Purification of a 107 kilodalton (kDa) casein kinase G substrate from thyroid cytosol.
A 107 kDa (pp107) casein kinase G (ck-G) substrate has been purified from mouse and beef thyroid cytosol; ck-G was purified from beef thyroid cytosol. Ck-G and pp107 were found to co-elute on DEAE cellulose chromatography at approximately 300 mM NaCl. Ck-G and pp107 were separated by spermine-agarose affinity chromatography; pp107 is eluted with a stepped gradient at 250 mM NaCl and ck-G is eluted at 500 mM NaCl. Ck-G was subsequently purified by casein-agarose and GTP-agarose affinity chromatography. The 107 kDa protein was purified using heparin-agarose affinity chromatography. Phosphorylation of purified pp107 by ck-G was stimulated by spermine (ED50 = 0.2 mM) and inhibited by low concentrations of heparin (0.1-5 micrograms/ml). The Km and Vmax for the reaction were 1.46 microM and 32.2 nmoles P transferred/20 min/mg protein, respectively; 1 mole pp107 incorporated 0.81 mole phosphorus. pp107 was found to be an acidic substrate with a pI of 3.87 and was absorbed to wheat-germ agglutinin-agarose. The specificity of pp107 phosphorylation was studied using diacylglycerol-activated calcium/phospholipid-dependent protein kinase C, calcium-activated calmodulin-dependent protein kinase, and the catalytic subunit of cAMP-dependent protein kinase A. Phosphorylation of pp107 by the other protein kinases tested never exceeded 4% of that of ck-G. Our data show that pp107 is an acidic glycoprotein which may serve as a high-affinity and specific substrate for ck-G.
Levasseur
S
S
Department of Medicine, Cook County Hospital, Chicago, IL 60612.
Poleck
T
T
Friedman
Y
Y
Burke
G
G
D000818:Animals; D047388:Casein Kinases; D005260:Female; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D013961:Thyroid Gland / Q000032:analysis*
D011506:Proteins; D011494:Protein Kinases; D047388:Casein Kinases