti-Inflammatory Agents ,Pharmaceutical Science ,Plasma protein binding ,Thiophenes ,Protein Serine-Threonine Kinases ,Biochemistry ,Mice ,Structure-Activity Relationship ,Protein structure ,TANK-binding kinase 1 ,Interferon β ,In vivo ,Drug Discovery ,Structure–activity relationship ,Animals ,Humans ,Binding site ,Molecular Biology ,Protein Kinase Inhibitors ,Binding Sites ,Kinase ,Chemistry ,Organic Chemistry ,I-kappa B Kinase ,Protein Structure, Tertiary ,Molecular Docking Simulation ,Pyrimidines ,Drug Design ,Molecular Medicine ,Caco-2 Cells ,Protein Binding - Abstract
The design, synthesis and structure-activity relationships of a novel series of 2,4-diamino-5-cyclopropyl pyrimidines is described. Starting from BX795, originally reported to be a potent inhibitor of PDK1, we have developed compounds with improved selectivity and drug-like properties. These compounds have been evaluated in a range of cellular and in vivo assays, enabling us to probe the putative role of the TBK1/IKKe pathway in inflammatory diseases.
- Published
- 2012
39. The IkappaB Kinase Family Phosphorylates the Parkinson’s Disease Kinase LRRK2 at Ser935 and Ser910 during Toll-Like Receptor Signaling
- Author
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Hwanguen Choi, Huaibin Cai, Jiazhen Zhang, Patrick G. A. Pedrioli, Nathanael S. Gray, Philip Cohen, Kristopher Clark, Simon Arthur, Nicolas Dzamko, Francisco Inesta-Vaquera, Dario R. Alessi, Chengsong Xie, and Li Tan
- Subjects
IκB kinase ,environment and public health ,Biochemistry ,Mice ,0302 clinical medicine ,TANK-binding kinase 1 ,Molecular Cell Biology ,Serine ,Signaling in Cellular Processes ,Phosphorylation ,Mice, Knockout ,0303 health sciences ,Toll-like receptor ,Multidisciplinary ,Kinase ,Mechanisms of Signal Transduction ,Toll-Like Receptors ,Antibodies, Monoclonal ,Parkinson Disease ,Signaling in Selected Disciplines ,LRRK2 ,Innate Immunity ,Signaling Cascades ,Cell biology ,Enzymes ,I-kappa B Kinase ,Medicine ,Cytokines ,Signal transduction ,Research Article ,Signal Transduction ,Science ,Immunology ,Biology ,Protein Serine-Threonine Kinases ,Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 ,Signaling Pathways ,Cell Line ,Enzyme Regulation ,03 medical and health sciences ,Lipopeptides ,Animals ,Humans ,030304 developmental biology ,Macrophages ,I-Kappa-B Kinase ,Immunity ,nervous system diseases ,enzymes and coenzymes (carbohydrates) ,Gene Expression Regulation ,Myeloid Differentiation Factor 88 ,Clinical Immunology ,030217 neurology & neurosurgery - Abstract
Mutations in leucine-rich repeat kinase 2 (LRRK2) are strongly associated with late-onset autosomal dominant Parkinson's disease. LRRK2 is highly expressed in immune cells and recent work points towards a link between LRRK2 and innate immunity. Here we demonstrate that stimulation of the Toll-Like Receptor (TLR) pathway by MyD88-dependent agonists in bone marrow-derived macrophages (BMDMs) or RAW264.7 macrophages induces marked phosphorylation of LRRK2 at Ser910 and Ser935, the phosphorylation sites that regulate the binding of 14-3-3 to LRRK2. Phosphorylation of these residues is prevented by knock-out of MyD88 in BMDMs, but not the alternative TLR adaptor protein TRIF. Utilising both pharmacological inhibitors, including a new TAK1 inhibitor, NG25, and genetic models, we provide evidence that both the canonical (IKKα and IKKβ) and IKK-related (IKKe and TBK1) kinases mediate TLR agonist induced phosphorylation of LRRK2 in vivo. Moreover, all four IKK members directly phosphorylate LRRK2 at Ser910 and Ser935 in vitro. Consistent with previous work describing Ser910 and Ser935 as pharmacodynamic biomarkers of LRRK2 activity, we find that the TLR independent basal phosphorylation of LRRK2 at Ser910 and Ser935 is abolished following treatment of macrophages with LRRK2 kinase inhibitors. However, the increased phosphorylation of Ser910 and Ser935 induced by activation of the MyD88 pathway is insensitive to LRRK2 kinase inhibitors. Finally, employing LRRK2-deficient BMDMs, we present data indicating that LRRK2 does not play a major role in regulating the secretion of inflammatory cytokines induced by activation of the MyD88 pathway. Our findings provide the first direct link between LRRK2 and the IKKs that mediate many immune responses. Further work is required to uncover the physiological roles that phosphorylation of LRRK2 by IKKs play in controlling macrophage biology and to determine how phosphorylation of LRRK2 by IKKs impacts upon the use of Ser910 and Ser935 as pharmacodynamic biomarkers.
