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Kinases are known to have kinase activity independent functions. To gain further insights into potential kinase-independent functions of SLK/STK2, we have developed a kinase-dead allele, SLKK63R using in vivo CRISPR/Cas technology. Our studies show that blastocysts homozygote for SLKK63R do not develop into viable mice. However, heterozygotes are viable and fertile with no overt phenotypes. Analyses of mouse embryonic fibroblasts show that expression of SLKK63R results in a 50% decrease in kinase activity in heterozygotes. In contrast to previous studies, our data show that SLK does not form homodimers and that the kinase defective allele does not act in a dominant negative fashion. Expression of SLKK63R leads to altered Rac1 and RhoA activity, increased stress fiber formation and delayed focal adhesion turnover. Our data support a previously observed role for SLK in cell migration and suggest that at least 50% kinase activity is sufficient for embryonic development. • Mice homozygote for kinase dead SLK are not viable but heterozygotes are apparently normal. • Mouse embryonic fibroblasts expressing kinase inactive SLK show defects in focal adhesion turnover and motility. • Expression of kinase inactive SLK in mouse embryonic fibroblasts induces RhoA activity and stress fibers but inhibits Rac1. [ABSTRACT FROM AUTHOR]

It has been suggested that the liver allograft can protect the kidney allograft from antibody mediated rejection in simultaneous liver/kidney transplant (SLK) recipients by reducing preexisting donor specific antibodies (DSA) via adsorption of DSA by the liver allograft. Recently, the SLK allocation system was altered to provide a kidney safety net to those who do not recover native kidney function after liver transplant. However, the kidney transplant under the safety net creates a theoretical challenge for sensitized patients as the liver graft may not be able to adsorb human leukocyte antigen (HLA) antibodies against the kidney under the safety net because the liver and kidney grafts are from different donors and may carry different HLA antigens. This prompts us to examine levels of non-donor specific HLA antibodies in SLK recipients in our hospital. We found that levels of both DSA and non-DSA decreased post SLK transplant. The presence of preexisting DSA was also not associated with kidney graft survival and antibody mediated rejection in SLK recipients. Our results indicate that the liver transplant can reduce non-DSA, which may increase the pool of compatible kidneys offered under the safety net program for sensitized patients. [ABSTRACT FROM AUTHOR]

Over the past 20 years, the Ste20-like kinase (SLK; also known as STK2) has emerged as a central regulator of cytoskeletal dynamics. Reorganization of the cytoskeleton is necessary for a plethora of biological processes including apoptosis, proliferation, migration, tissue repair and signaling. Several studies have also uncovered a role for SLK in disease progression and cancer. Here, we review the recent findings in the SLK field and summarize the various roles of SLK in different animal models and discuss the biochemical mechanisms regulating SLK activity. Together, these studies have revealed multiple roles for SLK in coupling cytoskeletal dynamics to cell growth, in muscle repair and in negative-feedback loops critical for cancer progression. Furthermore, the ability of SLK to regulate some systems appears to be kinase activity independent, suggesting that it may be an important scaffold for signal transduction pathways. These various findings reveal highly complex functions and regulation patterns of SLK in development and disease, making it a potential therapeutic target. [ABSTRACT FROM AUTHOR]

Purpose: Nocardia keratitis is a rare type of infectious keratitis and may mimic other corneal diseases and lead to delay in diagnosis. This case illustrates how Nocardia often escapes accurate diagnosis due to its insidious onset, variable clinical manifestations, and unusual characteristics on cultures. Observation: The patient presented with an epithelial defect and superior pannus and scarring, which was misdiagnosed as superior limbic keratoconjunctivitis (SLK) and herpes simplex virus (HSV) keratitis. Repeat corneal scraping cultures, smears, and conjunctival biopsy were necessary to elucidate the diagnosis. It can be effectively treated with the intravenous preparation of trimethoprim-sulfamethoxazole 80 mg/mL (brand name SEPTRA) used topically as eye drops. Conclusion: The diagnosis of Nocardia keratitis relies on a high clinical suspicion and a prompt corneal scraping with culture. Due to its potential for rapid resolution with early therapy, it is important to isolate Nocardia early in its disease course. Importance: Topical amikacin had been the standard of care for Nocardia keratitis for many years. However, recently there is increasing resistance of Nocardia to amikacin. SEPTRA offers an alternative therapy. Nocardia keratitis mimics other infectious and inflammatory etiologies so rapid diagnosis and treatment is critical in the prevention of long-term complications.

