Hormonal control of protein phosphorylation in turkey erythrocytes. Phosphorylation by cAMP-dependent and Ca2+-dependent protein kinases of distinct sites in goblin, a high molecular weight protein of the plasma membrane

Abstract

In previous studies, a correlation was observed between isoproterenol-responsive Na+-K+ co-transport in turkey erythrocytes and increased phosphorylation of goblin, an Mr = 230,000 protein of the turkey erythrocyte plasma membrane. The phosphorylation of specific sites in goblin has now been analyzed by tryptic fingerprinting. Three major phosphopeptides were detected in goblin prepared from intact, 32P-labeled erythrocytes. One of the peptides 1, was maximally phosphorylated in the absence of hormonal agents. Two additional peptides, 2 and 3, were phosphorylated only following exposure of cells to the beta-adrenergic agonist isoproterenol, to cAMP plus isobutylmethylxanthine, or to cholera toxin. In cells stimulated by isoproterenol, phosphorylation of goblin peptides 2 and 3 could be selectively and completely reversed by subsequent addition of the beta-adrenergic antagonist propranolol. Addition of either cAMP or of Ca2+ plus calmodulin to purified turkey erythrocyte plasma membranes increased incorporation of 32P into goblin. Peptides 2 and 3 of goblin were phosphorylated by addition to the membranes of cAMP or of purified cAMP-dependent protein kinase. Two additional goblin peptides, 4 and 5, were phosphorylated in the plasma membrane preparation by addition of purified calmodulin plus Ca2+, whereas peptides 2 and 3 of goblin were not phosphorylated under these conditions. Peptide 1 did not incorporate 32P in the plasma membranes under any condition tested. Both calmodulin and cAMP-dependent protein kinase were identified directly in turkey erythrocytes. The three major phosphopeptides of goblin phosphorylated in intact cells (peptides 1, 2, and 3) contained phosphothreonine and represented distinct phosphorylation sites. In contrast, the two phosphopeptides of goblin phosphorylated in plasma membranes by addition of Ca+/calmodulin (peptides 4 and 5) contained phosphoserine. It is concluded that goblin, a plasma membrane protein possibly involved in the hormonal regulation of Na+-K+ co-transport, contains at least 3 distinct threonine residues and 1 or more serine residues which serve as specific substrates for three or more distinct protein kinases of the turkey erythrocyte, namely a cAMP-dependent enzyme, a Ca2+/calmodulin-dependent enzyme, and a third enzyme with undetermined regulatory control.