- Published
- 2012
40. The TRAF-associated protein TANK facilitates cross-talk within the IκB kinase family during Toll-like receptor signaling
- Author
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Osamu Takeuchi, Shizuo Akira, Kristopher Clark, and Philip Cohen
- Subjects
cells ,Autoimmunity ,IκB kinase ,Biology ,Protein Serine-Threonine Kinases ,Ligands ,environment and public health ,Mice ,TANK-binding kinase 1 ,Animals ,Phosphorylation ,skin and connective tissue diseases ,Adaptor Proteins, Signal Transducing ,Mice, Knockout ,Multidisciplinary ,Protein-Serine-Threonine Kinases ,Kinase ,Macrophages ,Toll-Like Receptors ,Models, Immunological ,Receptor Cross-Talk ,Biological Sciences ,Cell biology ,I-kappa B Kinase ,Mice, Inbred C57BL ,enzymes and coenzymes (carbohydrates) ,Adaptor Proteins, Vesicular Transport ,TRIF ,Myeloid Differentiation Factor 88 ,Interferon Regulatory Factor-3 ,Signal transduction ,biological phenomena, cell phenomena, and immunity ,Signal Transduction - Abstract
Toll-like receptor (TLR) ligands that signal via TIR-domain-containing adapter-inducing IFNβ (TRIF) activate the IκB kinase (IKK)-related kinases, TRAF associated NFκB activator (TANK)-binding kinase-1 (TBK1) and IKKε, which then phosphorylate IRF3 and induce the production of IFNβ. Here we show that TBK1 and IKKε are also activated by TLR ligands that signal via MyD88. Notably, the activation of IKKε is rapid, transient, and it precedes a more prolonged activation of TBK1. The MyD88- and TRIF-dependent signaling pathways activate the IKK-related kinases by two signaling pathways. One is mediated by the canonical IKKs, whereas the other culminates in the autoactivation of the IKK-related kinases. Once activated, TBK1/IKKε then phosphorylate and inhibit the canonical IKKs. The negative regulation of the canonical IKKs by the IKK-related kinases occurs in both the TRIF- and MyD88-dependent TLR pathways, whereas IRF3 phosphorylation is restricted to the TRIF-dependent signaling pathway. We have discovered that the activation of IKKε is abolished, the activation of TBK1 is reduced, and the interaction between the IKK-related kinases and the canonical IKKs is suppressed in TANK −/− macrophages, preventing the IKK-related kinases from negatively regulating the canonical IKKs. In contrast, IRF3 phosphorylation and IFNβ production was normal in TANK −/− macrophages. Our results demonstrate a key role for TANK in enabling the canonical IKKs and the IKK-related kinases to regulate each other, which is required to limit the strength of TLR signaling and ultimately, prevent autoimmunity.
