Insights into the regulation of the human COP9 signalosome catalytic subunit,CSN5/Jab1

The COP9 (Constitutive photomorphogenesis 9) signalosome (CSN), a large multiprotein complex that resembles the 19S lid of the 26S proteasome, plays a central role in the regulation of the E3-cullin RING ubiquitin ligases (CRLs). The catalytic activity of the CSN complex, carried by subunit 5 (CSN5/Jab1), resides in the deneddylation of the CRLs that is the hydrolysis of the cullin-neural precursor cell expressed developmentally downregulated gene 8 (Nedd8)isopeptide bond. Whereas CSN-dependent CSN5 displays isopeptidase activity, it is intrinsically inactive in other physiologically relevant forms. Here we analyze the crystal structure of CSN5 in its catalytically inactive form to illuminate the molecular basis for its activation state. We show that CSN5 presents a catalytic domain that brings essential elements to understand its activity control. Although the CSN5 active site is catalytically competent and compatible with di-isopeptide binding, the Ins-1 segment obstructs access to its substrate-binding site, and structural rearrangements are necessary for the Nedd8-binding pocket formation. Detailed study of CSN5 by molecular dynamics unveils signs of flexibility and plasticity of the Ins-1 segment. These analyses led to the identification of a molecular trigger implicated in the active/inactive switch that is sufficient to impose on CSN5 an active isopeptidase state. We show that a single mutation in the Ins-1 segment restores biologically relevant deneddylase activity. This study presents detailed insights into CSN5 regulation. Additionally, a dynamic monomer-dimer equilibrium exists both in vitro and in vivo and may be functionally relevant.     

Crystal structure and versatile functional roles of the COP9 signalosome subunit 1

