title: Characterization of the cytoplasmic proline-directed protein kinase in proliferative cells and tissues as a heterodimer comprised of p34cdc2 and p58cyclin A.
abstract: Site-specific analysis of tyrosine hydroxylase phosphorylation in rat pheochromocytoma led previously to the identification of a novel growth factor-sensitive serine/threonine protein kinase, designated proline-directed protein kinase (PDPK). In this article we describe further the activation, purification, subunit configuration, and biochemical characteristics of this cytoplasmic enzyme system. In human A431 epidermoid carcinoma cells PDPK activity was found to be stimulated by epidermal growth factor in a dose-dependent, time-dependent manner. The PDPK purified from the cytosol of mouse FM3A mammary carcinoma cells exhibited the same chromatographic behavior and biochemical properties as the tyrosine hydroxylase-associated enzyme purified originally from rat pheochromocytoma. The presence of p34cdc2 was ultimately detected in all active fractions of highly purified PDPK by Western blotting and immunoprecipitation; however, it was determined that this catalytic subunit is complexed with a 58-kDa regulatory subunit that is clearly distinct from that of the "growth-associated" M phase-specific histone H1 kinase (i.e. cyclin B). The 58 kDa regulatory subunit of PDPK was identified by direct immunoblotting as a mammalian A-type cyclin. Furthermore, the p58cyclin A subunit of PDPK was found to be phosphorylated on tyrosine residues in vivo and in vitro, the latter of which resulted in a significant increase in PDPK activity. Additional distinctions between this growth factor-sensitive PDPK (p34cdc2-p58cyclin A) and the M phase-specific histone H1 kinase (p34cdc2-p62cyclin B-p13suc1) are identified on the basis of chromatographic behavior, enzyme kinetics, and physicochemical properties. Based on these findings, it is proposed that PDPK represents a unique complex of the p34cdc2 protein kinase which is active in the cytoplasm of proliferative cells, is regulated differently from the M phase-specific histone H1 kinase by phosphorylation reactions, and is modulated selectively by growth factors.
authors.affiliation: Division of Orthopaedic Surgery, Childrens Hospital of Los Angeles, California 90054-0700.
authors.forename: F L
authors.initials: FL
authors.lastname: Hall
authors.forename: R K
authors.initials: RK
authors.lastname: Braun
authors.forename: K
authors.initials: K
authors.lastname: Mihara
authors.forename: Y K
authors.initials: YK
authors.lastname: Fung
authors.forename: N
authors.initials: N
authors.lastname: Berndt
authors.forename: D A
authors.initials: DA
authors.lastname: Carbonaro-Hall
authors.forename: P R
authors.initials: PR
authors.lastname: Vulliet
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002455:Cell Division; D002851:Chromatography, High Pressure Liquid; D016213:Cyclins / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology*; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D038461:Proline-Directed Protein Kinases; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
chemical_list: D016213:Cyclins; D004815:Epidermal Growth Factor; D011494:Protein Kinases; D038461:Proline-Directed Protein Kinases; D016203:CDC2 Protein Kinase