- Published
- 2011
41. Novel cross-talk within the IKK family controls innate immunity
- Author
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Edward G. McIver, Lorna Plater, Mark Peggie, Jeffrey B. Madwed, Joanne Hough, Philip Cohen, Erick R. R. Young, Kristopher Clark, and Sorcek Ronald J
- Subjects
Morpholines ,IκB kinase ,Biology ,environment and public health ,Biochemistry ,Cell Line ,TANK-binding kinase 1 ,Piperidines ,Humans ,ASK1 ,skin and connective tissue diseases ,CHUK ,Molecular Biology ,MAPK14 ,Sulfonamides ,Molecular Structure ,Kinase ,Tumor Necrosis Factor-alpha ,Cell Biology ,Immunity, Innate ,Cell biology ,Toll-Like Receptor 3 ,Toll-Like Receptor 4 ,enzymes and coenzymes (carbohydrates) ,Gene Expression Regulation ,Cancer research ,I-kappa B Proteins ,biological phenomena, cell phenomena, and immunity ,Signal transduction ,IRF3 ,Cyclobutanes ,Interleukin-1 ,Signal Transduction - Abstract
Members of the IKK {IκB [inhibitor of NF-κB (nuclear factor κB)] kinase} family play a central role in innate immunity by inducing NF-κB- and IRF [IFN (interferon) regulatory factor]-dependent gene transcription programmes required for the production of pro-inflammatory cytokines and IFNs. However, the molecular mechanisms that activate these protein kinases and their complement of physiological substrates remain poorly defined. Using MRT67307, a novel inhibitor of IKKϵ/TBK1 (TANK {TRAF [TNF (tumour-necrosis-factor)-receptor-associated factor]-associated NF-κB activator}-binding kinase 1) and BI605906, a novel inhibitor of IKKβ, we demonstrate that two different signalling pathways participate in the activation of the IKK-related protein kinases by ligands that activate the IL-1 (interleukin-1), TLR (Toll-like receptor) 3 and TLR4 receptors. One signalling pathway is mediated by the canonical IKKs, which directly phosphorylate and activate IKKϵ and TBK1, whereas the second pathway appears to culminate in the autocatalytic activation of the IKK-related kinases. In contrast, the TNFα-induced activation of the IKK-related kinases is mediated solely by the canonical IKKs. In turn, the IKK-related kinases phosphorylate the catalytic subunits of the canonical IKKs and their regulatory subunit NEMO (NF-κB essential modulator), which is associated with reduced IKKα/β activity and NF-κB-dependent gene transcription. We also show that the canonical IKKs and the IKK-related kinases not only have unique physiological substrates, such as IκBα, p105, RelA (IKKα and IKKβ) and IRF3 (IKKϵ and TBK1), but also have several substrates in common, including the catalytic and regulatory (NEMO and TANK) subunits of the IKKs themselves. Taken together, our studies reveal that the canonical IKKs and the IKK-related kinases regulate each other by an intricate network involving phosphorylation of their catalytic and regulatory (NEMO and TANK) subunits to balance their activities during innate immunity.
- Published
- 2010
42. The alpha-kinases TRPM6 and TRPM7, but not eEF-2 kinase, phosphorylate the assembly domain of myosin IIA, IIB and IIC
- Author
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Frank N. van Leeuwen, Edwin Lasonder, Alexey G. Ryazanov, Maxim V. Dorovkov, Kristopher Clark, Carl G. Figdor, and Jeroen Middelbeek
- Subjects