The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) plays key roles in many biological processes, such as repression of photomorphogenesis in plants and protein subcellular localization, DNA-damage response, and NF-κB activation in mammals. It is an evolutionarily conserved eight-protein complex with subunits CSN1 to CSN8 named following the descending order of molecular weights. Here, we report the crystal structure of the largest CSN subunit, CSN1 from Arabidopsis thaliana (atCSN1), which belongs to the Proteasome, COP9 signalosome, Initiation factor 3 (PCI) domain containing CSN subunit family, at 2.7 Å resolution. In contrast to previous predictions and distinct from the PCI-containing 26S proteasome regulatory particle subunit Rpn6 structure, the atCSN1 structure reveals an overall globular fold, with four domains consisting of helical repeat-I, linker helix, helical repeat-II, and the C-terminal PCI domain. Our small-angle X-ray scattering envelope of the CSN1–CSN7 complex agrees with the EM structure of the CSN alone (apo-CSN) and suggests that the PCI end of each molecule may mediate the interaction. Fitting of the CSN1 structure into the CSN–Skp1-Cul1-Fbox (SCF) EM structure shows that the PCI domain of CSN1 situates at the hub of the CSN for interaction with several other subunits whereas the linker helix and helical repeat-II of CSN1 contacts SCF using a conserved surface patch. Furthermore, we show that, in human, the C-terminal tail of CSN1, a segment not included in our crystal structure, interacts with IκBα in the NF-κB pathway. Therefore, the CSN complex uses multiple mechanisms to hinder NF-κB activation, a principle likely to hold true for its regulation of many other targets and pathways.The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) is a more than 300-kDa complex that was first identified as a negative regulator of Constitutive Photomorphogenesis (COP) in plants (1, 2). In the subsequent years, the highly conserved protein complex was also found in fungi (3, 4), Caenorhabditis elegans (5), Drosophila melanogaster (6), and mammals (7, 8). The most studied function of the CSN complex in eukaryotes is the regulation of protein degradation through two pathways, deneddylation (9–11) and deubiquitination (12, 13). In the deneddylation pathway, the CSN complex can influence the cullin-RING ligase activity by removing Nedd8, a ubiquitin-like protein, from a cullin (9, 14). On the other hand, the CSN complex can also suppress cullin activity through recruitment of the deubiquitination enzyme USP15 (12) or Ubp12p, the Schizosaccharomyces pombe ortholog of human USP15 (13). Other functions of the CSN complex identified in mammalian cells include regulating the phosphorylation of ubiquitin–proteasome pathway substrates through CSN-associated kinases (7, 15–18). Overall, the CSN complex appears to be a key player in protein subcellular localization (19, 20), DNA-damage response (21), NF-κB activation (22), development, and cell cycle control (23, 24). Thus, the functions of the CSN complex are beyond the regulation of light-dependent reaction in plants.The CSN complex in most of the species contains eight subunits named CSN1 to CSN8, in order of decreasing size. All eight subunits share homologous sequences with “lid” components of the 26S proteasome regulatory particle and the eukaryotic translation initiation factor 3 (eIF3) (7, 25). Among these eight subunits, CSN6 and catalytic CSN5 contain a conserved MPN-domain (MOV34, Pad1N-terminal) (26), and the rest of the CSN subunits bear a PCI-domain (Proteasome, COP9 signalosome, Initiation factor eIF3). The MPN-domain within CSN5 contains a metal chelating site and forms the catalytic region of the isopeptidase for deneddylation (27). Recently, the crystal structures of the CSN6–MPN domain and the CSN5 subunit have been revealed (28, 29). Interestingly, amino acids 97–131, a flexible segment within the CSN5–MPN domain, were proven to be essential in regulating the isopeptidase states of CSN5 (29). PCI is an ∼200-amino acid domain, beginning with an N-terminal helical bundle arrangement and ending with a globular winged-helix subdomain (30, 31). A number of interactions between PCI domains of CSN subunits have been identified by the yeast two-hybrid system (32, 33). Dessau et al. reported the crystallographic data of the PCI domain of Arabidopsis thaliana subunit 7, and their in vitro studies also suggested that the PCI domain mediates and stabilizes protein–protein interactions within the complex (34).Although many speculated on how the CSN subunits interact with each other and come into a functional unit, the architecture of the CSN complex was accessed by electron microscopy (EM) (35, 36) and native mass spectrometry approaches (37). These studies confirmed structural similarities among CSN, the proteasome lid, and eIF3. Furthermore, the CSN appears to contain two dominant subcomplexes, CSN1/2/3/8 and CSN 4/5/6/7 (37), which correspond to the large and the small segments, respectively, in an EM study of the CSN alone (apo-CSN) (36). An EM study of the CSN in complex with an Skp1-Cul1-Fbox (SCF) E3 ligase was also reported, showing reciprocal regulation between CSN and SCF (38). To date, unfortunately, there is no high-resolution mapping on these subunit interactions.To further define the CSN structure and to study its functional significance, we feel the need to obtain structures of CSN subunits at an atomic level. In our study, we used Arabidopsis thaliana CSN1 (atCSN1) as a guide to facilitate our understandings of the PCI-containing CSN subunits. The atCSN1, encoded in the chromosome 3, has 441 amino acids that are 45% identical in sequence to Homo sapiens CSN1. Among all of the subunits of the complex, CSN1 is known to be the longest and to play a crucial role in complex integrity (39–41). Here, we report the crystal structure of atCSN1 and describe its integration within the complex as well as its interaction with IκBα in the NF-κB signaling pathway.     

Morphogenesis checkpoint kinase Swe1 is the executor of lipolysis-dependent cell-cycle progression

