Slap negatively regulates Src mitogenic function but does not revert Src-induced cell morphology changes

Src-like adapter protein (Slap) is a recently identified protein that negat ively regulates mitogenesis in murine fibroblasts (S. Roche, G. Alonso, A. Kazlausakas, V, M. Dixit, S, A. Courtneidge, and A. Pandey Curr, Biol. 8:97 5-978, 1998) and comprises an SH3 and SH2 domain with striking identity to the corresponding Src domains. In light of this, we sought to investigate w hether Slap could be an antagonist of all Src functions, Like Src, Slap was found to be myristylated in vivo and largely colocalized with Src when coe xpressed in Cos7 cells. Microinjection of a Slap-expressing construct into quiescent NIH 3T3 cells inhibited platelet-derived growth factor (PDGF)-ind uced DNA synthesis, and the inhibition was rescued by the transcription fac tor c-Myc but not by c-Jun/c-Fos expression. Fyn (or Src) overexpression ov errides the G(1)/S block induced by both SrcK- and a Slap mutant with a del etion of its C terminus (Slap Delta C), but not the block induced bg Slap o r Slap Delta SH3, implying that the C terminus is a noncompetitive inhibito r of Src mitogenic function. Furthermore, a chimeric adapter comprising Src Delta K fused to the Slap C terminus (Src/SlapC) also inhibited Src functi on during the PDGF response in a noncompetitive manner, as Src coexpression could not rescue PDGF signaling, Slap, however, did not reverse deregulate d Src-induced cell transformation, as it was unable to inhibit depolymeriza tion of actin stress fibers while still being able to inhibit SrcY527F-indu ced DNA synthesis, This was attributed to a distinct Slap SH3 binding speci ficity, since the chimeric Slap/SrcSH3 molecule, in which the Slap SH3 was replaced by the Src SH3 sequence, substantially restored stress fiber forma tion. Indeed, three amino acids important for ligand binding in Src SH3 wer e replaced in the Slap SH3 sequence; Slap SH3 did not bind to the Src SH3 p artners p85 alpha, Shc, and Sam68 in vitro, and the chimeric tyrosine kinas e Slap/SrcK composed of Slap Delta C fused to the SH2 linker kinase sequenc e of Src, was not regulated in vivo. Furthermore, the Src SH3 domain is req uired for signaling during mitogenesis and since Slap/SrcK behaved as a dom inant negative in the PDGF mitogenic response when microinjected into quies cent fibroblasts, We conclude that Slap is a negative regulator of Src duri ng mitogenesis involving both the SH2 and the C terminus domains in a nonco mpetitive manner, but it does not regulate all Src function due to specific SH3 binding substrates.