Tyrosine phosphorylation of protein kinase CK2 by Src-related tyrosine kinases correlates with increased catalytic activity.
Casein kinase-2 (CK2) is a pleiotropic and constitutively active serine/threonine protein kinase composed of two catalytic (alpha and/or alpha') and two regulatory beta-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 beta-subunits, are readily phosphorylated by the Src family protein tyrosine kinases Lyn and c-Fgr to a stoichiometry approaching 2 mol phosphotyrosine/mol CK2alpha with a concomitant 3-fold increase in catalytic activity. We also show that endogenous CK2alpha 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 CK2alpha 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 CK2alpha that had been phosphoradiolabelled by incubation with [(32)P]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 Tyr(255) in vitro. The identification of Tyr(255) as a phosphorylated residue was also supported by MS sequencing of both the phosphorylated and non-phosphorylated 248-268 tryptic fragments from CK2alpha and by on-target phosphatase treatment. A CK2alpha mutant in which Tyr(255) was replaced by phenylalanine proved less susceptible to phosphorylation and refractory to stimulation by c-Fgr.
Donella-Deana
Arianna
A
Dipartimento di Chimica Biologica and CRIBI, Centro Nazionale delle Ricerche, Institute of Neuroscience, University of Padova, Viale G. Colombo 3, Italy.
Cesaro
Luca
L
Sarno
Stefania
S
Ruzzene
Maria
M
Brunati
Anna Maria
AM
Marin
Oriano
O
Vilk
Greg
G
Doherty-Kirby
Amanda
A
Lajoie
Gilles
G
Litchfield
David W
DW
Pinna
Lorenzo A
LA
D020816:Amino Acid Motifs; D000818:Animals; D047390:Casein Kinase II; D002384:Catalysis; D020134:Catalytic Domain; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D019169:Jurkat Cells; D009154:Mutation; D010750:Phosphoproteins / Q000032:analysis; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D011743:Pyrimidines / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014357:Trypsin / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
C412373:AG 1879; D004791:Enzyme Inhibitors; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D011743:Pyrimidines; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; C053102:proto-oncogene proteins c-fgr; D019061:src-Family Kinases; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D014357:Trypsin