G. Manes et al., Slap negatively regulates Src mitogenic function but does not revert Src-induced cell morphology changes, MOL CELL B, 20(10), 2000, pp. 3396-3406
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.