In animal cells, the centrosome nucleates and anchors microtubules (MT), forming their radial array. During interphase centrosome-derived MT, aster can either team up with other MT network or function in an autonomous manner. What is the function of the centrosome-derived MT aster? We suggested that it might play an important role in the formation of the primary cilium, the organelle obligatorily associated with the centrosome. PCM-1 (PeriCentriolar Matrix 1) protein, which participates in the organization of the primary cilium, is a part of pericentiolar satellites. They are transported to the centrosome along MTs by the motor protein dynein in a complex with its cofactor dynactin. Previously, we showed that SLK/LOSK phosphorylated the p150Glued subunit of dynactin, thus promoting its centrosomal targeting followed by its participation in the retention of microtubules. Here, we found that under the repression of SLK/LOSK activity, the PCM-1 protein lost its pericentrosomal localization and was being dispersed throughout the cytoplasm. Despite that the alanine and glutamine mutants of p150Glued had opposite effects on PCM-1 localization, they associated with PCM-1 to the same extent. The occurrence of primary cilia also significantly decreased when SLK/LOSK was repressed. These defects also correlated with a disturbance of the long-range transport in cells, whereas dynein-depending motility was intact. Treatment with the GSK-3β kinase inhibitor also resulted in the loss of the centrosome-derived MT aster, dispersion of PCM-1 over the cytoplasm, and reduction of primary cilia occurrence. Thus, kinases involved in the centrosome-derived MT aster regulation can indirectly control the formation of primary cilia in cells. [ABSTRACT FROM AUTHOR]

Introduction and aim. Many transplant programs have expanded eligibility to include patients previously ineligible because of advanced age. Outcomes of simultaneous liver-kidney transplantation (SLK) in recipients with advanced age are not known.Material and methods. Data from patients undergoing transplantation between 2002 and 2015 were obtained from the UNOS Standard Analysis and Research file.Results. SLK recipients aged ≥ 65 years (N = 677), SLK recipients aged < 65 years (N = 4517), and recipients of liver transplant alone(LTA) aged ≥ 65 years(N = 8495) were compared. Recipient characteristics were similar between the SLK groups. Similar patient and graft survival were observed in SLK recipients aged ≥ 65 years compared to SLK recipients aged < 65 years and LTA recipients aged ≥ 65 years. Importantly, in a subgroup analysis, superior survival was seen in the SLK group aged ≥ 65 years compared to LTA recipients aged ≥ 65 years who underwent dialysis in the week prior to transplantation (p < 0.001). A prediction model of patient survival was developed for the SLK group aged ≥ 65 years with predictors including: age ≥ 70 years (3 points), calculated MELD score (-1 to 2 points), and recipient ventilator status at the time of SLK (4 points). The risk score predicted patient survival, with a significantly inferior survival seen in patients with a score ≥ 4 (p < 0.001).Conclusions. Age should not be used as a contraindication for SLK transplantation. The validated scoring system provides a guide for patient selection and can be used when evaluating elderly patients for SLK transplantation listing.

