Cyclic nucleotide-independent protein kinases from rabbit reticulocytes. Purification and characterization of protease-activated kinase II.
A cyclic nucleotide-independent protein kinase, protease-activated kinase II, which incorporates up to four phosphates into 40 S ribosomal protein S6, has been purified from the postribosomal supernatant of rabbit reticulocytes. Protease-activated kinase II was purified as an inactive proenzyme by chromatography on DEAE-cellulose, phosphocellulose, Sephadex G-150, and hydroxylapatite. The enzyme was activated in vitro by limited digestion with trypsin or chymotrypsin. No other mode of activation for protease-activated kinase II in vitro was identified. The proenzyme had a molecular weight of 80,000 as measured by gel filtration; following tryptic digestion, the molecular weight of the activated protein kinase was 45,000-55,000. Protease-activated kinase II required Mg2+ for activity but was inhibited by other divalent cations, monovalent cations, and fluoride ion. ATP was the phosphoryl donor in the phosphorylation reaction; GTP had no effect. In vitro, multiple phosphorylation of S6 was observed with some phosphate incorporated into S10. Phosphorylation of S6 by protease-activated kinase II has been shown to be stimulated in serum-starved 3T3-L1 cells by insulin (Perisic, O., and Traugh, J. A. (1983) J. Biol. Chem. 258, 9589-9592) and in reticulocytes by altering the pH of the incubation medium (Perisic, O., and Traugh, J. A. (1983) J. Biol. Chem. 258, 13998-14002.
D000818:Animals; D002413:Cations, Divalent; D002918:Chymotrypsin / Q000378:metabolism; D004789:Enzyme Activation; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010766:Phosphorylation; D011494:Protein Kinases / Q000097:blood* / Q000302:isolation & purification; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology*; D013696:Temperature; D014357:Trypsin / Q000378:metabolism
D002413:Cations, Divalent; D011494:Protein Kinases; C039767:protease-activated kinase II; D002918:Chymotrypsin; D014357:Trypsin; D008274:Magnesium