A role for the receptor-like protein tyrosine phosphatase alpha (PTPalpha) in regulating the kinase activity of Src family members has been proposed because ectopic expression of PTPalpha enhances the dephosphorylation and activation of Src and Fyn [1] [2] [3]. We have generated mice lacking catalytically active PTPalpha to address the question of whether PTPalpha is a physiological activator of Src and Fyn, and to investigate its other potential functions in the context of the whole animal. Mice homozygous for the targeted PTPalpha allele (PTPalpha-/-) and lacking detectable PTPalpha protein exhibited no gross phenotypic defects. The kinase activities of Src and Fyn were significantly reduced in PTPalpha-/- mouse brain and primary embryonic fibroblasts, and this correlated with enhanced phosphorylation of the carboxy-terminal regulatory Tyr527 of Src in PTPalpha-/- mice. Thus, PTPalpha is a physiological positive regulator of the tyrosine kinases Src and Fyn. Increased tyrosine phosphorylation of several unidentified proteins was also apparent in PTPalpha-/- mouse brain lysates. These may be PTPalpha substrates or downstream signaling proteins. Taken together, the results indicate that PTPalpha has a dual function as a positive and negative regulator of tyrosine phosphorylation events, increasing phosphotyrosyl proteins through activation of Src and Fyn, and directly or indirectly removing tyrosine phosphate from other unidentified proteins.

We investigated the molecular and cellular actions of receptor protein tyrosine phosphatase (PTP) α in integrin signaling using immortalized fibroblasts derived from wild-type and PTPα-deficient mouse embryos. Defects in PTPα−/− migration in a wound healing assay were associated with altered cell shape and focal adhesion kinase (FAK) phosphorylation. The reduced haptotaxis to fibronectin (FN) of PTPα−/− cells was increased by expression of active (but not inactive) PTPα. Integrin-mediated formation of src–FAK and fyn–FAK complexes was reduced or abolished in PTPα−/− cells on FN, concomitant with markedly reduced phosphorylation of FAK at Tyr397. Reintroduction of active (but not inactive) PTPα restored FAK Tyr-397 phosphorylation. FN-induced cytoskeletal rearrangement was retarded in PTPα−/− cells, with delayed filamentous actin stress fiber assembly and focal adhesion formation. This mimicked the effects of treating wild-type fibroblasts with the src family protein tyrosine kinase (Src-PTK) inhibitor PP2. These results, together with the reduced src/fyn tyrosine kinase activity in PTPα−/− fibroblasts (Ponniah et al., 1999; Su et al., 1999), suggest that PTPα functions in integrin signaling and cell migration as an Src-PTK activator. Our paper establishes that PTPα is required for early integrin-proximal events, acting upstream of FAK to affect the timely and efficient phosphorylation of FAK Tyr-397.

We have investigated the role of the mammalian Son of sevenless 1 (Sosl) protein in growth factor signaling in vivo by generating mice and cell lines that lacked the Sosl protein. Homozygous null embryos were smaller than normal, died mid-gestation with cardiovascular and yolk sac defects, and their fibroblasts showed reduced mitogen-activated protein kinase activation in response to epidermal growth factor (EGF). An intercross of mice mutant for Sosl and the EGF receptor {EGFR) demonstrated that a heterozygous mutation in Sosl dominantly enhanced the phenotype of a weak allele of the EGFR allele {wa-2). These animals had distinctive eye defects that closely resembled those seen in mice that were null for the EGFR or its ligand, TGFa. Our findings provide the first demonstration of a functional requirement for Sosl in growth factor signaling in vivo. They also show that the genetic test of enhancement of weak receptor allele by heterozygous mutation in one component represents a powerful tool for analyzing the ras pathway in mammals.[Key Words: Son of sevenless; EGF receptor; protein tyrosine kinase receptor signaling ras; gene targeting] Received September 27, 1996; revised version accepted December 17, 1996.Son of sevenless (Sos) proteins are guanine nucleotide exchange factors (GEFs) that catalyze the activation of ras proteins by facilitating GDP-GTP exchange. Hu mans and mice each have two unlinked Sos genes {Sosl, Sos2

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Ras GTPase-activating protein of 120 kDa (p120GAP) consists of a hydrophobic Gly-Ala-Pro-rich stretch and src homology 2 and 3 (SH2/SH3) domains in the N-terminal half, and a Ras GTPase-activating domain at the C-terminus. In order to evaluate the potential for cell-growth regulation of the N-terminal region of p120GAP, we isolated three distinct clones of rat 3Y1 fibroblast that express either the SH2/SH3 regions alone, the N-terminal half, or the whole p120GAP. Clones that express the SH2-SH3-SH2 regions of 37 kDa (p37SH2/3) at a level of only 15-30% that of endogenous p120GAP, but not clones expressing complete p120GAP or its N-terminal half of 55 kDa (p55GAP-N), showed significant growth-enhancing properties, including a higher saturation density and increased uptake of 2-deoxyglucose. Clones expressing p37SH2/3 or p55GAP-N maintained high levels of tyrosine-phosphorylated p190 and p62, both of which bind the SH2 domain of p120GAP, while clones expressing the whole p120GAP showed no tyrosine phosphorylation of p62. Furthermore, in the presence of a phorbol ester, only the clones expressing p37SH2/3 showed increased tyrosine phosphorylation of p62 and c-fos expression. These clones also showed the ability of colony formation in soft agar. These results indicate that the N-terminal domain of p120GAP consists of two regions with differential growth-enhancing activities and suggest that the transforming potential of SH2/SH3 regions is blocked by the N-terminal hydrophobic Gly-Ala-Pro stretch.