Adapter Protein Shc Regulates Janus Kinase 3 Phosphorylation

Although constitutive activation of Janus kinase 3 (Jak3) leads to different cancers, the mechanism of trans-molecular regulation of Jak3 activation is not known. Previously we reported that Jak3 interactions with adapter protein p52ShcA (Shc) facilitate mucosal homeostasis. In this study, we characterize the structural determinants that regulate the interactions between Jak3 and Shc and demonstrate the trans-molecular mechanism of regulation of Jak3 activation by Shc. We show that Jak3 autophosphorylation was the rate-limiting step during Jak3 trans-phosphorylation of Shc where Jak3 directly phosphorylated two tyrosine residues in Src homology 2 (SH2) domain and one tyrosine residue each in calponin homology 1 (CH1) domain and phosphotyrosine interaction domain (PID) of Shc. Direct interactions between mutants of Jak3 and Shc showed that although FERM domain of Jak3 was sufficient for binding to Shc, CH1 and PID domains of Shc were responsible for binding to Jak3. Functionally Jak3 was autophosphorylated under IL-2 stimulation in epithelial cells. However, Shc recruited tyrosine phosphatases SHP2 and PTP1B to Jak3 and thereby dephosphorylated Jak3. Thus we not only characterize Jak3 interaction with Shc, but also demonstrate the molecular mechanism of intracellular regulation of Jak3 activation where Jak3 interactions with Shc acted as regulators of Jak3 dephosphorylation through direct interactions of Shc with both Jak3 and tyrosine phosphatases.     

Protein Tyrosine Phosphatase 1B Regulates Pyruvate Kinase M2 Tyrosine Phosphorylation

Protein-tyrosine phosphatase 1B (PTP1B) is a physiological regulator of glucose homeostasis and adiposity and is a drug target for the treatment of obesity and diabetes. Here we identify pyruvate kinase M2 (PKM2) as a novel PTP1B substrate in adipocytes. PTP1B deficiency leads to increased PKM2 total tyrosine and Tyr¹⁰⁵ phosphorylation in cultured adipocytes and in vivo. Substrate trapping and mutagenesis studies identify PKM2 Tyr-105 and Tyr-148 as key sites that mediate PTP1B-PKM2 interaction. In addition, in vitro analyses illustrate a direct effect of Tyr-105 phosphorylation on PKM2 activity in adipocytes. Importantly, PTP1B pharmacological inhibition increased PKM2 Tyr-105 phosphorylation and decreased PKM2 activity. Moreover, PKM2 Tyr-105 phosphorylation is regulated nutritionally, decreasing in adipose tissue depots after high-fat feeding. Further, decreased PKM2 Tyr-105 phosphorylation correlates with the development of glucose intolerance and insulin resistance in rodents, non-human primates, and humans. Together, these findings identify PKM2 as a novel substrate of PTP1B and provide new insights into the regulation of adipose PKM2 activity.     

Macrophage mesenchymal migration requires podosome stabilization by filamin A

Filamin A (FLNa) is a cross-linker of actin filaments and serves as a scaffold protein mostly involved in the regulation of actin polymerization. It is distributed ubiquitously, and null mutations have strong consequences on embryonic development in humans, with organ defects which suggest deficiencies in cell migration. We have reported previously that macrophages, the archetypal migratory cells, use the protease- and podosome-dependent mesenchymal migration mode in dense three-dimensional environments, whereas they use the protease- and podosome-independent amoeboid mode in more porous matrices. Because FLNa has been shown to localize to podosomes, we hypothesized that the defects seen in patients carrying FLNa mutations could be related to the capacity of certain cell types to form podosomes. Using strategies based on FLNa knock-out, knockdown, and rescue, we show that FLNa (i) is involved in podosome stability and their organization as rosettes and three-dimensional podosomes, (ii) regulates the proteolysis of the matrix mediated by podosomes in macrophages, (iii) is required for podosome rosette formation triggered by Hck, and (iv) is necessary for mesenchymal migration but dispensable for amoeboid migration. These new functions assigned to FLNa, particularly its role in mesenchymal migration, could be directly related to the defects in cell migration described during the embryonic development in FLNa-defective patients.     

