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 compartment are described in the literature, and this prompted us to try to identify at least the more dominant ones. When solubilized bovine and murine microsomes were phosphorylated with protein kinase CK2 and [32P]ATP and separated on SDS-PAGE, the corresponding autoradiogram showed three dominant 32P-labeled proteins. These three [32P]phosphoproteins were identified as calcium-binding proteins (CaBP) 1, 2, and 4 after purification on a MonoQ column followed by SDS-PAGE, proteolytic cleavage and subsequent amino acid sequencing of the purified 32P-labeled peptides. All three were also phosphorylated by an endogenous kinase, found by us to be of the CK2 type. This kinase phosphorylated CaBP1 N-terminally at serine 427. Of the three proteins, only CaBP4 was previously known to be a substrate of CK2. The newly identified substrates CaBP 1 and 2 are members of the thioredoxin family and have a signal tetrapeptide in the C-terminal of the protein for retention in the 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 grown in the presence of [32P] 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 in microsomes.