Caldesmon phosphorylation in intact human platelets by cAMP-dependent protein kinase and protein kinase C

Caldesmon is a calmodulin- and actin-binding protein present in both smooth and non-muscle tissue. The present study demonstrates that platelet caldesmon is a substrate for cAMP-dependent protein kinase (protein kinase A). Purified platelet caldesmon has an apparent molecular mass of 82 kDa on sodium dodecyl sulfate-polyacrylamide gels and can be phosphorylated in vitro by the catalytic subunit of protein kinase A to a level of 2 mol of phosphate/mol of caldesmon. Phosphorylation of caldesmon by protein kinase A results in a shift in the apparent molecular mass of the protein to 86 kDa. When caldesmon was immunoprecipitated from intact platelets treated with prostacyclin (PGI2) the same shift in apparent molecular mass of caldesmon was observed. Comparison of two-dimensional tryptic phosphopeptide maps of caldesmon phosphorylated in vitro by protein kinase A with caldesmon immunoprecipitated from intact platelets verified that protein kinase A was responsible for the observed increase in caldesmon phosphorylation in PGI2-treated platelets. The present study demonstrates that although caldesmon is basally phosphorylated in the intact platelet, activation of protein kinase A by PGI2 results in the significant incorporation of phosphate into two new sites. In addition, the effects of phorbol ester, collagen, and thrombin on caldesmon phosphorylation were also examined. Although phorbol ester treatment results in a significant increase in caldesmon phosphorylation apparently by protein kinase C, treatment of intact platelets with thrombin or collagen does not result in an increase in caldesmon phosphorylation.     

The effects of caldesmon on smooth muscle heavy actomeromyosin ATPase activity and binding of heavy meromyosin to actin

Caldesmon was purified to homogeneity from both chicken gizzard and bovine aortic smooth muscles. Caldesmon purified from bovine aorta was slightly larger than caldesmon purified from chicken gizzards (Mr = 140,000) when the two were compared electrophoretically. Caldesmon bound tightly to actin saturating at a molar ratio of 1 caldesmon monomer per 6.6 actin monomers. Ca2+-calmodulin appeared to reduce the affinity of caldesmon for actin. Caldesmon was also a potent inhibitor of heavy actomeromyosin ATPase activity producing a maximal effect at a ratio of 1 caldesmon monomer per 7-10 actin monomers. This effect was also antagonized by Ca2+-calmodulin. While caldesmon inhibited heavy actomeromyosin ATPase activity, it greatly enhanced binding of both unphosphorylated and phosphorylated heavy meromyosin to actin in the presence of MgATP, reducing the Kd for binding by a factor of 40 for each form of heavy meromyosin. Although we did identify a Ca2+-calmodulin-stimulated "caldesmon kinase" activity in caldesmon preparations purified under nondenaturing conditions, we observed no effect of phosphorylation (2 mol of PO4/mol of caldesmon) on the capacity to inhibit heavy actomeromyosin ATPase activity. Our results suggest that caldesmon could serve some role in smooth muscle function by enhancing cross-bridge affinity while inhibiting actomyosin ATPase activity.     

Properties of caldesmon isolated from chicken gizzard.

Chicken gizzard smooth muscle contains two major calmodulin-binding proteins: caldesmon (11.1 microM; Mr 141 000) and myosin light-chain kinase (4.6 microM; Mr 136 000), both of which are associated with the contractile apparatus. The amino acid composition of caldesmon is distinct from that of myosin light-chain kinase and is characterized by a very high glutamic acid content (25.5%), high contents of lysine (13.6%) and arginine (10.3%), and a low aromatic amino acid content (2.4%). Caldesmon lacked myosin light-chain kinase and phosphatase activities and did not compete with either myosin light-chain kinase or cyclic nucleotide phosphodiesterase (both calmodulin-dependent enzymes) for available calmodulin, suggesting that calmodulin may have distinct binding sites for caldesmon on the one hand and myosin light-chain kinase and cyclic nucleotide phosphodiesterase on the other. Consistent with the lack of effect of caldesmon on myosin phosphorylation, caldesmon did not affect the assembly or disassembly of myosin filaments in vitro. As previously shown [Ngai & Walsh (1984) J. Biol. Chem. 259, 13656-13659], caldesmon can be reversibly phosphorylated. The phosphorylation and dephosphorylation of caldesmon were further characterized and the Ca2+/calmodulin-dependent caldesmon kinase was purified; kinase activity correlated with a protein of subunit Mr 93 000. Caldesmon was not a substrate of myosin light-chain kinase or phosphorylase kinase, both calmodulin-activated protein kinases.     

