Csk phosphorylation and inactivation in vitro by the cAMP-dependent protein kinase.
Csk is a protein tyrosine kinase that phosphorylates other protein tyrosine kinases of the Src family and down-regulates their activities. It is not known how Csk is regulated. We investigated the possibility that Csk is regulated through phosphorylation by examining if Csk can serve as an in vitro substrate for a panel of protein kinases. We found that Csk was phosphorylated by the cAMP-dependent protein kinase (PKA), but not by protein kinase C, Src, or the fibroblast growth factor receptor kinase. Csk phosphorylation in vitro by PKA is on a serine residue(s) and can reach a stoichiometry of approximately 0.6 mol phosphate per mole of enzyme. Furthermore, incubation with PKA in the presence of ATP and magnesium ion results in a time-dependent decrease in Csk kinase activity. A six-fold decrease in Csk activity (expressed as Vmax/Km ratio) was achieved due to a threefold increase in its Km and a twofold decrease in its Vmax value within 1 h of incubation with the catalytic subunit of PKA and ATP-Mg. Both phosphorylation and inactivation by PKA were blocked by a PKA-specific inhibitor. Csk mutants with a deleted SH2 or SH3 domain retained their ability to be phosphorylated and inactivated by PKA, indicating that the phosphorylation site is located within the catalytic domain. These studies suggest that the cAMP-dependent protein kinase can regulate Csk activity.
Sun
G
G
Department of Neuro-Oncology, University of Texas M. D. Anderson Cancer Center, Houston 77030, USA.
Ke
S
S
Budde
R J
RJ
D000595:Amino Acid Sequence; D000081247:CSK Tyrosine-Protein Kinase; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D019061:src-Family Kinases
D004791:Enzyme Inhibitors; D010455:Peptides; D011994:Recombinant Proteins; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C