Elongation Factor 2 Kinase ,Myosin light-chain kinase ,Age-related aspects of cancer [ONCOL 2] ,TRPM6 ,Molecular Sequence Data ,Biophysics ,TRPM7 ,TRPM Cation Channels ,macromolecular substances ,Biology ,Protein Serine-Threonine Kinases ,Biochemistry ,Cell Line ,Metabolism, transport and motion [NCMLS 2] ,Protein structure ,Structural Biology ,Immune Regulation [NCMLS 2] ,Translational research [ONCOL 3] ,Myosin ,Genetics ,Humans ,Amino Acid Sequence ,Phosphorylation ,Molecular Biology ,Peptide sequence ,Myosin II ,Myosin Type II ,Protein-Serine-Threonine Kinases ,Nonmuscle Myosin Type IIB ,Myosin Heavy Chains ,Kinase ,Nonmuscle Myosin Type IIA ,Wild type ,Immunotherapy, gene therapy and transplantation [UMCN 1.4] ,Cell Biology ,Protein Structure, Tertiary ,Mitochondrial medicine [IGMD 8] ,Cellular energy metabolism [UMCN 5.3] ,α-Kinase ,Immunity, infection and tissue repair [NCMLS 1] - Abstract
TRPM6 and TRPM7 encode channel-kinases. While these channels share electrophysiological properties and cellular functions, TRPM6 and TRPM7 are non-redundant genes raising the possibility that the kinases have distinct substrates. Here, we demonstrate that TRPM6 and TRPM7 phosphorylate the assembly domain of myosin IIA, IIB and IIC on identical residues. Whereas phosphorylation of myosin IIA is restricted to the coiled-coil domain, TRPM6 and TRPM7 also phosphorylate the non-helical tails of myosin IIB and IIC. TRPM7 does not phosphorylate eukaryotic elongation factor-2 (eEF-2) and myosin II is a poor substrate for eEF-2 kinase. In conclusion, TRPM6 and TRPM7 share exogenous substrates among themselves but not with functionally distant α-kinases.Structured summaryMINT-6700314:GNA1 (uniprotkb:Q96EK6) and GNA1 (uniprotkb:Q96EK6) bind (MI:0407) by X-ray crystallography (MI:0114)
- Published
- 2008
43. Interplay between TRP channels and the cytoskeleton in health and disease
- Author
-
Jeroen Middelbeek, Kristopher Clark, and Frank N. van Leeuwen
- Subjects
Histology ,Age-related aspects of cancer [ONCOL 2] ,Biology ,Kidney ,Models, Biological ,Pathology and Forensic Medicine ,Transient receptor potential channel ,Transient Receptor Potential Channels ,Immune Regulation [NCMLS 2] ,Translational research [ONCOL 3] ,Animals ,Drosophila Proteins ,Humans ,Mechanotransduction ,Cell adhesion ,Cytoskeleton ,Hearing Disorders ,Actin ,Vision, Ocular ,Endothelial Cells ,Immunotherapy, gene therapy and transplantation [UMCN 1.4] ,Cell Biology ,General Medicine ,Cell biology ,Ion homeostasis ,Tubulin ,Drosophila melanogaster ,biology.protein ,Kidney Diseases ,Visual phototransduction - Abstract
Contains fulltext : 69664.pdf (Publisher’s version ) (Closed access) Transient receptor potential (TRP) channels are a family of cation channels that play a key role in ion homeostasis and cell volume regulation. In addition, TRP channels are considered universal integrators of sensory information required for taste, vision, hearing, touch, temperature, and the detection of mechanical force. Seminal investigations exploring the molecular mechanisms of phototransduction in Drosophila have demonstrated that TRP channels operate within macromolecular complexes closely associated with the cytoskeleton. More recent evidence shows that mammalian TRP channels similarly connect to the cytoskeleton to affect cytoskeletal organization and cell adhesion via ion-transport-dependent and -independent mechanisms. In this review, we discuss new insights into the interplay between TRP channels and the cytoskeleton and provide recent examples of such interactions in different physiological systems.