In situ phosphorylation of human platelet myosin heavy and light chains by protein kinase C

Treatment of human platelets with 162 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in phosphorylation of a number of peptides, including myosin heavy chain and the 20-kDa myosin light chain. The site phosphorylated on the myosin heavy chain was localized by two-dimensional peptide mapping to a serine residue(s) in a single major tryptic phosphopeptide. This phosphopeptide co-migrated with a tryptic peptide that was produced following in vitro phosphorylation of platelet myosin heavy chain using protein kinase C. The sites phosphorylated in the 20-kDa myosin light chain in intact cells were analyzed by two-dimensional mapping of tryptic peptides and found to correspond to Ser1 and Ser2 in the turkey gizzard myosin light chain. In vitro phosphorylation of purified human platelet myosin by protein kinase C showed that in addition to Ser1 and Ser2, a third site corresponding to Thr9 in turkey gizzard myosin light chain is also phosphorylated. The phosphorylatable myosin light chains from human platelets were found to consist of two major isoforms present in approximately equal amounts, but differing in their molecular weights and isoelectric points. A third, minor isoform was also visualized by two-dimensional gel electrophoresis. Following treatment with TPA, both the mono- and diphosphorylated forms of each isoform could be visualized, and the sites of phosphorylation were identified. The phosphate content rose from negligible amounts found prior to treatment with TPA to 1.2 mol of phosphate/mol of myosin light chain and 0.7 mol of phosphate/mol of myosin heavy chain following treatment. These results suggest that TPA mediates phosphorylation of both myosin light and heavy chains in intact platelets by activation of protein kinase C.     

Phosphorylation of smg p21, a ras p21-like GTP-binding protein, by cyclic AMP-dependent protein kinase in a cell-free system and in response to prostaglandin E1 in intact human platelets

We have separated multiple small Mr GTP-binding proteins (G proteins) from bovine brain membranes by several column chromatographies and purified to near homogeneity four of them, including a novel Mr 24,000 G protein (smg p25A), a novel Mr 22,000 G protein (smg p21), the rho protein (rho p20), and the c-Ki-ras protein (c-Ki-ras p21). Among these small Mr G proteins, only smg p21 is phosphorylated stoichiometrically by cAMP-dependent protein kinase (protein kinase A), and c-Ki-ras p21 is phosphorylated to a small extent by protein kinase A in a cell-free system. None of smg p25A, rho p20, and other partially purified small Mr G proteins is phosphorylated by protein kinase A. Neither smg p21 nor other small Mr G proteins are phosphorylated by protein kinase C. About 1 mol of phosphate is maximally incorporated into 1 mol of smg p21 by protein kinase A. Only serine residue(s) are phosphorylated. The guanosine 5'-3-O-(thio) triphosphate (GTP gamma S)-bound and GDP-bound forms of smg p21 are phosphorylated with the same reaction velocity. The phosphorylation of smg p21 affects neither its GTP gamma S-binding nor GTPase activity. smg p21 is found in human platelets, and this human platelet smg p21 is also phosphorylated by protein kinase A at the same site(s) as bovine brain smg p21 in a cell-free system. When intact human platelets are stimulated by prostaglandin E1 known to elevate the cAMP level, four proteins with apparent Mr values of 240,000, 50,000, 24,000, and 22,000 are phosphorylated. These four proteins are also phosphorylated by the action of dibutyryl cAMP but not by the action of thrombin, Ca2+ ionophore A23187, or 12-O-tetradecanoylphorbol-13-acetate. Among the four proteins, the Mr 22,000 protein is identified as smg p21. The site(s) of phosphorylation of smg p21 by protein kinase A in a cell-free system are identical to that phosphorylated in response to prostaglandin E1 in intact platelets. These results indicate that among many small Mr G proteins, smg p21 is selectively phosphorylated by protein kinase A and that this G protein is also phosphorylated by this protein kinase in response to prostaglandin E1 in intact human platelets.     

