Casein kinase II phosphorylation of signal sequence receptor alpha and the associated membrane chaperone calnexin.
Signal sequence receptor alpha (SSR alpha) and calnexin are major calcium-binding proteins of the endoplasmic reticulum (ER) which are implicated in chaperone functions. They were identified as major membrane substrates after in vitro phosphorylation of ER membranes with [gamma-32P]GTP (Wada, I., Rindress, D., Cameron, P. H., Ou, W. J., Doherty, J.-J., II, Louvard, D., Bell, A. W., Dignard, D., Thomas, D. Y., and Bergeron, J. J. M. (1991) J. Biol. Chem. 266, 19599-19610). Using purified SSR alpha and associated calnexin as substrates, we have attempted to identify the kinase(s) responsible for their phosphorylation. A salt extract from canine pancreatic ER membranes and cytosol possessed SSR alpha kinase activity which showed identical chromatographic behavior through phosphocellulose, DEAE-Sepharose, and hydroxylapatite purification protocols. Final purification was effected from the cytosol with three polypeptides of 38, 36, and 28 kDa detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On the basis of primary sequence analysis of the three subunits of the purified kinase and the reconstitution of phosphorylation of SSR alpha and associated calnexin in heat-inactivated ER membranes by the addition of the purified kinase we conclude that the ER-associated kinase responsible for the GTP phosphorylation of SSR alpha and associated calnexin is casein kinase II.
Ou
W J
WJ
Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada.
Thomas
D Y
DY
Bell
A W
AW
Bergeron
J J
JJ
D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D037281:Calnexin; D047390:Casein Kinase II; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D004285:Dogs; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D006882:Hydroxyapatites; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010179:Pancreas / Q000201:enzymology*; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear; D018000:Receptors, Peptide; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D002135:Calcium-Binding Proteins; D006882:Hydroxyapatites; D008562:Membrane Glycoproteins; D010750:Phosphoproteins; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D018000:Receptors, Peptide; C066973:signal sequence receptor; D037281:Calnexin; D017886:Durapatite; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases