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JAK2 is cytokine-activated non-receptor tyrosine kinase. Although JAK2 is mainly localized at the plasma membrane, it is also present on the centrosome. In this study, we demonstrated that JAK2 localization to the centrosome depends on the SH2 domain and intact kinase activity. We created JAK2 mutants deficient in centrosomal localization ΔSH2, K882E and (ΔSH2, K882E). We showed that JAK2 WT clone strongly enhances cell proliferation as compared to control cells while JAK2 clones ΔSH2, K882E and (ΔSH2, K882E) proliferate slower than JAK2 WT cells. These mutant clones also progress much slower through the cell cycle as compared to JAK2 WT clone and the enhanced proliferation of JAK2 WT cells is accompanied by increased S −> G2 progression. Both the SH2 domain and the kinase activity of JAK2 play a role in prolactin-dependent activation of JAK2 substrate STAT5. We showed that JAK2 is an important regulator of centrosome function as the SH2 domain of JAK2 regulates centrosome amplification. The cells overexpressing ΔSH2 and (ΔSH2, K-E) JAK2 have almost three-fold the amplified centrosomes of WT cells. In contrast, the kinase activity of JAK2 is dispensable for centrosome amplification. Our observations provide novel insight into the role of SH2 domain and kinase activity of JAK2 in centrosome localization of JAK2 and in the regulation of cell growth and centrosome biogenesis.

Background Recent studies have linked certain single nucleotide polymorphisms in the leucine-rich repeat kinase 2 (LRRK2) gene with Parkinson’s disease (PD). Among the mutations, LRRK2 c.4883G>C (R1628P) variant was identified to have a significant association with the risk of PD in ethnic Han-Chinese populations. But the molecular pathological mechanisms of R1628P mutation in PD is still unknown. Principle Findings Unlike other LRRK2 mutants in the Roc-COR-Kinase domain, the R1628P mutation didn’t alter the LRRK2 kinase activity and promote neuronal death directly. LRRK2 R1628P mutation increased the binding affinity of LRRK2 with Cyclin-dependent kinase 5 (Cdk5). Interestingly, R1628P mutation turned its adjacent amino acid residue S1627 on LRRK2 protein to a novel phosphorylation site of Cdk5, which could be defined as a typical type II (+) phosphorylation-related single nucleotide polymorphism. Importantly, we showed that the phosphorylation of S1627 by Cdk5 could activate the LRRK2 kinase, and neurons ectopically expressing R1628P displayed a higher sensitivity to 1-methyl-4-phenylpyridinium, a bioactive metabolite of environmental toxin MPTP, in a Cdk5-dependent manner. Conclusion Our data indicate that Parkinson-related LRRK2 mutation R1628P leads to Cdk5 phosphorylation of LRRK2 at S1627, which would upregulate the kinase activity of LRRK2 and consequently cause neuronal death.

MAP/microtubule-affinity regulating kinases (MARK1-4) are members of the AMPK family of Ser/Thr-specific kinases, which phosphorylate substrates at consensus LXRXXSXXXL motifs. Within microtubule-associated proteins, MARKs also mediate phosphorylation of variant KXGS or ζXKXGSXXNΨ motifs, interfering with the ability of tau and MAP2/4 to bind to microtubules. Here we show that, although MARKs and the closely related salt-inducible kinases (SIKs) phosphorylate substrates with consensus AMPK motifs comparably, MARKs are more potent in recognizing variant ζXKXGSXXNΨ motifs on cellular tau. In studies to identify regions of MARKs that confer catalytic activity towards variant sites, we found that the C-terminal kinase associated-1 (KA1) domain in MARK1-3 mediates binding to microtubule-associated proteins CLASP1/2; but this interaction is dispensable for ζXKXGSXXNΨ phosphorylation. Mutational analysis of MARK2 revealed that the N-terminal kinase domain of MARK2 is sufficient for phosphorylation of both consensus and variant ζXKXGSXXNΨ sites. Within this domain, the KLDpT activation loop motif promotes MARK2 activity both intracellularly and in vitro, but has no effect on SIK2 activity. As KLDpT is conserved in all vertebrates MARKs, we conclude that this sequence is crucial for MARK-dependent regulation of cellular polarity.

[Background]The mechanism of cardiac energy production against sustained pressure overload remains to be elucidated. [Methods and Results]We generated cardiac-specific kinase-dead (kd) calcium/calmodulin-dependent protein kinase kinase-β (CaMKKβ) transgenic (α-MHC CaMKKβ[kd] TG) mice using α-myosin heavy chain (α-MHC) promoter. Although CaMKKβ activity was significantly reduced, these mice had normal cardiac function and morphology at baseline. Here, we show that transverse aortic binding (TAC) in α-MHC CaMKKβ[kd] TG mice led to accelerated death and left ventricular (LV) dilatation and dysfunction, which was accompanied by significant clinical signs of heart failure. CaMKKβ downstream signaling molecules, including adenosine monophosphate-activated protein kinase (AMPK), were also suppressed in α-MHC CaMKKβ[kd] TG mice compared with wild-type (WT) mice. The expression levels of peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, which is a downstream target of both of CaMKKβ and calcium/calmodulin kinases, were also significantly reduced in α-MHC CaMKKβ[kd] TG mice compared with WT mice after TAC. In accordance with these findings, mitochondrial morphogenesis was damaged and creatine phosphate/β-ATP ratios assessed by magnetic resonance spectroscopy were suppressed in α-MHC CaMKKβ[kd] TG mice compared with WT mice after TAC. [Conclusions]These data indicate that CaMKKβ exerts protective effects on cardiac adaptive energy pooling against pressure-overload possibly through phosphorylation of AMPK and by upregulation of PGC-1α. Thus, CaMKKβ may be a therapeutic target for the treatment of heart failure.

Background: The non-canonical NF-kappa B activating kinase IKK alpha, encoded by CHUK (conserved-helix-loop-helix-ubiquitous-kinase), has been reported to modulate pro- or anti-inflammatory responses, cellular survival and cellular differentiation. Here, we have investigated the mechanism of action of IKK alpha as a novel effector of human and murine chondrocyte extracellular matrix (ECM) homeostasis and differentiation towards hypertrophy. Methodology/Principal Findings: IKK alpha expression was ablated in primary human osteoarthritic (OA) chondrocytes and in immature murine articular chondrocytes (iMACs) derived from IKK alpha(f/f):CreERT2 mice by retroviral-mediated stable shRNA transduction and Cre recombinase-dependent Lox P site recombination, respectively. MMP-10 was identified as a major target of IKK alpha in chondrocytes by mRNA profiling, quantitative RT-PCR analysis, immunohistochemistry and immunoblotting. ECM integrity, as assessed by type II collagen (COL2) deposition and the lack of MMP-dependent COL2 degradation products, was enhanced by IKK alpha ablation in mice. MMP-13 and total collagenase activities were significantly reduced, while TIMP-3 (tissue inhibitor of metalloproteinase-3) protein levels were enhanced in IKK alpha-deficient chondrocytes. IKK alpha deficiency suppressed chondrocyte differentiation, as shown by the quantitative inhibition of. Alizarin red staining and the reduced expression of multiple chondrocyte differentiation effectors, including Runx2, Col10a1 and Vegfa,. Importantly, the differentiation of IKK alpha-deficient chondrocytes was rescued by a kinase-dead IKK alpha protein mutant. Conclusions/Significance: IKK alpha acts independent of its kinase activity to help drive chondrocyte differentiation towards a hypertrophic-like state. IKK alpha positively modulates ECM remodeling via multiple downstream targets (including MMP-10 and TIMP-3 at the mRNA and post-transcriptional levels, respectively) to maintain maximal MMP-13 activity, which is required for ECM remodeling leading to chondrocyte differentiation. Chondrocytes are the unique cell component in articular cartilage, which are quiescent and maintain ECM integrity during tissue homeostasis. In OA, chondrocytes reacquire the capacity to proliferate and differentiate and their activation results in pronounced cartilage degeneration. T eta nu sigma, our findings are also of potential relevance for defining the onset and/or progression of OA disease.

The creatine kinase (CK) phosphagen system is fundamental to cellular energy homeostasis. Cardiomyocytes express three CK isoforms, namely the mitochondrial sarcomeric CKMT2 and the cytoplasmic CKM and CKB. We hypothesized that augmenting CK in vitro would preserve cell viability and function and sought to determine efficacy of the various isoforms. The open reading frame of each isoform was cloned into pcDNA3.1, followed by transfection and stable selection in human embryonic kidney cells (HEK293). CKMT2- CKM- and CKB-HEK293 cells had increased protein and total CK activity compared to non-transfected cells. Overexpressing any of the three CK isoforms reduced cell death in response to 18h hypoxia at 1% O2 followed by 2h re-oxygenation as assayed using propidium iodide: by 33% in CKMT2, 47% in CKM and 58% in CKB compared to non-transfected cells (P

Src family kinases (SFK) control multiple processes during brain development and function. We show here that the phosphoprotein associated with glycosphigolipid-enriched microdomains (PAG)/Csk binding protein (Cbp) modulates SFK activity in the brain. The timing and localization of PAG expression overlap with Fyn and Src, both of which we find associated to PAG. We demonstrate in newborn (P1) mice that PAG negatively regulates Src family kinases (SFK). P1 Pag1(-/-) mouse brains show decreased recruitment of Csk into lipid rafts, reduced phosphorylation of the inhibitory tyrosines within SFKs, and an increase in SFK activity of >/ = 50%. While in brain of P1 mice, PAG and Csk are highly and ubiquitously expressed, little Csk is found in adult brain suggesting altered modes of SFK regulation. In adult brain Pag1-deficiency has no effect upon Csk-distribution or inhibitory tyrosine phosphorylation, but kinase activity is now reduced (-20-30%), pointing to the development of a compensatory mechanism that may involve PSD93. The distribution of the Csk-homologous kinase CHK is not altered. Importantly, since the activities of Fyn and Src are decreased in adult Pag1(-/-) mice, thus presenting the reversed phenotype of P1, this provides the first in vivo evidence for a Csk-independent positive regulatory function for PAG in the brain.

Nucleoside diphosphate kinase (NDK), which has the same sequence as oncoprotein (OP) in humans, can induce nucleoside triphosphates in DNA replication by maintenance of the deoxynucleotide triphosphate (dNTP’s) and is known to be regulated by viral infection in the shrimp Litopenaeus vannamei. This paper describes the relationship between NDK and white spot syndrome virus (WSSV) infection. The recombinant NDK was produced by a prokaryotic expression system. WSSV copy numbers and mRNA levels of IE1 and VP28 were significantly increased in shrimp injected with recombinant NDK at 72 h after WSSV infection. After synthesizing dsRNA-NDK and confirming the efficacy of NDK silencing, we recorded the cumulative mortality of WSSV-infected shrimp injected with NDK and dsRNA-NDK. A comparison between the results demonstrated that silencing NDK delayed the death of shrimps. These findings indicate that NDK has an important role influencing the replication of WSSV replication in shrimp. Furthermore, NDK may have potential target as a new therapeutic strategy against WSSV infection in shrimp.

Tyrosine kinase 2 (TYK2) is a member of the Janus kinase (JAK) family and is involved in cytokine signalling. In vitro analyses suggest that TYK2 also has kinase-independent, i.e., non-canonical, functions. We have generated gene-targeted mice harbouring a mutation in the ATP-binding pocket of the kinase domain. The Tyk2 kinase-inactive (Tyk2(K923E)) mice are viable and show no gross abnormalities. We show that kinase-active TYK2 is required for full-fledged type I interferon- (IFN) induced activation of the transcription factors STAT1-4 and for the in vivo antiviral defence against viruses primarily controlled through type I IFN actions. In addition, TYK2 kinase activity was found to be required for the protein's stability. An inhibitory function was only observed upon over-expression of TYK2(K923E)in vitro. Tyk2(K923E) mice represent the first model for studying the kinase-independent function of a JAK in vivo and for assessing the consequences of side effects of JAK inhibitors.

Background Protein-tyrosine kinases (PTKs) regulate virtually all biological processes. PTKs phosphorylate substrates in a sequence-specific manner and relatively short peptide sequences determine selectivity. Here, we developed new technology to determine PTK activity profiles using peptide arrays. The zebrafish is an excellent model system to investigate signaling in the whole organism, given its wealth of genetic tools, including morpholino-mediated knock down technology. We used zebrafish embryo lysates to determine PTK activity profiles, thus providing the unique opportunity to directly compare the effect of protein knock downs on PTK activity profiles on the one hand and phenotypic changes on the other. Methodology We used multiplex arrays of 144 distinct peptides, spotted on a porous substrate, allowing the sample to be pumped up and down, optimizing reaction kinetics. Kinase reactions were performed using complex zebrafish embryo lysates or purified kinases. Peptide phosphorylation was detected by fluorescent anti-phosphotyrosine antibody binding and the porous chips allowed semi-continuous recording of the signal. We used morpholinos to knock down protein expression in the zebrafish embryos and subsequently, we determined the effects on the PTK activity profiles. Results and Conclusion Reproducible PTK activity profiles were derived from one-day-old zebrafiish embryos. Morpholino-mediated knock downs of the Src family kinases, Fyn and Yes, induced characteristic phenotypes and distinct changes in the PTK activity profiles. Interestingly, the peptide substrates that were less phosphorylated upon Fyn and Yes knock down were preferential substrates of purified Fyn and Yes. Previously, we demonstrated that Wnt11 knock down phenocopied Fyn/Yes knock down. Interestingly, Wnt11 knock down induced similar changes in the PTK activity profile as Fyn/Yes knock down. The control Nacre/Mitfa knock down did not affect the PTK activity profile significantly. Our results indicate that the novel peptide chip technology can be used to unravel kinase signaling pathways in vivo.

Hematopoietic progenitor kinase 1 (HPK1), a hematopoietic cell-specific Ste20-related serine/threonine kinase, is a negative regulator of signal transduction in immune cells, including T cells, B cells, and dendritic cells (DCs). In mice, HPK1 deficiency subverts inhibition of the anti-tumor immune response and is associated with functional augmentation of anti-tumor T cells. We have used a potent, small molecule HPK1 inhibitor, Compound 1, to investigate the effects of pharmacological intervention of HPK1 kinase activity in immune cells. Compound 1 enhanced Th1 cytokine production in T cells and fully reverted immune suppression imposed by the prostaglandin E2(PGE2) and adenosine pathways in human T cells. Moreover, the combination of Compound 1 with pembrolizumab, a humanized monoclonal antibody against the programmed cell death protein 1 (PD-1), demonstrated a synergistic effect, resulting in enhanced interferon (IFN)-γ production. Collectively, our results suggest that blocking HPK1 kinase activity with small molecule inhibitors alone or in combination with checkpoint blockade may be an attractive approach for the immunotherapy of cancer.

Ataxia-telangiectasia (A-T) is a neurodegenerative disease caused by mutation of the A-T mutated (ATM) gene. ATM encodes a protein kinase that is activated by DNA damage and phosphorylates many proteins, including those involved in DNA repair, cell cycle control, and apoptosis. Characteristic biological and molecular functions of ATM observed in mammals are conserved in Drosophila melanogaster. As an example, conditional loss-of-function ATM alleles in flies cause progressive neurodegeneration through activation of the innate immune response. However, unlike in mammals, null alleles of ATM in flies cause lethality during development. With the goals of understanding biological and molecular roles of ATM in a whole animal and identifying candidate therapeutics for A-T, we performed a screen of 2400 compounds, including FDA-approved drugs, natural products, and bioactive compounds, for modifiers of the developmental lethality caused by a temperature-sensitive ATM allele (ATM8) that has reduced kinase activity at non-permissive temperatures. Ten compounds reproducibly suppressed the developmental lethality of ATM8 flies, including Ronnel, which is an organophosphate. Ronnel and other suppressor compounds are known to cause mitochondrial dysfunction or to inhibit the enzyme acetylcholinesterase, which controls the levels of the neurotransmitter acetylcholine, suggesting that detrimental consequences of reduced ATM kinase activity can be rescued by inhibiting the function of mitochondria or increasing acetylcholine levels. We carried out further studies of Ronnel because, unlike the other compounds that suppressed the developmental lethality of homozygous ATM8 flies, Ronnel was toxic to the development of heterozygous ATM8 flies. Ronnel did not affect the innate immune response of ATM8 flies, and it further increased the already high levels of DNA damage in brains of ATM8 flies, but its effects were not harmful to the lifespan of rescued ATM8 flies. These results provide new leads for understanding the biological and molecular roles of ATM and for the treatment of A-T.