Phosphorylation of caldesmon in arterial smooth muscle

We have isolated caldesmon (Mr = 145,000), by immunoprecipitation, from [32P]orthophosphate-loaded porcine carotid arteries. In resting muscles, caldesmon was phosphorylated to 0.45 mol of PO4/mol protein, while the 20,000-dalton myosin regulatory light chain (LC20) was phosphorylated to less than 0.05 mol/mol. After stimulation by KCl (110 mM) for 75 min and phorbol 12,13-dibutyrate (PDBu, 1 microM) for 60 min, caldesmon phosphorylation levels rose to 0.96 and 1.1 mol/mol, respectively. LC20 phosphorylation increased to 0.49 mol/mol at 1 min of stimulation by KCl and decreased to 0.17 mol/mol at 60 min. With PDBu, phosphate incorporation into LC20 rose only slightly, reaching 0.09 mol/mol after 90 min. Muscles contracted with histamine (10 microM) or ouabain (1 microM) also demonstrated elevated levels of phosphate incorporation into caldesmon. In these muscles, LC20 phosphorylation levels were less than 0.05 mol/mol. Three major phosphopeptides of indistinguishable mobility were identified on maps of caldesmon from resting, KCl-stimulated, and PDBu-stimulated muscles. There was, however, little similarity between the phosphopeptide maps of caldesmon phosphorylated in intact tissue and maps of purified caldesmon phosphorylated in vitro by protein kinase C (Ca2+/phospholipid-dependent enzyme) or Ca2+/calmodulin kinase II.     

Vascular smooth muscle caldesmon

Caldesmon, a major actin- and calmodulin-binding protein, has been identified in diverse bovine tissues, including smooth and striated muscles and various nonmuscle tissues, by denaturing polyacrylamide gel electrophoresis of tissue homogenates and immunoblotting using rabbit anti-chicken gizzard caldesmon. Caldesmon was purified from vascular smooth muscle (bovine aorta) by heat treatment of a tissue homogenate, ion-exchange chromatography, and affinity chromatography on a column of immobilized calmodulin. The isolated protein shared many properties in common with chicken gizzard caldesmon: immunological cross-reactivity, Ca2+-dependent interaction with calmodulin, Ca2+-independent interaction with F-actin, competition between actin and calmodulin for caldesmon binding only in the presence of Ca2+, and inhibition of the actin-activated Mg2+-ATPase activity of smooth muscle myosin without affecting the phosphorylation state of myosin. Maximal binding of aorta caldesmon to actin occurred at 1 mol of caldesmon: 9-10 mol of actin, and binding was unaffected by tropomyosin. Half-maximal inhibition of the actin-activated myosin Mg2+-ATPase occurred at approximately 1 mol of caldesmon: 12 mol of actin. This inhibition was also unaffected by tropomyosin. Caldesmon had no effect on the Mg2+-ATPase activity of smooth muscle myosin in the absence of actin. Bovine aorta and chicken gizzard caldesmons differed in several respects: Mr (149,000 for bovine aorta caldesmon and 141,000 for chicken gizzard caldesmon), extinction coefficient (E1%280nm = 19.5 and 5.0 for bovine aorta and chicken gizzard caldesmon, respectively), amino acid composition, and one-dimensional peptide maps obtained by limited chymotryptic and Staphylococcus aureus V8 protease digestion. In a competitive enzyme-linked immunosorbent assay, using anti-chicken gizzard caldesmon, a 174-fold molar excess of bovine aorta caldesmon relative to chicken gizzard caldesmon was required for half-maximal inhibition. These studies establish the widespread tissue and species distribution of caldesmon and indicate that vascular smooth muscle caldesmon exhibits physicochemical differences yet structural and functional similarities to caldesmon isolated from chicken gizzard.     

Characterization of mitotically phosphorylated caldesmon.

