Studies of calcium binding proteins 1 and 2 and protein kinase CK2

Sammanfattning: The endoplasmic reticulum (ER) is a major intracellular compartment of eukaryotic cells and is the site of the synthesis and post-translational modification of both secretory and membrane glycoproteins. Upon cell rupture vesicles of several kinds are formed from cell membranes, mainly derived from ER and are called microsomes. Solubilized microsomes were phosphorylated with various exogenous protein kinases using radiolabelled ATP. After phosphorylation with protein kinase CK2 three dominant phosphoproteins were found. These phosphoproteins were identified as calcium binding proteins 1, 2 and 4 (CaBP1, CaBP2 and CaBP4). CaBP1 and CaBP2 have not earlier been described as substrates for CK2. All three were earlier shown to be luminal proteins of the ER, with an ER-retention signal motif. This motif (KDEL/KBEL) interacts with a receptor, the erd2 receptor, which is located in the pre-Golgi complex. Analyzes were performed as to whether the phosphorylation of CaBP1 and 2 by CK2 influenced their binding to the erd2 receptor. The whole receptor sequence was mapped by synthesizing overlapping carrier-bound peptides to which both unphosphorylated and phosphorylated CaBPs were bound. The phosphorylation did notinfluence the specific binding, however one specific site with high affinity (KIWK) was identified. The finding of this site strengthened one of the predicted topological models. Protein kinase CK2 was also found endogenously in the microsomal preparation. This fraction was further purified in order to clarify if the kinase CK2 could phosphorylate its substrates (CaBPs) in intact microsomes exclusively derived from ER. Since the CaBPs were found to protrude from the BR membrane they were considered to be accessible to cytosolic kinases and might therefore be their physiological substrates.Protein kinase CK2 is an ubiquitous serine/threonine specific protein kinase required for viability, and for cell cycle progression. A hallmark of CK2 is its extreme sensitivity to inhibition by heparin. A kinetic study of the inhibitory effect of CK2 by heparin of different chain length was done using both casein as well as a specific peptide as substrates. The inhibitory effect increased with the length of the heparin fragments and a breakpoint was obtained at eight-mers where an enhanced increase in inhibitory activity occurred. The mode of inhibition was found to be different depending on whether the substrate was a peptide or a protein.