Identification, assay, and purification of a Cdc2-activating threonine-161 protein kinase from human cells.
The biological activities of cyclin-dependent, proline-directed protein kinases (PDPKs) are highly regulated by a complex series of protein phosphorylation/dephosphorylation reactions involving both catalytic and regulatory subunits. In this paper we report on the enzymatic activation of p34cdc2/p58Cyclin A PDPK by a protein kinase present in human cells that targets threonine-161 of Cdc2. An assay for this Cdc2 kinase-kinase (PK161) was developed and specific enzyme activity was detected in a variety of mammalian cells and tissues. PK161 activity was rapidly stimulated by epidermal growth factor in human A431 epidermoid carcinoma cells. The development of an assay selective for PK161 phosphotransferase activity afforded the partial purification of the enzyme from human Wilms' tumors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of highly purified enzyme preparations revealed the presence of phosphoproteins migrating at approximately 42-44 and approximately 95 kDa, respectively, which correlated with enzyme activity upon fast-protein liquid chromatography gel permeation chromatography. Further purification was accomplished by immobilized peptide substrate affinity chromatography. The ability of PK161 to phosphorylate and activate p34cdc2/p58Cyclin A PDPK was confirmed by the use of purified recombinant subunits. Polyclonal antibodies directed against the Xenopus MO15 gene product (a putative Cdc2-activating kinase) cross-reacted with the purified 42- to 44-kDa phosphoprotein, thus identifying the catalytic subunit of human PK161 as a human homologue of Xenopus p40MO15. Subsequent immunoprecipitation experiments with metabolically labeled A431 cells identified a approximately 95-kDa phosphoprotein that coprecipitated with the approximately 42-kDa catalytic subunit. Taken together, these findings identify a human Cdc2-activating kinase as a growth factor-responsive enzyme system that may participate in the acute activation of cyclin-dependent protein kinases observed in mammalian somatic cells.
Williams
R T
RT
Research Institute of Childrens Hospital Los Angeles, University of Southern California School of Medicine 90027.
Wu
L
L
Carbonaro-Hall
D A
DA
Hall
F L
FL
D000818:Animals; D001792:Blood Platelets / Q000201:enzymology; D016203:CDC2 Protein Kinase / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007163:Immunosorbent Techniques; D007700:Kinetics; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins; D013912:Threonine / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D009396:Wilms Tumor / Q000201:enzymology
D011994:Recombinant Proteins; D013912:Threonine; D004815:Epidermal Growth Factor; D011494:Protein Kinases; D016203:CDC2 Protein Kinase