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.
Highlights
From the +Divisionof Orthopaedic Surgery, the IlDivision of HematologylOncology and Ophthalmology, **Department of Pediatrics, Childrens Hospitalof Los Angeles, Universityof Southern California School of Medicine and School of Pharmacy, Los Angeles
prolinedirected protein kinase (PDPK) is thpe58cyc'i"Asubunit of PDPK was found activated rapidly byboth epidermal growth factor (EGF) and NGFprionliferative PC12 to be phosphorylated on tyrosine residuesin vivo and pheochromocytoma cells [3,4].In addition to phosphorylating in vitro, the latter of which resulted in a significant tyrosine hydroxylase, PDPK readily phosphorylates the neuincrease in PDPK activity
Recent acids(minusMet-1) of rattyrosine hydroxylase [4].Thispeptide studies have demonstrated that this consensus sequence is selectivefor PDPK butappears to be phosphorylated, under certain circumstances, by the "growth-associated" M phase-specific histone H1 kinase. this striking similarity of phosphorylation site specificity is counterposed by major differences in subcellular substrate (100 p ~ w)as incubated with the enzyme preparations for 20 min at 30 "C ina reaction mixture containing100mM Tris acetate, pH 7.5,lOmM magnesium acetate, and100 p M ATP
Summary
Protein KinaseAssay-The proline-directed protein kinasaectivity of various tissues, extracts, and column fractionswas assayed in the presence of [-y-"P]ATP, utilizing a synthetic peptide substrate (TH2") with a primary sequence that is identical to the first 16 amino istic turn or hinge region of its substrate proteins. Maintained in exponential growth in RPMI 1640 medium supplementedwith 10% fetal bovine serum.HistoneH1kinase was extracted from human HL60 promyelocytic leukemic cells by lysis in hypotonic lysis buffer 40), 10 plof anti-~34"~a"n' tiserum was added, and the mixture was incubated for 2 h on ice. The immune complexes were collected by the addition of 10 pl of a (1:l) suspension of protein A-Sepharose beads, the pellets were washed with IP buffer followed by kinase reaction buffer 1 .o noprecipitation of PDPK activity was performed by incubating the EGF CONCENTRATION (ng/ml) diluted enzyme preparations with increasing ammounts of anti-~34"~ (expressed protein) antisera followed by assay of both the resulting supernatants and thweashed protein A-Sepharose beads under standard assay conditions
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