- Published
- 2008
44. TRPM7 regulates myosin IIA filament stability and protein localization by heavy chain phosphorylation
- Author
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Nick A. Morrice, Frank N. van Leeuwen, Edwin Lasonder, Jeroen Middelbeek, Anne R. Bresnick, Alexey G. Ryazanov, Natalya G. Dulyaninova, Kristopher Clark, and Carl G. Figdor
- Subjects
Myosin light-chain kinase ,Age-related aspects of cancer [ONCOL 2] ,Molecular Sequence Data ,TRPM Cation Channels ,macromolecular substances ,Biology ,Article ,Cell Line ,Myosin head ,Metabolism, transport and motion [NCMLS 2] ,Mice ,Phosphoserine ,Structural Biology ,Immune Regulation [NCMLS 2] ,Translational research [ONCOL 3] ,Myosin ,Chlorocebus aethiops ,Animals ,Humans ,Amino Acid Sequence ,Phosphorylation ,Cytoskeleton ,Molecular Biology ,Actin ,Conserved Sequence ,Myosin Heavy Chains ,Nonmuscle Myosin Type IIA ,Immunotherapy, gene therapy and transplantation [UMCN 1.4] ,Subcellular localization ,Protein subcellular localization prediction ,Cell biology ,Kinetics ,Mitochondrial medicine [IGMD 8] ,Phosphothreonine ,Biochemistry ,Mutation ,MYH7 ,Cellular energy metabolism [UMCN 5.3] ,Sequence Alignment ,Immunity, infection and tissue repair [NCMLS 1] - Abstract
Contains fulltext : 70900.pdf (Publisher’s version ) (Closed access) Deregulation of myosin II-based contractility contributes to the pathogenesis of human diseases, such as cancer, which underscores the necessity for tight spatial and temporal control of myosin II activity. Recently, we demonstrated that activation of the mammalian alpha-kinase TRPM7 inhibits myosin II-based contractility in a Ca(2+)- and kinase-dependent manner. However, the molecular mechanism is poorly defined. Here, we demonstrate that TRPM7 phosphorylates the COOH-termini of both mouse and human myosin IIA heavy chains--the COOH-terminus being a region that is critical for filament stability. Phosphorylated residues were mapped to Thr1800, Ser1803 and Ser1808. Mutation of these residues to alanine and that to aspartic acid lead to an increase and a decrease, respectively, in myosin IIA incorporation into the actomyosin cytoskeleton and accordingly affect subcellular localization. In conclusion, our data demonstrate that TRPM7 regulates myosin IIA filament stability and localization by phosphorylating a short stretch of amino acids within the alpha-helical tail of the myosin IIA heavy chain.
- Published
- 2007
45. Myosin II and mechanotransduction: a balancing act
- Author
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Michiel Langeslag, Carl G. Figdor, Kristopher Clark, and Frank N. van Leeuwen
- Subjects
rho GTP-Binding Proteins ,Age-related aspects of cancer [ONCOL 2] ,Myosin light-chain kinase ,Cellular differentiation ,TRPM Cation Channels ,macromolecular substances ,Biology ,Mechanotransduction, Cellular ,Extracellular matrix ,Immune Regulation [NCMLS 2] ,Translational research [ONCOL 3] ,Myosin ,Animals ,Humans ,Neoplasm Invasiveness ,Mechanotransduction ,Cell adhesion ,Myosin-Light-Chain Kinase ,Myosin Type II ,Cell growth ,Cell adhesion molecule ,S100 Proteins ,Immunotherapy, gene therapy and transplantation [UMCN 1.4] ,Cell Differentiation ,Cell Biology ,Actomyosin ,Cell biology ,rac GTP-Binding Proteins ,Immunity, infection and tissue repair [NCMLS 1] - Abstract
Contains fulltext : 51627.pdf (Publisher’s version ) (Closed access) Adherent cells respond to mechanical properties of the surrounding extracellular matrix. Mechanical forces, sensed at specialized cell-matrix adhesion sites, promote actomyosin-based contraction within the cell. By manipulating matrix rigidity and adhesion strength, new roles for actomyosin contractility in the regulation of basic cellular functions, including cell proliferation, migration and stem cell differentiation, have recently been discovered. These investigations demonstrate that a balance of forces between cell adhesion on the outside and myosin II-based contractility on the inside of the cell controls many aspects of cell behavior. Disturbing this balance contributes to the pathogenesis of various human diseases. Therefore, elaborate signaling networks have evolved that modulate myosin II activity to maintain tensional homeostasis. These include signaling pathways that regulate myosin light chain phosphorylation as well as myosin II heavy chain interactions.