TGFβ promotes podocyte hypertrophy and expression of matrix proteins in fibrotic kidney diseases such as diabetic nephropathy. Both mTORC1 and mTORC2 are hyperactive in response to TGFβ in various renal diseases. Deptor is a component of mTOR complexes and a constitutive inhibitor of their activities. We identified that deptor downregulation by TGFβ maintains hyperactive mTOR in podocytes. To unravel the mechanism, we found that TGFβ -initiated noncanonical signaling controls deptor inhibition. Pharmacological inhibitor of PI 3 kinase, Ly 294002 and pan Akt kinase inhibitor MK 2206 prevented the TGFβ induced downregulation of deptor, resulting in suppression of both mTORC1 and mTORC2 activities. However, specific isoform of Akt involved in this process is not known. We identified Akt2 as predominant isoform expressed in kidney cortex, glomeruli and podocytes. TGFβ time-dependently increased the activating phosphorylation of Akt2. Expression of dominant negative PI 3 kinase and its signaling inhibitor PTEN blocked Akt2 phosphorylation by TGFβ. Inhibition of Akt2 using a phospho-deficient mutant that inactivates its kinase activity, as well as siRNA against the kinase markedly diminished TGFβ -mediated deptor suppression, its association with mTOR and activation of mTORC1 and mTORC2. Importantly, inhibition of Akt2 blocked TGFβ -induced podocyte hypertrophy and expression of the matrix protein fibronectin. This inhibition was reversed by the downregulation of deptor. Interestingly, we detected increased phosphorylation of Akt2 concomitant with TGFβ expression in the kidneys of diabetic rats. Thus, our data identify previously unrecognized Akt2 kinase as a driver of TGFβ induced deptor downregulation and sustained mTORC1 and mTORC2 activation. Furthermore, we provide the first evidence that deptor downstream of Akt2 contributes to podocyte hypertrophy and matrix protein expression found in glomerulosclerosis in different renal diseases.

Osteoclasts are large multinucleated cells responsible for bone resorption. Excessive inflammatory activation of osteoclasts leads to bony erosions, which are the hallmark of several diseases such as rheumatoid arthritis (RA). Salt-inducible kinases (SIK) constitute a subfamily of kinases comprising three members (SIK1, -2, and -3). Inhibition of SIK kinase activity induces an anti-inflammatory phenotype in macrophages. Since osteoclasts originate from precursors of macrophage origin, we hypothesized a role of SIK in osteoclastogenesis. We analyzed SIK1, -2 and -3 expression and function in osteoclast differentiation using the mouse macrophage cell line RAW264.7 and bone marrow-derived macrophages (BMM). We show that all three SIK are expressed in fully differentiated osteoclasts and that in BMM-derived osteoclasts there is an increased expression of SIK1 and SIK3 proteins. Interestingly, the pan-SIK inhibitor HG-9-91-01 significantly inhibited osteoclastogenesis by dose dependently reducing osteoclast differentiation markers (i.e. CathepsinK, MMP-9 and TRAP) and bone resorbing activity. Analysis of the signaling pathways activated by RANKL in RAW cells showed that SIK inhibitors did not affect RANKL-induced ERK1/2, JNK, p38 or NF-κB activation, but induced a significant downregulation in c-Fos and NFATc1 protein levels, the two main transcription factors involved in the regulation of osteoclast-specific genes. Moreover, SIK inhibition partially increased the proteasome-mediated degradation of c-Fos. SIK2 and SIK3 knockout RAW cells were generated by the CRISPR/Cas9 approach. SIK2 KO and, to a lesser extent, SIK3 KO recapitulated the effect of SIK small molecule inhibitor, thus confirming the specificity of the effect of SIK inhibition on the reduction of osteoclastogenesis. Overall, our results support the notion that the SIK signaling pathway plays a significant role among the check-points controlling osteoclastogenesis. SIK kinase inhibitors could thus represent a potential novel therapy to prevent bone erosions.

Mutations within the LRRK2 gene have been identified in Parkinson's disease (PD) patients and have been implicated in the dysfunction of several cellular pathways. Here, we explore how pathogenic mutations and the inhibition of LRRK2 kinase activity affect cytoskeleton dynamics in mouse and human cell systems. We generated and characterized a novel transgenic mouse model expressing physiological levels of human wild type and G2019S-mutant LRRK2. No neuronal loss or neurodegeneration was detected in midbrain dopamine neurons at the age of 12 months. Postnatal hippocampal neurons derived from transgenic mice showed no alterations in the seven parameters examined concerning neurite outgrowth sampled automatically on several hundred neurons using high content imaging. Treatment with the kinase inhibitor LRRK2-IN-1 resulted in no significant changes in the neurite outgrowth. In human fibroblasts we analyzed whether pathogenic LRRK2 mutations change cytoskeleton functions such as cell adhesion. To this end we compared the adhesion characteristics of human skin fibroblasts derived from six PD patients carrying one of three different pathogenic LRRK2 mutations and from four age-matched control individuals. The mutant LRRK2 variants as well as the inhibition of LRRK2 kinase activity did not reveal any significant cell adhesion differences in cultured fibroblasts. In summary, our results in both human and mouse cell systems suggest that neither the expression of wild type or mutant LRRK2, nor the inhibition of LRRK2 kinase activity affect neurite complexity and cellular adhesion.

ALK inhibitor crizotinib has shown potent antitumor activity in children with refractory Anaplastic Large Cell Lymphoma (ALCL) and the opportunity to include ALK inhibitors in first-line therapies is oncoming. However, recent studies suggest that crizotinib-resistance mutations may emerge in ALCL patients. In the present study, we analyzed ALK kinase domain mutational status of 36 paediatric ALCL patients at diagnosis to identify point mutations and gene aberrations that could impact on NPM-ALK gene expression, activity and sensitivity to small-molecule inhibitors. Amplicon ultra-deep sequencing of ALK kinase domain detected 2 single point mutations, R335Q and R291Q, in 2 cases, 2 common deletions of exon 23 and 25 in all the patients, and 7 splicing-related INDELs in a variable number of them. The functional impact of missense mutations and INDELs was evaluated. Point mutations were shown to affect protein kinase activity, signalling output and drug sensitivity. INDELs, instead, generated kinase-dead variants with dominant negative effect on NPM-ALK kinase, in virtue of their capacity of forming non-functional heterocomplexes. Consistently, when co-expressed, INDELs increased crizotinib inhibitory activity on NPM-ALK signal processing, as demonstrated by the significant reduction of STAT3 phosphorylation. Functional changes in ALK kinase activity induced by both point mutations and structural rearrangements were resolved by molecular modelling and dynamic simulation analysis, providing novel insights into ALK kinase domain folding and regulation. Therefore, these data suggest that NPM-ALK pre-therapeutic mutations may be found at low frequency in ALCL patients. These mutations occur randomly within the ALK kinase domain and affect protein activity, while preserving responsiveness to crizotinib.

