Selective modulation of band 4.1 binding to erythrocyte membranes by protein kinase C

We have studied the effects of band 4.1 phosphorylation on its association with red cell inside-out vesicles stripped of all peripheral proteins. Band 4.1 bound to these vesicles in a saturable manner, and binding was characterized by a linear Scatchard plot with an apparent Kd of 1-2 x 10(-7) M. Phosphorylation of band 4.1 by purified protein kinase C reduced its ability to bind to membranes, resulting in a reduction in the apparent binding capacity of the membrane by 60-70% but little or no change in the apparent Kd of binding. By contrast, phosphorylation of band 4.1 by cAMP-dependent kinase had no effect on membrane binding. Digestion of the stripped inside-out vesicles with trypsin cleaved 100% of the cytoplasmic domain of band 3 but had little or no effect on glycophorin. Binding of band 4.1 to these digested vesicles was reduced by 70%. Phosphorylation of band 4.1 by protein kinase C had no effect on its binding to the digested vesicles, suggesting that the cytoplasmic domain of band 3 contained the phosphorylation-sensitive binding sites. This was confirmed by direct measurement of band 4.1 binding to the purified cytoplasmic domain of band 3. Phosphorylation of band 4.1 by protein kinase C reduced its binding to the purified 43-kDa domain by as much as 90%, while phosphorylation by cAMP-dependent kinase was without effect. These results show a selective effect of protein kinase C phosphorylation on the binding of band 4.1 to one of its membrane receptors, band 3, and suggest a mechanism whereby one of the key red cell-skeletal membrane associations may be modulated.     

Phosphorylation of purified rat brain Na+ channel reconstituted into phospholipid vesicles by protein kinase C.

Phosphorylation of voltage-sensitive Na+ channels in neurons by protein kinase C slows Na+ channel inactivation and reduces peak Na+ currents. Na+ channels purified from rat brain and reconstituted into phospholipid vesicles under conditions that restore Na+ channel function were rapidly phosphorylated by protein kinase C on their 260-kDa alpha subunit. The phosphorylation reaction required Ca2+, diolein, and phosphatidylserine for activation of protein kinase C, and the rate of phosphorylation of reconstituted Na+ channels was 3- to 4-fold faster than for Na+ channels in detergent solution. Phosphorylation was on serine residues in three distinct tryptic phosphopeptides designated A, B, and C. Up to 2.5 mol of phosphate were incorporated per mol of Na+ channel. Following maximum phosphorylation by protein kinase C, cAMP-dependent protein kinase was able to incorporate more than 2.25 mol of phosphate per mol of Na+ channel indicating that these two kinases phosphorylate distinct sites. However, prior phosphorylation by cAMP-dependent protein kinase prevented phosphorylation of phosphopeptide B indicating that both kinases phosphorylate the site in this peptide. Phosphopeptide B shown here to be phosphorylated by protein kinase C and phosphopeptide 7 previously shown to be phosphorylated by cAMP-dependent protein kinase co-migrate on two-dimensional phosphopeptide maps and evidently are identical. The reduction in peak Na+ currents caused by both protein kinase C and cAMP-dependent protein kinase may result from phosphorylation of this single common site.     

Phosphorylation of myocardial fructose-6-phosphate,2-kinase: fructose-2,6-bisphosphatase by cAMP-dependent protein kinase and protein kinase C. Activation by phosphorylation and amino acid sequences of the phosphorylation sites

