Specific Stimulation of c‐Fgr Kinase by Tyrosine‐Phosphorylated (Poly)Peptides

Abstract

Hematopoietic lineage cell-specific HSI protein is converted into a substrate for c-Fgr by previous Syk-mediated phosphorylation, at site(s) that bind to the SH2 domain of c-Fgr [Ruzzene, M., Biochemistry 35, 5327-53321. Here we show that a phosphopeptide derived from one such site, HS1-(320-329)-phosphopeptide (PEGDYpEEVLE), enhances up to tenfold, in a dose-dependent manner, the catalytic activity of c-Fgr either assayed with peptide substrates or evaluated as intermolecular autophosphorylation of c-Fgr itself. The dephosphorylated HS1-(320-329)-peptide is totally ineffective, while the stimulatory efficacy of other phosphopeptides derived from the polyoma virus middle T antigen-(393 -402) sequence, c-Src, and c-Fgr autophosphorylation sites, and the C-terminal c-Src site (Tyr527) is variable and correlates reasonably well with the predicted affinity for the c-Fgr SH2 domain. Stimulation of c-Fgr catalytic activity is also promoted by the full-length HSI protein, previously tyrosine phosphorylated by Syk, and is accounted for by an increased V,,,,, while the K,,, values are unchanged. If the normal activator of c-Fgr kinase, Mg", is replaced by MnZ+, stimulation by HS 1 -(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 autophosphorylated c-Fgr to be stimulated by HS L(320-329)-phosphopeptide, support the view that stimulation of c-Fgr by phosphopeptide is not or is not entirely a consequence of increased autophosphorylation. Interestingly, neither Syk and C-terminal Src kinase nor three other members of the Src family (Lyn, Lck, and Fyn) are susceptible to stimulation by phosphopeptide, as observed with c-Fgr. These data support the notion that c-Fgr undergoes a unique mechanism of activation promoted by tyrosine-phosphorylated polypeptide that binds to its SH2 domain. This suggests that such a mode of regulation is peculiar of protein-tyrosine kinases committed to the secondary phosphorylation of sequentially phosphorylated proteins.Keywords: protein-tyrosine kinase : c-Fgr: Src homology domain 2 ; phosphotyrosine peptide; HS1 protein.Src-related tyrosine kinases are members of a family of nonreceptor enzymes that includes not less than ten proteins, which all display a remarkable homology and share a common structure: a poorly conserved N-terminal segment (unique domain), followed by a Src homology 3 (SH3) and a Src homology 2 (SH2) domain, a catalytic domain (SHl), and a C-terminal tail. Two highly conserved in vivo tyrosine phosphorylation sites are known: an autophosphorylation site (Tyr416 in chicken c-Src) and a C-terminal phosphorylation site (Tyr527). This latter site, once phosphorylated by C-terminal Src kinase (CSK) (Okada and Nakagawa, 1989;Okada et al., 1991), plays a negative regulatory role through its interaction with the SH2 domain (Cantley et al