Inhibition of the catalytic activity of cell adhesion kinase beta by protein-tyrosine phosphatase-PEST-mediated dephosphorylation

Protein-tyrosine phosphatase (PTP)-PEST is a cytoplasmic tyrosine phosphatase that can bind and dephosphorylate the focal adhesion-associated proteins p130(CAS) and paxillin. Focal adhesion kinase (FAK) and cell adhesion kinase beta (CAKbeta)/PYK2/CADTK/RAFTK are protein-tyrosine kinases that can colocalize with, bind to, and induce tyrosine phosphorylation of p130(CAS) and paxillin. Thus, we considered the possibility that these kinases might be substrates for PTP-PEST. Using a combination of substrate-trapping assays and overexpression of PTP-PEST in mammalian cells, CAKbeta was found to be a substrate for PTP-PEST. Both the major autophosphorylation site of CAKbeta (Tyr(402)) and activation loop tyrosine residues, Tyr(579) and Tyr(580), were targeted for dephosphorylation by PTP-PEST. Dephosphorylation of CAKbeta by PTP-PEST dramatically inhibited CAKbeta kinase activity. In contrast, FAK was a poor substrate for PTP-PEST, and treatment with PTP-PEST had no effect on FAK kinase activity. Tyrosine phosphorylation of paxillin, which is greatly enhanced by CAKbeta overexpression, was dramatically reduced upon coexpression of PTP-PEST. Finally, endogenous PTP-PEST and endogenous CAKbeta were found to localize to similar cellular compartments in epithelial and smooth muscle cells. These results suggest that CAKbeta is a substrate of PTP-PEST and that FAK is a poor PTP-PEST substrate. Further, PTP-PEST can negatively regulate CAKbeta signaling by inhibiting the catalytic activity of the kinase.     

Hic-5, a paxillin homologue, binds to the protein-tyrosine phosphatase PEST (PTP-PEST) through its LIM 3 domain

The Hic-5 protein is encoded by a transforming growth factor-beta1- and hydrogen peroxide-inducible gene, hic-5, and has striking similarity to paxillin, especially in their C-terminal LIM domains. Like paxillin, Hic-5 is localized in focal adhesion plaques in association with focal adhesion kinase in cultured fibroblasts. We carried out yeast two-hybrid screening to identify cellular factors that form a complex with Hic-5 using its LIM domains as a bait, and we identified a cytoplasmic tyrosine phosphatase (PTP-PEST) as one of the partners of Hic-5. These two proteins are associated in mammalian cells. From in vitro binding experiments using deletion and point mutations, it was demonstrated that the essential domain in Hic-5 for the binding was LIM 3. As for PTP-PEST, one of the five proline-rich sequences found on PTP-PEST, Pro-2, was identified as the binding site for Hic-5 in in vitro binding assays. Paxillin also binds to the Pro-2 domain of PTP-PEST. In conclusion, Hic-5 may participate in the regulation of signaling cascade through its interaction with distinct tyrosine kinases and phosphatases.     

The noncatalytic domain of protein-tyrosine phosphatase-PEST targets paxillin for dephosphorylation in vivo

The noncatalytic domain of protein-tyrosine phosphatase (PTP)-PEST contains a binding site for the focal adhesion-associated protein paxillin. This binding site has been narrowed to a 52-residue sequence that is composed of two nonoverlapping, weak paxillin binding sites. The PTP-PEST binding site on paxillin has been mapped to the two carboxyl-terminal LIM (lin11, isl-1, and mec-3) domains. Transient expression of PTP-PEST reduced tyrosine phosphorylation of p130(cas), as anticipated. A PTP-PEST mutant defective for binding p130(cas) does not cause a reduction in its tyrosine phosphorylation in vivo. Expression of PTP-PEST also caused a reduction of phosphotyrosine on paxillin. Expression of mutants of PTP-PEST with deletions in the paxillin-binding site did not associate with paxillin in vivo and failed to cause a reduction in the phosphotyrosine content of paxillin. These results demonstrate that paxillin can serve as a PTP-PEST substrate in vivo and support the model that a noncatalytic domain interaction recruits paxillin to PTP-PEST to facilitate its dephosphorylation.     

