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.