Smooth muscle cells (SMCs) are key regulators of vascular disease and circulating smooth muscle progenitor cells may play important roles in vascular repair or remodelling. We developed enhanced protocols to derive smooth muscle progenitors from murine bone marrow and tested whether factors that are increased in atherosclerotic plaques, namely platelet-derived growth factor—BB (PDGF-BB) and monomeric collagen, can influence the smooth muscle specific differentiation, proliferation, and survival of mouse bone marrow-derived progenitor cells. During a 21 day period of culture, bone marrow cells underwent a marked increase in expression of the SMC markers α-SMA (1.93 ± 0.15 vs. 0.0008 ± 0.0003 (ng/ng GAPDH) at 0 d), SM22-α (1.50 ± 0.27 vs. 0.005 ± 0.001 (ng/ng GAPDH) at 0 d) and SM-MHC (0.017 ± 0.004 vs. 0.001 ± 0.001 (ng/ng GAPDH) at 0 d). Bromodeoxyuridine (BrdU) incorporation experiments showed that in early culture, the smooth muscle progenitor subpopulation could be identified by high proliferative rates prior to the expression of smooth muscle specific markers. Culture of fresh bone marrow or smooth muscle progenitor cells with PDGF-BB suppressed the expression of α-SMA and SM22-α, in a rapidly reversible manner requiring PDGF receptor kinase activity. Progenitors cultured on polymerized collagen gels demonstrated expression of SMC markers, rates of proliferation and apoptosis similar to that of cells on tissue culture plastic; in contrast, cells grown on monomeric collagen gels displayed lower SMC marker expression, lower growth rates (319 ± 36 vs. 635 ± 97 cells/mm2), and increased apoptosis (5.3 ± 1.6% vs. 1.0 ± 0.5% (Annexin 5 staining)). Our data shows that the differentiation and survival of smooth muscle progenitors are critically affected by PDGF-BB and as well as the substrate collagen structure.

Activated Akt has been previously implicated in acting on RS domain-containing proteins. However, it has been questioned whether its action is direct or it is mediated by co-existing SR kinase activity. To address this issue we studied in detail the phosphorylation of Lamin B Receptor (LBR) by Akt. Using synthetic peptides and a set of recombinant proteins expressing mutants of the LBR RS domain we now demonstrate that while all serines of the RS domain represent more or less equal phosphoacceptor sites for SRPK1, Ser80 and Ser82 are mainly targeted by Akt. 3D-modeling combined with molecular dynamics (MD) simulations show that amongst short, overlapping LBR RS-containing peptides complying with the minimum Akt recognition consensus sequence, only those bearing phosphosites either at Ser80 or Ser82 are able to fit into the active site of Akt, at least as effectively as its known substrate, GSK3-β. Combined our results provide evidence that Akt kinases directly phosphorylate an RS domain-containing protein and that both the residues N-terminal the phosphosite and at position +1 are essential for Akt specificity, with the latter substrate position being compatible with the arginine residue of RS-repeats.

The genes PRKACA and PRKACB encode the principal catalytic (C) subunits of protein kinase A (PKA) Cα and Cβ, respectively. Cα is expressed in all eukaryotic tissues examined and studies of Cα knockout mice demonstrate a crucial role for Cα in normal physiology. We have sequenced exon 2 through 10 of PRKACA from the genome of 498 Norwegian donors and extracted information about PRKACA mutations from public databases. We identified four interesting nonsynonymous point mutations, Arg45Gln, Ser109Pro, Gly186Val, and Ser263Cys, in the Cα1 splice variant of the kinase. Cα variants harboring the different amino acid mutations were analyzed for kinase activity and regulatory (R) subunit binding. Whereas mutation of residues 45 and 263 did not alter catalytic activity or R subunit binding, mutation of Ser109 significantly reduced kinase activity while R subunit binding was unaltered. Mutation of Cα Gly186 completely abrogated kinase activity and PKA type I but not type II holoenzyme formation. Gly186 is located in the highly conserved DFG motif of Cα and mutation of this residue to Val was predicted to result in loss of binding of ATP and Mg2+, which may explain the kinetic inactivity. We hypothesize that individuals born with mutations of Ser109 or Gly186 may be faced with abnormal development and possibly severe disease. © 2012 Søberg et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The Ste20-like kinase, SLK, has diverse cellular functions. SLK mediates organ development, cell cycle progression, cytoskeletal remodeling, cytokinesis, and cell survival. Expression and activity of SLK are enhanced in renal ischemia-reperfusion injury, and overexpression of SLK was shown to induce apoptosis in cultured glomerular epithelial cells (GECs) and renal tubular cells, as well as GEC/podocyte injury in vivo. The SLK protein consists of a N-terminal catalytic domain and an extensive C-terminal domain, which contains coiled-coils. The present study addresses the regulation of SLK activity. Controlled dimerization of the SLK catalytic domain enhanced autophosphorylation of SLK at T183 and S189, which are located in the activation segment. The full-length ectopically- and endogenously-expressed SLK was also autophosphorylated at T183 and S189. Using ezrin as a model SLK substrate (to address exogenous kinase activity), we demonstrate that dimerized SLK 1-373 or full-length SLK can effectively induce activation-specific phosphorylation of ezrin. Mutations in SLK, including T183A, S189A or T193A reduced T183 or S189 autophosphorylation, and showed a greater reduction in ezrin phosphorylation. Mutations in the coiled-coil region of full-length SLK that impair dimerization, in particular I848G, significantly reduced ezrin phosphorylation and tended to reduce autophosphorylation of SLK at T183. In experimental membranous nephropathy in rats, proteinuria and GEC/podocyte injury were associated with increased glomerular SLK activity and ezrin phosphorylation. In conclusion, dimerization via coiled-coils and phosphorylation of T183, S189 and T193 play key roles in the activation and signaling of SLK, and provide targets for novel therapeutic approaches.

Mitogen Activated Protein Kinases (MAPKs) are a class of serine/threonine kinases that regulate a number of different cellular activities including cell proliferation, differentiation, survival and even death. The pathogen Entamoeba histolytica possess a single homologue of a typical MAPK gene (EhMAPK) whose identification was previously reported by us but its functional implications remained unexplored. EhMAPK, the only mitogen-activated protein kinase from the parasitic protist Entamoeba histolytica with Threonine-X-Tyrosine (TXY) phosphorylation motif was cloned, expressed in E. coli and functionally characterized under different stress conditions. The expression profile of EhMAPK at the protein and mRNA level remained similar among untreated, heat shocked and hydrogen peroxide-treated samples in all cases of dose and time. But a significant difference was obtained in the phosphorylation status of the protein in response to different stresses. Heat shock at 43°C or 0.5 mM H(2)O(2) treatment enhanced the phosphorylation status of EhMAPK and augmented the kinase activity of the protein whereas 2.0 mM H(2)O(2) treatment induced dephosphorylation of EhMAPK and loss of kinase activity. 2.0 mM H(2)O(2) treatment reduced parasite viability significantly but heat shock and 0.5 mM H(2)O(2) treatment failed to adversely affect E. histolytica viability. Therefore, a distinct possibility that activation of EhMAPK is associated with stress survival in E. histolytica is seen. Our study also gives a glimpse of the regulatory mechanism of the protein under in vivo conditions. Since the parasite genome lacks any typical homologue of mammalian MEK, the dual specificity kinases which are the upstream activators of MAPK, indications of the existence of some alternate regulatory mechanisms of the EhMAPK activity is perceived. These may include the autophosphorylation activity of the protein itself in combination with some upstream phosphatases which are not yet identified.

Background: The mTOR inhibitor rapamycin has anti-tumor activity across a variety of human cancers, including hepatocellular carcinoma. However, resistance to its growth inhibitory effects is common. We hypothesized that hepatic cell lines with varying rapamycin responsiveness would show common characteristics accounting for resistance to the drug. Methodology/Principal Findings: We profiled a total of 13 cell lines for rapamycin-induced growth inhibition. The nontumorigenic rat liver epithelial cell line WB-F344 was highly sensitive while the tumorigenic WB311 cell line, originally derived from the WB-F344 line, was highly resistant. The other 11 cell lines showed a wide range of sensitivities. Rapamycin induced inhibition of cyclin E–dependent kinase activity in some cell lines, but the ability to do so did not correlate with sensitivity. Inhibition of cyclin E–dependent kinase activity was related to incorporation of p27 Kip1 into cyclin E–containing complexes in some but not all cell lines. Similarly, sensitivity of global protein synthesis to rapamycin did not correlate with its anti-proliferative effect. However, rapamycin potently inhibited phosphorylation of two key substrates, ribosomal protein S6 and 4E-BP1, in all cases, indicating that the locus of rapamycin resistance was downstream from inhibition of mTOR Complex 1. Microarray analysis did not disclose a unifying mechanism for rapamycin resistance, although the glycolytic pathway was downregulated in all four cell lines studied. Conclusions/Significance: We conclude that the mechanisms of rapamycin resistance in hepatic cells involve alterations of signaling downstream from mTOR and that the mechanisms are highly heterogeneous, thus predicting that maintaining or promoting sensitivity will be highly challenging.