Phosphorylation of pure fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase from bovine heart by cAMP-dependent protein kinase and protein kinase C was investigated. The major enzyme form (subunit Mr of 58,000) was rapidly phosphorylated by both cAMP-dependent protein kinase and protein kinase C, incorporating 0.8 and 1.0 mol/mol of subunit, respectively. The rate of phosphorylation of the heart enzyme by cAMP-dependent protein kinase was 10 times faster than that of the rat liver enzyme. The minor enzyme (subunit Mr of 54,000), however, was phosphorylated only by protein kinase C and was phosphorylated much more slowly with a phosphate incorporation of less than 0.1 mol/mol of subunit. Phosphorylation by either cAMP-dependent protein kinase or protein kinase C activated the enzyme, but each phosphorylation affected different kinetic parameters. Phosphorylation by cAMP-dependent protein kinase lowered the Km value for fructose 6-phosphate from 87 to 42 microM without affecting the Vmax, whereas the phosphorylation by protein kinase C increased the Vmax value from 55 to 85 milliunits/mg without altering the Km value. The phosphorylated peptides were isolated, and their amino acid sequences were determined. The phosphorylation sites for both cAMP-dependent protein kinase and protein kinase C were located in a single peptide whose sequence was Arg-Arg-Asn-Ser-(P)-Phe-Thr-Pro-Leu-Ser-Ser-Ser-Asn-Thr(P)-Ile-Arg-Arg-Pro. The seryl residue nearest the N terminus was the residue specifically phosphorylated by cAMP-dependent protein kinase, whereas the threonine residue nearest the C terminus was phosphorylated by protein kinase C.     

Protein kinase C of human erythrocytes phosphorylates bands 4.1 and 4.9

Addition of 10 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) to intact human erythrocytes results in rapid phosphorylation of two cytoskeletal components, bands 4.1 and 4.9. The synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol, shows a similar effect, while the biologically inactive phorbol ester, 4 alpha-phorbol didecanoate, fails to enhance phosphorylation. That TPA and 1-oleoyl-2-acetylglycerol stimulate this phosphorylation suggests that protein kinase C is being activated. In the presence of TPA, bands 4.1 and 4.9 incorporate 1.5 mol Pi/mol protein and 1.2 mol Pi/mol protein, respectively. The pattern and extent of phosphorylation shows that it is not due to cAMP-dependent protein kinases, which also phosphorylate bands 4.1 and 4.9. Ca2+-phospholipid-dependent protein kinase activity is demonstrable in the soluble fraction of erythrocytes, and has been partially purified (2200-fold) from the hemolysate by affinity chromatography (Uchida and Filburn, 1984. J. Biol. Chem. 259, 12311-12314). The affinity purified erythrocyte kinase has a 42 A Stokes' radius and phosphorylates purified bands 4.1 and 4.9 in vitro in a Ca2+- and phospholipid-dependent manner. These results show that human erythrocytes contain protein kinase C, and that band 4.1 and 4.9 are the major endogenous substrates for this kinase.     

Phosphorylation of L-type pyruvate kinase by a Ca2+/calmodulin-dependent protein kinase

Rat liver L-type pyruvate kinase was phosphorylated in vitro by a Ca2+/calmodulin-dependent protein kinase purified from rabbit liver. The calmodulin (CaM)-dependent kinase catalyzed incorporation of up to 1.7 mol of 32P/mol of pyruvate kinase subunit; maximum phosphorylation was associated with a 3.0-fold increase in the K0.5 for P-enolpyruvate. This compares to incorporation of 0.7 to 1.0 mol of 32P/mol catalyzed by the cAMP-dependent protein kinase with a 2-fold increase in K0.5 for P-enolpyruvate. When [32P]pyruvate kinase, phosphorylated by the CaM-dependent protein kinase, was subsequently incubated with 5 mM ADP and cAMP-dependent protein kinase (kinase reversal conditions), 50-60% of the 32PO4 was removed from pyruvate kinase, but the K0.5 for P-enolpyruvate decreased only 20-30%. Identification of 32P-amino acids after partial acid hydrolysis showed that the CaM-dependent protein kinase phosphorylated both threonyl and seryl residues (ratio of 1:2, respectively) whereas the cAMP-dependent protein kinase phosphorylated only seryl groups. The two phosphorylation sites were present in the same 3-4-kDa CNBr fragment located near the amino terminus of the enzyme subunit. These results indicate that the CaM-dependent protein kinase catalyzed phosphorylation of L-type pyruvate kinase at two discrete sites. One site is apparently the same serine which is phosphorylated by the cAMP-dependent protein kinase. The second site is a unique threonine residue whose phosphorylation also inactivates pyruvate kinase by elevating the K0.5 for P-enolpyruvate. These results may account for the Ca2+-dependent phosphorylation of pyruvate kinase observed in isolated hepatocytes.     

