EVIDENCE THAT CASEIN KINASE-2 PHOSPHORYLATES HEPATIC-MICROSOMAL CALCIUM-BINDING PROTEIN-1 AND PROTEIN-2 BUT NOT PROTEIN-3

We have extensively purified three of the hepatic microsomal intralume nal Ca2+-binding proteins, CBP1, CBP2, and CBP3, which were originally described by Van et al. [(1989) J. Biol. Chem. 264, 17494-17501]. The se apparently homogeneous preparations showed only single Ca-45(2+) bi nding bands. On the basis of the peptide sequence, CBP2 was found to b e highly homologous with the previously described protein ERp72. Simil arly, CBP3 was identical to calreticulin and CBP1 had some homology to calmodulin. Contrary to the report of Van et al. (1989), we found tha t CBP2 had little thiol:protein disulfide oxidoreductase activity. Of the three purified preparations, only CBP2 exhibited apparent intrinsi c protein kinase activity. This activity was found to be due to contam ination of the CBP2 preparation by an extremely low concentration of t ightly bound casein kinase 2 (CK2). In line with this observation, the phosphorylation was inhibited by heparin, removed by antibody to CK2, and stimulated by spermine. Furthermore, CBP2 was readily phosphoryla ted irt vitro by added CK2 but only slowly phosphorylated by several o ther protein kinases. Thus, the persistence of CK2 in a highly purifie d preparation of CBP2 along with several other lines of evidence prese nted in this study might suggest that the protein CBP2 is a physiologi cally relevant substrate for CK2. Furthermore, these data suggest that CK2 might be localized in the lumen of the endoplasmic reticulum and that the phosphorylation of CBP2 in the lumen may play a role in the c haperone activity attributed to this protein.