Molecular features underlying the sequential phosphorylation of HS1 protein and its association with c-Fgr protein-tyrosine kinase

The hematopoietic lineage cell-specific protein HS1 was shown to undergo a process of sequential phosphorylation both in vitro and in vitro, which is synergistically mediated by Syk and Src family protein-tyrosine kinases and essential for B cell antigen receptor-mediated apoptosis, We have now iden tified tyrosine 222 as the HS1 residue phosphorylated by the Src family pro tein kinases c-Fgr and Lyn, and we show that a truncated form of HS1 (HS1-2 08-401) lacking the N-terminal putative DNA binding region and the C-termin al Src homology 3 (SH3) domain is still able to undergo all the steps of se quential phosphorylation as efficiently as full-length HS1. We also show th at a stable association of phospho-HS1 with c-Fgr through its SH2 domain re quires previous autophosphorylation of the kinase and is prevented by subse quent phosphorylation of Tyr-222. Kinetic studies with HS1 and its truncate d forms previously phosphorylated by Syk and with a peptide substrate repro ducing the sequence around tyrosine 222 support the view that efficient pho sphorylation of HS1 by Src family protein kinases entirely relies on TyrP S H2 domain interaction with negligible, if any, contribution of local specif icity determinants. Our data indicate that the proline-rich region of HS1 b ordered by tyrosyl residues affected by Syk and Src family kinases represen ts a functional domain designed to undergo a process of sequential phosphor ylation.