ISOLATION AND CHARACTERIZATION OF A STRUCTURAL HOMOLOG OF HUMAN PRK2 FROM RAT-LIVER - DISTINGUISHING SUBSTRATE AND LIPID ACTIVATOR SPECIFICITIES

A homologue of human protein kinase C (PKC)-related kinase-2, PRK2, wh ich had previously escaped identification in normal mammalian tissues, was isolated from rat liver as the protease-activated kinase (PAK) or iginally named PAK-2. The 130-kDa cytosolic enzyme was purified to hom ogeneity and shown by tryptic peptide and reverse transcriptase- polym erase chain reaction (RT-PCR)-amplified rat cDNA sequence analyses to be structurally related to the 116-kDa rat hepatic PAK-1/protein kinas e N (PKN) and, even more closely (95% sequence identity) to the 130-kD a human PKC-related kinase, PRK2. Rat myeloma RNA was used as the RT-P CR template because of its relative abundance in PAR-2/PRK2 mRNA compa red with liver and other rat tissues. The catalytic properties of PAK- 2/PRK2 in many respects resembled those of hepatic PAK-1/PKN, but were distinguished by more favorable kinetics with several peptide substra tes, and greater sensitivity to PKC pseudosubstrate and polybasic amin o acid inhibitors. PAK-2/PRK2 was also activated by lipids, particular ly cardiolipin and to a lesser extent by other acidic phospholipids an d unsaturated fatty acids. Cardiolipin activation was most evident wit h autophosphorylation and histone H2B phosphorylation, but only margin ally evident with the favored ribosomal S6-(229-239) peptide substrate for the protease-activated kinase activity. It was concluded that PAK -2 is the rat homologue of human PRK2, with biochemical properties dis tinct from although overlapping those of the PAK-1/PKN/PRK1 isoform.