Purification of catalytic subunit of low density lipoprotein receptor kinase and identification of heat-stable activator protein.
Bovine adrenal cortex contains a high molecular weight casein kinase II-like enzyme (Mr 500,000) that phosphorylates a specific serine residue in the cytoplasmic domain of the low density lipoprotein (LDL) receptor (Kishimoto, A., Brown, M. S., Slaughter, C. A., and Goldstein, J. L. (1987) J Biol. Chem. 262, 1344-1351). In the current paper, we provide evidence to suggest that this 500-kDa kinase can be dissociated into two subunits, a catalytic subunit and an activator subunit, by treatment with 1 M NaCl. The catalytic subunit was purified to homogeneity (greater than 100,000-fold) using affinity chromatography on GTP-agarose plus several other chromatography steps. It had an Mr of 50,000 by gel filtration and 35,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The catalytic subunit phosphorylated casein actively, but it phosphorylated the LDL receptor with only low affinity. The affinity for the LDL receptor was increased 10-fold (saturation at 10 nM LDL receptor) by addition of a second protein that was released from a high molecular weight 500-kDa complex by 1 M NaCl. This activator protein (Mr 120,000 by gel filtration) was extremely heat stable but was destroyed by trypsin. It appeared to be required in stoichiometric amounts with relation to the LDL receptor. It did not increase the ability of the 50-kDa subunit to phosphorylate casein nor did it activate phosphorylation of the LDL receptor or casein by classic casein kinase II. The current data raise the possibility that the specificity of the 500-kDa LDL receptor kinase is attributable to a heat-stable activator subunit that binds to the LDL receptor and thereby renders it a better substrate for the catalytic subunit of the kinase.
D000302:Adrenal Cortex / Q000201:enzymology*; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D004789:Enzyme Activation; D006358:Hot Temperature; D007700:Kinetics; D008970:Molecular Weight; D011494:Protein Kinases / Q000302:isolation & purification*; D011973:Receptors, LDL / Q000378:metabolism*
D011973:Receptors, LDL; D011494:Protein Kinases