Chemokine (C-C motif) receptor 8 (CCR8), the chemokine receptor for chemokine (C-C motif) ligand 1 (CCL1), is expressed in T-helper type-2 lymphocytes and peritoneal macrophages (PMφ) and is involved in various pathological conditions, including peritoneal adhesions. However, the role of CCR8 in inflammatory responses is not fully elucidated. To investigate the function of CCR8 in macrophages, we compared cytokine secretion from mouse PMφ or bone marrow-derived macrophages (BMMφ) stimulated with various Toll-like receptor (TLR) ligands in CCR8 deficient (CCR8- /-) and wild-type (WT) mice. We found that CCR8-/- PMφ demonstrated attenuated secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 when stimulated with lipopolysaccharide (LPS). In particular, LPS-induced IL-10 production absolutely required CCR8. CCR8-dependent cytokine secretion was characteristic of PMφ but not BMMφ. To further investigate this result, we selected the small molecule compound R243 from a library of compounds with CCR8-antagonistic effects on CCL1-induced Ca2+ flux and CCL1-driven PMφ aggregation. Similar to CCR8-/- PMφ, R243 attenuated secretion of TNF-α, IL-6, and most strikingly IL-10 from WT PMφ, but not BMMφ. CCR8-/- PMφ and R243-treated WT PMφ both showed suppressed c-jun N-terminal kinase activity and nuclear factor-κB signaling after LPS treatment when compared with WT PMφ. A c-Jun signaling pathway inhibitor also produced an inhibitory effect on LPS-induced cytokine secretion that was similar to that of CCR8 deficiency or R243 treatment. As seen in CCR8-/- mice, administration of R243 attenuated peritoneal adhesions in vivo. R243 also prevented hapten-induced colitis. These results are indicative of cross talk between signaling pathways downstream of CCR8 and TLR-4 that induces cytokine production by PMφ. Through use of CCR8-/- mice and the new CCR8 inhibitor, R243, we identified a novel macrophage innate immune response pathway that involves a chemokine receptor.

Extracellular signals are translated and amplified via cascades of serially switched protein kinases, MAP kinases (MAPKs). One of the MAP pathways, the classical RAS/RAF/MEK/ERK pathway, transduces signals from receptor tyrosine kinases and plays a central role in regulation of cell proliferation. RAF kinases (A-, B- and C-RAF) function atop of this cascade and convert signals emanating from conformational change of RAS GTPases into their kinase activity, which in turn phosphorylates their immediate substrate, MEK. Disregulated kinase activity of RAF can result in tumor formation, as documented for many types of cancer, predominantly melanomas and thyroid carcinomas (B-RAF). A-RAF is the least characterized RAF, possibly due to its low intrinsic kinase activity and comparatively mild phenotype of A-RAF knockout mice. Nevertheless, the unique phenotype of araf -/- mice, showed predominantly neurological abnormalities such as cerebellum disorders, suggesting that A-RAF participates in a specific process not complemented by activities of B- and CRAF. Here we describe the role of A-RAF in membrane trafficking and identify its function in a specific step of endocytosis. This work led to the discovery of a C-terminally truncated version of A-RAF, AR149 that strongly interfered with cell growth and polarization in yeast and with endocytosis and actin polymerization in mammalian cells. As this work was in progress two splicing isoforms of ARAF, termed DA-RAF1 and DA-RAF2 were described that act as natural inhibitors of RAS-ERK signaling during myogenic differentiation (Yokoyama et al., 2007). DA-RAF2 contains the first 153 aa of A-RAF and thus is nearly identical with AR149. AR149 localized specifically to the recycling endosomal compartments as confirmed by colocalization and coimmunoprecipitation with ARF6. Expression of AR149 interferes with recycling of endocytosed transferrin (Tfn) and with actin polymerization. The endocytic compartment, where internalized Tfn is trapped, was identified as ARF6- and RAB11- positive endocytic vesicles. We conclude that the inhibition of Tfn trafficking in the absence of A-RAF or under overexpression of AR149 occurs between tubular- and TGNassociated recycling endosomal compartments. siRNA-mediated depletion of endogenous A-RAF or inhibition of MEK by U0126 mimic the AR149 overexpression phenotype, supporting a role of ARAF regulated ERK signalling at endosomes that is controlled by AR149 and targets ARF6. Our data additionally suggest EFA6 as a partner of A-RAF during activation of ARF6. The novel findings on the A-RAF localization and the interaction with ARF6 have led to a new model of ARAF function were A-RAF via activation of ARF6 controls the recycling of endocytic vesicles.Endocytosis and rapid recycling of synaptic vesicles is critically important for the physiological function of neurons. The finding, that A-RAF regulates endocytic recycling open a new perspective for investigation of the role of A-RAF in the nervous system.

The transcriptional activity of the integrated HIV provirus is dependent on the chromatin organization of the viral promoter and the transactivator Tat. Tat recruits the cellular pTEFb complex and interacts with several chromatin-modifying enzymes, including the histone acetyltransferases p300 and PCAF. Here, we examined the interaction of Tat with activation-dependent histone kinases, including the p90 ribosomal S6 kinase 2 (RSK2). Dominant-negative RSK2 and treatment with a small-molecule inhibitor of RSK2 kinase activity inhibited the transcriptional activity of Tat, indicating that RSK2 is important for Tat function. Reconstitution of RSK2 in cells from subjects with a genetic defect in RSK2 expression (Coffin-Lowry syndrome) enhanced Tat transactivation. Tat interacted with RSK2 and activated RSK2 kinase activity in cells. Both properties were lost in a mutant Tat protein (F38A) that is deficient in HIV transactivation. Our data identify a novel reciprocal regulation of Tat and RSK2 function, which might serve to induce early changes in the chromatin organization of the HIV LTR.

G protein-Coupled Receptors (GPCRs) kinases (GRKs) play a crucial role in regulating cardiac hypertrophy. Recent data from our lab has shown that, following ventricular pressure overload, GRK5, a primary cardiac GRK, facilitates maladaptive myocyte growth via novel nuclear localization. In the nucleus, GRK5's newly discovered kinase activity on histone deacetylase 5 induces hypertrophic gene transcription. The mechanisms governing the nuclear targeting of GRK5 are unknown. We report here that GRK5 nuclear accumulation is dependent on Ca(2+)/calmodulin (CaM) binding to a specific site within the amino terminus of GRK5 and this interaction occurs after selective activation of hypertrophic Gq-coupled receptors. Stimulation of myocytes with phenylephrine or angiotensinII causes GRK5 to leave the sarcolemmal membrane and accumulate in the nucleus, while the endothelin-1 does not cause nuclear GRK5 localization. A mutation within the amino-terminus of GRK5 negating CaM binding attenuates GRK5 movement from the sarcolemma to the nucleus and, importantly, overexpression of this mutant does not facilitate cardiac hypertrophy and related gene transcription in vitro and in vivo. Our data reveal that CaM binding to GRK5 is a physiologically relevant event that is absolutely required for nuclear GRK5 localization downstream of hypertrophic stimuli, thus facilitating GRK5-dependent regulation of maladaptive hypertrophy.

PAF complex (PAFc) is an RNA polymerase II associated factor that controls diverse steps of transcription. Although it is generally associated with actively transcribed genes, a repressive PAFc has also been suggested. Here, we report that PAFc regulates the transition from transcription initiation to transcription elongation. PAFc repressed IL-6-induced, but not TNF-alpha-induced, immediate early gene expression. PAFc constitutively associated with the 5'-coding region of the c-Fos locus, then transiently dissociated upon IL-6 stimulation. When CTR9, a component of PAFc, was depleted, higher levels of serine 5-phosphorylated or serine 2-phosphorylated forms of RNA Polymerase II were associated with the unstimulated c-Fos locus. We also observed an increased association of CDK9, a kinase component of the pTEF-b elongation factor, with the c-Fos locus in the CTR9-depleted condition. Furthermore, association of negative elongation factor, NELF, which is required to proceed to the elongation phase, was significantly reduced by CTR9 depletion, whereas elongation factor SPT5 recruitment was enhanced by CTR9 depletion. Finally, the chromatin association of CTR9 was specifically controlled by IL-6-induced kinase activity, because a JAK2 kinase inhibitor, AG-490, blocked its association. In conclusion, our data suggest that PAFc controls the recruitment of NELF and SPT5 to target loci in a signal- and locus-specific manner.

