SPECIFIC STIMULATION OF C-FGR KINASE BY TYROSINE-PHOSPHORYLATED (POLY)PEPTIDES - POSSIBLE IMPLICATION IN THE SEQUENTIAL MODE OF PROTEIN-PHOSPHORYLATION

Hematopoietic lineage cell-specific HS1 protein is converted into a su bstrate for c-Fgr by previous Syk-mediated phosphorylation, at site(s) that bind to the SH2 domain of c-Fgr [Ruzzene, M., Brunati, A. M., Ma rin, O., Donella-Deana, A. & Pinna, L. A. (1996) Biochemistry 35, 5327 -5332]. Here we show that a phosphopeptide derived from one such site, HS1-(320-329)-phosphopeptide (PEGDYpEEVLE), enhances up to tenfold, i n a dose-dependent manner, the catalytic activity of c-Fgr either assa yed with peptide substrates or evaluated as intermolecular autophospho rylation of c-Fgr itself. The dephosphorylated HS1-(320-329)-peptide i s totally ineffective, while the stimulatory efficacy of other phospho peptides derived from the polyoma virus middle T antigen-(393-402) seq uence, c-Src, and c-Fgr autophosphorylation sites, and the C-terminal c-Src site (Tyr527) is variable and correlates reasonably well with th e predicted affinity for the c-Fgr SH2 domain. Stimulation of c-Fgr ca talytic activity is also promoted by the full-length HS1 protein, prev iously tyrosine phosphorylated by Syk, and is accounted for by an incr eased V-max while the K-m values are unchanged. If the normal activato r of c-Fgr kinase, Mg2+, is replaced by Mn2+, stimulation by HS1-(320- 329)-phosphopeptide is still observable with peptide substrates, while autophosphorylation is, in contrast, inhibited by the phosphopeptide. These findings, in conjunction with the ability of previously autopho sphorylated c-Fgr to be stimulated by HS1-(320-329)-phosphopeptide, su pport the view that stimulation of c-Fgr by phosphopeptide is not or i s not entirely a consequence of increased autophosphorylation. Interes tingly, neither Syk and C-terminal Src kinase nor three other members of the Src family (Lyn, Lck, and Fyn) are susceptible to stimulation b y phosphopeptide, as observed with c-Fgr. These data support the notio n that c-Fgr undergoes a unique mechanism of activation promoted by ty rosine-phosphorylated polypeptide that binds to its SH2 domain. This s uggests that such a mode of regulation is peculiar of protein-tyrosine kinases committed to the secondary phosphorylation of sequentially ph osphorylated proteins.