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Molecular and Cellular Biology, May 2000, p. 3396-3406, Vol. 20, No. 10
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Slap Negatively Regulates Src Mitogenic Function but Does Not Revert Src-Induced Cell Morphology Changes

Gaël Manes, Paul Bello, and Serge Roche*

Centre de Recherche de Biochimie Macromoléculaire, Centre National de la Recherche Scientifique UPR-1086, 34293 Montpellier, France

Received 15 September 1999/Returned for modification 4 November 1999/Accepted 7 February 2000

Src-like adapter protein (Slap) is a recently identified protein that negatively regulates mitogenesis in murine fibroblasts (S. Roche, G. Alonso, A. Kazlausakas, V. M. Dixit, S. A. Courtneidge, and A. Pandey, Curr. Biol. 8:975-978, 1998) and comprises an SH3 and SH2 domain with striking identity to the corresponding Src domains. In light of this, we sought to investigate whether 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 coexpressed in Cos7 cells. Microinjection of a Slap-expressing construct into quiescent NIH 3T3 cells inhibited platelet-derived growth factor (PDGF)-induced DNA synthesis, and the inhibition was rescued by the transcription factor c-Myc but not by c-Jun/c-Fos expression. Fyn (or Src) overexpression overrides the G1/S block induced by both SrcK- and a Slap mutant with a deletion of its C terminus (SlapDelta C), but not the block induced by Slap or SlapDelta SH3, implying that the C terminus is a noncompetitive inhibitor of Src mitogenic function. Furthermore, a chimeric adapter comprising SrcDelta K fused to the Slap C terminus (Src/SlapC) also inhibited Src function during the PDGF response in a noncompetitive manner, as Src coexpression could not rescue PDGF signaling. Slap, however, did not reverse deregulated Src-induced cell transformation, as it was unable to inhibit depolymerization of actin stress fibers while still being able to inhibit SrcY527F-induced DNA synthesis. This was attributed to a distinct Slap SH3 binding specificity, since the chimeric Slap/SrcSH3 molecule, in which the Slap SH3 was replaced by the Src SH3 sequence, substantially restored stress fiber formation. Indeed, three amino acids important for ligand binding in Src SH3 were replaced in the Slap SH3 sequence; Slap SH3 did not bind to the Src SH3 partners p85alpha , Shc, and Sam68 in vitro, and the chimeric tyrosine kinase Slap/SrcK, composed of SlapDelta C fused to the SH2 linker kinase sequence of Src, was not regulated in vivo. Furthermore, the Src SH3 domain is required for signaling during mitogenesis and since Slap/SrcK behaved as a dominant negative in the PDGF mitogenic response when microinjected into quiescent fibroblasts. We conclude that Slap is a negative regulator of Src during mitogenesis involving both the SH2 and the C terminus domains in a noncompetitive manner, but it does not regulate all Src function due to specific SH3 binding substrates.

* Corresponding author. Mailing address: CRBM, CNRS UPR-1086, 1919 route de Mende, 34293 Montpellier, France. Phone: (33) 467 61 33 73. Fax: (33) 467 52 15 59. E-mail: roche{at}crbm.cnrs-mop.fr.

Molecular and Cellular Biology, May 2000, p. 3396-3406, Vol. 20, No. 10
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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