Frank-ter Haar Syndrome Protein Tks4 Regulates Epidermal Growth Factor-dependent Cell Migration

Mutations in the SH3PXD2B gene coding for the Tks4 protein are responsible for the autosomal recessive Frank-ter Haar syndrome. Tks4, a substrate of Src tyrosine kinase, is implicated in the regulation of podosome formation. Here, we report a novel role for Tks4 in the EGF signaling pathway. In EGF-treated cells, Tks4 is tyrosine-phosphorylated and associated with the activated EGF receptor. This association is not direct but requires the presence of Src tyrosine kinase. In addition, treatment of cells with LY294002, an inhibitor of PI 3-kinase, or mutations of the PX domain reduces tyrosine phosphorylation and membrane translocation of Tks4. Furthermore, a PX domain mutant (R43W) Tks4 carrying a reported point mutation in a Frank-ter Haar syndrome patient showed aberrant intracellular expression and reduced phosphoinositide binding. Finally, silencing of Tks4 was shown to markedly inhibit HeLa cell migration in a Boyden chamber assay in response to EGF or serum. Our results therefore reveal a new function for Tks4 in the regulation of growth factor-dependent cell migration.     

Discovery of Novel Irreversible Inhibitors of Interleukin (IL)-2-inducible Tyrosine Kinase (Itk) by Targeting Cysteine 442 in the ATP Pocket

IL-2-inducible tyrosine kinase (Itk) plays a key role in antigen receptor signaling in T cells and is considered an important target for anti-inflammatory drug discovery. In order to generate inhibitors with the necessary potency and selectivity, a compound that targeted cysteine 442 in the ATP binding pocket and with an envisaged irreversible mode of action was designed. We incorporated a high degree of molecular recognition and specific design features making the compound suitable for inhaled delivery. This study confirms the irreversible covalent binding of the inhibitor to the kinase by x-ray crystallography and enzymology while demonstrating potency, selectivity, and prolonged duration of action in in vitro biological assays. The biosynthetic turnover of the kinase was also examined as a critical factor when designing irreversible inhibitors for extended duration of action. The exemplified Itk inhibitor demonstrated inhibition of both T_H1 and T_H2 cytokines, was additive with fluticasone propionate, and inhibited cytokine release from human lung fragments. Finally, we describe an in vivo pharmacodynamic assay that allows rapid preclinical development without animal efficacy models.     

Tyrosine Phosphorylation on Spleen Tyrosine Kinase (Syk) Is Differentially Regulated in Human and Murine Platelets by Protein Kinase C Isoforms

Protein kinase C (PKC) isoforms differentially regulate platelet functional responses downstream of glycoprotein VI (GPVI) signaling, but the role of PKCs regulating upstream effectors such as Syk is not known. We investigated the role of PKC on Syk tyrosine phosphorylation using the pan-PKC inhibitor GF109203X (GFX). GPVI-mediated phosphorylation on Syk Tyr-323, Tyr-352, and Tyr-525/526 was rapidly dephosphorylated, but GFX treatment inhibited this dephosphorylation on Tyr-525/526 in human platelets but not in wild type murine platelets. GFX treatment did not affect tyrosine phosphorylation on FcRγ chain or Src family kinases. Phosphorylation of Lat Tyr-191 and PLCγ2 Tyr-759 was also increased upon treatment with GFX. We evaluated whether secreted ADP is required for such dephosphorylation. Exogenous addition of ADP to GFX-treated platelets did not affect tyrosine phosphorylation on Syk. FcγRIIA- or CLEC-2-mediated Syk tyrosine phosphorylation was also potentiated with GFX in human platelets. Because potentiation of Syk phosphorylation is not observed in murine platelets, PKC-deficient mice cannot be used to identify the PKC isoform regulating Syk phosphorylation. We therefore used selective inhibitors of PKC isoforms. Only PKCβ inhibition resulted in Syk hyperphosphorylation similar to that in platelets treated with GFX. This result indicates that PKCβ is the isoform responsible for Syk negative regulation in human platelets. In conclusion, we have elucidated a novel pathway of Syk regulation by PKCβ in human platelets.     

