Molecular Features Underlying the Sequential Phosphorylation of HS1 Protein and Its Association with c-Fgr Protein-tyrosine Kinase

Abstract

The hematopoietic lineage cell-specific protein HS1 was shown to undergo a process of sequential phosphorylation both in vitro and in vivo, which is synergistically mediated by Syk and Src family protein-tyrosine kinases and essential for B cell antigen receptor-mediated apoptosis. We have now identified tyrosine 222 as the HS1 residue phosphorylated by the Src family protein kinases c-Fgr and Lyn, and we show that a truncated form of HS1 (HS1-208-401) lacking the N-terminal putative DNA binding region and the C-terminal Src homology 3 (SH3) domain is still able to undergo all the steps of sequential phosphorylation as efficiently as fulllength HS1. We also show that a stable association of phospho-HS1 with c-Fgr through its SH2 domain requires previous autophosphorylation of the kinase and is prevented by subsequent phosphorylation of Tyr-222. Kinetic studies with HS1 and its truncated forms previously phosphorylated by Syk and with a peptide substrate reproducing the sequence around tyrosine 222 support the view that efficient phosphorylation of HS1 by Src family protein kinases entirely relies on TyrP-SH2 domain interaction with negligible, if any, contribution of local specificity determinants. Our data indicate that the proline-rich region of HS1 bordered by tyrosyl residues affected by Syk and Src family kinases represents a functional domain designed to undergo a process of sequential phosphorylation. HS11 is an intracellular protein with expression that is limited to hematopoietic and lymphoid cells (1). The gene sequence shows that is composed of 486 amino acids with a predicted M r of ϳ54 kDa. In contrast, HS1 has been constantly isolated as a protein with an apparent M r of ϳ75 kDa, as judged from SDS-PAGE, giving rise to the suggestion that it might be heavily affected by post-translational modifications, e.g. glycosylation (1). The amino acid sequence of HS1 contains a variety of structurally significant motifs, including an N-terminal region responsible for the binding of mitochondrial protein HS1-associated protein X-1 (2), followed by four 37-amino acids repeats that can form a helix-turn-helix structure frequently found in the DNA binding domain of various transcriptional factors (1); this region contains three putative phosphatidylinositol 4,5-bisphosphate binding motifs and has been suggested to be involved in F-actin binding (3), a proline-rich region localized to the C-terminal central moiety, which may represent an SH3 binding motif and is ending with a stretch of proline-glutamate repeats, and an SH3 domain located at the C-terminal extremity (see schematic representation of HS1 in Fig. 1).Evidence that HS1 plays a role in the receptor-mediated apoptosis and proliferative responses was provided by the analysis of HS1-deficient mice (4) and WEH1-231 B lymphoma cells (5-7). The observation that HS1 is readily tyrosine phosphorylated after B cell antigen receptor cross-linking, in parallel with activation of Src family kinases (8, 9), is also consistent with a role for HS1 in signal transduct...

Lyn deficiency reduces GATA-1, EKLF and STAT5, and induces extramedullary stress erythropoiesis

“…Other studies have confirmed the association of Lyn with the Epo receptor and its participation in downstream signaling, including phosphorylation of STAT5 (Chin et al, 1998;Arai et al, 2001). Lyn is also activated by Epo in CD34 þ cells maturing along the erythroid lineage (Harashima et al, 2002), and can phosphorylate protein Band 3 in erythrocytes (Brunati et al, 2000).…”

SDF1α‐induced interaction of the adapter proteins Nck and HS1 facilitates actin polymerization and migration in T cells

“…In detail, the HS1 sites Y 378 and Y 397 are phosphorylated by Syk in vitro [36] and presumably by ZAP70 upon TCR ligation [27]. Besides these tyrosine motifs, in vitro phosphorylation of Y 222 (YKKT) [36][37][38] and Y 198 (YGIQ) [39] was documented. However, since the sequence of these phospho-sites pretty much differs from the Nck SH2 consensus-binding sequence, an interaction with the Nck SH2 domain is rather unlikely.…”

Cortactin: coupling membrane dynamics to cortical actin assembly

“…HS1 di ers from cortactin in that it contains only three complete cortactin repeats ( Figure 1), and that the helical and proline-rich domains are inverted with respect to cortactin . In spite of these di erences, HS1 shares functional similarities with cortactin, serving as a Srcfamily tyrosine kinase substrate (Takemoto et al, 1995;Brunati et al, 1999) as well as playing a unique and essential role in antigen-receptor induced B cell clonal expansion .…”