Abstract We have previously shown that the Ste20-like kinase SLK interacts directly with the LIM domain-binding protein 1, Ldb1. Ldb1 knock down in murine fibroblasts activates SLK and enhances cell migration. To investigate the effect of Ldb1 deletion in ErbB2/HER2-driven tumorigenesis, Ldb1 conditional mice were crossed into MMTV-NIC mice, expressing the Neu oncogene and Cre recombinase from a bi-cistronic transgene. Our results show that Ldb1 is expressed in the mammary epithelium and that deletion of Ldb1 does not impair mammary gland development. Although high levels of Ldb1 can be correlated with poor prognosis in HER2+ breast cancers, Ldb1 ablation does not affect Neu-induced tumor progression in transgenic mice. Surprisingly, Ldb1 deletion did not affect SLK kinase activity in primary tumors or established cell lines. Nevertheless, Ldb1-deficient tumor cells showed enhanced mesenchymal and migratory characteristics in vitro. However, Ldb1-null cells failed to colonize the lungs of wildtype female mice when injected into the tail vein. Together our results show that Ldb1 is dispensable for mammary gland development and Neu-induced tumor progression but required for dissemination at secondary sites. Furthermore, our data also highlight contrasting cell line behaviours observed from in vivo and in vitro assays. Graphical abstract Unlabelled Image Highlights • Ldb1 deletion does not affect normal mammary gland development. • Ldb1 deletion does not affect Neu-induced tumorigenesis. • Ldb1 deletion enhances in vitro invasion but inhibits lung colonization in vivo. • Ldb1 deletion does not modulate SLK kinase activity. [ABSTRACT FROM AUTHOR]

How cells specify morphologically distinct plasma membrane domains is poorly understood. Prior work has shown that restriction of microvilli to the apical aspect of epithelial cells requires the localized activation of the membrane-F-actin linking protein ezrin. Using an in vitro system, we now define a multi-step process whereby the kinase LOK specifically phosphorylates ezrin to activate it. Binding of PIP2 to ezrin induces a conformational change permitting the insertion of the LOK C-terminal domain to wedge apart the membrane and F-actin-binding domains of ezrin. The N-terminal LOK kinase domain can then access a site 40 residues distal from the consensus sequence that collectively direct phosphorylation of the appropriate threonine residue. We suggest that this elaborate mechanism ensures that ezrin is only phosphorylated at the plasma membrane, and with high specificity by the apically localized kinase LOK.

Introduction and aim. Many transplant programs have expanded eligibility to include patients previously ineligible because of advanced age. Outcomes of simultaneous liver-kidney transplantation (SLK) in recipients with advanced age are not known. Material and methods. Data from patients undergoing transplantation between 2002 and 2015 were obtained from the UNOS Standard Analysis and Research file. Results. SLK recipients aged ≥ 65 years (N = 677), SLK recipients aged < 65 years (N = 4517), and recipients of liver transplant alone(LTA) aged ≥ 65 years(N = 8495) were compared. Recipient characteristics were similar between the SLK groups. Similar patient and graft survival were observed in SLK recipients aged ≥ 65 years compared to SLK recipients aged < 65 years and LTA recipients aged ≥ 65 years. Importantly, in a subgroup analysis, superior survival was seen in the SLK group aged ≥ 65 years compared to LTA recipients aged ≥ 65 years who underwent dialysis in the week prior to transplantation (p < 0.001). A prediction model of patient survival was developed for the SLK group aged ≥ 65 years with predictors including: age ≥ 70 years (3 points), calculated MELD score (-1 to 2 points), and recipient ventilator status at the time of SLK (4 points). The risk score predicted patient survival, with a significantly inferior survival seen in patients with a score ≥ 4 (p < 0.001). Conclusions. Age should not be used as a contraindication for SLK transplantation. The validated scoring system provides a guide for patient selection and can be used when evaluating elderly patients for SLK transplantation listing. [ABSTRACT FROM AUTHOR]