Phosphorylation of smooth muscle myosin light chain kinase by protein kinase C. Comparative study of the phosphorylated sites

Smooth muscle myosin light chain kinase is phosphorylated in vitro by protein kinase C purified from human platelets. When myosin light chain kinase which has calmodulin bound is phosphorylated by protein kinase C, 0.8-1.1 mol of phosphate is incorporated per mol of myosin light chain kinase with no effect on its enzyme activity. Phosphorylation of myosin light chain kinase with no calmodulin bound results in the incorporation of 2-2.4 mol of phosphate and significantly decreases the rate of myosin light chain kinase activity. The decrease in myosin light chain kinase activity is due to a 3.3-fold increase in the concentration of calmodulin necessary for the half-maximal activation of myosin light chain kinase. The sites phosphorylated by protein kinase C and the catalytic subunit of cAMP-dependent protein kinase were compared by two-dimensional peptide mapping following extensive tryptic digestion of phosphorylated myosin light chain kinase. The single site phosphorylated by protein kinase C when calmodulin is bound to myosin light chain kinase (site 3) is different from that phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (site 1). The additional site that is phosphorylated by protein kinase C when calmodulin is not bound appears to be the same site phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (site 2). These studies confirm the important role of site 2 in binding calmodulin to myosin light chain kinase. Sequential studies using both protein kinase C and the catalytic subunit of cAMP-dependent protein kinase suggest that the phosphorylation of site 1 also plays a part in decreasing the affinity of myosin light chain kinase for calmodulin.     

Phosphorylation of smooth muscle myosin light chain kinase by Ca2+-activated, phospholipid-dependent protein kinase

Protein kinase C incorporates phosphate into two sites of myosin light chain kinase (MLC-kinase) in the absence of calmodulin. Phosphorylation is all but abolished in the presence of Ca2+ and calmodulin, suggesting that both sites of phosphorylation are close to the calmodulin binding site. The phosphorylation of MLC-kinase results in an approximately 10-fold increase in the dissociation constant of MLC-kinase for calmodulin. Following phosphorylation (2 mol/mol of enzyme) of MLC-kinase by protein kinase C, an additional 2 mol of phosphate can be incorporated into the MLC-kinase apoenzyme by the cAMP-dependent protein kinase. Different maps of phosphopeptides were obtained by tryptic hydrolysis from MLC-kinase preparations phosphorylated by each kinase. The phosphorylation sites for the cAMP-dependent kinase were located in a fragment of approximately 25,000 daltons. In contrast the phosphorylation sites for protein kinase C are found in a much smaller tryptic peptide. These results suggest that the phosphorylation sites on MLC-kinase are different for protein kinase C and for cAMP-dependent protein kinase. However, phosphorylation in both regions results in a reduced affinity for calmodulin.     

Phosphorylation of the androgen receptor by a nuclear cAMP-independent protein kinase

The androgen receptor was purified from rat ventral prostate. The purified receptor migrated as a single band of mol. wt. 87000 on SDS-polyacrylamide gels, had a kd for R-1881 (17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one) binding as 6 nM, and sedimentation coefficient of 4.5 S. Phosphorylation of the purified receptor was studied by incubating it with [gamma-32P]ATP in the presence of several purified protein kinases including cAMP-dependent protein kinase, and four cAMP-independent protein kinases (which were active towards substrates such as phosvitin and casein). Phosphorylation of the 87000 mol. wt. androgen receptor protein occurred only in the presence of a nuclear cAMP-independent protein kinase (of the N2 type). No auto-phosphorylation of the receptor was detected. The results indicate that the androgen receptor is a phosphoprotein. Further, phosphorylation of the androgen receptor by only a specific nuclear cAMP-independent protein kinase may be important in determining the dynamics of its function.     