Cell growth and division requires the precise duplication of cellular DNA content but also of membranes and organelles. Knowledge about the cell-cycle–dependent regulation of membrane and storage lipid homeostasis is only rudimentary. Previous work from our laboratory has shown that the breakdown of triacylglycerols (TGs) is regulated in a cell-cycle–dependent manner, by activation of the Tgl4 lipase by the major cyclin-dependent kinase Cdc28. The lipases Tgl3 and Tgl4 are required for efficient cell-cycle progression during the G1/S (Gap1/replication phase) transition, at the onset of bud formation, and their absence leads to a cell-cycle delay. We now show that defective lipolysis activates the Swe1 morphogenesis checkpoint kinase that halts cell-cycle progression by phosphorylation of Cdc28 at tyrosine residue 19. Saturated long-chain fatty acids and phytosphingosine supplementation rescue the cell-cycle delay in the Tgl3/Tgl4 lipase-deficient strain, suggesting that Swe1 activity responds to imbalanced sphingolipid metabolism, in the absence of TG degradation. We propose a model by which TG-derived sphingolipids are required to activate the protein phosphatase 2A (PP2A^Cdc55) to attenuate Swe1 phosphorylation and its inhibitory effect on Cdc28 at the G1/S transition of the cell cycle.The eukaryotic cell cycle is a highly coordinated and conserved process. In addition to DNA replication, one of the major requirements for the cell to progress through the cell cycle is the precise duplication of membrane-enclosed organelles and other cellular components before cell division. Knowledge about the mechanisms regulating (membrane) lipid homeostasis during the cell cycle is scarce (1), however several levels of evidence suggest regulation of key enzymes of lipid metabolism in a cell-cycle–dependent manner. The PAH1-encoded phosphatidic acid (PA) phosphatase (Pah1), a key enzyme of triacylglycerol (TG) synthesis that provides the TG precursor diacylglycerol (DG), is phosphorylated and inactivated by the cyclin-dependent kinases Cdc28 and Pho85–Pho80 (2, 3). Kurat et al. showed that Tgl4, next to Tgl3, one of the two major TG lipases in yeast and the ortholog of mammalian ATGL (4, 5), is also phosphorylated by Cdc28. In contrast to Pah1, however, Tgl4 is activated by Cdc28 (6). This inverse regulation of Pah1 and Tgl4 by Cdc28-dependent phosphorylation led to the model by which the TG content oscillates during the cell cycle: On the one hand, TG synthesis serves as a buffer for excess de novo generated fatty acids (FAs), and on the other hand, in times of increased demand—that is, at the onset of bud formation and bud growth—Tgl4-catalyzed lipolysis becomes active to provide TG-derived precursors for membrane lipid synthesis (6).TG and membrane phospholipids share the same intermediates, PA and DG; PA is generated by sequential acylation of glycerol-3-phosphate, reactions that mostly take place in the endoplasmic reticulum (ER) membrane (7). The dephosphorylation of PA to DG by the PAH1-encoded PA phosphatase Pah1 is the major regulator of cellular TG synthesis in yeast (8), similar to its mammalian ortholog, lipin (9). According to this central role, TG content in PAH1-deficient yeast cells is decreased by 70–90%; the source of the residual TG in these mutants is currently unclear. TG synthesis from DG requires one additional acylation step that is catalyzed by the DGA1-encoded acyl-CoA–dependent DG acyltransferase (10, 11) and the phospholipid-dependent acyltransferase, encoded by LRO1 (7). Alternatively, in the presence of the phospholipid precursors, ethanolamine and/or choline, DG may be converted into phospholipids via the Kennedy pathway (7). Thus, net TG synthesis in growing cells is determined by multiple factors, including the availability of FAs, presence of lipid precursors, and the activities of PA phosphatase and the DG acyltransferases. Degradation of TG in yeast is governed by the major lipid droplet (LD)-associated lipases, encoded by TGL3 and TGL4 (4, 12); both enzymes belong to the patatin-domain–containing family of proteins, members of which play a crucial role in lipid homeostasis also in mammals (13). Multiple additional lipases exist in yeast, but their specific function and contribution to TG homeostasis may be