Protein tyrosine phosphatase-PEST regulates focal adhesion disassembly, migration, and cytokinesis in fibroblasts. (1/68)

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.  (+info)

Hic-5, a paxillin homologue, binds to the protein-tyrosine phosphatase PEST (PTP-PEST) through its LIM 3 domain. (2/68)

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.  (+info)

Intact LIM 3 and LIM 4 domains of paxillin are required for the association to a novel polyproline region (Pro 2) of protein-tyrosine phosphatase-PEST. (3/68)

The focal adhesion protein p130(Cas) was identified as a substrate for the protein-tyrosine phosphatase (PTP)-PEST, and the specificity of this interaction is mediated by a dual mechanism involving a Src homology 3 domain-mediated binding and PTP domain recognition. Recently, paxillin was also demonstrated to interact with PTP-PEST (Shen, Y., Schneider, G., Cloutier, J. F., Veillette, A., and Schaller, M. D. (1998) J. Biol. Chem. 273, 6474-6481). In the present study, we show that amino acids 344-397 of PTP-PEST are sufficient for the binding to paxillin. We demonstrate that a proline-rich segment of PTP-PEST (Pro 2), 355PPEPHPVPPILTPSPPSAFP374, is essential for this interaction in vivo. Furthermore, mutation of proline residues within the Pro 2 motif reveal that proline 362 is critical for the binding of paxillin. Conversely, using deletion and point mutants of paxillin, LIM 3 and 4 domains were both found to be necessary for binding of PTP-PEST. Finally, using a "substrate trapping" approach, we demonstrate that, unlike p130(Cas), paxillin is not a substrate for PTP-PEST. In conclusion, we show that a novel proline-rich motif found in PTP-PEST serves as a ligand for the LIM domains of paxillin. Interestingly, the focal adhesion targeting of paxillin is mediated by LIM 3. Thus, we propose that PTP-PEST, by a competition with the ligand of paxillin in the focal adhesion complex, could contribute to the removal of paxillin from the adhesion sites and consequently promote focal adhesion turnover.  (+info)

The noncatalytic domain of protein-tyrosine phosphatase-PEST targets paxillin for dephosphorylation in vivo. (4/68)

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.  (+info)

Crucial role of the amino-terminal tyrosine residue 42 and the carboxyl-terminal PEST domain of I kappa B alpha in NF-kappa B activation by an oxidative stress. (5/68)

Activation of transcription factor NF-kappa B involves the signal-dependent degradation of basally phosphorylated inhibitors such as I kappa B alpha. In response to proinflammatory cytokines or mitogens, the transduction machinery has recently been characterized, but the activation mechanism upon oxidative stress remains unknown. In the present work, we provide several lines of evidence that NF-kappa B activation in a T lymphocytic cell line (EL4) by hydrogen peroxide (H2O2) did not involve phosphorylation of the serine residues 32 and 36 in the amino-terminal part of I kappa B alpha. Indeed, mutation of Ser32 and Ser36 blocked IL-1 beta- or PMA-induced NF-kappa B activation, but had no effect on its activation by H2O2. Although I kappa B alpha was phosphorylated upon exposure to H2O2, tyrosine residue 42 and the C-terminal PEST (proline-glutamic acid-serine-threonine) domain played an important role. Indeed, mutation of tyrosine 42 or serine/threonine residues of the PEST domain abolished NF-kappa B activation by H2O2, while it had no effect on activation by IL-1 beta or PMA-ionomycin. This H2O2-inducible phosphorylation was not dependent on I kappa B kinase activation, but could involve casein kinase II, because an inhibitor of this enzyme (5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole) blocks NF-kappa B activation. H2O2-induced I kappa B alpha phosphorylation was followed by its degradation by calpain proteases or through the proteasome. Taken together, our findings suggest that NF-kappa B activation by H2O2 involves a new mechanism that is totally distinct from those triggered by proinflammatory cytokines or mitogens.  (+info)

Cytoskeletal protein PSTPIP1 directs the PEST-type protein tyrosine phosphatase to the c-Abl kinase to mediate Abl dephosphorylation. (6/68)

A search for c-Abl interacting proteins resulted in the recovery of PSTPIP1, originally identified as a binding protein of the PEST-type protein tyrosine phosphatases (PTP). PSTPIP1 was phosphorylated by c-Abl, and growth factor-induced PSTPIP1 phosphorylation was diminished in Abl null fibroblasts. PSTPIP1 was able to bridge c-Abl to the PEST-type PTPs. Several experiments suggest that the PEST-type PTPs negatively regulate c-Abl activity: c-Abl was hyperphosphorylated in PTP-PEST-deficient cells; disruption of the c-Abl-PSTPIP1-PEST-type PTP ternary complex by overexpression of PSTPIP1 mutants increased c-Abl phosphotyrosine content; and PDGF-induced c-Abl kinase activation was prolonged in PTP-PEST-deficient cells. Dephosphorylation of c-Abl by PEST-type PTP represents a novel mechanism by which c-Abl activity is regulated.  (+info)

Activation of the focal adhesion kinase signaling pathway by structural alterations in the carboxyl-terminal region of c-Crk II. (7/68)

The Crk II adaptor protein encodes an SH2/SH3-domain containing adaptor protein with an SH2-SH3-SH3 domain structure that transmits signals from tyrosine kinases. The two SH3 domains are separated by a 54 amino acid linker region, whose length is highly conserved in xenopus, chicken, and mamalian Crk II proteins. To gain a better understanding into the role of the C-terminal region of Crk, we generated a series of C-terminal SH3 domain and SH3 linker mutants and examined their role in tyrosine kinase pathways. Expression of point mutations in the C-terminal SH3 domain (W276K Crk), at the tyrosine phosphorylation site (Y222F Crk II), or truncation of the entire C-terminus (Crk I or Crk Delta242), all increased c-Abl binding to the N-terminal SH3 domain of Crk and, where relevant, increased Tyr(222) phosphorylation. Deletion analysis of c-Crk II also revealed the presence of a C-terminal segment important for trans-activation of FAK. Such mutants, Crk Delta255 or Crk Delta242 Extended Linker (Crk Delta242([EL])), characterized by a disruption in the SH3 linker/C-terminal SH3 boundary, induced robust hyperphosphorylation of focal adhesion kinase (FAK) on Tyr(397), hyperphosphorylation of focal adhesion proteins p130(cas) and paxillin and increased focal adhesion formation in NIH3T3 cells. The effects of Crk Delta242([EL]) could be abrogated by co-expression of dominant negative c-Src or the protein tyrosine phosphatase PTP-PEST, but not by dominant negative Abl. Our results suggest that the C-terminal region of Crk contains negative regulatory elements important for both Abl and FAK dependent signal pathways, and offers a paradigm for an autoinhibitory region in the SH3 linker/C-terminal SH3 domain.  (+info)

Inhibition of the catalytic activity of cell adhesion kinase beta by protein-tyrosine phosphatase-PEST-mediated dephosphorylation. (8/68)

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.  (+info)