The intersectin 1 (ITSN1) scaffold stimulates Ras activation on endocytic vesicles without activating classic Ras effectors. The identification of Class II phosphatidylinositol 3-kinase beta, PI3KC2β, as an ITSN1 target on vesicles and the presence of a Ras binding domain (RBD) in PI3KC2β suggests a role for Ras in PI3KC2β activation. Here, we demonstrate that nucleotide-free Ras negatively regulates PI3KC2β activity. PI3KC2β preferentially interacts in vivo with dominant-negative (DN) Ras, which possesses a low affinity for nucleotides. PI3KC2β interaction with DN Ras is disrupted by switch 1 domain mutations in Ras as well as RBD mutations in PI3KC2β. Using purified proteins, we demonstrate that the PI3KC2β-RBD directly binds nucleotide-free Ras in vitro and that this interaction is not disrupted by nucleotide addition. Finally, nucleotide-free Ras but not GTP-loaded Ras inhibits PI3KC2β lipid kinase activity in vitro. Our findings indicate that PI3KC2β interacts with and is regulated by nucleotide-free Ras. These data suggest a novel role for nucleotide-free Ras in cell signaling in which PI3KC2β stabilizes nucleotide-free Ras and that interaction of Ras and PI3KC2β mutually inhibit one another.

The interaction of viral proteins with host-cellular proteins elicits the activation of cellular signal transduction pathways and possibly leads to viral pathogenesis as well as cellular biological events. Apoptotic signals induced by DNA-damage are remarkably up-regulated by Friend leukemia virus (FLV) exclusively in C3H hosts; however, the mechanisms underlying the apoptosis enhancement and host-specificity are unknown. Here, we show that C3H mouse-derived hematopoietic cells originally express higher levels of the minichromosome maintenance (MCM) 2 protein than BALB/c- or C57BL/6-deriverd cells, and undergo more frequent apoptosis following doxorubicin-induced DNA-damage in the presence of the FLV envelope protein gp70. Dual transfection with gp70/Mcm2 reproduced doxorubicin-induced apoptosis even in BALB/c-derived 3T3 cells. Immunoprecipitation assays using various deletion mutants of MCM2 revealed that gp70 bound to the nuclear localization signal (NLS) 1 (amino acids 18-24) of MCM2, interfered with the function of NLS2 (amino acids 132-152), and suppressed the normal nuclear-import of MCM2. Cytoplasmic MCM2 reduced the activity of protein phosphatase 2A (PP2A) leading to the subsequent hyperphosphorylation of DNA-dependent protein kinase (DNA-PK). Phosphorylated DNA-PK exhibited elevated kinase activity to phosphorylate P53, thereby up-regulating p53-dependent apoptosis. An apoptosis-enhancing domain was identified in the C-terminal portion (amino acids 703-904) of MCM2. Furthermore, simultaneous treatment with FLV and doxorubicin extended the survival of SCID mice bearing 8047 leukemia cells expressing high levels of MCM2. Thus, depending on its subcellular localization, MCM2 plays different roles. It participates in DNA replication in the nucleus as shown previously, and enhances apoptosis in the cytoplasm.

The epidermal growth factor receptor (EGFR) is involved in many cancers and EGFR has been heavily pursued as a drug target. Drugs targeting EGFR have shown promising clinical results for several cancer types. However, resistance to EGFR inhibitors often occurs, such as with KRAS mutant cancers, therefore new methods of targeting EGFR are needed. The juxtamembrane (JXM) domain of EGFR is critical for receptor activation and targeting this region could potentially be a new method of inhibiting EGFR. We hypothesized that the structural role of the JXM region could be mimicked by peptides encoding a JXM amino acid sequence, which could interfere with EGFR signaling and consequently could have anti-cancer activity. A peptide encoding EGFR 645-662 conjugated to the Tat sequence (TE-64562) displayed anti-cancer activity in multiple human cancer cell types with diminished activity in non-EGFR expressing cells and non-cancerous cells. In nude mice, TE-64562 delayed MDA-MB-231 tumor growth and prolonged survival, without inducing toxicity. TE-64562 induced non-apoptotic cell death after several hours and caspase-3-mediated apoptotic cell death with longer treatment. Mechanistically, TE-64562 bound to EGFR, inhibited its dimerization and caused its down-regulation. TE-64562 reduced phosphorylated and total EGFR levels but did not inhibit kinase activity and instead prolonged it. Our analysis of patient data from The Cancer Genome Atlas supported the hypothesis that down-regulation of EGFR is a potential therapeutic strategy, since phospho- and total-EGFR levels were strongly correlated in a large majority of patient tumor samples, indicating that lower EGFR levels are associated with lower phospho-EGFR levels and presumably less proliferative signals in breast cancer. Akt and Erk were inhibited by TE-64562 and this inhibition was observed in vivo in tumor tissue upon treatment with TE-64562. These results are the first to indicate that the JXM domain of EGFR is a viable drug target for several cancer types.

Transforming growth factor-β1 (TGF-β1) is an important anti-inflammatory cytokine that modulates and resolves inflammatory responses. Recent studies have demonstrated that inflammation enhances neoplastic risk and potentiates tumor progression. In the evolution of cancer, pro-inflammatory cytokines such as IL-1β must overcome the anti-inflammatory effects of TGF-β to boost pro-inflammatory responses in epithelial cells. Here we show that IL-1β or Lipopolysaccharide (LPS) suppresses TGF-β-induced anti-inflammatory signaling in a NF-κB-independent manner. TRAF6, a key molecule in IL-1β signaling, mediates this suppressive effect through interaction with the type III TGF-β receptor (TβRIII), which is TGF-β-dependent and requires type I TGF-β receptor (TβRI) kinase activity. TβRI phosphorylates TβRIII at residue S829, which promotes the TRAF6/TβRIII interaction and consequent sequestration of TβRIII from the TβRII/TβRI complex. Our data indicate that IL-1β enhances the pro-inflammatory response by suppressing TGF-β signaling through TRAF6-mediated sequestration of TβRIII, which may be an important contributor to the early stages of tumor progression.

Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG) stimulate innate immune responses. Phosphoinositide 3-kinase (PI3K) has been implicated in CpG-induced immune activation; however, its precise role has not yet been clarified. CpG-induced production of IL-10 was dramatically increased in macrophages deficient in PI3Kγ (p110γ(-/-)). By contrast, LPS-induced production of IL-10 was unchanged in the cells. CpG-induced, but not LPS-induced, IL-10 production was almost completely abolished in SCID mice having mutations in DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Furthermore, wortmannin, an inhibitor of DNA-PKcs, completely inhibited CpG-induced IL-10 production, both in wild type and p110γ(-/-) cells. Microscopic analyses revealed that CpG preferentially localized with DNA-PKcs in p110γ(-/-) cells than in wild type cells. In addition, CpG was preferentially co-localized with the acidic lysosomal marker, LysoTracker, in p110γ(-/-) cells, and with an early endosome marker, EEA1, in wild type cells. Over-expression of p110γ in Cos7 cells resulted in decreased acidification of CpG containing endosome. A similar effect was reproduced using kinase-dead mutants, but not with a ras-binding site mutant, of p110γ. Thus, it is likely that p110γ, in a manner independent of its kinase activity, inhibits the acidification of CpG-containing endosomes. It is considered that increased acidification of CpG-containing endosomes in p110γ(-/-) cells enforces endosomal escape of CpG, which results in increased association of CpG with DNA-PKcs to up-regulate IL-10 production in macrophages.

