Activation of Protein-tyrosine Phosphatase SH-PTP2 by a Tyrosine-based Activation Motif of a Novel Brain Molecule

Abstract

BIT (a brain immunoglobulin-like molecule with tyrosine-based activation motifs) is a brain-specific membrane protein which has two cytoplasmic TAMs (tyrosine-based activation motifs). Using the Far Western blotting technique, we detected association of a 70-kDa protein with the tyrosine-phosphorylated TAMs of BIT. A mouse brain cDNA library in gt11 was screened for this association, and two positive clones encoding tyrosine phosphatase SH-PTP2 were isolated. SH-PTP2 has two SH2 domains and is believed to function as a positive mediator in receptor tyrosine kinase signaling. SH-PTP2 and BIT were coimmunoprecipitated from phosphorylated rat brain lysate, and BIT was a major tyrosine-phosphorylated protein associated with SH-PTP2 in this lysate. This interaction was also observed in Jurkat T cells transfected with BIT cDNA depending on tyrosine phosphorylation of BIT. Bisphosphotyrosyl peptides corresponding to BIT-TAMs stimulated SH-PTP2 activity 33-35-fold in vitro, indicating that two SH2 domains of SH-PTP2 simultaneously interact with two phosphotyrosines of BIT-TAM. Our findings suggest that the tyrosine phosphorylation of BIT results in stimulation of the signal transduction pathway promoted by SH-PTP2 and that BIT is probably a major receptor molecule in the brain located just upstream of SH-PTP2.Protein tyrosine phosphorylation plays an important role in signal transduction and regulates a wide range of cellular processes. Protein-tyrosine kinases and protein-tyrosine phosphatases are highly expressed in the central nervous system, consistent with the importance of tyrosine phosphorylation in neural function (1).BIT 1 (a brain immunoglobulin-like molecule with tyrosinebased activation motifs) is a novel immune antigen receptorlike molecule of the brain.2 This molecule is composed of an antigen receptor-like extracellular domain, a transmembrane domain, and a cytoplasmic region containing two variants of TAM (tyrosine-based activation motif) that was recently designated ITAM (immunoreceptor TAM). This cytoplasmic motif contains two tyrosine phosphorylation sites. TAM was originally described in the immune system where it plays a crucial role in the activation responses of B and T cells (2-5). BIT is one of major substrates of protein-tyrosine kinase(s) in crude brain suspensions 2 and is widely distributed in the brain in synapse-rich regions and in some nerve fibers.3 These findings suggest that the tyrosine phosphorylation of TAMs in BIT may be involved in neural signal transduction. Recent studies in the immune system have demonstrated that the oligomerization of TAMs allows the phosphorylation of two tyrosine residues found in this motif and these phosphotyrosine residues act as a bidentate docking site for the paired Src homology 2 (SH2) domains present in the cytoplasmic tyrosine kinases, Syk and ZAP-70, believed to be indispensable for initiation of the signaling cascade (6 -8). From these investigations, we predicted that TAMs of BIT may recruit tyrosine kinases containing paired SH2 domains to the ...

SHP-2 binds to Tyr763 and Tyr1009 in the PDGF β-receptor and mediates PDGF-induced activation of the Ras/MAP kinase pathway and chemotaxis

“…One possible explanation is that the two SH2 domains of SHP-2 have di erent binding speci®city. When comparing the sequences surrounding naturally occuring association sites for SHP-2, Tyr763 is quite similar to the association sites found in a number of other molecules (Table 1) (Case et al, 1994;Fujioka et al, 1996;Jackson et al, 1997;Ohnishi et al, 1996;Ottinger et al, 1998;Sugimoto et al, 1994). Many of these molecules have an alanine residue in position +1, an acidic residue in position +2, followed by either leucine or isoleucine in position +3.…”

“…It has previously been shown that Tyr1009 in the PDGF breceptor binds to SHP-2 (Kazlauskas et al, 1993), and that the association between the SH2 domains of SHP-2 and its target proteins triggers activation of the phosphatase activity of SHP-2 Pluskey et al, 1995). Simultaneous occupancy of both SH2 domains by phosphopeptides is required for full activation of the phosphatase activity of SHP-2 (Eck et al, 1996;Jackson et al, 1997;Ohnishi et al, 1996;Pluskey et al, 1995). This suggests that additional autophosphorylation sites in the PDGF b-receptor might contribute to the activation of SHP-2.…”

Inhibition of EGFR-mediated phosphoinositide-3-OH kinase (PI3-K) signaling and glioblastoma phenotype by Signal-Regulatory Proteins (SIRPs)

“…A consensus cytoplasmic sequence found in these inhibitory receptors, referred to as the immunoreceptor tyrosine-based inhibitory motif or ITIM, binds to the SH2 domaincontaining phosphotyrosine phosphatases (PTPs) SHP1 and SHP2. More recent e orts (Fujioka et al, 1996;Ohnishi et al, 1996;Kharitonenkov et al, 1997) led to the isolation and cloning of new inhibitory receptors in neurons and epithelial cells based on their binding to SHP2. Signal-Regulatory Proteins [SIRPs (Kharitonenkov et al, 1997); murine homolog SHPS-1, or SHP substrate 1 (Fujioka et al, 1996)], a family of membrane glycoproteins and putative substrates of the SHP1 and SHP2 phosphatases, are unique members of the inhibitory receptor family whose members are ubiquitously expressed.…”

Lysophosphatidic acid-induced association of SHP-2 with SHPS-1: roles of RHO, FAK, and a SRC family kinase

“…We have recently characterized a membrane glycoprotein, SHPS-1 (SHP substrate-1) Noguchi et al, 1996;Yamao et al, 1997) (also known as SIRP1 (Kharitonenkov et al, 1997) or BIT (Ohnishi et al, 1996)), whose extracellular region contains three homologous immunoglobulin-like domains and multiple N-linked glycosylation sites. The cytoplasmic region of SHPS-1 contains four tyrosine residues followed by XX(L/V/I) sequences (Y408ADL, Y432ASI, Y449ADL and Y473ASV), which represent potential tyrosine phosphorylation sites.…”

“…SHPS-1 appears to be a docking protein, such as IRS-1, Gab-1 (Holgado-Madruga et al, 1996) and DOS (Herbst et al, 1996), that recruits SHP-2 from the cytosol to a site near the plasma membrane in response to various growth factors or cell adhesion. The synthetic phosphotyrosyl peptide containing sequence surrounding Tyr 449 and Tyr 473 increases the phosphatase activity of SHP-2 in vitro, suggesting that the binding of SHP-2 to tyrosine-phosphorylated SHPS-1 may stimulate the PTPase activity of SHP-2 (Ohnishi et al, 1996). After activation induced by binding to SHPS-1, SHP-2 may dephosphorylate and dissociate from SHPS-1, stimulating the RAS ± MAP kinase cascade through dephosphorylation of an unknown target protein.…”