- Published
- 2006
46. Activation of TRPM7 channels by phospholipase C-coupled receptor agonists
- Author
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Kristopher Clark, Frank N. van Leeuwen, Wouter H. Moolenaar, Kees Jalink, and Michiel Langeslag
- Subjects
Age-related aspects of cancer [ONCOL 2] ,Patch-Clamp Techniques ,TRPM Cation Channels ,Biology ,Protein Serine-Threonine Kinases ,Bradykinin ,Biochemistry ,chemistry.chemical_compound ,Neuroblastoma ,Immune Regulation [NCMLS 2] ,Translational research [ONCOL 3] ,TRPM7 ,Cell Line, Tumor ,Lysophosphatidic acid ,Cell Adhesion ,Fluorescence Resonance Energy Transfer ,Humans ,Magnesium ,Patch clamp ,Cell adhesion ,Receptor ,Molecular Biology ,Phospholipase C ,Thrombin ,Immunotherapy, gene therapy and transplantation [UMCN 1.4] ,Cell Biology ,Cell biology ,chemistry ,Type C Phospholipases ,Second messenger system ,Calcium ,Signal transduction ,Lysophospholipids ,Immunity, infection and tissue repair [NCMLS 1] ,Protein Binding ,Signal Transduction - Abstract
Contains fulltext : 51829.pdf (Publisher’s version ) (Open Access) TRPM7 is a ubiquitously expressed nonspecific cation channel that has been implicated in cellular Mg(2+) homeostasis. We have recently shown that moderate overexpression of TRPM7 in neuroblastoma N1E-115 cells elevates cytosolic Ca(2+) levels and enhances cell-matrix adhesion. Furthermore, activation of TRPM7 by phospholipase C (PLC)-coupled receptor agonists caused a further increase in intracellular Ca(2+) levels and augmented cell adhesion and spreading in a Ca(2+)-dependent manner (1). Regulation of the TRPM7 channel is not well understood, although it has been reported that PIP(2) hydrolysis closes the channel. Here we have examined the regulation of TRPM7 by PLC-coupled receptor agonists such as bradykinin, lysophosphatidic acid, and thrombin. Using FRET assays for second messengers, we have shown that the TRPM7-dependent Ca(2+) increase closely correlates with activation of PLC. Under non-invasive "perforated patch clamp" conditions, we have found similar activation of TRPM7 by PLC-coupled receptor agonists. Although we could confirm that, under whole-cell conditions, the TRPM7 currents were significantly inhibited following PLC activation, this PLC-dependent inhibition was only observed when [Mg(2+)](i) was reduced below physiological levels. Thus, under physiological ionic conditions, TRPM7 currents were activated rather than inhibited by PLC-activating receptor agonists.
- Published
- 2006
47. TRPM7, a novel regulator of actomyosin contractility and cell adhesion
- Author
-
Bart van Leeuwen, Kristopher Clark, Alexey G. Ryazanov, Carl G. Figdor, Kees Jalink, Michiel Langeslag, Wouter H. Moolenaar, Leonie Ran, and Frank N. van Leeuwen
- Subjects
Age-related aspects of cancer [ONCOL 2] ,TRPM Cation Channels ,macromolecular substances ,Biology ,Protein Serine-Threonine Kinases ,General Biochemistry, Genetics and Molecular Biology ,Article ,Cell Line ,Contractility ,Focal adhesion ,Mice ,Immune Regulation [NCMLS 2] ,Translational research [ONCOL 3] ,Myosin ,Cell Adhesion ,Animals ,Humans ,Immunoprecipitation ,Phosphorylation ,Cytoskeleton ,Cell adhesion ,Molecular Biology ,General Immunology and Microbiology ,General Neuroscience ,Nonmuscle Myosin Type IIA ,Phosphotransferases ,Immunotherapy, gene therapy and transplantation [UMCN 1.4] ,Adhesion ,Actomyosin ,Cell biology ,Microscopy, Fluorescence ,Calcium ,Microbial pathogenesis and host defense [UMCN 4.1] ,Cytokinesis ,Intracellular ,Immunity, infection and tissue repair [NCMLS 1] - Abstract
Contains fulltext : 51410.pdf (Publisher’s version ) (Closed access) Actomyosin contractility regulates various cell biological processes including cytokinesis, adhesion and migration. While in lower eukaryotes, alpha-kinases control actomyosin relaxation, a similar role for mammalian alpha-kinases has yet to be established. Here, we examined whether TRPM7, a cation channel fused to an alpha-kinase, can affect actomyosin function. We demonstrate that activation of TRPM7 by bradykinin leads to a Ca(2+)- and kinase-dependent interaction with the actomyosin cytoskeleton. Moreover, TRPM7 phosphorylates the myosin IIA heavy chain. Accordingly, low overexpression of TRPM7 increases intracellular Ca2+ levels accompanied by cell spreading, adhesion and the formation of focal adhesions. Activation of TRPM7 induces the transformation of these focal adhesions into podosomes by a kinase-dependent mechanism, an effect that can be mimicked by pharmacological inhibition of myosin II. Collectively, our results demonstrate that regulation of cell adhesion by TRPM7 is the combined effect of kinase-dependent and -independent pathways on actomyosin contractility.