Mitosis-specific phosphorylation by cdc2 kinase causes nonmuscle caldesmon to dissociate from microfilaments (Yamashiro, S., Yamakita, Y., Ishikawa, R., and Matsumura, F. (1990) Nature 344, 675-678; Yamashiro, S., Yamakita, Y., Hosoya, H., and Matsumura, F. (1991) Nature 349, 169-172). To explore the function of mitosis-specific phosphorylation of caldesmon, in vivo- and in vitro-phosphorylated caldesmons have been characterized. We have found that both in vivo and in vitro phosphorylation of caldesmon causes similar changes in the properties, including reduction in actin, calmodulin, and myosin binding of caldesmon, and a decrease in the inhibition of actomyosin ATPase by caldesmon. Rat non-muscle caldesmon is phosphorylated in vitro up to a ratio of 7 mol/mol of protein. Actin-binding constants of both a high affinity (K a = 1.2 x 10(7) M-1) and a low affinity (K a = 1 x 10(6) M-1) site of unphosphorylated caldesmon are reduced to less than 10(5) M-1 with 5 mol of phosphate incorporation per mol of protein. Actin-bound caldesmon can be phosphorylated by cdc2 kinase, which results in the dissociation of caldesmon from F-actin. Caldesmon has a second myosin-binding site in the C terminus, in addition to the N terminus myosin-binding domain previously reported, because the bacterially expressed C terminus of caldesmon shows binding to myosin. Phosphorylation of the C-terminal fragments decreases their myosin-binding affinity as observed with intact caldesmon. These results suggest that caldesmon loses most of its in vitro functions during mitosis as a result of phosphorylation, which may be required for the reorganization of microfilaments during mitosis.     

Autophosphorylation of smooth-muscle caldesmon.

Caldesmon, a major actin- and calmodulin-binding protein of smooth muscle, has been implicated in regulation of the contractile state of smooth muscle. The isolated protein can be phosphorylated by a co-purifying Ca2+/calmodulin-dependent protein kinase, and phosphorylation blocks inhibition of the actomyosin ATPase by caldesmon [Ngai & Walsh (1987) Biochem. J. 244, 417-425]. We have examined the phosphorylation of caldesmon in more detail. Several lines of evidence indicate that caldesmon itself is a kinase and the reaction is an intermolecular autophosphorylation: (1) caldesmon (141 kDa) and a 93 kDa proteolytic fragment of caldesmon can be separated by ion-exchange chromatography: both retain caldesmon kinase activity, which is Ca2+/calmodulin-dependent; (2) chymotryptic digestion of caldesmon generates a Ca2+/calmodulin-independent form of caldesmon kinase; (3) caldesmon purified to electrophoretic homogeneity retains caldesmon kinase activity, and elution of enzymic activity from a fast-performance-liquid-chromatography ion-exchange column correlates with caldesmon of Mr 141,000; (4) caldesmon is photoaffinity-labelled with 8-azido-[alpha-32P]ATP; labelling is inhibited by ATP, GTP and CTP, indicating a lack of nucleotide specificity; (5) caldesmon binds tightly to Affi-Gel Blue resin, which recognizes proteins having a dinucleotide fold. Autophosphorylation of caldesmon occurs predominantly on serine residues (83.3%), with some threonine (16.7%) and no tyrosine phosphorylation. Autophosphorylation is site-specific: 98% of the phosphate incorporated is recovered in a 26 kDa chymotryptic peptide. Complete tryptic/chymotryptic digestion of this phosphopeptide followed by h.p.l.c. indicates three major phosphorylation sites. Caldesmon exhibits a high degree of substrate specificity: apart from autophosphorylation, brain synapsin I is the only good substrate among many potential substrates examined. These observations indicate that caldesmon may regulate its own function (inhibition of the actomyosin ATPase) by Ca2+/calmodulin-dependent autophosphorylation. Furthermore, caldesmon may regulate other cellular processes, e.g. neurotransmitter release, through the Ca2+/calmodulin-dependent phosphorylation of other proteins such as synapsin I.     

Protein kinase C phosphorylates caldesmon77 and vimentin and enhances albumin permeability across cultured bovine pulmonary artery endothelial cell monolayers.