Role of Tyrosine Phosphorylation in the Muscarinic Activation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride (Cl⁻) channel, which plays an important role in physiological anion and fluid secretion, and is defective in several diseases. Although its activation by PKA and PKC has been studied extensively, its regulation by receptors is less well understood. To study signaling involved in CFTR activation, we measured whole-cell Cl⁻ currents in BHK cells cotransfected with GPCRs and CFTR. In cells expressing the M3 muscarinic acetylcholine receptor, the agonist carbachol (Cch) caused strong activation of CFTR through two pathways; the canonical PKA-dependent mechanism and a second mechanism that involves tyrosine phosphorylation. The role of PKA was suggested by partial inhibition of cholinergic stimulation by the specific PKA inhibitor Rp-cAMPS. The role of tyrosine kinases was suggested by Cch stimulation of 15SA-CFTR and 9CA-CFTR, mutants that lack 15 PKA or 9 PKC consensus sequences and are unresponsive to PKA or PKC stimulation, respectively. Moreover the residual Cch response was sensitive to inhibitors of the Pyk2 and Src tyrosine kinase family. Our results suggest that tyrosine phosphorylation acts on CFTR directly and through inhibition of the phosphatase PP2A. Results suggest that PKA and tyrosine kinases contribute to CFTR regulation by GPCRs that are expressed at the apical membrane of intestinal and airway epithelia.     

Direct Regulation of Nephrin Tyrosine Phosphorylation by Nck Adaptor Proteins

The transmembrane protein nephrin is a key component of the kidney slit diaphragm that contributes to the morphology of podocyte foot processes through signaling to the underlying actin cytoskeleton. We have recently reported that tyrosine phosphorylation of the cytoplasmic tail of nephrin facilitates recruitment of Nck SH2/SH3 adaptor proteins and subsequent actin remodeling and that phosphorylation of the Nck binding sites on nephrin is decreased during podocyte injury. We now demonstrate that Nck directly modulates nephrin phosphorylation through formation of a signaling complex with the Src family kinase Fyn. The ability of Nck to enhance nephrin phosphorylation is compromised in the presence of a Src family kinase inhibitor and when the SH3 domains of Nck are mutated. Furthermore, induced loss of Nck expression in podocytes in vivo is associated with a rapid reduction in nephrin tyrosine phosphorylation. Our results suggest that Nck may facilitate dynamic signaling events at the slit diaphragm by promoting Fyn-dependent phosphorylation of nephrin, which may be important in the regulation of foot process morphology and response to podocyte injury.     

Regulation of the Tyrosine Kinase Pyk2 by Calcium Is through Production of Reactive Oxygen Species in Cytotoxic T Lymphocytes

Pyk2 was identified as a Ca²⁺-dependent kinase, however, the regulation of Pyk2 by Ca²⁺ in T cells remains controversial. We found that Ca²⁺ mobilization preferentially induced Pyk2 phosphorylation in cytotoxic T lymphocytes (CTL). Furthermore, Pyk2 phosphorylation in CTL was not absolutely Ca²⁺ dependent but relied on the strength of T cell receptor stimulation. Ionomycin-stimulated Pyk2 phosphorylation did not require calmodulin activity, because phosphorylation was not inhibited by the calmodulin inhibitor W7, and we detected no Ca²⁺-regulated association between Pyk2 and calmodulin. Ca²⁺-stimulated Pyk2 phosphorylation was dependent on Src-family kinase activity, even at the Pyk2 autophosphorylation site. We sought to identify a Ca²⁺-regulated pathway that could trigger Pyk2 phosphorylation in T cells and found that ionomycin stimulated the production of reactive oxygen species and an H₂O₂ scavenger inhibited ionomycin-induced Pyk2 phosphorylation. Additionally, H₂O₂ induced strong Erk activation and ionomycin-stimulated Pyk2 phosphorylation was Erk dependent. These data support the conclusion that Ca²⁺ mobilization induces the production of reactive oxygen species, which in turn activate the Erk pathway, leading to Src-family kinase-dependent Pyk2 phosphorylation. Our data demonstrate that Pyk2 is not a Ca²⁺-dependent kinase in T cells but instead, increased intracellular Ca²⁺ induces Pyk2 phosphorylation through production of reactive oxygen species. These findings are consistent with the possibility that Pyk2 acts as an early sensor of numerous extracellular signals that trigger a Ca²⁺ flux and/or reactive oxygen species to amplify tyrosine phosphorylation signaling events.     