Phosphorylation of a Wiscott-Aldrich syndrome protein-associated signal complex is critical in osteoclast bone resorption

The activities of different kinases have been correlated to the phosphorylation of Wiscott-Aldrich syndrome protein (WASP) by studies in multiple cell systems. The purpose of this study was to elucidate the regulatory mechanisms involved in WASP phosphorylation and the resulting sealing ring formation in osteoclasts. The phosphorylation state of WASP and WASP-interacting proteins was determined in osteoclasts treated with osteopontin or expressing either constitutively active or kinase-defective Src by adenovirus-mediated delivery. In vitro kinase analysis of WASP immunoprecipitates exhibited phosphorylation of c-Src, PYK2, WASP, protein-tyrosine phosphatase (PTP)-PEST, and Pro-Ser-Thr phosphatase-interacting protein (PSTPIP). Phosphorylation of these proteins was increased in osteopontin-treated and constitutively active Src-expressing osteoclasts. Pulldown analysis with glutathione S-transferase-fused proline-rich regions of PTP-PEST revealed coprecipitation of WASP, PYK2, c-Src, and PSTPIP proteins with the N-terminal region (amino acids 294-497) of PTP-PEST. Similarly, interaction of the same signaling proteins, as well as PTP-PEST, was observed with glutathione S-transferase-fused proline-rich regions of WASP. Furthermore, osteopontin stimulation or constitutively active Src expression resulted in serine phosphorylation and inhibition of WASP-associated PTP-PEST. The inhibition of PTP-PEST was accompanied by an increase in tyrosine phosphorylation of WASP and other associated signaling proteins. Experiments with an inhibitor to phosphatase and small interference RNA to PTP-PEST confirmed the involvement of PTP-PEST in sealing ring formation and bone resorption. WASP, which is identified in the sealing ring of resorbing osteoclasts, also demonstrates colocalization with c-Src, PYK2, PSTPIP, and PTP-PEST in immunostaining analyses. Our findings suggest that both tyrosine kinase(s) and the tyrosine phosphatase PTP-PEST coordinate the formation of the sealing ring and thus the bone-resorbing function of osteoclasts.     

Protein tyrosine phosphatase-PEST regulates focal adhesion disassembly, migration, and cytokinesis in fibroblasts

In this article, we show that, in transfected COS-1 cells, protein tyrosine phosphatase (PTP)-PEST translocates to the membrane periphery following stimulation by the extracellular matrix protein fibronectin. When plated on fibronectin, PTP-PEST (-/-) fibroblasts display a strong defect in motility. 3 h after plating on fibronectin, the number and size of vinculin containing focal adhesions were greatly increased in the homozygous PTP-PEST mutant cells as compared with heterozygous cells. This phenomenon appears to be due in part to a constitutive increase in tyrosine phosphorylation of p130(CAS), a known PTP-PEST substrate, paxillin, which associates with PTP-PEST in vitro, and focal adhesion kinase (FAK). Another effect of this constitutive hyperphosphorylation, consistent with the focal adhesion regulation defect, is that (-/-) cells spread faster than the control cell line when plated on fibronectin. In the PTP-PEST (-/-) cells, an increase in affinity for the SH2 domains of Src and Crk towards p130(CAS) was also observed. In (-/-) cells, we found a significant increase in the level of tyrosine phosphorylation of PSTPIP, a cleavage furrow-associated protein that interacts physically with all PEST family members. An effect of PSTPIP hyperphosphorylation appears to be that some cells remain attached at the site of the cleavage furrow for an extended period of time. In conclusion, our data suggest PTP-PEST plays a dual role in cell cytoskeleton organization, by promoting the turnover of focal adhesions required for cell migration, and by directly or indirectly regulating the proline, serine, threonine phosphatase interacting protein (PSTPIP) tyrosine phosphorylation level which may be involved in regulating cleavage furrow formation or disassembly during normal cell division.     

Caspase-3 regulates catalytic activity and scaffolding functions of the protein tyrosine phosphatase PEST, a novel modulator of the apoptotic response