Cytoskeletal protein (CSP) interactions are critical to the contractile response in muscle and non-muscle cells. Current concepts suggest that activation of the contractile apparatus occurs through selective phosphorylation by specific cellular kinase systems. Because the Ca(2+)-phospholipid-dependent protein kinase C (PKC) is involved in the regulation of a number of key endothelial cell responses, the hypothesis that PKC modulates endothelial cell contraction and monolayer permeability was tested. Phorbol myristate acetate (PMA), a direct PKC activator, and alpha-thrombin, a receptor-mediated agonist known to increase endothelial cell permeability, both induced rapid, dose-dependent activation and translocation of PKC in bovine pulmonary artery endothelial cells (BPAEC), as assessed by gamma-[32P]ATP phosphorylation of H1 histone in cellular fractions. This activation was temporally associated with evidence of agonist-mediated endothelial cell contraction as demonstrated by characteristic changes in cellular morphology. Agonist-induced activation of the contractile apparatus was associated with increases in BPAEC monolayer permeability to albumin (approximately 200% increase with 10(-6) MPMA, approximately 400% increase with 10(-8) M alpha-thrombin). To more closely examine the role of PKC in activation of the contractile apparatus, PKC-mediated phosphorylation of two specific CSPs, the actin- and calmodulin-binding protein, caldesmon77, and the intermediate filament protein, vimentin, was assessed. In vitro phosphorylation of both caldesmon and vimentin was demonstrated by addition of exogenous, purified BPAEC PKC to unstimulated BPAEC homogenates, to purified bovine platelet caldesmon77, or to purified smooth muscle caldesmon150. Caldesmon77 and vimentin phosphorylation were observed in intact [32P]-labeled BPAEC monolayers stimulated with either PMA or alpha-thrombin, as detected by immunoprecipitation. In addition, BPAEC pretreatment with the PKC inhibitor, staurosporine, prevented alpha-thrombin- and PMA-induced phosphorylation of both cytoskeletal proteins, attenuated morphologic evidence of contraction, and abolished agonist-induced barrier dysfunction. These results demonstrate that agonist-stimulated PKC activity results in cytoskeletal protein phosphorylation in BPAEC monolayer, an event which occurs in concert with agonist-mediated endothelial cell contraction and resultant barrier dysfunction.     

Phosphorylation of vascular smooth muscle caldesmon by endogenous kinase.

Caldesmon was phosphorylated up to 1.2 molPi/mol using a partially purified endogenous kinase fraction. The phosphorylation site was within the C-terminal 99 amino acids. We were also able to phosphorylate caldesmon incorporated into native and synthetic smooth muscle thin filaments. Phosphorylation did not alter caldesmon binding to actin or inhibition of actomyosin ATPase. It also did not change Ca2+ sensitivity in native thin filaments. Phosphorylated caldesmon bound to myosin less than unphosphorylated caldesmon, especially when the myosin was also not phosphorylated. This work did not support the hypothesis that caldesmon function is modulated by phosphorylation.     

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.     

Characterization of the phosphorylation sites in the chicken and bovine myristoylated alanine-rich C kinase substrate protein, a prominent cellular substrate for protein kinase C

Little is known about the important cellular substrates for protein kinase C and their potential roles in mediating protein kinase C-dependent processes. We evaluated the protein kinase C phosphorylation sites in a major cellular substrate for the kinase, a protein of apparent Mr 80,000 in bovine and 60,000 in chicken tissues; we have recently determined the primary sequences of these proteins and tentatively named them the myristoylated alanine-rich C kinase substrates. The proteins were purified to apparent homogeneity from bovine and chicken brains, phosphorylated with protein kinase C, digested with trypsin, and the phosphopeptides purified and sequenced. Four distinct phosphopeptides were identified from both the bovine and chicken proteins. Two of the phosphorylated serines were contained in the repeated motif FSFKK, one in the sequence LSGF, and one in the sequence SFK. All four sites were contained within a basic domain of 25 amino acids which was identical in the chicken and bovine proteins. All of the sites phosphorylated in the cell-free system appeared to be phosphorylated in intact cells; an additional site may have been present in the proteins from intact cells. The identity of the phosphorylation site domains from two proteins of overall 65% amino acid sequence identity suggests a potential role for this domain in the physiological function of the myristoylated alanine-rich C kinase substrate proteins.     

Identification of the site phosphorylated by casein kinase II in smooth muscle caldesmon

Phosphorylation of avian gizzard caldesmon by casein kinase II was investigated. The enzyme incorporates about 1 mol of phosphate per mol of caldesmon. All sites of phosphorylation are located in short chymotryptic peptides with Mr 25-27 kDa or in the short N-terminal peptide formed after cleavage of chicken gizzard caldesmon at Cys153. The primary structure of the tryptic peptide containing the main site of duck gizzard caldesmon phosphorylation is S-E-V-N-A-Q-N-X-V-A-E-D-E-T-K, where X is an unidentified residue, presumed to be phosphoserine. Thus, Ser73 is the main site phosphorylated by casein kinase II in avian gizzard caldesmon.     

