IDENTIFICATION, ASSAY, AND PURIFICATION OF A CDC2-ACTIVATING THREONINE-161 PROTEIN-KINASE FROM HUMAN-CELLS

The biological activities of cyclin-dependent, proline-directed protei n kinases (PDPKs) are highly regulated by a complex series of protein phosphorylation/dephosphorylation reactions involving both catalytic a nd regulatory subunits. In this paper we report on the enzymatic activ ation of p34(cdc2)/p58(Cyelin A) PDPK by a protein kinase present in h uman cells that targets threonine-181 of Cdc2. An assay for this Cdc2 kinase-kinase (PK161) was developed and specific enzyme activity was d etected in a variety of mammalian cells and tissues. PK161 activity wa s rapidly stimulated by epidermal growth factor in human A431 epidermo id carcinoma cells. The development of an assay selective for PK161 ph osphotransferase activity afforded the partial purification of the enz yme from human Wilms' tumors. Sodium dodecyl sulfate-polyacrylamide ge l electrophoresis and silver staining of highly purified enzyme prepar ations revealed the presence of phosphoproteins migrating at similar t o 42-44 and similar to 95 kDa, respectively, which correlated with enz yme activity upon fast-protein liquid chromatography gel permeation ch romatography. Further purification was accomplished by immobilized pep tide substrate affinity chromatography. The ability of PK 161 to phosp horylate and activate p34(cdc2)/p58(Cyclin A) PDPK was confirmed by th e use of purified recombinant subunits. Polyclonal antibodies directed against the Xenopus MO15 gene product (a putative Cdc2-activating kin ase) cross-reacted with the purified 42- to 44-kDa phosphoprotein, thu s identifying the catalytic subunit of human PK161 as a human homologu e of Xenopus p40(MO15). Subsequent immunoprecipitation experiments wit h metabolically labeled A431 cells identified a similar to 95-kDa phos phoprotein that coprecipitated with the similar to 42-kDa catalytic su bunit. Taken together, these findings identify a human Cdc2-activating kinase as a growth factor-responsive enzyme system that may participa te in the acute activation of cyclin-dependent protein kinases observe d in mammalian somatic cells. (C) 1994 Academic Press, Inc.