The protein tyrosine phosphatase PEST (PTP-PEST) is involved in the regulation of the actin cytoskeleton. Despite the emerging functions attributed to both PTPs and the actin cytoskeleton in apoptosis, the involvement of PTP-PEST in apoptotic cell death remains to be established. Using several cell-based assays, we showed that PTP-PEST participates in the regulation of apoptosis. As apoptosis progressed, a pool of PTP-PEST localized to the edge of retracting lamellipodia. Expression of PTP-PEST also sensitized cells to receptor-mediated apoptosis. Concertedly, specific degradation of PTP-PEST was observed during apoptosis. Pharmacological inhibitors, immunodepletion experiments, and in vitro cleavage assays identified caspase-3 as the primary regulator of PTP-PEST processing during apoptosis. Caspase-3 specifically cleaved PTP-PEST at the (549)DSPD motif and generated fragments, some of which displayed increased catalytic activity. Moreover, caspase-3 regulated PTP-PEST interactions with paxillin, leupaxin, Shc, and PSTPIP. PTP-PEST acted as a scaffolding molecule connecting PSTPIP to additional partners: paxillin, Shc, Csk, and activation of caspase-3 correlated with the modulation of the PTP-PEST adaptor function. In addition, cleavage of PTP-PEST facilitated cellular detachment during apoptosis. Together, our data demonstrate that PTP-PEST actively contributes to the cellular apoptotic response and reveal the importance of caspases as regulators of PTPs in apoptosis.     

Macrophage Fusion Is Controlled by the Cytoplasmic Protein Tyrosine Phosphatase PTP-PEST/PTPN12

Macrophages can undergo cell-cell fusion, leading to the formation of multinucleated giant cells and osteoclasts. This process is believed to promote the proteolytic activity of macrophages toward pathogens, foreign bodies, and extracellular matrices. Here, we examined the role of PTP-PEST (PTPN12), a cytoplasmic protein tyrosine phosphatase, in macrophage fusion. Using a macrophage-targeted PTP-PEST-deficient mouse, we determined that PTP-PEST was not needed for macrophage differentiation or cytokine production. However, it was necessary for interleukin-4-induced macrophage fusion into multinucleated giant cells in vitro. It was also needed for macrophage fusion following implantation of a foreign body in vivo. Moreover, in the RAW264.7 macrophage cell line, PTP-PEST was required for receptor activator of nuclear factor kappa-B ligand (RANKL)-triggered macrophage fusion into osteoclasts. PTP-PEST had no impact on expression of fusion mediators such as β-integrins, E-cadherin, and CD47, which enable macrophages to become fusion competent. However, it was needed for polarization of macrophages, migration induced by the chemokine CC chemokine ligand 2 (CCL2), and integrin-induced spreading, three key events in the fusion process. PTP-PEST deficiency resulted in specific hyperphosphorylation of the protein tyrosine kinase Pyk2 and the adaptor paxillin. Moreover, a fusion defect was induced upon treatment of normal macrophages with a Pyk2 inhibitor. Together, these data argue that macrophage fusion is critically dependent on PTP-PEST. This function is seemingly due to the ability of PTP-PEST to control phosphorylation of Pyk2 and paxillin, thereby regulating cell polarization, migration, and spreading.     

Growth hormone-induced alteration in ErbB-2 phosphorylation status in 3T3-F442A fibroblasts

The growth hormone receptor (GHR), a cytokine receptor superfamily member, requires the JAK2 tyrosine kinase for signaling. We now examine functional interactions between growth hormone (GH) and epidermal growth factor (EGF) in 3T3-F442A fibroblasts. Although EGF enhanced ErbB-2 tyrosine phosphorylation, GH, while causing retardation of its migration on SDS-polyacrylamide gel electrophoresis, decreased ErbB-2's tyrosine phosphorylation. GH-induced retardation was reversed by treatment of anti-ErbB-2 precipitates with both alkaline phosphatase and protein phosphatase 2A, suggesting that GH induced serine/threonine phosphorylation of ErbB-2. Both GH-induced shift in ErbB-2 migration and GH-induced MAP kinase activation were unaffected by a protein kinase C inhibitor but were blocked by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (MEK1) inhibitor, PD98059. Notably, leukemia inhibitory factor, but not interferon-gamma, also promoted ErbB-2 shift and mitogen-activated protein kinase activation. Cotreatment with EGF and GH versus EGF alone resulted in a 35% decline in acute ErbB-2 tyrosine 1248 autophosphorylation, a marked decline (approximately 50%) in DNA synthesis, and substantially decreased cyclin D1 expression. We conclude that in 3T3-F442A cells, 1) the GH-induced decrease in ErbB-2 tyrosine phosphorylation correlates with MEK1/mitogen-activated protein kinase activity and 2) GH antagonizes EGF-induced DNA synthesis and cyclin D1 expression in a pattern consistent with its alteration in ErbB-2 phosphorylation status.     

