Comparison of tyrosine protein kinases in membrane fractions from mouse liver and Ehrlich ascites tumor.
Tubulin was phosphorylated mainly at tyrosine residues by membranes from mouse liver and Ehrlich ascites tumor with ATP in the presence of MnCl2, ZnCl2, NaVO3, and Nonidet P-40 in an epidermal growth factor (EGF)- and insulin-independent manner. The tyrosine tubulin kinase activity in the tumor membranes is comparable to the activity in liver membranes. Two tyrosine tubulin kinases, namely I (Mr = 64,000) and II (Mr = 46,000), were purified 5-17-fold and were free of the EGF receptor and the insulin receptor. Phosphorylation of endogenous proteins produced major alkali-resistant phosphoproteins of 56 and 53 kDa in kinase I preparations and of 46 and 37 kDa in kinase II preparations. These kinases phosphorylated tubulin stoichiometrically at tyrosine and had a preference of alpha-subunit to beta-subunit of tubulin. Apparent Km values of kinases I and II for tubulin were about 4 and 8 microM and for ATP were about 2 and 4 microM, respectively. Thiol reagents inhibited their reactions. N alpha-p-Tosyl-L-lysine chloromethyl ketone stimulated the reactions at 1 mM but inhibited them at 5 mM. Although kinases I and II also phosphorylated angiotensin II, tyrosine-glutamate copolymers, and heavy chains of anti-pp60src IgG, they differed from each other in preferences for the substrates. More than 95% of the enzyme activities was not immunoprecipitated with the anti-pp60src IgG which cross-reacts with pp60c-src. In comparison with the corresponding liver enzymes, tumor enzymes were more stable to heat and incorporated more phosphate into tubulin, but showed lower activity toward angiotensin II and anti-pp60src IgG.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology*; D002417:Cattle; D002499:Centrifugation, Density Gradient; D006358:Hot Temperature; D007074:Immunoglobulin G / Q000032:analysis; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D051379:Mice; D008970:Molecular Weight; D015688:Oncogene Protein pp60(v-src); D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D012191:Retroviridae Proteins / Q000276:immunology; D012995:Solubility; D014404:Tubulin / Q000378:metabolism
D007074:Immunoglobulin G; D007527:Isoenzymes; D011506:Proteins; D012191:Retroviridae Proteins; D014404:Tubulin; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src)