Response Gene to Complement 32 Protein Promotes Macrophage Phagocytosis via Activation of Protein Kinase C Pathway

Macrophage phagocytosis plays an important role in host defense. The molecular mechanism, especially factors regulating the phagocytosis, however, is not completely understood. In the present study, we found that response gene to complement 32 (RGC-32) is an important regulator of phagocytosis. Although RGC-32 is induced and abundantly expressed in macrophage during monocyte-macrophage differentiation, RGC-32 appears not to be important for this process because RGC-32-deficient bone marrow progenitor can normally differentiate to macrophage. However, both peritoneal macrophages and bone marrow-derived macrophages with RGC-32 deficiency exhibit significant defects in phagocytosis, whereas RGC-32-overexpressed macrophages show increased phagocytosis. Mechanistically, RGC-32 is recruited to macrophage membrane where it promotes F-actin assembly and the formation of phagocytic cups. RGC-32 knock-out impairs F-actin assembly. RGC-32 appears to interact with PKC to regulate PKC-induced phosphorylation of F-actin cross-linking protein myristoylated alanine-rich protein kinase C substrate. Taken together, our results demonstrate for the first time that RGC-32 is a novel membrane regulator for macrophage phagocytosis.     

Binding of the Receptor Tyrosine Kinase TrkB to the Neural Cell Adhesion Molecule (NCAM) Regulates Phosphorylation of NCAM and NCAM-dependent Neurite Outgrowth

Recognition molecules and neurotrophins play important roles during development and maintenance of nervous system functions. In this study, we provide evidence that the neural cell adhesion molecule (NCAM) and the neurotrophin receptor TrkB directly interact via sequences in their intracellular domains. Stimulation of TrkB by brain-derived neurotrophic factor leads to tyrosine phosphorylation of NCAM at position 734. Mutation of this tyrosine to phenylalanine completely abolishes tyrosine phosphorylation of NCAM by TrkB. Moreover, the knockdown of TrkB in hippocampal neurons leads to a reduction of NCAM-induced neurite outgrowth. Transfection of NCAM-deficient hippocampal neurons with mutated NCAM carrying an exchange of tyrosine by phenylalanine at position 734 leads to promotion of NCAM-induced neurite outgrowth in comparison with that observed after transfection with wild-type NCAM, whereas a reduction of neurite outgrowth was observed after transfection with mutated NCAM, which carries an exchange of tyrosine by glutamate that mimics the phosphorylated tyrosine. Our observations indicate a functional relationship between TrkB and NCAM.     

Phosphorylation of Tyrosine Hydroxylase by Cyclic GMP-Dependent Protein Kinase

Tyrosine hydroxylase purified from rat pheochromocytoma was phosphorylated and activated by purified cyclic GMP-dependent protein kinase as well as by cyclic AMP-dependent protein kinase catalytic subunit. The extent of activation was correlated with the degree of phosphate incorporated into the enzyme. Comparable stoichiometric ratios (0.6 mol phosphate/mol tyrosine hydroxylase subunit) were obtained at maximal concentrations of either cyclic AMP-dependent or cyclic GMP-dependent protein kinases. The enzymes appeared to mediate the phosphorylation of the same residue based on the observation that incorporation was not increased when both enzymes were present. The major tryptic phosphopeptide obtained from tyrosine hydroxylase phosphorylated by each protein kinase exhibited an identical retention time following HPLC. The purified phosphopeptides also exhibited identical isoelectric points. These data provide support for the notion that the protein kinases are phosphorylating the same residue of tyrosine hydroxylase.     