The Ste20-like kinase SLK plays a pivotal role in cell migration and focal adhesion turnover and is regulated by the LIM domain-binding proteins Ldb1 and Ldb2. These adapter proteins have been demonstrated to interact with LMO4 in the organization of transcriptional complexes. Therefore, we have assessed the ability of LMO4 to also interact and regulate SLK activity. Our data show that LMO4 can directly bind to SLK and activate its kinase activity in vitro and in vivo. LMO4 can be co-precipitated with SLK following the induction of cell migration by scratch wounding and Cre-mediated deletion of LMO4 in conditional LMO4 fl/fl fibroblasts inhibits cell migration and SLK activation. Deletion of LMO4 impairs Ldb1 and SLK recruitment to the leading edge of migrating cells. Supporting this, Src/Yes/Fyn-deficient cells (SYF) expressing very low levels of LMO4 do not recruit SLK to the leading edge. Re-expression of wildtype Myc–LMO4 in SYF cells, but not a mutant version, restores SLK localization and kinase activity. Overall, our data suggest that activation of SLK by haptotactic signals requires its recruitment to the leading edge by LMO4 in a Src-dependent manner. Furthermore, this establishes a novel cytosolic role for the transcriptional co-activator LMO4. [ABSTRACT FROM AUTHOR]

It has been suggested that the liver allograft can protect the kidney allograft from antibody mediated rejection in simultaneous liver/kidney transplant (SLK) recipients by reducing preexisting donor specific antibodies (DSA) via adsorption of DSA by the liver allograft. Recently, the SLK allocation system was altered to provide a kidney safety net to those who do not recover native kidney function after liver transplant. However, the kidney transplant under the safety net creates a theoretical challenge for sensitized patients as the liver graft may not be able to adsorb human leukocyte antigen (HLA) antibodies against the kidney under the safety net because the liver and kidney grafts are from different donors and may carry different HLA antigens. This prompts us to examine levels of non-donor specific HLA antibodies in SLK recipients in our hospital. We found that levels of both DSA and non-DSA decreased post SLK transplant. The presence of preexisting DSA was also not associated with kidney graft survival and antibody mediated rejection in SLK recipients. Our results indicate that the liver transplant can reduce non-DSA, which may increase the pool of compatible kidneys offered under the safety net program for sensitized patients., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Protein kinase autophosphorylation of activation segment residues is a common regulatory mechanism in phosphorylation-dependent signalling cascades. However, the molecular mechanisms that guarantee specific and efficient phosphorylation of these sites have not been elucidated. Here, we report on three novel and diverse protein kinase structures that reveal an exchanged activation segment conformation. This dimeric arrangement results in an active kinase conformation in trans, with activation segment phosphorylation sites in close proximity to the active site of the interacting protomer. Analytical ultracentrifugation and chemical cross-linking confirmed the presence of dimers in solution. Consensus substrate sequences for each kinase showed that the identified activation segment autophosphorylation sites are non-consensus substrate sites. Based on the presented structural and functional data, a model for specific activation segment phosphorylation at non-consensus substrate sites is proposed that is likely to be common to other kinases from diverse subfamilies. [ABSTRACT FROM AUTHOR]

Over the past 20 years, the Ste20-like kinase (SLK; also known as STK2) has emerged as a central regulator of cytoskeletal dynamics. Reorganization of the cytoskeleton is necessary for a plethora of biological processes including apoptosis, proliferation, migration, tissue repair and signaling. Several studies have also uncovered a role for SLK in disease progression and cancer. Here, we review the recent findings in the SLK field and summarize the various roles of SLK in different animal models and discuss the biochemical mechanisms regulating SLK activity. Together, these studies have revealed multiple roles for SLK in coupling cytoskeletal dynamics to cell growth, in muscle repair and in negative-feedback loops critical for cancer progression. Furthermore, the ability of SLK to regulate some systems appears to be kinase activity independent, suggesting that it may be an important scaffold for signal transduction pathways. These various findings reveal highly complex functions and regulation patterns of SLK in development and disease, making it a potential therapeutic target. [ABSTRACT FROM AUTHOR]

