Phosphorylase kinase phosphorylates the calmodulin-binding regulatory regions of neuronal tissue-specific proteins B-50 (GAP-43) and neurogranin.
Neuronal tissue-specific proteins B-50 (GAP-43, neuromodulin) and neurogranin are phosphorylated by phosphorylase kinase with stoichiometries of 0.4 and 0.5 mol of phosphate/mol of protein, respectively. The apparent Km and kcat values determined at pH 8.2 for neurogranin phosphorylation are 28.4 microM and 139.3 min-1, respectively, and for B-50 phosphorylation are 22.8 microM and 33.2 min-1, respectively. As a substrate of phosphorylase kinase, phosphorylase is approximately 44 and approximately 13 times better than B-50 and neurogranin, respectively. Both proteins are better substrates of protein kinase C than of phosphorylase kinase and are phosphorylated on a single site by phosphorylase kinase. The sequence analyses of tryptic phosphopeptides isolated from neurogranin and B-50 phosphorylated by phosphorylase kinase revealed the same amino acid sequence, IQASF, indicating that phosphorylase kinase phosphorylates the calmodulin-binding regulatory regions of B-50 and neurogranin previously known to be phosphorylated by protein kinase C (Coggins, P. J., and Zwiers, H. (1989) J. Neurochem. 53, 1895-1901; Baudier, J., Deloulme, J. C., Dorsselaer, A. V., Black, D., and Matthes, W. D. (1991) J. Biol. Chem. 266, 229-237). In rat brain synaptosomes, a relatively high phosphorylase kinase specific activity is detected, and approximately 32% activity is associated with synaptic membranes where B-50 is localized. In rat brain homogenate and synaptosomal membranes, phosphorylation of a protein that co-migrates with B-50 on SDS-polyacrylamide gel electrophoresis is enhanced in the presence of exogenous phosphorylase kinase.
Paudel
H K
HK
Department of Medical Biochemistry, Faculty of Medicine, University of Calgary, Alberta, Canada.
Zwiers
H
H
Wang
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D019922:GAP-43 Protein; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D051579:Neurogranin; D010750:Phosphoproteins / Q000378:metabolism*; D010764:Phosphorylase Kinase / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D013574:Synaptosomes / Q000201:enzymology
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D019922:GAP-43 Protein; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; C496651:Nrgn protein, rat; D010750:Phosphoproteins; D051579:Neurogranin; D010764:Phosphorylase Kinase