Isolation and characterization of a structural homologue of human PRK2 from rat liver. Distinguishing substrate and lipid activator specificities.
A homologue of human protein kinase C (PKC)-related kinase-2, PRK2, which had previously escaped identification in normal mammalian tissues, was isolated from rat liver as the protease-activated kinase (PAK) originally named PAK-2. The 130-kDa cytosolic enzyme was purified to homogeneity and shown by tryptic peptide and reverse transcriptase- polymerase chain reaction (RT-PCR)-amplified rat cDNA sequence analyses to be structurally related to the 116-kDa rat hepatic PAK-1/protein kinase N (PKN) and, even more closely (95% sequence identity) to the 130-kDa human PKC-related kinase, PRK2. Rat myeloma RNA was used as the RT-PCR template because of its relative abundance in PAK-2/PRK2 mRNA compared 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 substrates, and greater sensitivity to PKC pseudosubstrate and polybasic amino acid inhibitors. PAK-2/PRK2 was also activated by lipids, particularly cardiolipin and to a lesser extent by other acidic phospholipids and unsaturated fatty acids. Cardiolipin activation was most evident with autophosphorylation and histone H2B phosphorylation, but only marginally 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 distinct from although overlapping those of the PAK-1/PKN/PRK1 isoform.
Yu
W
W
Russell Grimwade School of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria, 3052, Australia.
Liu
J
J
Morrice
N A
NA
Wettenhall
R E
RE
D000595:Amino Acid Sequence; D000818:Animals; D005227:Fatty Acids / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008055:Lipids / Q000494:pharmacology*; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010743:Phospholipids / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D011915:Rats, Inbred BUF; D016415:Sequence Alignment; D013379:Substrate Specificity
D005227:Fatty Acids; D008055:Lipids; D010743:Phospholipids; C061570:protein kinase N; D011493:Protein Kinase C