PHOSPHORYLATION OF CABP1 AND CABP2 BY PROTEIN-KINASE CK2

Several proteins in the mammalian endoplasmic reticulum are substrates for protein kinases. Many unidentified phosphoproteins from this comp artment are described in the literature, and this prompted us to try t o identify at least the more dominant ones. When solubilized bovine an d murine microsomes were phosphorylated with protein kinase CK2 and [P -32] ATP and separated on SDS-PAGE, the corresponding autoradiogram sh owed three dominant P-32-labeled proteins. These three [P-32] phosphop roteins were identified as calcium-binding proteins (CaBP) 1, 2, and 4 after purification on a MonoQ column followed by SDS-PAGE, proteolyti c cleavage and subsequent amino acid sequencing of the purified P-32-l abeled peptides, AIL three were also phosphorylated by an endogenous k inase, found by us to be of the CK2 type. This kinase phosphorylated C aBP1 N-terminally at serine 427. Of the three proteins, only CaBP4 was previously known to be a substrate of CK2. The newly identified subst rates CaBP 1 and 2 are members of the thioredoxin family and have a si gnal tetrapeptide in the C-terminal of the protein for retention in th e ER. Serines and/or threonines in the C-terminal were phosphorylated in CaBP1 when the endogenous CK2 was used as protein kinase. A protein with the same molecular mass as CaBP1 on SDS-PAGE was phosphorylated when intact hepatocytes were groan in the presence of [P-32]phosphate. The in vitro phosphorylation with protein kinase CK2 can be used as a specific and sensitive method for identification of CaBP1, 2, and 4 i n microsomes.