Purification and characterization of a novel proline-directed protein kinase from bovine brain

A novel protein kinase which phosphorylates a synthetic peptide substrate (RRPDAHRTPNRAF) has been purified approximately 200,000-fold from bovine brain. This peptide contains the consensus sequence for phosphorylation by the p34^cdc2 kinase. The purification procedure took advantage of the phenomenon that this novel brain kinase, in partially purified extracts, chromatographed on a gel filtration column as a high molecular weight complex which dissociated in buffer containing 1 M NaC1. The purified native enzyme was estimated to be approximately 63,000, and displayed two bands of M_r = 33,000 and 25,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On Western immunoblot, the M_r = 33,000 peptide reacted strongly with antibodies specific for a conserved amino-terminal sequence, weakly with antibodies to the conserved PSTAIRE sequence, and not at all with antibodies to the carboxyl terminus, of HeLa cell p34^cdc2. The brain kinase and p34^cdc2 were similar in displaying good activity toward the parent peptide substrate, but no activity toward peptide analogues in which the -T-P- motif was substituted with either -T-G- or -T-A-. Both kinases showed marked preference in phosphorylating a peptide derived from H1 histone (KTPKKAKKPKTPKKAKKL), and both kinases could be phosphorylated by the src-family tyrosine kinase, p56^lyn, purified from bovine spleen. However, the brain kinase did not co-purify with a subunit having a molecular weight corresponding to known cyclins, nor did it undergo specific interaction with p13^suc1 beads, suggesting that this enzyme is distinct from p34^cdc2.