MEKK1 is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates the MAPK JNK and is required for microtubule inhibitor-induced apoptosis in B cells. Here, we find that apoptosis induced by actin disruption via cytochalasin D and by the protein phosphatase 1/2A inhibitor okadaic acid also requires MEKK1 activation. To elucidate the functional requirements for activation of the MEKK1-dependent apoptotic pathway, we created mutations within MEKK1. MEKK1-deficient cells were complemented with MEKK1 containing mutations in either the ubiquitin interacting motif (UIM), plant homeodomain (PHD), caspase cleavage site or the kinase domain at near endogenous levels of expression and tested for their sensitivity to each drug. We found that both the kinase activity and the PHD domain of MEKK1 are required for JNK activation and efficient induction of apoptosis by drugs causing cytoskeletal disruption. Furthermore, we discovered that modification of MEKK1 and its localization depends on the integrity of the PHD.

Background: NFkB signaling is of paramount importance in the regulation of apoptosis, proliferation, and inflammatory responses during human development and homeostasis, as well as in many human cancers. Receptor Tyrosine Kinases (RTKs), including the Fibroblast Growth Factor Receptors (FGFRs) are also important in development and disease. However, a direct relationship between growth factor signaling pathways and NFkB activation has not been previously described, although FGFs have been known to antagonize TNFa-induced apoptosis. Methodology/Principal Findings: Here, we demonstrate an interaction between FGFR4 and IKKb (Inhibitor of NFkB Kinase b subunit), an essential component in the NFkB pathway. This novel interaction was identified utilizing a yeast two-hybrid screen [1] and confirmed by coimmunoprecipitation and mass spectrometry analysis. We demonstrate tyrosine phosphorylation of IKKb in the presence of activated FGFR4, but not kinase-dead FGFR4. Following stimulation by TNFa (Tumor Necrosis Factor a) to activate NFkB pathways, FGFR4 activation results in significant inhibition of NFkB signaling as measured by decreased nuclear NFkB localization, by reduced NFkB transcriptional activation in electophoretic mobility shift assays, and by inhibition of IKKb kinase activity towards the substrate GST-IkBa in in vitro assays. FGF19 stimulation of endogenous FGFR4 in TNFa-treated DU145 prostate cancer cells also leads to a decrease in IKKb activity, concomitant reduction in NFkB nuclear localization, and reduced apoptosis. Microarray analysis demonstrates that FGF19 + TNFa treatment of DU145 cells, in comparison with TNFa alone, favors proliferative genes while downregulating genes involved in apoptotic responses and NFkB signaling. Conclusions/Significance: These results identify a compelling link between FGFR4 signaling and the NFkB pathway, and reveal that FGFR4 activation leads to a negative effect on NFkB signaling including an inhibitory effect on proapoptotic signaling. We anticipate that this interaction between an RTK and a component of NFkB signaling will not be limited to FGFR4 alone.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are a common cause of autosomal dominant familial Parkinson's disease (PD). LRRK2 encodes a multi-domain protein containing GTPase and kinase enzymatic domains. Disease-associated mutations in LRRK2 variably influence enzymatic activity with the common G2019S variant leading to enhanced kinase activity. Mutant LRRK2 induces neuronal toxicity through a kinase-dependent mechanism suggesting that kinase activity is important for mediating the pathogenic effects of LRRK2 mutations. A number of LRRK2 kinase substrates have been identified in vitro but whether they represent authentic physiological substrates in mammalian cells or tissues is not yet clear. The eukaryotic initiation factor 4E (eIF4E)-binding protein, 4E-BP1, was recently identified as a potential substrate of LRRK2 kinase activity in vitro and in Drosophila with phosphorylation occurring at Thr37 and Thr46. Here, we explore a potential interaction of LRRK2 and 4E-BP1 in mammalian cells and brain. We find that LRRK2 can weakly phosphorylate 4E-BP1 in vitro but LRRK2 overexpression is not able to alter endogenous 4E-BP1 phosphorylation in mammalian cells. In mammalian neurons LRRK2 and 4E-BP1 display minimal co-localization, whereas the subcellular distribution, protein complex formation and covalent post-translational modification of endogenous 4E-BP1 are not altered in the brains of LRRK2 knockout or mutant LRRK2 transgenic mice. In the brain, the phosphorylation of 4E-BP1 at Thr37 and Thr46 does not change in LRRK2 knockout or mutant LRRK2 transgenic mice, nor is 4E-BP1 phosphorylation altered in idiopathic or G2019S mutant PD brains. Collectively, our results suggest that 4E-BP1 is neither a major nor robust physiological substrate of LRRK2 in mammalian cells or brain.

The anticancer agent 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine (laromustine), upon decomposition in situ, yields methyl isocyanate and the chloroethylating species 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE). 90CE has been shown to kill tumor cells via a proposed mechanism that involves interstrand DNA cross-linking. However, the role of methyl isocyanate in the antineoplastic function of laromustine has not been delineated. Herein, we show that 1,2-bis(methylsulfonyl)-1-[(methylamino)carbonyl]hydrazine (101MDCE), an analog of laromustine that generates only methyl isocyanate, activates ASK1-JNK/p38 signaling in endothelial cells (EC). We have previously shown that ASK1 forms a complex with reduced thioredoxin (Trx1) in resting EC, and that the Cys residues in ASK1 and Trx1 are critical for their interaction. 101MDCE dissociated ASK1 from Trx1, but not from the phosphoserine-binding inhibitor 14-3-3, in whole cells and in cell lysates, consistent with the known ability of methyl isocyanate to carbamoylate free thiol groups of proteins. 101MDCE had no effect on the kinase activity of purified ASK1, JNK, or the catalytic activity of Trx1. However, 101MDCE, but not 90CE, significantly decreased the activity of Trx reductase-1 (TrxR1). We conclude that methyl isocyanate induces dissociation of ASK1 from Trx1 either directly by carbamoylating the critical Cys groups in the ASK1-Trx1 complex or indirectly by inhibiting TrxR1. Furthermore, 101MDCE (but not 90CE) induced EC death through a non-apoptotic (necroptotic) pathway leading to inhibition of angiogenesis in vitro. Our study has identified methyl isocyanates may contribute to the anticancer activity in part by interfering with tumor angiogenesis.

PB1-F2, a protein encoded by a second open reading frame of the influenza virus RNA segment 2, has emerged as a modulator of lung inflammatory responses but the molecular mechanisms underlying this are only poorly understood. Here we show that PB1-F2 inhibits the activation of NF-κB dependent signalling pathways in luciferase reporter assays. PB1-F2 proteins from four different viruses interact with IKKβ in yeast two-hybrid assays and by co-immunoprecipitation. PB1-F2 expression did not inhibit IKKβ kinase activity or NF-κB translocation into the nucleus, but NF-κB binding to DNA was severely impaired in PB1-F2 transfected cells as assessed by Electrophoretic Mobility Shift Assay. Neither the N-terminal 57 amino acid truncated forms nor the C-terminus of PB1-F2 were able to inhibit NF-κB dependent signalling, indicating that the full length protein is necessary for the inhibition.

RIG-I-like receptors (RLR) are intracellular sensors utilized by nearly all cell types for recognition of viral RNA, initiation of antiviral defense, and induction of type I interferons (IFN). TBK1 is a critical kinase implicated in RLR-dependent IFN transcription. Posttranslational modification of TBK1 by K63-linked ubiquitin is required for RLR driven signaling. However, the TBK1 ubiquitin acceptor sites and the function of ubiquitinated TBK1 in the signaling cascade are unknown. We now show that TBK1 is ubiquitinated on residues K69, K154, and K372 in response to infection with RNA virus. The K69 and K154 residues are critical for innate antiviral responses and IFN production. Ubiquitinated TBK1 recruits the downstream adaptor NEMO through ubiquitin binding domains. The assembly of the NEMO/TBK1 complex on the mitochondrial protein MAVS leads to activation of TBK1 kinase activity and phosphorylation of the transcription factor, interferon response factor 3. The combined results refine current views of RLR signaling, define the role of TBK1 polyubiquitination, and detail the mechanisms involved in signalosome assembly.

