Activation of a cyclic AMP-independent protein kinase by an endogenous ATP-requiring protease from lymphosarcoma cells.
The activation of a cyclic AMP-independent protein kinase by an endogenous protease is described. The H4 phosphotransferase (Masaracchia, R. A., Kemp, B., and Walsh, D. A. (1977) J. Biol. Chem. 252, 7109-7117) from lymphosarcoma cells was isolated in a nonactive form. Activation required ATP and Mg2+ and was shown to be time-dependent. Although Mn2+ was capable of substituting for Mg2+ in the protein kinase reaction, no activation was observed when Mn2+ replaced Mg2+. The protein substrate histone H4 inhibited phosphotransferase activation at concentrations greater than 60 microM. The inhibition was complete in the presence of 100 microM H4. Comparable concentrations of bovine serum albumin did not inhibit the activation. The selective dependence on Mg2+ suggested separate activating and phosphotransferase activities. This was confirmed by heat denaturation in which the activation reaction was shown to be more sensitive to heat inactivation than was the phosphotransferase reaction. The activating enzyme was separated from the protein kinase by column chromatofocusing in the pH range 7-4. The pI of the activating enzyme was greater than 7.0. The pI values of the activated and nonactivated phosphotransferase were 4.8 and 5.3, respectively. The apparent molecular weight of the nonactivated phosphotransferase was 68,000; the activated enzyme was eluted from an S-200 Sephadex column with an apparent Mr = 52,000. Despite many similarities to a protease-activated Ca2+/phospholipid-dependent enzyme isolated from lymphocytes (Ogawa, Y., Takai, Y., Kawahara, Y., Kimura, S., and Nishizuka, Y. (1981) J. Immunol. 127, 1369-1374), the H4 phosphotransferase was not activated by Ca2+, phospholipids, or any combination thereof.
D049069:ATP-Dependent Proteases; D000818:Animals; D000242:Cyclic AMP / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation; D006360:Heat-Shock Proteins; D006657:Histones / Q000378:metabolism; D007700:Kinetics; D008228:Lymphoma, Non-Hodgkin / Q000201:enzymology*; D051379:Mice; D008807:Mice, Inbred BALB C; D011494:Protein Kinases / Q000378:metabolism*; D012513:Sarcoma, Experimental / Q000201:enzymology; D012697:Serine Endopeptidases
D006360:Heat-Shock Proteins; D006657:Histones; D000242:Cyclic AMP; D011494:Protein Kinases; D010450:Endopeptidases; D049069:ATP-Dependent Proteases; D012697:Serine Endopeptidases