Role of protein kinase C in the regulation of rat liver glycogen synthase

Rat liver glycogen synthase was phosphorylated by purified protein kinase C in a Ca2+- and phospholipid-dependent fashion to 1-1.4 mol PO4/subunit. Analysis of the 32P-labeled tryptic peptides derived from the phosphorylated synthase by isoelectric focusing and two-dimensional peptide mapping revealed the presence of a major radioactive peptide. The sites in liver synthase phosphorylated by protein kinase C appears to be different from those phosphorylated by other kinases. Prior phosphorylation of the synthase by protein kinase C has no significant effect on the subsequent phosphorylation by glycogen synthase (casein) kinase-1 or kinase Fa, but prevents the synthase from further phosphorylation by cAMP-dependent protein kinase, Ca2+/calmodulin-dependent protein kinase, phosphorylase kinase, or casein kinase-2. Additive phosphorylation of liver glycogen synthase can be observed by the combination of protein kinase C with the former set of kinases but not with the latter. Phosphorylation of liver synthase by protein kinase C alone did not cause an inactivation nor did the combination of this kinase with glycogen synthase (casein) kinase-1 or kinase Fa produce a synergistic effect on the inactivation of the synthase. Based on these findings we conclude that the phorbol ester-induced inactivation of glycogen synthase previously observed in hepatocytes cannot be accounted for entirely by the activation of protein kinase C.     

Phosphorylation of bovine cardiac C-protein by protein kinase C

C-protein, a thick filament-associated protein, has been isolated from bovine myocardium and found to be a substrate in vitro of the Ca2+- and phospholipid-dependent protein kinase (protein kinase C). Incorporation of approximately 1.6 mol Pi/mol C-protein was observed. This phosphorylation was dependent on both Ca2+ and a phospholipid (L-alpha-phosphatidyl-L-serine was used). Phosphate incorporation specifically into C-protein was verified by SDS-polyacrylamide gel electrophoresis and autoradiography and was almost exclusively into serine residues (86.9%), with only a small amount of phosphothreonine (13.1%) and no phosphotyrosine being detected. Two-dimensional thin-layer electrophoresis of a chymotryptic digest of phosphorylated C-protein indicated site specificity of phosphorylation. Cardiac C-protein is known to be a substrate of cAMP-dependent protein kinase both in vitro and in vivo (Jeacocke, S.A. and England, P.J. (1980) FEBS Lett. 122, 129-132). Isolated bovine cardiac C-protein was rapidly phosphorylated, to the extent of 5 mol/mol, by the purified catalytic subunit of cAMP-dependent protein kinase. Phosphorylation catalyzed by these two protein kinases was not additive, suggesting that the sites phosphorylated by protein kinase C are also phosphorylated by cAMP-dependent protein kinase. Chicken cardiac muscle has also been shown to contain a Ca2+, calmodulin-dependent protein kinase which phosphorylates C-protein (Hartzell, H.C. and Glass, D.B. (1984) J. Biol. Chem. 259, 15587-15596). The physiological role of cardiac C-protein may therefore be subject to regulation by multiple protein kinases.     

Phosphorylation of the cardiac ryanodine receptor by cAMP-dependent protein kinase

The phosphorylation of canine cardiac and skeletal muscle ryanodine receptors by the catalytic subunit of cAMP-dependent protein kinase has been studied. A high-molecular-weight protein (Mr 400,000) in cardiac microsomes was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. A monoclonal antibody against the cardiac ryanodine receptor immunoprecipitated this phosphoprotein. In contrast, high-molecular-weight proteins (Mr 400,000-450,000) in canine skeletal microsomes isolated from extensor carpi radialis (fast) or superficial digitalis flexor (slow) muscle fibers were not significantly phosphorylated. In agreement with these findings, the ryanodine receptor purified from cardiac microsomes was also phosphorylated by cAMP-dependent protein kinase. Phosphorylation of the cardiac ryanodine receptor in microsomal and purified preparations occurred at the ratio of about one mol per mol of ryanodine-binding site. Upon phosphorylation of the cardiac ryanodine receptor, the levels of [3H]ryanodine binding at saturating concentrations of this ligand increased by up to 30% in the presence of Ca2+ concentrations above 1 microM in both cardiac microsomes and the purified cardiac ryanodine receptor preparation. In contrast, the Ca2+ concentration dependence of [3H]ryanodine binding did not change significantly. These results suggest that phosphorylation of the ryanodine receptor by cAMP-dependent protein kinase may be an important regulatory mechanism for the calcium release channel function in the cardiac sarcoplasmic reticulum.     