restricted to specific growth conditions (7, 14, 15).Absence of lipolysis in mutants lacking TGL3 and TGL4 results in up to threefold elevated levels of TG and reduced levels of phosphatidylcholine and sphingolipids (4, 12, 16, 17), indicating that TG breakdown provides precursors for these lipids or generates some regulatory factors required for their synthesis. The rate of phosphatidylinositol (PI) synthesis after readdition of inositol to inositol-starved cells is reduced by 50% in lipase-deficient cells; the boost of PI synthesis under inositol refeeding conditions is completely abolished if de novo FA synthesis is additionally blocked in the lipase mutants by the inhibitor cerulenin (18). These data clearly demonstrate the requirements for TG breakdown, in addition to de novo FA synthesis, to generate precursors for membrane lipids. As a consequence of defective lipolysis, entry of quiescent cells into vegetative growth is significantly delayed; thus, TG breakdown is particularly important for promoting exit from the stationary phase and entry into the gap1 (G1) phase of the cell cycle (4, 6, 19).Progression through the cell cycle is regulated by specific checkpoint pathways that ensure completion of crucial events and execute a halt under nonconducive conditions. Checkpoint mechanisms slow down or arrest the cell cycle to enable cells to fix damage or to obtain the required metabolites before proceeding and are as such important for the integrity of cell division (20–22). According to this critical function in quality control, mutations in checkpoint genes in mammals have been linked to cancer predisposition and progression. The first discovered cell-cycle checkpoint in Schizosaccharomyces pombe that regulates entry into mitosis is executed by the Wee1 kinase (23, 24), which delays mitosis by phosphorylating and inhibiting cyclin-dependent kinase Cdk1 (25). Conversely, the phosphatase Cdc25 promotes entry into mitosis by removing the inhibitory phosphorylation of Cdk1 (26–28). The budding yeast orthologs of Wee1 and Cdc25 are called Swe1 and Mih1, and their key functions in regulating Cdk1 activity are highly conserved (29, 30). Swe1 phosphorylates Cdk1 (encoded by CDC28 in budding yeast) at the tyrosine 19 residue and inhibits its kinase activity (29, 31, 32); the Mih1 phosphatase removes this inhibitory phosphorylation initiating G2/M cell-cycle progression (26). The Swe1 and Cdk1/Cdc28 kinases operate in an autoregulatory loop in which Swe1 is initially phosphorylated and activated by Cdk1/Cdc28 that is associated with mitotic cyclins; subsequently, activated Swe1 phosphorylates and inhibits Cdk1/Cdc28 (33). The initial phosphorylation of Swe1 is opposed by the protein phosphatase 2A (PP2A) with its catalytic subunits Pph21 or Pph22 and the regulatory subunit Cdc55 (PP2A^Cdc55), which sets a threshold, limiting the activation of Swe1 by Cdk1/Cdc28 in early mitosis (34, 35). Loss of the regulatory subunit Cdc55 leads to hyperactivation of Swe1 (35); after the initial phosphorylation of Swe1 in early mitosis, subsequent phosphorylation events trigger full hyperphosphorylation of Swe1 (33), which leads to its ubiquitin-mediated degradation (36, 37). Of note, regulation of Cdk1/Cdc28 by the G1 cyclin Cln2 plays an important role in actin cytoskeleton polarization and the localized delivery of secretory vesicles, which contribute membrane lipids to the developing bud, thus linking cell surface growth to the cell cycle (38).Despite its proposed role as a gap2 phase (G2) checkpoint regulator, we now show that Swe1 kinase is responsible for the G1/S (Gap1/replication phase) cell-cycle delay in mutants defective in TG lipolysis by phosphorylating Cdk1/Cdc28 at tyrosine 19. Deletion of Swe1 in the tgl3 tgl4 lipase mutant restores normal cell-cycle progression; similarly, supplementation of mutant cells with saturated FAs (myristic acid, palmitic acid) or a precursor of sphingolipid synthesis, phytosphingosine (PHS), suppress the cell-cycle delay in the lipase mutants. These data suggest that Swe1 is a lipid-regulated kinase that is activated in the absence of specific lipids, presumably sphingolipids, and halts G1/S transition by phosphorylating Cdk1/Cdc28 in lipase-deficient cells that exit from the G0 phase of the cell cycle.     