Tyrosine Phosphorylation of Extracellular Signal-Regulated Protein Kinase 4 in Response to Growth Factors

Abstract: Extracellular signal-regulated protein kinases (ERKs) are members of the mitogen-activated protein kinase family that are rapidly phosphorylated and activated in response to various extracellular stimuli, including growth factors. Of these, the ERK1 and ERK2 forms are by far the most abundant and the most studied. Much less is known about other ERK forms, including one previously designated ERK4 on the basis of its cross-reactivity with ERK1 and ERK2. We report here that ERK4 in rat PC12 pheochromocytoma cells can be immunoprecipitated by anti-ERK antiserum R2 and have used this reagent to characterize this species further. We find that ERK4 rapidly becomes tyrosine-phosphorylated in response to nerve growth factor (NGF) and epidermal growth factor (EGF) and, to a lesser degree, in response to insulin and a permeant cyclic AMP analogue. As in the case of ERK1 and ERK2, tyrosine phosphorylation of ERK4 occurs by a ras-dependent pathway in response to NGF and EGF and shows prolonged kinetics for NGF but not EGF treatment. Recognition by multiple antisera directed against various domains of ERK1 supports classification of ERK4 within the ERK family; however, two-dimensional gel analysis clearly distinguishes ERK4 from isoforms of ERK1. These findings thus reveal an additional member of the ERK family that is responsive to growth factors and that could play a distinct role in intracellular signaling.     

Structural Basis for the Binding Specificity of Human Recepteur d'Origine Nantais (RON) Receptor Tyrosine Kinase to Macrophage-stimulating Protein

Recepteur d''origine nantais (RON) receptor tyrosine kinase and its ligand, serum macrophage-stimulating protein (MSP), play important roles in inflammation, cell growth, migration, and epithelial to mesenchymal transition during tumor development. The binding of mature MSPαβ (disulfide-linked α- and β-chains) to RON ectodomain modulates receptor dimerization, followed by autophosphorylation of tyrosines in the cytoplasmic receptor kinase domains. Receptor recognition is mediated by binding of MSP β-chain (MSPβ) to the RON Sema. Here we report the structure of RON Sema-PSI-IPT₁ (SPI₁) domains in complex with MSPβ at 3.0 Å resolution. The MSPβ serine protease-like β-barrel uses the degenerate serine protease active site to recognize blades 2, 3, and 4 of the β-propeller fold of RON Sema. Despite the sequence homology between RON and MET receptor tyrosine kinase and between MSP and hepatocyte growth factor, it is well established that there is no cross-reactivity between the two receptor-ligand systems. Comparison of the structure of RON SPI₁ in complex with MSPβ and that of MET receptor tyrosine kinase Sema-PSI in complex with hepatocyte growth factor β-chain reveals the receptor-ligand selectivity determinants. Analytical ultracentrifugation studies of the SPI₁-MSPβ interaction confirm the formation of a 1:1 complex. SPI₁ and MSPαβ also associate primarily as a 1:1 complex with a binding affinity similar to that of SPI₁-MSPβ. In addition, the SPI₁-MSPαβ ultracentrifuge studies reveal a low abundance 2:2 complex with ∼10-fold lower binding affinity compared with the 1:1 species. These results support the hypothesis that the α-chain of MSPαβ mediates RON dimerization.     

