Ser/Thr phosphorylation of hematopoietic specific protein 1 (HS1). Implication of protein kinase CK2

Abstract

Hematopoietic lineage cell-specific protein 1 (HS1), a tyrosine multiphosphorylated protein implicated in receptor-mediated apoptosis and proliferative responses, is shown here to become Ser/ Thr phosphorylated upon incubation of platelets with radiolabeled inorganic phosphate. The in vivo Ser/ Thr phosphorylation of HS1 is enhanced by okadaic acid and reduced by specific inhibitors of casein kinase (CK)2. In vitro, HS1 is an excellent substrate for either CK2 a subunit alone (K m 47 nm) or CK2 holoenzyme, tested in the presence of polylysine (K m 400 nm). Phosphorylation reaches a stoichiometry of about 2 mol phosphate per mol HS1 and occurs mainly at threonyl residue(s), mostly located in the N-terminal region, but also at seryl residue(s) residing in the central core of the molecule (208±402), as judged from experiments with deleted forms of HS1.Ser/ Thr phosphorylation of HS1, either induced in vivo by okadaic acid or catalysed in vitro by CK2, potentiates subsequent phosphorylation at tyrosyl residues. These data indicate the possibility that regulation of HS1 may also be under the control of Ser/ Thr phosphorylation, and suggest that in quiescent cells CK2 could play a role in inducing constitutive Tyr phosphorylation of HS1 in the absence of stimuli that activate the protein tyrosine kinase pathway.Keywords: HS1; CK2; casein kinase 2; phosphorylation; tyrosine kinase.HS1 is an intracellular protein whose expression is limited to hematopoietic and lymphoid cells [1]. Despite the fact that it is composed of 486 amino acids and has a predicted M r of about 54 000, HS1 is isolated as a protein of about 75 kDa as judged from SDS/PAGE. We have recently shown that this discrepancy is not due to post-translational modifications (e.g. glycosylation) but rather to its highly asymmetric rod shaped structure conferred by its central proline-rich domain [2]. The aminoacid sequence of HS1 contains a variety of structurally significant motifs, including: an N-terminal region responsible for the binding of mitochondrial protein HAX-1 [3], followed by four 37 amino-acid repeats that can form a helix-turn-helix structure frequently found in the DNA binding domain of various transcription factors [1]; a proline-rich region located at the C-terminus of the central moiety and ending with a stretch of proline-glutamate repeats; and an SH3 domain located at the C-terminal extremity.HS1 is readily tyrosine phosphorylated upon B-cell receptor cross-linking, in parallel with the activation of Src family kinases [4,5]. In vitro studies however, have revealed that HS1 is not a substrate for the Src-related kinases c-Fgr, Lyn and Fyn unless it has been phosphorylated previously by the protein tyrosine kinase Syk (not related to Src) [6,7]. Syk-mediated phosphorylation affects tyrosyl residues located just upstream from the SH3 domain domain (notably Tyr378 and Try397) and generates high-affinity binding sites for Src family SH2 domains. This provides the structural basis for subsequent association to and secondary phosphorylation by ...

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