The immunogenicity of Bcr-Abl expressing dendritic cells is dependent on the Bcr-Abl kinase activity and dominated by Bcr-Abl regulated antigens

In Ph(+) chronic myeloid leukemia (CML), the constitutively active Bcr-Abl kinase leads to the up-regulation and activation of multiple genes, which may subsequently result in the expression of leukemia-associated antigens. In this study, we investigated the immunogenicity of Bcr-Abl-regulated antigens by stimulating CD8(+) T lymphocytes with autologous dendritic cells transfected with RNA coding for Bcr-Abl wild-type or a kinase-deficient mutant. Significant HLA class I-restricted T-cell responses were detected against antigens regulated by the Bcr-Abl kinase, but not toward the Bcr-Abl protein itself. The T-cell repertoire of a patient with CML in major molecular remission due to imatinib mesylate was also dominated by T cells directed against Bcr-Abl-regulated antigens. These results encourage the development of immunotherapeutic approaches against Bcr-Abl-regulated antigens for the treatment of CML patients with residual disease following therapy with Bcr-Abl kinase inhibitors.     

The Btk tyrosine kinase is a major target of the Bcr-Abl inhibitor dasatinib

Dasatinib is a small-molecule kinase inhibitor used for the treatment of imatinib-resistant chronic myelogenous leukemia (CML). We have analyzed the kinases targeted by dasatinib by using an unbiased chemical proteomics approach to detect binding proteins directly from lysates of CML cells. Besides Abl and Src kinases, we have identified the Tec kinases Btk and Tec, but not Itk, as major binders of dasatinib. The kinase activity of Btk and Tec, but not of Itk, was inhibited by nanomolar concentrations of dasatinib in vitro and in cultured cells. We identified the gatekeeper residue as the critical determinant of dasatinib susceptibility. Mutation of Thr-474 in Btk to Ile and Thr-442 in Tec to Ile conferred resistance to dasatinib, whereas mutation of the corresponding residue in Itk (Phe-435) to Thr sensitized the otherwise insensitive Itk to dasatinib. The configuration of this residue may be a predictor for dasatinib sensitivity across the kinome. Analysis of mast cells derived from Btk-deficient mice suggested that inhibition of Btk by dasatinib may be responsible for the observed reduction in histamine release upon dasatinib treatment. Furthermore, dasatinib inhibited histamine release in primary human basophils and secretion of proinflammatory cytokines in immune cells. The observed inhibition of Tec kinases by dasatinib predicts immunosuppressive (side) effects of this drug and may offer therapeutic opportunities for inflammatory and immunological disorders.     

The serum- and glucocorticoid-induced kinase is a physiological mediator of aldosterone action

Aldosterone plays a major role in regulating sodium and potassium flux in epithelial tissues such as kidney and colon. Recent evidence suggests that serum- and glucocorticoid-regulated kinase (SGK) is induced by aldosterone and acts as a key mediator of aldosterone action in epithelial tissues. Induction of SGK messenger RNA (mRNA) has previously been shown within 30 min of addition of supraphysiological doses of aldosterone to XENOPUS: A6 cells and within 4 h in rat kidney in vivo. In this study we determined the time course of SGK induction, at doses of aldosterone in the physiological range, in rat kidney and colon, using Northern and Western blot analyses and in situ hybridization and determined concurrent changes in urinary sodium and potassium excretion by Kagawa bioassay. On Northern blot analysis, SGK mRNA levels were significantly elevated in both kidney and colon 60 min after the injection of aldosterone. SGK protein in late distal colon was significantly elevated 2 and 4 h after aldosterone treatment. In situ hybridization showed SGK mRNA to be induced in renal collecting ducts and distal tubular elements in both cortex and medulla by doses of aldosterone of 0.1 microg/100 g BW or more within 30 min of steroid treatment. Significant changes in urinary composition were similarly seen with an aldosterone dose of 0.1 microg/100 g BW from 90 min after aldosterone injection. The early onset of SGK induction in kidney and colon and the correlation with urinary changes in terms of both time course and dose response suggest that SGK plays an important role in mediating the effects of aldosterone on sodium homeostasis in vivo.     

Osteopontin is a novel downstream target of SOX9 with diagnostic implications for progression of liver fibrosis in humans

Osteopontin (OPN) is an important component of the extracellular matrix (ECM), which promotes liver fibrosis and has been described as a biomarker for its severity. Previously, we have demonstrated that Sex-determining region Y-box 9 (SOX9) is ectopically expressed during activation of hepatic stellate cells (HSC) when it is responsible for the production of type 1 collagen, which causes scar formation in liver fibrosis. Here, we demonstrate that SOX9 regulates OPN. During normal development and in the mature liver, SOX9 and OPN are coexpressed in the biliary duct. In rodent and human models of fibrosis, both proteins were increased and colocalized to fibrotic regions in vivo and in culture-activated HSCs. SOX9 bound a conserved upstream region of the OPN gene, and abrogation of Sox9 in HSCs significantly decreased OPN production. Hedgehog (Hh) signaling has previously been shown to regulate OPN expression directly by glioblastoma (GLI) 1. Our data indicate that in models of liver fibrosis, Hh signaling more likely acts through SOX9 to modulate OPN. In contrast to Gli2 and Gli3, Gli1 is sparse in HSCs and is not increased upon activation. Furthermore, reduction of GLI2, but not GLI3, decreased the expression of both SOX9 and OPN, whereas overexpressing SOX9 or constitutively active GLI2 could rescue the antagonistic effects of cyclopamine on OPN expression. Conclusion: These data reinforce SOX9, downstream of Hh signaling, as a core factor mediating the expression of ECM components involved in liver fibrosis. Understanding the role and regulation of SOX9 during liver fibrosis will provide insight into its potential modulation as an antifibrotic therapy or as a means of identifying potential ECM targets, similar to OPN, as biomarkers of fibrosis. (HEPATOLOGY 2012;56:1108-1116).     