Paxillin enables attachment-independent tyrosine phosphorylation of focal adhesion kinase and transformation by RAS

Paxillin and HIC5 are closely related adapter proteins that regulate cell migration and are tyrosine-phosphorylated by focal adhesion kinase (FAK). Paxillin, HIC5, and FAK tyrosine phosphorylation increase upon cell attachment and decrease upon detachment from extracellular matrix. Unexpectedly, we found that although FAK tyrosine phosphorylation in attached cells did not require paxillin, in detached fibroblasts there was remaining FAK tyrosine phosphorylation that required expression of paxillin and was not supported by HIC5. The support of attachment-independent FAK tyrosine phosphorylation required the paxillin LIM domains and suggested that paxillin might facilitate oncogenic transformation. Paxillin but not HIC5 augmented anchorage-independent cell proliferation induced by RAS. Both anchorage-independent FAK tyrosine phosphorylation and RAS-induced colony formation required multiple docking sites on paxillin, including LD4 (docking sites for FAK-Src and GIT1/2-PIX-NCK-PAK complex), LD5, and all four carboxyl-terminal LIM domains (that bind tubulin and PTP-PEST). Analysis using paxillin mutants dissociated domains of paxillin that are required for regulation of cell migration from domains that are required for anchorage-independent cell proliferation and demonstrated essential functions of the paxillin LIM domains that are not found in HIC5 LIM domains. These results highlight the role of paxillin in facilitating attachment-independent signal transduction implicated in cancer.     

The Phosphatase PTP-PEST/PTPN12 Regulates Endothelial Cell Migration and Adhesion,but Not Permeability,and Controls Vascular Development and Embryonic Viability

Protein-tyrosine phosphatase (PTP)-PEST (PTPN12) is ubiquitously expressed. It is essential for normal embryonic development and embryonic viability in mice. Herein we addressed the involvement of PTP-PEST in endothelial cell functions using a combination of genetic and biochemical approaches. By generating primary endothelial cells from an inducible PTP-PEST-deficient mouse, we found that PTP-PEST is not needed for endothelial cell differentiation and proliferation or for the control of endothelial cell permeability. Nevertheless, it is required for integrin-mediated adhesion and migration of endothelial cells. PTP-PEST-deficient endothelial cells displayed increased tyrosine phosphorylation of Cas, paxillin, and Pyk2, which were previously also implicated in integrin functions. By eliminating PTP-PEST in endothelial cells in vivo, we obtained evidence that expression of PTP-PEST in endothelial cells is required for normal vascular development and embryonic viability. Therefore, PTP-PEST is a key regulator of integrin-mediated functions in endothelial cells seemingly through its capacity to control Cas, paxillin, and Pyk2. This function explains at least in part the essential role of PTP-PEST in embryonic development and viability.     

JNK regulates cell migration through promotion of tyrosine phosphorylation of paxillin

The adaptor protein paxillin plays an important role in cell migration. Although the c-Jun amino-terminal kinase (JNK) phosphorylation of paxillin on Ser 178 has been found to be critical for cell migration, the precise mechanism by which JNK regulates cell migration is still not very clear. Here, the migration of human corneal epithelial (HCE) cells was used to determine which signaling pathways are involved in EGF-induced paxillin phosphorylation. Paxillin was phosphorylated on Tyr 31 and Tyr 118 after induction of migration by EGF in HCE cells. Specific inhibition of JNK activation by inhibitor SP600125 or overexpression of a dominant-negative JNK mutant not only blocked EGF-induced cell migration, but also eliminated tyrosine phosphorylation of paxillin on Tyr 31 and Tyr 118. HCE cells overexpressing paxillin-S178A mutant also exhibited lower mobility, and reduced phosphorylation of Tyr 31 and Tyr 118. However, paxillin-S178A-inhibited cell migration can be rescued by overexpression of paxillin-Y31E/Y118E mutant. Importantly, inhibition of JNK by SP600125 or overexpression of paxillin-S178A mutant prevented the association of FAK with paxillin. Taken together, these results suggest that phosphorylation of paxillin on Ser 178 by JNK is required for the association of paxillin with FAK, and subsequent tyrosine phosphorylation of paxillin.     