Effect of 2,3-diphosphoglycerate on the phosphorylation of protein 4.1 by protein kinase C.

We have previously shown that 2,3-diphosphoglycerate (2,3-DPG) inhibits the phosphorylation of erythrocyte membrane cytoskeletal proteins by endogenous casein kinases. Here, we report that 2,3-DPG stimulates the phosphorylation of protein 4.1 by protein kinase C. Studies with red cell membrane preparations showed that while the phosphorylation of most of the membrane proteins by endogenous membrane-bound kinases and purified kinase C was inhibited by 2,3-DPG, the phosphorylation of protein 4.1 was slightly enhanced by the metabolite. The effect of 2,3-DPG was further examined using purified protein 4.1 preparations. Our results indicate that 2,3-DPG stimulates both the rate and the extent of phosphorylation of purified protein 4.1 by kinase C. The amount of phosphate incorporated was found to double to 2 mol of phosphate per mole of protein 4.1 in the presence of 10 mM 2,3-DPG. The increase in phosphorylation was distributed over all phosphorylation sites as revealed by an analysis of the labeling patterns of phosphopeptides resolved by high performance liquid chromatography, but a significantly higher incorporation was detected in two of the phosphopeptides. The stimulatory effect of 2,3-DPG on the phosphorylation of protein 4.1 was observed only with kinase C. Phosphorylation by the cytosolic erythrocyte casein kinase and the cyclic AMP-dependent protein kinase was inhibited by 2,3-DPG. Moreover, the stimulatory effect of 2,3-DPG seemed to be unique to the phosphorylation of protein 4.1 since a similar effect had not been observed with other protein kinase C substrates. Our results suggest that 2,3-DPG may play an important role in the regulation of cytoskeletal interactions.     

Identification of a new in vitro substrate of tyrosine protein kinase

Recent studies in our laboratory [Tokuda, M., Khanna, N.C., Aurora, A., & Waisman, D. M. (1986) Biochem. Biophys. Res. Commun. 139, 910-917] have identified in membranes of rat spleen two tyrosine protein kinases named TPK-I and TPK-II. In this paper the identification of the Ca2+ binding protein CAB-48 as a major in vitro substrate of TPK-II is reported. TPK-II catalyzed the incorporation of 0.73 mol of phosphate/mol of CAB-48. Phosphoamino acid analysis revealed that phosphorylation of CAB-48 was specific for tyrosine residues. Phosphorylation of CAB-48 by TPK-I (rat spleen), protein kinase C, casein kinase I, casein kinase II, cAMP-dependent protein kinase, or calcium calmodulin dependent protein kinase was not observed.     