- Published
- 2006
48. P089 Identification of the first protein kinase inhibitors that switch macrophages from pro-inflammatory to anti-inflammatory cytokine production
- Author
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Patrick G. A. Pedrioli, Simon Arthur, Yosua Adi Kristariyanto, Kirsty F. MacKenzie, K. Petkevicius, Kristopher Clark, Edward G. McIver, Mark Peggie, Jinwei Zhang, Nathanael S. Gray, and Philip Cohen
- Subjects
Kinase ,medicine.medical_treatment ,Immunology ,Inflammation ,Hematology ,Biology ,Biochemistry ,Regulatory macrophages ,Cell biology ,Cytokine ,medicine ,Immunology and Allergy ,Macrophage ,Cytokine secretion ,medicine.symptom ,Protein kinase A ,Molecular Biology ,MAPK14 - Abstract
Introduction Macrophages acquire strikingly different properties that enable them to play key roles during the initiation, propagation and resolution of inflammation. Classically-activated macrophages produce pro-inflammatory mediators that help to combat invading pathogens and respond to tissue damage in the host, whereas regulatory macrophages produce high levels of anti-inflammatory molecules, such as interleukin (IL)-10, and low levels of pro-inflammatory cytokines, like IL-12, and are important for the resolution of inflammatory responses. A central problem in this area is to understand how the formation of regulatory macrophages can be promoted at sites of inflammation to prevent and/or alleviate chronic inflammatory and autoimmune diseases. Methods Bone-marrow derived macrophages were treated with protein kinase inhibitors prior to stimulation with Toll-like receptor (TLR) agonists. mRNA synthesis was measured by qPCR and cytokine secretion was measured using the BIOPLEX system. To identify the relevant substrate, the effects of the protein kinase inhibitors on the phosphoproteome were measured by LC-MS/MS. Subsequently, the repertoire of phosphorylation sites were mapped and their effects on protein function were analyzed using luciferase assays. Finally, the targets of the protein kinase inhibitors were validated in mouse knock-outs. Results We have discovered a novel mechanism that restricts the formation of regulatory macrophages. This has led us to identify the first small-molecule inhibitors of protein kinases capable of inducing all the hallmarks of regulatory macrophages, including the production of high levels of IL-10 and IL-1ra. The molecular mechanism underlying the effects of these compounds will be presented. Conclusion The remarkable effects of these protein kinase inhibitors on macrophage function suggest a novel approach for the development of anti-inflammatory drugs.