NS-187, a potent and selective dual Bcr-Abl/Lyn tyrosine kinase inhibitor, is a novel agent for imatinib-resistant leukemia

Although the Abelson (Abl) tyrosine kinase inhibitor imatinib mesylate has improved the treatment of breakpoint cluster region-Abl (Bcr-Abl)-positive leukemia, resistance is often reported in patients with advanced-stage disease. Although several Src inhibitors are more effective than imatinib and simultaneously inhibit Lyn, whose overexpression is associated with imatinib resistance, these inhibitors are less specific than imatinib. We have identified a specific dual Abl-Lyn inhibitor, NS-187 (elsewhere described as CNS-9), which is 25 to 55 times more potent than imatinib in vitro. NS-187 is also at least 10 times as effective as imatinib in suppressing the growth of Bcr-Abl-bearing tumors and markedly extends the survival of mice bearing such tumors. The inhibitory effect of NS-187 extends to 12 of 13 Bcr-Abl proteins with mutations in their kinase domain but not to T315I. NS-187 also inhibits Lyn without affecting the phosphorylation of Src, Blk, or Yes. These results suggest that NS-187 may be a potentially valuable novel agent to combat imatinib-resistant Philadelphia-positive (Ph+) leukemia.     

Molecular mechanisms of endothelin-1-induced cell-cycle progression: involvement of extracellular signal-regulated kinase, protein kinase C, and phosphatidylinositol 3-kinase at distinct points

Although it is well established that endothelin-1 (ET-1) has not only vasoconstrictive effects but also mitogenic effects, which seem to be implicated in vascular remodeling, little is known about the molecular mechanisms by which ET-1 induces cell-cycle progression. In this study, we examined the effects of ET-1 on the cell-cycle regulatory machinery, including cyclins, cyclin-dependent kinase (cdk), and cdk inhibitors in NIH3T3 cells. ET-1 increased cyclin D1 protein (5.1+/-1.9-fold increase, 8 hours after stimulation, P<0.05), cdk4 kinase activity (2.8+/-0. 5-fold increase, 12 hours after stimulation, P<0.01), and cdk2 kinase activity (2.1+/-0.4-fold increase, 16 hours after stimulation, P<0.05) in a time- and dose-dependent manner. ET-1-induced increase in cyclin D1 protein, and cdk4 kinase activity was not significantly inhibited by an inhibitor of the mitogen-activated protein kinase kinase 1/2, PD98059, nor by the protein kinase C inhibitor calphostin C, whereas ET-1-induced upregulation of cyclin D1 protein and cdk4 kinase activity was significantly inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002. In contrast, ET-1-induced activation of cdk2 kinase was significantly inhibited by PD98059, calphostin C, and LY294002. ET-1 increased 3H-thymidine uptake in a time-dependent fashion (0 hours, 4216+/-264 cpm per well; 8 hours, 5025+/-197 cpm per well; 16 hours, 9239+/-79 cpm per well, P<0.001 versus 0 hours). ET-1-induced increase in 3H-thymidine uptake was significantly inhibited by PD98059, calphostin C, and LY294002. These results suggest that ET-1-induced cell-cycle progression is, at least in part, mediated by the extracellular signal-regulated kinase, protein kinase C, and phosphatidylinositol 3-kinase and that those pathways may be involved in the progression of the cell cycle at distinct points.     

Protein kinase D is a novel mediator of cardiac troponin I phosphorylation and regulates myofilament function

Protein kinase D (PKD) is a serine kinase whose myocardial substrates are unknown. Yeast 2-hybrid screening of a human cardiac library, using the PKD catalytic domain as bait, identified cardiac troponin I (cTnI), myosin-binding protein C (cMyBP-C), and telethonin as PKD-interacting proteins. In vitro phosphorylation assays revealed PKD-mediated phosphorylation of cTnI, cMyBP-C, and telethonin, as well as myomesin. Peptide mass fingerprint analysis of cTnI by liquid chromatography-coupled mass spectrometry indicated PKD-mediated phosphorylation of a peptide containing Ser22 and Ser23, the protein kinase A (PKA) targets. Ser22 and Ser23 were replaced by Ala, either singly (Ser22Ala or Ser23Ala) or jointly (Ser22/23Ala), and the troponin complex reconstituted in vitro, using wild-type or mutated cTnI together with wild-type cardiac troponin C and troponin T. PKD-mediated cTnI phosphorylation was reduced in complexes containing Ser22Ala or Ser23Ala cTnI and completely abolished in the complex containing Ser22/23Ala cTnI, indicating that Ser22 and Ser23 are both targeted by PKD. Furthermore, troponin complex containing wild-type cTnI was phosphorylated with similar kinetics and stoichiometry (approximately 2 mol phosphate/mol cTnI) by both PKD and PKA. To determine the functional impact of PKD-mediated phosphorylation, Ca2+ sensitivity of tension development was studied in a rat skinned ventricular myocyte preparation. PKD-mediated phosphorylation did not affect maximal tension but produced a significant rightward shift of the tension-pCa relationship, indicating reduced myofilament Ca2+ sensitivity. At submaximal Ca2+ activation, PKD-mediated phosphorylation also accelerated isometric crossbridge cycling kinetics. Our data suggest that PKD is a novel mediator of cTnI phosphorylation at the PKA sites and may contribute to the regulation of myofilament function.     