The malignant development and poor prognosis of gliomas are associated with a high degree of invasion and a high recurrence rate. However, the molecular mechanism underlying the invasiveness of glioma remains to be elucidated. Ste20- like kinase (SLK) is one of the members of the Ste20 family, which has been implicated in cellular migration and invasion. In this study, we intended to explore the expression of SLK significantly related to clinicopathologic stages of gliomas. Immunohistochemical staining and western blot analysis demonstrated that SLK was highly expressed in human glioma tissues and cell lines. Kaplan-Meier analysis revealed that poor survival was associated with high SLK expression. The inhibition of SLK by RNA interference significantly suppressed the invasion ability of glioma, and on protein level, knock- down of SLK leaded to an up-regulation of E-cadherin and a down-regulation of Vimentin in glioma cells. Collectively, this research shed light on mechanisms of invasion and progression of malignant gliomas and suggested that SLK may be a potential therapeutic strategy for glioma.

Invasion can be stimulated in vitro using the soluble ligand transforming growth factor-β (TGFβ) to induce a process called epithelial-to-mesenchymal transition (EMT) characterized by cell-cell junction breakdown and an invasive phenotype. We have previously demonstrated a role for Ste20-like kinase SLK cell migration and invasion. Here we show that SLK depletion in NMuMG mammary epithelial cells significantly impairs their TGFβ-induced migration and invasion. Immunofluorescence studies show that a fraction of SLK localizes to E-cadherin-positive adherens junction and that SLK impairs the breakdown of cell-cell contacts. We find that SLK-depleted cultures express significantly lower levels of vimentin protein as well as Snai1 and E-cadherin mRNA levels following TGF-β treatment. Surprisingly, our data show that SLK depletion does not affect the activation and nuclear translocation of Smad3. Furthermore, we show that expression of a dominant negative kinase does not impair tight junction breakdown and rescues Snai1 mRNA expression levels. Together these data suggest that SLK plays a novel role in TGFβ-induced EMT, independent of Smads, in a kinase activity-independent manner., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

How cells specify morphologically distinct plasma membrane domains is poorly understood. Prior work has shown that restriction of microvilli to the apical aspect of epithelial cells requires the localized activation of the membrane-F-actin linking protein ezrin. Using an in vitro system, we now define a multi-step process whereby the kinase LOK specifically phosphorylates ezrin to activate it. Binding of PIP 2 to ezrin induces a conformational change permitting the insertion of the LOK C-terminal domain to wedge apart the membrane and F-actin-binding domains of ezrin. The N-terminal LOK kinase domain can then access a site 40 residues distal from the consensus sequence that collectively direct phosphorylation of the appropriate threonine residue. We suggest that this elaborate mechanism ensures that ezrin is only phosphorylated at the plasma membrane, and with high specificity by the apically localized kinase LOK.

Acute kidney injury (AKI) occurs commonly in patients with advanced cirrhosis and negatively impacts pre- and post-transplant outcomes. Physiologic changes that occur in patients with decompensated cirrhosis with ascites, place these patients at high risk of AKI. The most common causes of AKI in cirrhosis include prerenal injury, acute tubular necrosis (ATN), and the hepatorenal syndrome (HRS), accounting for more than 80% of AKI in this population. Distinguishing between these causes is particularly important for prognostication and treatment. Treatment of Type 1 HRS with vasoconstrictors and albumin improves short term survival and renal function in some patients while awaiting liver transplantation. Patients with HRS who fail to respond to medical therapy or those with severe renal failure of other etiology may require renal replacement therapy. Simultaneous liver kidney transplant (SLK) is needed in many of these patients to improve their post-transplant outcomes. However, the criteria to select patients who would benefit from SLK transplantation are based on consensus and lack strong evidence to support them. In this regard, novel serum and/or urinary biomarkers such as neutrophil gelatinase-associated lipocalin, interleukins-6 and 18, kidney injury molecule-1, fatty acid binding protein, and endothelin-1 are emerging with a potential for accurately differentiating common causes of AKI. Prospective studies are needed on the use of these biomarkers to predict accurately renal function recovery after liver transplantation alone in order to optimize personalized use of SLK.