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.
Highlights
In previous studies, a correlation was observed be- as specific substrates for three morore distinct protein tween isoproterenol-responsive Na+-K+co-transport in kinases of the turkey erythrocyte, namely a CAMP
Goblin is a major protein of M, = 230,000 which is tightly associated with the plasma membrane of the turkey erythrocyte [1].Exposure of intact erythrocytes to ("'P)Pi leads to incorporation of '"P into many proteins of the turkey erythrocyte, but phosphorylationof goblin is enhanced by subsequent exposure of the cells to isoproterenol, or to otheragents which elevateintracellularcAMP [1,2,3]
Individual phosphorylation sites of goblin, a high molecular weight protein of the turkey erythrocyte plasma membrane, have been identified by tryptic fingerprinting of the protein isolated from SDS-polyacrylamide gels
Summary
PHOSPHORYLATION BY CAMP-DEPENDENT AND Ca2'-DEPENDENT PROTEIN KINASES OF DISTINCT SITES IN GOBLIN, A HIGH MOLECULAR WEIGHT PROTEIN OF T H E PLASMA MEMBRANE*. Hormone-responsive Na+-K' copeptides of goblin phosphorylated in plasma mem- transport in intactturkeyerythrocyteshas beenfound to branes by addition of Ca2+/calmodulin(peptides 4 and correlatequalitatively withhormone-responsive phosphate. In the experiment shoiwnnFig. 6, plasma membranes (50 pgof protein) were preincubated for 1 min a t 30°C in a buffer containing 10 mM Tris-HC1 (pH 7.4), 10 mMMgC12 and, as indicated, 20 p~ CAMP,or 1pgof bovine brain calmodulin and 200 p~ CaC12. Samples were dried a t 45°C in a vacuum desiccator, resuspended in phoresis of the proteins of intact cells or of purified plasma mem- 8.7%acetic acid, 2.5% formic acid (pH 1.9),spotted 3 cm from the end branes was carried out by the method of Laemmli [23] in 4% polyacrylamide gel slabs (Fig. L4) or in lineargradients of 5 to 10% polyacrylamide(Fig. 6) of 1.4-mm thickness.
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