Phosphorylation of rat liver glycogen synthase by phosphorylase kinase

Phosphorylation of rat liver glycogen synthase by rabbit skeletal muscle phosphorylase kinase results in the incorporation of approximately 0.8-1.2 mol of PO4/subunit. Analyses of the tryptic peptides by isoelectric focusing and thin layer chromatography reveal the presence of two major 32P-labeled peptides. Similar results were obtained when the synthase was phosphorylated by rat liver phosphorylase kinase. This extent of phosphorylation does not result in a significant change in the synthase activity ratio. In contrast, rabbit muscle glycogen synthase is readily inactivated by rabbit muscle phosphorylase kinase; this inactivation is further augmented by the addition of rabbit muscle cAMP-dependent protein kinase or cAMP-independent synthase (casein) kinase-1. Addition of cAMP-dependent protein kinase after initial phosphorylation of liver synthase with phosphorylase kinase, however, does not result in an inactivation or additional phosphorylation. The lack of additive phosphorylation under this condition appears to result from the phosphorylation of a common site by these two kinases. Partial inactivation of liver synthase can be achieved by sequential phosphorylation with phosphorylase kinase followed by synthase (casein) kinase-1. Under this assay condition, the phosphate incorporation into the synthase is additively increased and the synthase activity ratio (-glucose-6-P/+glucose-6-P) is reduced from 0.95 to 0.6. Nevertheless, if the order of the addition of these two kinases is reversed, neither additive phosphorylation nor inactivation of the synthase is observed. Prior phosphorylation of the synthase by phosphorylase kinase transforms the synthase such that it becomes a better substrate for synthase (casein) kinase-1 as evidenced by a 2- to 4-fold increase in the rate of phosphorylation. This increased rate of phosphorylation of the synthase appears to result from the rapid phosphorylation of a site neighboring that previously phosphorylated by phosphorylase kinase.     

Regulation of adrenergic receptor function by phosphorylation. II. Effects of agonist occupancy on phosphorylation of alpha 1- and beta 2-adrenergic receptors by protein kinase C and the cyclic AMP-dependent protein kinase

The present study was undertaken to determine the ability of protein kinase C and protein kinase A to directly phosphorylate the purified alpha 1- and beta 2-adrenergic receptors (AR). Both the catalytic subunit of protein kinase A and the protein kinase C, purified from bovine heart and pig brain, respectively, are able to phosphorylate the purified alpha 1-AR from DDT1 MF-2 smooth muscle cells. Occupancy of the receptor by an alpha 1 agonist, norepinephrine (100 microM), increases the rate of phosphorylation by protein kinase C but not by protein kinase A. The maximum stoichiometry of phosphorylation obtained is not affected by the agonist and reached 3 mol of PO4/mol of receptor for protein kinase C and 1 mol of PO4/mol of receptor for protein kinase A. The phosphopeptide maps of the trypsinized alpha 1-AR phosphorylated by each kinase differ drastically. The beta 2-AR purified from hamster lungs can also be phosphorylated by the two kinases. In contrast to the alpha 1-AR, the occupancy of the beta 2-AR by the agonist isoproterenol (20 microM) increases the rate of phosphorylation of the beta 2-AR by protein kinase A but not by protein kinase C. The maximum amount of phosphate incorporated into the receptor is not affected in either case by the agonist and reaches 1 mol of PO4/mol of receptor with protein kinase A and 0.4 mol of PO4/mol of receptor with protein kinase C. The phosphopeptide maps of the trypsinized receptor phosphorylated by either kinase reveal similar profiles. Thus, both alpha 1-AR and beta 2-AR are substrates for protein kinase A and protein kinase C. Agonist occupancy of the two receptors facilitates their phosphorylation only by the protein kinase coupled to their own signal transduction pathway. These observations suggest that "feedback" and "cross-system" phosphorylation may represent distinct and differently regulated mechanisms of modulation of receptor function.     

Phosphorylation reduces the affinity of protein 4.1 for spectrin

The phosphorylation of protein 4.1 by the membrane kinase and casein kinase A has been investigated. Each of these kinases catalyzed the incorporation of 2 mol of phosphate per mole of protein 4.1. The presence of both kinases in the reaction mixture did not lead to an increase in the incorporation of phosphates into the protein. An analysis of the acid hydrolysis products of the 32P-labeled protein 4.1 indicated that the radioactivities were distributed between phosphothreonine and phosphoserine in a ratio of about 2 to 1. The effects of phosphorylation on the binding of protein 4.1 to spectrin were investigated by using sucrose density gradient centrifugation. The affinity of protein 4.1 for spectrin was reduced about 5-fold, from a KD of 2 X 10(-6) M to a KD of 9.4 X 10(-6) M, by phosphorylation. The phosphorylation of spectrin, on the other hand, appeared to increase slightly its affinity for protein 4.1. The results suggest that phosphorylation may lead to a relaxation of the cytoskeletal network and the formation of a more flexible membrane structure that is important to red cell function.     