The p38 MAPK signaling pathway plays a pivotal role in inflammation. Targeting p38 MAPK may be a potential strategy for the treatment of inflammatory diseases. In the present study, we show that a novel chromone derivative, DCO-6, significantly reduced lipopolysaccharide (LPS)-induced production of nitric oxide, IL-1β and IL-6, decreased the levels of iNOS, IL-1β and IL-6 mRNA expression in both RAW264.7 cells and mouse primary peritoneal macrophages, and inhibited LPS-induced activation of p38 MAPK but not of JNK, ERK. Moreover, DCO-6 specifically inhibited TLR4-dependent p38 activation without directly inhibiting its kinase activity. LPS-induced production of intracellular reactive oxygen species (ROS) was remarkably impaired by DCO-6, which disrupted the formation of the TRAF6-ASK1 complex. Administering DCO-6 significantly protected mice from LPS-induced septic shock in parallel with the inhibition of p38 activation and ROS production. Our results indicate that DCO-6 showed anti-inflammatory properties through inhibition of ROS-dependent activation of TRAF6-ASK1-p38 pathway. Blockade of the upstream events required for p38 MAPK action by DCO-6 may provide a new therapeutic option in the treatment of inflammatory diseases.

Background: Programmed cell death is critical not only in adult tissue homeostasis but for embryogenesis as well. One of the earliest steps in development, formation of the proamniotic cavity, involves coordinated apoptosis of embryonic cells. Recent work from our group demonstrated that c-Src protein-tyrosine kinase activity triggers differentiation of mouse embryonic stem (mES) cells to primitive ectoderm-like cells. In this report, we identified Timeless (Tim), the mammalian ortholog of a Drosophila circadian rhythm protein, as a binding partner and substrate for c-Src and probed its role in the differentiation of mES cells. Methodology/Principal Findings: To determine whether Tim is involved in ES cell differentiation, Tim protein levels were stably suppressed using shRNA. Tim-defective ES cell lines were then tested for embryoid body (EB) formation, which models early mammalian development. Remarkably, confocal microscopy revealed that EBs formed from the Tim-knockdown ES cells failed to cavitate. Cells retained within the centers of the failed cavities strongly expressed the pluripotency marker Oct4, suggesting that further development is arrested without Tim. Immunoblots revealed reduced basal Caspase activity in the Tim-defective EBs compared to wild-type controls. Furthermore, EBs formed from Tim-knockdown cells demonstrated resistance to staurosporine-induced apoptosis, consistent with a link between Tim and programmed cell death during cavitation. Conclusions/Significance: Our data demonstrate a novel function for the clock protein Tim during a key stage of early development. Specifically, EBs formed from ES cells lacking Tim showed reduced caspase activity and failed to cavitate. As a consequence, further development was halted, and the cells present in the failed cavity remained pluripotent. These findings reveal a new function for Tim in the coordination of ES cell differentiation, and raise the intriguing possibility that circadian rhythms and early development may be intimately linked. © 2011 O'Reilly et al.

Arrestins were originally described as proteins recruited to ligand-activated, phosphorylated G protein-coupled receptors (GPCRs) to attenuate G protein-mediated signaling. It was later revealed that arrestins also mediate GPCR internalization and recruit a number of signaling proteins including, but not limited to, Src family kinases, ERK1/2, and JNK3. GPCR-arrestin binding and trafficking control the spatial and temporal activity of these multi-protein complexes. In previous reports, we concluded that N-formyl peptide receptor (FPR)-mediated apoptosis, which occurs upon receptor stimulation in the absence of arrestins, is associated with FPR accumulation in perinuclear recycling endosomes. Under these conditions, inhibition of Src kinase and ERK1/2 prevented FPR-mediated apoptosis. To better understand the role of Src kinase in this process, in the current study we employed a previously described arrestin-2 (arr2) mutant deficient in Src kinase binding (arr2-P91G/P121E). Unlike wild type arrestin, arr2-P91G/P121E did not inhibit FPR-mediated apoptosis, suggesting that Src binding to arrestin-2 prevents apoptotic signaling. However, in cells expressing this mutant, FPR-mediated apoptosis was still blocked by inhibition of Src kinase activity, suggesting that activation of Src independent of arrestin-2 binding is involved in FPR-mediated apoptosis. Finally, while Src kinase inhibition prevented FPR-mediated-apoptosis in the presence of arr2-P91G/P121E, it did not prevent FPR-arr2-P91G/P121E accumulation in the perinuclear recycling endosome. On the contrary, inhibition of Src kinase activity mediated the accumulation of activated FPR-wild type arrestin-2 in recycling endosomes without initiating FPR-mediated apoptosis. Based on these observations, we conclude that Src kinase has two independent roles following FPR activation that regulate both FPR-arrestin-2 signaling and trafficking.

Objective Experimental and clinical studies have shown that administration of insulin during reperfusion is cardioprotective, but the mechanisms underlying this effect are still unknown. In this study, the ability of insulin to protect apoptotic cardiomyocytes from hypoxia/reoxygenation injury using the sphingosine kinase/sphingosine 1-phosphate axis was investigated. Methods and Results Rat cardiomyocytes were isolated and subjected to hypoxia and reoxygenation. [γ-32P] ATP was used to assess sphingosine kinase activity. Insulin was found to increase sphingosine kinase activity. Immunocytochemistry and Western blot analysis showed changes in the subcellular location of sphingosine kinase 1 from cytosol to the membrane in cardiomyocytes. Insulin caused cardiomyocytes to accumulate of S1P in a dose-dependent manner. FRET efficiency showed that insulin also transactivates the S1P1 receptor. TUNEL staining showed that administration of insulin during reoxygenation could to reduce the rate of reoxygenation-induced apoptosis, which is a requirement for SphK 1 activity. It also reduced the rate of activation of the S1P receptor and inhibited hypoxia/reoxygenation-induced cell death in cardiomyocytes. Conclusion The sphingosine kinase 1/sphingosine 1-phosphate/S1P receptor axis is one pathway through which insulin protects rat cardiomyocytes from apoptosis induced by hypoxia/reoxygenation injury.

We examined expression of protease-activated receptors 2 (PAR2) and characterized their signaling pathways in rabbit gastric muscle cells. The PAR2 activating peptide SLIGRL (PAR2-AP) stimulated Gq, G13, Gi1, PI hydrolysis, and Rho kinase activity, and inhibited cAMP formation. Stimulation of PI hydrolysis was partly inhibited in cells expressing PAR2 siRNA, Gaq or Gai minigene and in cells treated with pertussis toxin, and augmented by expression of dominant negative regulator of G protein signaling (RGS4(N88S)). Stimulation of Rho kinase activity was abolished by PAR-2 or Ga13 siRNA, and by Ga13 minigene. PAR2-AP induced a biphasic contraction; initial contraction was selectively blocked by the inhibitor of PI hydrolysis (U73122) or MLC kinase (ML-9), whereas sustained contraction was selectively blocked by the Rho kinase inhibitor (Y27632). PAR2-AP induced phosphorylation of MLC20, MYPT1 but not CPI-17. PAR2-AP also caused a decrease in the association of NF-kB and PKA catalytic subunit: the effect of PAR2-AP was blocked by PAR2 siRNA or phosphorylation-deficient RhoA (RhoA(S188A)). PAR2-AP-induced degradation of IkBa and activation of NF-kB were abolished by the blockade of RhoA activity by Clostridium botulinum C3 exoenzyme suggesting RhoA-dependent activation of NF-kB. PAR2-AP-stimulated Rho kinase activity was significantly augmented by the inhibitors of PKA (myristoylated PKI), IKK2 (IKKIV) or NF-kB (MG132), and in cells expressing dominant negative mutants of IKK (IKK(K44A), IkBa (IkBa (S32A/S36A)) or RhoA(S188A), suggesting feedback inhibition of Rho kinase activity via PKA derived from NF-kB pathway. PAR2-AP induced phosphorylation of RhoA and the phosphorylation was attenuated in cells expressing phosphorylation-deficient RhoA(S188A). Our results identified signaling pathways activated by PAR2 to mediate smooth muscle contraction and a novel pathway for feedback inhibition of PAR2-stimulated RhoA. The pathway involves activation of the NF-kB to release catalytic subunit of PKA from its binding to IkBa and phosphorylation of RhoA at Ser(188).