A CARDIOLIPIN-ACTIVATED PROTEIN-KINASE FROM RAT-LIVER STRUCTURALLY DISTINCT FROM THE PROTEIN-KINASES-C

A cardiolipin- and protease-activated protein kinase (PAK) has been is olated from cytoplasmic extracts of rat liver. The enzyme (PAK-1) phos phorylates the ribosomal protein S6-(229-239) peptide analogue and can be activated by limited proteolysis. Partial amino acid sequences of tryptic peptides derived from both the purified 116-kDa PAK-1 holoenzy me and its active catalytic fragment reveal that the catalytic domain is most related (50-58% identity) to the protein kinase C family. PAK- 1 has protein and peptide substrate specificities distinct from those of known protein kinase C isoforms and is insensitive to inhibition by the protein kinase C-alpha-(19-31) pseudosubstrate peptide. Phosphati dylserine, diacylglycerol, and phorbol ester do not activate PAK-1 tow ard the S6 peptide substrate. However, other acidic phospholipids, the most effective being cardiolipin, activate PAK-1 to a similar extent as trypsin. The PAK-1 catalytic activities generated through activatio n by cardiolipin or limited proteolysis were kinetically similar, with K-m values of 3.6 and 3.4 mu M, respectively, for the S6-(229-239) pe ptide substrate. However, differences were observed in the catalytic a ctivities with protamine sulfate and the glycogen synthase-(1-12) pept ide analogue as substrates. It was concluded that PAK-1 is a phospholi pid regulated protein kinase with a primary structure, substrate speci ficity, and mechanism of regulation in vitro distinct from those of an y known member of the protein kinase C superfamily.