Phosphorylation of the acetylcholine receptor by protein kinase C and identification of the phosphorylation site within the receptor delta subunit

Purified acetylcholine receptor is rapidly and specifically phosphorylated by partially purified protein kinase C, the Ca2+/phospholipid-dependent enzyme. The receptor delta subunit is the major target for phosphorylation and is phosphorylated on serine residues to a final stoichiometry of 0.4 mol of phosphate/mol of subunit. Phosphorylation is dose-dependent with a Km value of 0.2 microM. Proteolytic digestion of the delta subunit phosphorylated by either protein kinase C or the cAMP-dependent protein kinase yielded a similar pattern of phosphorylated fragments. The amino acids phosphorylated by either kinase co-localized within a 15-kDa proteolytic fragment of the delta subunit. This fragment was visualized by immunoblotting with antibodies against a synthetic peptide corresponding to residues 354-367 of the receptor delta subunit. This sequence, which contains 3 consecutive serine residues, was recently shown to include the cAMP-dependent protein kinase phosphorylation site (Souroujon, M. C., Neumann, D., Pizzighella, S., Fridkin, M., and Fuchs, S. (1986) EMBO J. 5, 543-546). Concomitantly, the synthetic peptide 354-367 was specifically phosphorylated in a Ca2+- and phospholipid-dependent manner by protein kinase C. Furthermore, antibodies directed against this peptide inhibited phosphorylation of the intact receptor by protein kinase C. We thus conclude that both the cAMP-dependent protein kinase and protein kinase C phosphorylation sites reside in very close proximity within the 3 adjacent serine residues at positions 360, 361, and 362 of the delta subunit of the acetylcholine receptor.     

Phosphorylation of the porcine atrial muscarinic acetylcholine receptor by cyclic AMP dependent protein kinase

cAMP-dependent protein kinase, protein kinase C, cGMP-dependent protein kinase, smooth muscle myosin light-chain kinase, and phosphorylase kinase were examined with respect to their ability to phosphorylate porcine atrial muscarinic receptors (mAcChRs). Experiments were performed both in detergent solution and in a reconstituted system containing the mAcChR alone or in the presence of the purified porcine atrial inhibitor guanine nucleotide binding protein (Gi). Only cAMP-dependent protein kinase was capable of phosphorylating the receptor under any of the experimental conditions examined. Phosphorylation of the mAcChR in the detergent-solubilized state resulted in a loss of ligand binding sites that was reversible upon treatment with calcineurin in the presence of calcium and calmodulin. Upon reconstitution, the apparent stoichiometry of phosphorylation was increased by about 15-fold. Carbachol-stimulated covalent incorporation of phosphate was found only in the reconstituted system in the presence of Gi, suggesting that the large agonist-stimulated increase in phosphorylation observed in vivo [Kwatra, M. M., & Hosey, M. M. (1986) J. Biol. Chem. 261, 12429-12432] may in part result from a unique receptor conformation that occurs upon association with this protein. Ligand binding studies indicated that phosphorylation of the mAcChR in the detergent-solubilized or reconstituted state did not affect its interaction with carbachol or L-quinuclidinyl benzilate in vitro. Carbachol-induced stimulation of the GTPase activity of Gi in the reconstituted system was also unaffected by phosphorylation.     

Phosphorylation of cardiac troponin by guanosine 3':5'-monophosphate-dependent protein kinase.

Homogeneous cGMP-dependent protein kinase catalyzes the rapid incorporation of phosphate, specifically into the inhibitory subunit of purified cardiac troponin with a maximal incorporation of 1 mol of phosphate/mol of troponin. When troponin was incubated in the presence of both cGMP- and cAMP-dependent protein kinases, a maximal incorporation of 1 mol of phosphate/mol of troponin was observed which suggested phosphorylation of the same site by the two kinases. Both cyclic nucleotide-dependent kinases had similar Km values for troponin, but the Vmax value for the phosphorylation reaction catalyzed by cAMP-dependent protein kinase was 12-fold greater than the value obtained for cGMP-dependent protein kinase.