Phosphorylation events associated with different states of activation of a hepatic cardiolipin/protease-activated protein kinase. Structural identity to the protein kinase N-type protein kinases.
Cardiolipin- or protease-activated protein kinase, isolated from rat liver cytosol and originally named liver PAK-1, was found to be the natural form of protein kinase N (PKN) by comparing the sequences of 43 tryptic peptides of the purified liver enzyme and determining the corresponding liver cDNA sequence. These analyses also identified (i) Arg-546 as the major site of proteolytic activation, (ii) the protease resistance of the C-terminal extension beyond the catalytic domain, and (iii) in vivo stoichiometric phosphorylation of Thr-778 in the mature enzyme. Homology modeling of the catalytic domain indicated that phosphothreonine 778 functions as an anchoring site similar to Thr-197 in cAMP-dependent protein kinase, which stabilizes an active site compatible with preferred substrate sequences of PAK-1/PKN. Sigmoidal autophosphorylation kinetics and increased S6-(229-239) peptide kinase activity following preincubation with ATP suggested phosphorylation-dependent activation of PAK-1/PKN. The onset of activation corresponded with phosphorylation of the regulatory domain site Ser-377 (located within a spectrin homology region), followed by Thr-504 (within a limited protein kinase C homology region), and, to a lesser extent, Thr-64 (in the RhoA-binding region). Several additional sites in the hinge region adjacent to a PEST protein degradation signal were selectively autophosphorylated following cardiolipin activation. Overall, these observations suggest that the regulation of this class of protein kinase involves complex interactions among phosphorylation-, lipid-, and other ligand-dependent activation events.
Peng
B
B
Russell Grimwade School of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3052, Australia.
Morrice
N A
NA
Groenen
L C
LC
Wettenhall
R E
RE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002308:Cardiolipins / Q000737:chemistry* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D006801:Humans; D007525:Isoelectric Focusing; D008099:Liver / Q000201:enzymology*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D011915:Rats, Inbred BUF; D014357:Trypsin / Q000378:metabolism; D014981:Xenopus
D002308:Cardiolipins; D011494:Protein Kinases; C036447:protease activated kinase I; C061570:protein kinase N; D011493:Protein Kinase C; D014357:Trypsin