Tyrosine Phosphorylation Regulates the SH3-mediated Binding of the Wiskott-Aldrich Syndrome Protein to PSTPIP, a Cytoskeletal-associated Protein

Abstract

Wiskott-Aldrich syndrome is an X-linked hematopoietic disease that manifests itself in platelet deficiency and a compromised immune system. Analysis of hematopoietic cells from affected individuals reveals that mutations in the Wiskott-Aldrich syndrome protein (WASP) result in structural and functional abnormalities in the cell cortex, consistent with the suggestion that WASP is involved with regulation of the actin-rich cortical cytoskeleton. Here we report that WASP interacts with a recently described cytoskeletal-associated protein, PST-PIP, a molecule that is related to the Schizosaccharomyces pombe cleavage furrow regulatory protein, CDC15p. This association is mediated by an interaction between the PSTPIP SH3 domain and two polyproline-rich regions in WASP. Co-expression of PSTPIP with WASP in vivo results in a loss of WASP-induced actin bundling activity and co-localization of the two proteins, which requires the PSTPIP SH3 domain. Analysis of tyrosine phosphorylation of PSTPIP reveals that two sites are modified in response to v-Src co-transfection or pervanadate incubation. One of these tyrosines is found in the SH3 domain poly-proline recognition site, and mutation of this tyrosine to aspartate or glutamate to mimic this phosphorylation state results in a loss of WASP binding in vitro and a dissolution of co-localization in vivo. In addition, PSTPIP that is tyrosine phosphorylated in the SH3 domain interacts poorly with WASP in vitro. These data suggest that the PSTPIP and WASP interaction is regulated by tyrosine phosphorylation of the PSTPIP SH3 domain, and this binding event may control aspects of the actin cytoskeleton. PSTPIP is a recently described coiled-coil and SH3 domaincontaining protein that is homologous to CDC15p, a Schizosaccharomyces pombe phosphoprotein involved with the assembly of the cytokinetic cleavage furrow (1-3). PSTPIP is associated with protein-tyrosine phosphatase (PTP) 1 HSCF, a member of the PEST family of PTPs (4), via a critical tryptophan in the PSTPIP coiled-coil domain (2), and this association mediates the dephosphorylation of tyrosine residues in PSTPIP that are modified either by co-expression of the v-Src tyrosine kinase or in the presence of the PTP inhibitor pervanadate. Throughout interphase, the coiled-coil domain localizes PST-PIP to actin-rich regions of the cell, including the cortical cytoskeleton and lammelipodia, and the protein migrates to the cleavage furrow during cytokinesis. Overexpression of PSTPIP in mammalian cells induces filapodial extension and cell rounding, and induced expression of the mammalian protein in S. pombe results in a dominant negative inhibition of cytokinesis (1). These data suggest that PSTPIP is a tyrosine phosphorylated cytoskeletal regulatory protein that may be involved with the control of cytokinesis.Wiskott-Aldrich syndrome is an X-linked hematopoietic disease that presents with platelet abnormalities and immunodeficiency (5, 6), and mutations in the Wiskott-Aldrich syndrome protein (WASP) cause this syndrome (7-9). WAS...

Vascular Endothelial Growth Factor Causes Translocation of p47 to Membrane Ruffles through WAVE1

“…The WASP/suppressor of cAMP receptor family proteins hold potential for integrating such cytoskeletal and signaling responses. The more intensely studied WASP and N-WASP have well known effects on actin nucleation and in addition bind signaling proteins such as Src and Tec family tyrosine kinases and adapters such as Pro-Ser-Thr phosphatase-interacting protein, Grb2, and Nck (25)(26)(27)(28). Although WAVE1 has a clear role in the formation of Rac-induced ruffles and lamellipodia, its capacity to facilitate other signaling events is less clear.…”

PSTPIP Is a Substrate of PTP-PEST and Serves as a Scaffold Guiding PTP-PEST Toward a Specific Dephosphorylation of WASP

“…1A). This domain of PSTPIP has been reported to become tyrosine phosphorylated (19). Since several experiments using this antibody will aim at characterizing the tyrosine phosphorylation level of PSTPIP and its association with other proteins, we studied the anti-PSTPIP antibody in more detail.…”

“…Previously, PTP-HSCF, a protein restricted to hemapoietic stem cells, was shown to interact with the proline, serine, threonine-rich phosphatase interacting protein (PSTPIP) via its CTH domain (18). The discovery that WASP, the protein encoded by the gene mutated in the Wiskott-Aldrich syndrome, is a ligand for the SH3 domain of PSTPIP creates an intriguing link between PSTPIP and actin reorganization (19).…”

SLP-76 Coordinates Nck-Dependent Wiskott-Aldrich Syndrome Protein Recruitment with Vav-1/Cdc42-Dependent Wiskott-Aldrich Syndrome Protein Activation at the T Cell-APC Contact Site

“…Instead, the proline-rich region of WASP is necessary and sufficient to mediate recruitment. Many Src homology 3 (SH3) domain-containing proteins including Lck, Fyn, Btk, Itk, Nck, Grb2, Profilin, and proline, serine, threonine phosphatase interacting protein (PST-PIP) have been shown to interact with the proline-rich domain of WASP in vitro (23)(24)(25)(26)(27)(28)(29). However, the protein responsible for WASP recruitment during T cell signaling is not known.…”