The receptor-like kinase SERK3/BAK1 is a central regulator of innate immunity in plants

In pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), plant cell surface receptors sense potential microbial pathogens by recognizing elicitors called PAMPs. Although diverse PAMPs trigger PTI through distinct receptors, the resulting intracellular responses overlap extensively. Despite this, a common component(s) linking signal perception with transduction remains unknown. In this study, we identify SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK)3/brassinosteroid-associated kinase (BAK)1, a receptor-like kinase previously implicated in hormone signaling, as a component of plant PTI. In Arabidopsis thaliana, AtSERK3/BAK1 rapidly enters an elicitor-dependent complex with FLAGELLIN SENSING 2 (FLS2), the receptor for the bacterial PAMP flagellin and its peptide derivative flg22. In the absence of AtSERK3/BAK1, early flg22-dependent responses are greatly reduced in both A. thaliana and Nicotiana benthamiana. Furthermore, N. benthamiana Serk3/Bak1 is required for full responses to unrelated PAMPs and, importantly, for restriction of bacterial and oomycete infections. Thus, SERK3/BAK1 appears to integrate diverse perception events into downstream PAMP responses, leading to immunity against a range of invading microbes.     

The P21-activated kinase PAK4 is implicated in fatty-acid potentiation of insulin secretion downstream of free fatty acid receptor 1

Free fatty acid receptor 1 (FFA1/GPR40) plays a key role in the potentiation of glucose-stimulated insulin secretion by fatty acids in pancreatic β cells. We previously demonstrated that GPR40 signaling leads to cortical actin remodeling and potentiates the second phase of insulin secretion. In this study, we examined the role of p21 activated kinase 4 (PAK4), a known regulator of cytoskeletal dynamics, in GPR40-dependent potentiation of insulin secretion. The fatty acid oleate induced PAK4 phosphorylation in human islets, in isolated mouse islets and in the insulin secreting cell line INS832/13. However, oleate-induced PAK4 phosphorylation was not observed in GPR40-null mouse islets. siRNA-mediated knockdown of PAK4 in INS832/13 cells abrogated the potentiation of insulin secretion by oleate, whereas PAK7 knockdown had no effect. Our results indicate that PAK4 plays an important role in the potentiation of insulin secretion by fatty acids downstream of GPR40.     

Helicobacter pylori inhibits activity of cdc2 kinase and delays G2/M to G1 progression in gastric adenocarcinoma cell line

BACKGROUND: Helicobacter pylori is a bacterial pathogen strongly associated with ulcer diseases and gastric cancer. The bacterial-induced alteration of cell-cycle control in host cells may play a role in the pathogenetic mechanisms. The aims of this study were to define the effect of H. pylori on the G2/M to G1 transition in a gastric cell line. METHODS: Cultured gastric cells, AGS, were synchronized in the S/early G2 phase and treated with intact H. pylori. The cell-cycle distribution of AGS cells was determined by flow cytometry. The activity of cdc2 kinase, as well as of some parameters that affect the kinase activity, was also examined. RESULTS: H. pylori delays cell-cycle progression at the G2/M phase in AGS cells. The G2/M delay was associated with reduced activity of cdc2 kinase. Both down-regulation of cell-cycle regulators (p34cdc2, cyclin B1 and cdc25C) and decreased association between p34cdc2 and cyclin B1 were found to be associated with the activity of cdc2 kinase abated after the H. pylori infection. In addition, the H. pylori-induced G2/M delay required direct contact between the bacteria and host cells. CONCLUSIONS: H. pylori inhibits G2/M to G1 progression and causes a reduction of cell division in gastric epithelial cells.