Casein kinase-2 (CK2) is a pleiotropic and constitutively active serine/threonine protein kinase composed of two catalytic (α and/or α´) and two regulatory β-subunits, whose regulation is still not well understood. In the present study, we show that the catalytic subunits of human CK2, but not the regulatory β-subunits, are readily phosphorylated by the Src family protein tyrosine kinases Lyn and c-Fgr to a stoichiometry approaching 2 mol phosphotyrosine/mol CK2α with a concomitant 3-fold increase in catalytic activity. We also show that endogenous CK2α becomes tyrosine-phosphorylated in pervanadate-treated Jurkat cells. Both tyrosine phosphorylation and stimulation of activity are suppressed by the specific Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. By comparison, mutations giving rise to inactive forms of CK2α do not abrogate and, in some cases, stimulate Lyn and c-Fgr-dependent tyrosine phosphorylation of CK2. Several radiolabelled phosphopeptides could be resolved by HPLC, following tryptic digestion of CK2α that had been phosphoradiolabelled by incubation with [32P]ATP and c-Fgr. The most prominent phosphopeptide co-migrates with a synthetic peptide encompassing the 248–268 sequence, phosphorylated previously by c-Fgr at Tyr255 in vitro. The identification of Tyr255 as a phosphorylated residue was also supported by MS sequencing of both the phosphorylated and non-phosphorylated 248–268 tryptic fragments from CK2α and by on-target phosphatase treatment. A CK2α mutant in which Tyr255 was replaced by phenylalanine proved less susceptible to phosphorylation and refractory to stimulation by c-Fgr.
- casein kinase-2
- Src tyrosine kinase
- tyrosine phosphorylation
Abbreviations used: CK2, casein kinase-2; ESI, electrospray ionization; FSO2BzAdo, p-fluoro-sulphonylbenzoyl-5′-adenosine; 4HCCA, α-cyano-4-hydroxy cinnamic acid; MALDI–TOF, matrix-assisted laser-desorption ionization–time-of-flight; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; PTK, protein tyrosine kinase; RP, reverse phase; TBB, 4,5,6,7-tetrabromobenzo-2-azabenzimidazole; TFA, trifluoroacetic acid.
- The Biochemical Society, London ©2003