A novel non-canonical mechanism of regulation of MST3 (mammalian Sterile20-related kinase 3)

The canonical pathway of regulation of the GCK (germinal centre kinase) III subgroup member, MST3 (mammalian Sterile20-related kinase 3), involves a caspase-mediated cleavage between N-terminal catalytic and C-terminal regulatory domains with possible concurrent autophosphorylation of the activation loop MST3(Thr(178)), induction of serine/threonine protein kinase activity and nuclear localization. We identified an alternative 'non-canonical' pathway of MST3 activation (regulated primarily through dephosphorylation) which may also be applicable to other GCKIII (and GCKVI) subgroup members. In the basal state, inactive MST3 co-immunoprecipitated with the Golgi protein GOLGA2/gm130 (golgin A2/Golgi matrix protein 130). Activation of MST3 by calyculin A (a protein serine/threonine phosphatase 1/2A inhibitor) stimulated (auto)phosphorylation of MST3(Thr(178)) in the catalytic domain with essentially simultaneous cis-autophosphorylation of MST3(Thr(328)) in the regulatory domain, an event also requiring the MST3(341-376) sequence which acts as a putative docking domain. MST3(Thr(178)) phosphorylation increased MST3 kinase activity, but this activity was independent of MST3(Thr(328)) phosphorylation. Interestingly, MST3(Thr(328)) lies immediately C-terminal to a STRAD (Sterile20-related adaptor) pseudokinase-like site identified recently as being involved in binding of GCKIII/GCKVI members to MO25 scaffolding proteins. MST3(Thr(178)/Thr(328)) phosphorylation was concurrent with dissociation of MST3 from GOLGA2/gm130 and association of MST3 with MO25, and MST3(Thr(328)) phosphorylation was necessary for formation of the activated MST3-MO25 holocomplex.     

PTP-PEST: a protein tyrosine phosphatase regulated by serine phosphorylation.

The protein tyrosine phosphatase PTP-PEST is an 88 kDa cytosolic enzyme which is ubiquitously expressed in mammalian tissues. We have expressed PTP-PEST using recombinant baculovirus, and purified the protein essentially to homogeneity in order to investigate phosphorylation as a potential mechanism of regulation of the enzyme. PTP-PEST is phosphorylated in vitro by both cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC) at two major sites, which we have identified as Ser39 and Ser435. PTP-PEST is also phosphorylated on both Ser39 and Ser435 following treatment of intact HeLa cells with TPA, forskolin or isobutyl methyl xanthine (IBMX). Phosphorylation of Ser39 in vitro decreases the activity of PTP-PEST by reducing its affinity for substrate. In addition, PTP-PEST immunoprecipitated from TPA-treated cells displayed significantly lower PTP activity than enzyme obtained from untreated cells. Our results suggest that both PKC and PKA are capable of phosphorylating, and therefore inhibiting, PTP-PEST in vivo, offering a mechanism whereby signal transduction pathways acting through either PKA or PKC may directly influence cellular processes involving reversible tyrosine phosphorylation.     

Ras-induced and extracellular signal-regulated kinase 1 and 2 phosphorylation-dependent isomerization of protein tyrosine phosphatase (PTP)-PEST by PIN1 promotes FAK dephosphorylation by PTP-PEST

Protein tyrosine phosphatase (PTP)-PEST is a critical regulator of cell adhesion and migration. However, the mechanism by which PTP-PEST is regulated in response to oncogenic signaling to dephosphorylate its substrates remains unclear. Here, we demonstrate that activated Ras induces extracellular signal-regulated kinase 1 and 2-dependent phosphorylation of PTP-PEST at S571, which recruits PIN1 to bind to PTP-PEST. Isomerization of the phosphorylated PTP-PEST by PIN1 increases the interaction between PTP-PEST and FAK, which leads to the dephosphorylation of FAK Y397 and the promotion of migration, invasion, and metastasis of v-H-Ras-transformed cells. These findings uncover an important mechanism for the regulation of PTP-PEST in activated Ras-induced tumor progression.     