- Published
- 2012
49. Novel cross-talk within the IKK family controls innate immunity.
- Author
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Kristopher Clark, Mark Peggie, Lorna Plater, Ronald J. Sorcek, Erick R. R. Young, Jeffrey B. Madwed, Joanne Hough, Edward G. McIver, and Philip Cohen
- Subjects
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NF-kappa B , *IMMUNITY , *INTERFERONS , *GENETIC transcription , *INFLAMMATION , *CYTOKINES , *MOLECULAR biology , *PHOSPHORYLATION - Abstract
Members of the IKK {IκB [inhibitor of NF-κB (nuclear factor κB)] kinase} family play a central role in innate immunity by inducing NF-κB- and IRF [IFN (interferon) regulatory factor]-dependent gene transcription programmes required for the production of pro-inflammatory cytokines and IFNs. However, the molecular mechanisms that activate these protein kinases and their complement of physiological substrates remain poorly defined. Using MRT67307, a novel inhibitor of IKKϵ/TBK1 (TANK {TRAF [TNF (tumour-necrosis-factor)-receptor-associated factor]-associated NF-κB activator}-binding kinase 1) and BI605906, a novel inhibitor of IKKβ, we demonstrate that two different signalling pathways participate in the activation of the IKK-related protein kinases by ligands that activate the IL-1 (interleukin-1), TLR (Toll-like receptor) 3 and TLR4 receptors. One signalling pathway is mediated by the canonical IKKs, which directly phosphorylate and activate IKKϵ and TBK1, whereas the second pathway appears to culminate in the autocatalytic activation of the IKK-related kinases. In contrast, the TNFα-induced activation of the IKK-related kinases is mediated solely by the canonical IKKs. In turn, the IKK-related kinases phosphorylate the catalytic subunits of the canonical IKKs and their regulatory subunit NEMO (NF-κB essential modulator), which is associated with reduced IKKα/β activity and NF-κB-dependent gene transcription. We also show that the canonical IKKs and the IKK-related kinases not only have unique physiological substrates, such as IκBα, p105, RelA (IKKα and IKKβ) and IRF3 (IKKϵ and TBK1), but also have several substrates in common, including the catalytic and regulatory (NEMO and TANK) subunits of the IKKs themselves. Taken together, our studies reveal that the canonical IKKs and the IKK-related kinases regulate each other by an intricate network involving phosphorylation of their catalytic and regulatory (NEMO and TANK) subunits to balance their activities during innate immunity. [ABSTRACT FROM AUTHOR]
- Published
- 2011
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50. The alpha-kinase family: an exceptional branch on the protein kinase tree
- Author
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Kristopher Clark, Frank N. van Leeuwen, Hanka Venselaar, Jeroen Middelbeek, and Martijn A. Huynen
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Chemical and physical biology [NCMLS 7] ,Energy and redox metabolism [NCMLS 4] ,Genetics and epigenetic pathways of disease [NCMLS 6] ,Alpha-kinase family ,Review ,Signal transduction ,Biology ,SH3 domain ,Cellular and Molecular Neuroscience ,Protein phosphorylation ,Immune Regulation [NCMLS 2] ,Translational research [ONCOL 3] ,Animals ,Humans ,ASK1 ,c-Raf ,Protein kinase A ,Molecular Biology ,Serine/threonine-specific protein kinase ,Pharmacology ,Cyclin-dependent kinase 1 ,Kinase ,Cell Membrane ,Cell Biology ,Cell biology ,Mitochondrial medicine [IGMD 8] ,Biochemistry ,Atypical kinases ,Energy and redox metabolism Mitochondrial medicine [NCMLS 4] ,Molecular Medicine ,Protein Kinases - Abstract
Contains fulltext : 88992.pdf (Publisher’s version ) (Closed access) The alpha-kinase family represents a class of atypical protein kinases that display little sequence similarity to conventional protein kinases. Early studies on myosin heavy chain kinases in Dictyostelium discoideum revealed their unusual propensity to phosphorylate serine and threonine residues in the context of an alpha-helix. Although recent studies show that some members of this family can also phosphorylate residues in non-helical regions, the name alpha-kinase has remained. During evolution, the alpha-kinase domains combined with many different functional subdomains such as von Willebrand factor-like motifs (vWKa) and even cation channels (TRPM6 and TRPM7). As a result, these kinases are implicated in a large variety of cellular processes such as protein translation, Mg(2+) homeostasis, intracellular transport, cell migration, adhesion, and proliferation. Here, we review the current state of knowledge on different members of this kinase family and discuss the potential use of alpha-kinases as drug targets in diseases such as cancer. 01 maart 2010
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