Phosphorylation of synaptic membrane glycoproteins: the effects of Ca2+ and calmodulin.
Synaptic membranes were incubated with [gamma-32P]ATP, and glycoproteins were isolated by affinity chromatography on concanavalin A agarose. Glycoproteins accounted for 1.5-2.5% of the total 32P incorporated into synaptic membrane proteins. Ca2+ and calmodulin enhanced the phosphorylation of synaptic membrane glycoproteins approximately threefold. In the presence of Ca2+ and calmodulin, the rate of glycoprotein dephosphorylation was also increased three- to four-fold. Gel electrophoretic analysis identified several synaptic membrane glycoproteins that incorporated 32P, with the most highly labeled glycoprotein under basal phosphorylating conditions having an apparent Mr of 205,000 (gpiii). Ca2+ and calmodulin produced a marked increase in the phosphorylation of a glycoprotein with an apparent Mr of 180,000 (gpiv) and lesser increases in the labeling of three other glycoproteins. Membranes that had been labeled with [gamma-32P]ATP were extracted with Triton X-100 under conditions that yield a detergent-insoluble residue enriched in postsynaptic structures. The Triton X-100 insoluble residue accounted for 20-25% of the 32P associated with synaptic membrane glycoproteins. Gpiv and other glycoproteins, the phosphorylation of which was stimulated by calmodulin, were located exclusively in the Triton X-100 insoluble residue, whereas gpiii and other calmodulin-insensitive glycoproteins partitioned predominantly into the Triton X-100-soluble fraction. Phosphopeptide maps and phosphoamino acid analysis of gpiv isolated from synaptic membranes and a postsynaptic glycoprotein of apparent Mr of 180,000 (gp180) isolated from synaptic junctions indicated that the former protein was identical to the previously identified postsynaptic-specific gp180. In addition to phosphoserine and phosphothreonine, gpiv also contained phosphotyrosine, identifying it as a substrate for tyrosine-protein kinase as well as for Ca2+/calmodulin-dependent protein kinase.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000494:pharmacology*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000032:analysis / Q000378:metabolism*; D008562:Membrane Glycoproteins; D017830:Octoxynol; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D011092:Polyethylene Glycols / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D012995:Solubility; D013570:Synaptic Membranes / Q000187:drug effects / Q000378:metabolism*
D002147:Calmodulin; D006023:Glycoproteins; D008562:Membrane Glycoproteins; C030340:elastin microfibril interface located protein; D011092:Polyethylene Glycols; D000255:Adenosine Triphosphate; D017830:Octoxynol; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases; D002118:Calcium