Glycogen synthase kinase 3 and h-prune regulate cell migration by modulating focal adhesions

h-prune, which has been suggested to be involved in cell migration, was identified as a glycogen synthase kinase 3 (GSK-3)-binding protein. Treatment of cultured cells with GSK-3 inhibitors or small interfering RNA (siRNA) for GSK-3 and h-prune inhibited their motility. The kinase activity of GSK-3 was required for the interaction of GSK-3 with h-prune. h-prune was localized to focal adhesions, and the siRNA for GSK-3 or h-prune delayed the disassembly of paxillin. The tyrosine phosphorylation of focal adhesion kinase (FAK) and the activation of Rac were suppressed in GSK-3 or h-prune knocked-down cells. GSK-3 inhibitors suppressed the disassembly of paxillin and the activation of FAK and Rac. Furthermore, h-prune was highly expressed in colorectal and pancreatic cancers, and the positivity of the h-prune expression was correlated with tumor invasion. These results suggest that GSK-3 and h-prune cooperatively regulate the disassembly of focal adhesions to promote cell migration and that h-prune is useful as a marker for tumor aggressiveness.     

Regulation of mammalian STE20-like kinase 2 (MST2) by protein phosphorylation/dephosphorylation and proteolysis

Mammalian STE20-like kinase 2 (MST2), a member of the STE20-like kinase family, has been shown in previous studies to undergo proteolytic activation by caspase-3 during cell apoptosis. A few studies have also implicated protein phosphorylation reactions in MST2 regulation. In this study, we examined the mechanism of MST2 regulation with an emphasis on the relationship between caspase-3 cleavage and protein phosphorylation. Both the full-length MST2 and the caspase-3-truncated form of MST2 overexpressed in 293T cells exist in a phosphorylated state. On the other hand, the endogenous full-length MST2 from rat thymus or from proliferating cells is mainly unphosphorylated whereas the caspase-3-truncated endogenous MST2 from apoptotic cells is highly phosphorylated. Cell transfection studies using mutant MST2 constructs indicate that MST2 depends on the autophosphorylation of a unique threonine residue, Thr(180), for kinase activity. The autophosphorylation reaction shows strong dependence on MST2 concentration suggesting that it is an intermolecular reaction. While both the full-length MST2 and the caspase-3-truncated form of MST2 undergo autophosphorylation, the two forms of the phosphorylated MST2 display marked difference in susceptibility to protein phosphatases. The full-length phospho-MST2 is rapidly dephosphorylated by protein phosphatase 1 or protein phosphatase 2A whereas the truncated MST2 is remarkably resistant to the dephosphorylation. Based on the present results, a novel molecular mechanism for MST2 regulation in apoptotic cells is postulated. In normal cells, because of the low concentration and the ready reversal of the autophosphorylation by protein phosphatases, MST2 is present mainly in the unphosphorylated and inactive state. During cell apoptosis, MST2 is cleaved by caspase-3 and undergoes irreversible autophosphorylation, thus resulting in the accumulation of active MST2.     

Transglutaminase-2 in cell adhesion

Cell-matrix adhesion is a fundamental biological process that governs survival, migration, and proliferation of living eukaryotic cells. Paxillin is an important central player in a network of adhesome proteins that form focal adhesion complexes. Phosphorylation of tyrosine and serine residues in paxillin is critical for the coordinated sequential recruitment of other adaptor and kinase proteins to adhesion complexes. Recently, the phosphorylation of serine178 in paxillin has been shown to be vital for epithelial cell adhesion and migration. In vivo and in vitro evidence have shown that transglutaminase (TG)-2 positively regulates this phosphorylation. Here, we propose three possible mechanisms that may explain these observations. First, TG-2 itself may be an adhesome member directly interacting with paxillin in a non-covalent way. Second, TG-2 may cross link a mitogen-activated protein kinase kinase kinase (MAP3K), which eventually activates c-Jun N-terminal kinase (JNK), and the latter phosphorylates paxillin. Lastly, TG-2 may have intrinsic kinase activity that phosphorylates paxillin. Future studies investigating these hypotheses on TG-2-paxillin relationships are necessary in order to address this fundamental process in cell matrix adhesion signaling.     