Platelet-derived Growth Factor Differentially Regulates the Expression and Post-translational Modification of Versican by Arterial Smooth Muscle Cells through Distinct Protein Kinase C and Extracellular Signal-regulated Kinase Pathways

The synthesis of proteoglycans involves steps that regulate both protein and glycosaminoglycan (GAG) synthesis, but it is unclear whether these two pathways are regulated by the same or different signaling pathways. We therefore investigated signaling pathways involved in platelet-derived growth factor (PDGF)-mediated increases in versican core protein and GAG chain synthesis in arterial smooth muscle cells (ASMCs). PDGF treatment of ASMCs resulted in increased versican core protein synthesis and elongation of GAG chains attached to the versican core protein. The effects of PDGF on versican mRNA were blocked by inhibiting either protein kinase C (PKC) or the ERK pathways, whereas the GAG elongation effect of PDGF was blocked by PKC inhibition but not by ERK inhibition. Interestingly, blocking protein synthesis in the presence of cycloheximide abolished the PDGF effect, but not in the presence of xyloside, indicating that GAG synthesis that results from PKC activation is independent from de novo protein synthesis. PDGF also stimulated an increase in the chondroitin-6-sulfate to chondroitin-4-sulfate ratio of GAG chains on versican, and this effect was blocked by PKC inhibitors. These data show that PKC activation is sufficient to cause GAG chain elongation, but both PKC and ERK activation are required for versican mRNA core protein expression. These results indicate that different signaling pathways control different aspects of PDGF-stimulated versican biosynthesis by ASMCs. These data will be useful in designing strategies to interfere with the synthesis of this proteoglycan in various disease states.     

Cleavage of Mer Tyrosine Kinase (MerTK) from the Cell Surface Contributes to the Regulation of Retinal Phagocytosis

Phagocytosis of apoptotic cells by macrophages and spent photoreceptor outer segments (POS) by retinal pigment epithelial (RPE) cells requires several proteins, including MerTK receptors and associated Gas6 and protein S ligands. In the retina, POS phagocytosis is rhythmic, and MerTK is activated promptly after light onset via the αvβ5 integrin receptor and its ligand MFG-E8, thus generating a phagocytic peak. The phagocytic burst is limited in time, suggesting a down-regulation mechanism that limits its duration. Our previous data showed that MerTK helps control POS binding of integrin receptors at the RPE cell surface as a negative feedback loop. Our present results show that a soluble form of MerTK (sMerTK) is released in the conditioned media of RPE-J cells during phagocytosis and in the interphotoreceptor matrix of the mouse retina during the morning phagocytic peak. In contrast to macrophages, the two cognate MerTK ligands have an opposite effect on phagocytosis and sMerTK release, whereas the integrin ligand MFG-E8 markedly increases both phagocytosis and sMerTK levels. sMerTK acts as a decoy receptor blocking the effect of both MerTK ligands. Interestingly, stimulation of sMerTK release decreases POS binding. Conversely, blocking MerTK cleavage increased mostly POS binding by RPE cells. Therefore, our data suggest that MerTK cleavage contributes to the acute regulation of RPE phagocytosis by limiting POS binding to the cell surface.     

Involvement of c-Src Tyrosine Kinase Upstream of Class I Phosphatidylinositol (PI) 3-Kinases in Salmonella Enteritidis Rck Protein-mediated Invasion

The Salmonella outer membrane protein Rck mediates a Zipper entry mechanism controlled by tyrosine phosphorylation and class I phosphatidylinositol 3-kinase (PI 3-kinase). However, the underlying mechanism leading to this signaling cascade remains unclear. The present study showed that using Rck-coated beads or Rck-overexpressing Escherichia coli, Rck-mediated actin polymerization and invasion were blocked by PP2, a Src family tyrosine kinase inhibitor. In addition, phosphorylation of Src family kinases significantly increased after stimulation with Rck. The specific contribution of c-Src, one member of the Src family kinases, was demonstrated using c-Src-deficient fibroblasts or c-Src siRNA transfected epithelial cells. We also observed that Rck-mediated internalization led to the formation of a complex between c-Src and at least one tyrosine-phosphorylated protein. Furthermore, our results revealed that the c-Src signal molecule was upstream of PI 3-kinase during the Rck-mediated signaling pathway as Rck-mediated PI 3-kinase activation was blocked by PP2, and PI 3-kinase inhibitor had no effect on the Src phosphorylation. These results demonstrate the involvement of c-Src upstream of the PI 3-kinase in the Zipper entry process mediated by Rck.