Mechanism of oleic acid-induced myofibril disassembly in rat cardiomyocytes

This study investigated the mechanism of oleic acid (OA)-induced disassembly of myofibrils in cardiomyocytes. OA treatment disrupted myofibrils, as revealed by the disorganization of several sarcomeric proteins. Since focal adhesions (FAs) are implicated in myofibril assembly, we examined structural changes in FAs after OA treatment. Immunofluorescence studies with antibodies against FA proteins (vinculin, integrin beta1D, and paxillin) showed that FAs and costameres disintegrated or disappeared after OA treatment and that the changes in FA proteins occurred prior to myofibril disassembly. The effects of OA on FAs and myofibrils were reversed after removal of OA. OA decreased expression of integrin beta1D, paxillin, vinculin, and actin, and induced tyrosine dephosphorylation of FA kinase (FAK) and paxillin. These effects were blocked by pretreatment with sodium orthovanadate, a protein tyrosine phosphatase (PTP) inhibitor. This inhibitor also prevented OA-induced myofibril disassembly, indicating the involvement of PTP in myofibril disassembly. Furthermore, OA increased protein levels of PTP-PEST. The upregulation of this phosphatase correlated with the tyrosine dephosphorylation of paxillin and FAK, which are targets for PTP-PEST. In addition, OA decreased RhoA activity and the phosphorylation of cofilin, a downstream target of RhoA. Cofilin dephosphorylation increased its actin-severing activity and led to the depolymerization of F-actin, which might provide another potential mechanism for OA-induced myofibril disassembly.     

Regulation of the Src Kinase-associated Phosphoprotein 55 Homologue by the Protein Tyrosine Phosphatase PTP-PEST in the Control of Cell Motility

PTP-PEST is a cytosolic ubiquitous protein tyrosine phosphatase (PTP) that contains, in addition to its catalytic domain, several protein-protein interaction domains that allow it to interface with several signaling pathways. Among others, PTP-PEST is a key regulator of cellular motility and cytoskeleton dynamics. The complexity of the PTP-PEST interactome underscores the necessity to identify its interacting partners and physiological substrates in order to further understand its role in focal adhesion complex turnover and actin organization. Using a modified yeast substrate trapping two-hybrid system, we identified a cytosolic adaptor protein named Src kinase-associated phosphoprotein 55 homologue (SKAP-Hom) as a novel substrate of PTP-PEST. To confirm PTP-PEST interaction with SKAP-Hom, in vitro pull down assays were performed demonstrating that the PTP catalytic domain and Proline-rich 1 (P1) domain are respectively binding to the SKAP-Hom Y260 and Y297 residues and its SH3 domain. Subsequently, we generated and rescued SKAP-Hom-deficient mouse embryonic fibroblasts (MEFs) with WT SKAP-Hom, SKAP-Hom tyrosine mutants (Y260F, Y260F/Y297F), or SKAP-Hom SH3 domain mutant (W335K). Given the role of PTP-PEST, wound-healing and trans-well migration assays were performed using the generated lines. Indeed, SKAP-Hom-deficient MEFs showed a defect in migration compared with WT-rescued MEFs. Interestingly, the SH3 domain mutant-rescued MEFs showed an enhanced cell migration corresponding potentially with higher tyrosine phosphorylation levels of SKAP-Hom. These findings suggest a novel role of SKAP-Hom and its phosphorylation in the regulation of cellular motility. Moreover, these results open new avenues by which PTP-PEST regulates cellular migration, a hallmark of metastasis.     

Src family kinase involvement in muscarinic receptor-induced tyrosine phosphorylation in differentiated SH-SY5Y cells

Muscarinic receptor-mediated changes in protein tyrosine phosphorylation were examined in differentiated human neuroblastoma SH-SY5Y cells. Treatment of differentiated cells with 1 mM carbachol caused rapid increases in the tyrosine phosphorylation of focal adhesion kinase (FAK), Cas, and paxillin. The src family kinase-selective inhibitor PP1 reduced carbachol-stimulated tyrosine phosphorylation of FAK, Cas, and paxillin by 50 to 75%. In contrast, carbachol-stimulated activation of ERK1/2 was unaffected by PP1. Src family kinase activation by carbachol was further demonstrated by increased carbachol-induced tyrosine phosphorylation of the src-substrate, p120, and tyrosine phosphorylation of the src family kinase activation-associated autophosphorylation site. Site-specific FAK phosphotyrosine antibodies were used to determine that the carbachol-stimulated increase in the autophosphorylation of FAK was unaffected by pretreatment with PP1, whereas the carbachol-stimulated increase in the src family kinase-mediated phosphotyrosine of FAK was completely blocked by pretreatment with PP1. In SH-SY5Y cell lines stably overexpressing Fyn, the phosphotyrosine immunoreactivity of FAK was 625% that of control cells. Thus, muscarinic receptors activate protein tyrosine phosphorylation in differentiated cells, and the tyrosine phosphorylation of FAK, Cas, and paxillin, but not ERK1/2, is mediated by a src family tyrosine kinase activated in response to stimulation of muscarinic receptors.