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.     

Evidence against the regulation of caldesmon inhibitory activity by p42/p44erk mitogen-activated protein kinase in vitro and demonstration of another caldesmon kinase in intact gizzard smooth muscle.

The effect of direct phosphorylation by recombinant p44erk1 mitogen-activated protein kinase on the inhibitory activity of caldesmon and its C-terminal fragment H1 was studied in vitro. Neither inhibition of actin-tropomyosin activated ATPase of heavy meromyosin by caldesmon or H1, nor inhibition of the actin-tropomyosin motility over heavy meromyosin by H1 was significantly affected by the phosphorylation while only a moderate effect on the actin-activated component of heavy meromyosin ATPase inhibition was observed. Phosphopeptide mapping of caldesmon immunoprecipitated from [32P]PO4-labelled intact gizzard strips revealed that it is predominantly phosphorylated at mitogen-activated protein kinase sites in unstimulated tissue and that it is stimulated for 1 h with phorbol 12,13-dibutyrate. We find that phorbol 12,13-dibutyrate also induces a transitory phosphorylation of caldesmon peaking at 15 min after addition and this phosphorylation is not attributed to mitogen-activated protein kinase, protein kinase C, Ca2+/calmodulin-dependent kinase II or casein kinase II. We suggest that a yet unidentified kinase, rather than mitogen-activated protein kinase, may be involved in regulation of the caldesmon function in vivo.     

Vasodilator-stimulated protein phosphorylation in platelets is mediated by cAMP- and cGMP-dependent protein kinases

Vasodilators such as sodium nitroprusside, nitroglycerin and various prostaglandins are capable of inhibiting platelet aggregation associated with an increase of either cGMP or cAMP. In our studies with intact platelets, prostaglandin E1 and sodium nitroprusside stimulated the phosphorylation of several proteins which could be distinguished from proteins known to be phosphorylated by a calmodulin-regulated protein kinase or by protein kinase C. Prostaglandin E1 (10 microM) or dibutyryl cAMP (2 mM) stimulated the phosphorylation of proteins with apparent relative molecular masses, Mr, of 240,000, 68,000, 50,000, and 22,000 in intact platelets. These proteins were also phosphorylated in response to low concentrations (1-2 microM) of cAMP in a particulate fraction of platelets. In intact platelets, sodium nitroprusside (100 microM) and the 8-bromo derivative of cGMP (2 mM) increased the phosphorylation of one protein of Mr 50,000 which was also phosphorylated in response to low concentrations (1-2 microM) of cGMP in platelet membranes. An additional protein (Mr 24,000) appeared to be phosphorylated to a lesser degree in intact platelets by prostaglandin E1 and sodium nitroprusside. Since the phosphorylation of the protein of Mr 50,000 was stimulated both in intact platelets by cyclic-nucleotide-elevating agents and cyclic nucleotide analogs, as well as in platelet membranes by cyclic nucleotides, this phosphoprotein was analyzed by limited proteolysis, tryptic fingerprinting and phosphoamino acid analysis. These experiments indicated that the 50-kDa proteins phosphorylated by sodium nitroprusside and prostaglandin E1 were identical, and that the peptide of the 50-kDa protein phosphorylated by both agents was also the same as the peptide derived from the 50-kDa protein phosphorylated in platelet membranes by cGMP- and cAMP-dependent protein kinases, respectively. Regulation of protein phosphorylation mediated by cAMP- and cGMP-dependent protein kinases may be the molecular mechanism by which those vasodilators, capable of increasing either cAMP or cGMP, inhibit platelet aggregation.     

Phosphorylation of human platelet glycoprotein IIIa (GPIIIa). Dissociation from fibrinogen receptor activation and phosphorylation of GPIIIa in vitro.

Glycoprotein IIb-IIIa (GPIIb-IIIa) is the fibrinogen receptor on activated platelets. GPIIIa is phosphorylated in resting platelets and the incorporation of 32Pi increases with platelet activation. To address the functional significance of this modification, the stoichiometry of GPIIIa phosphorylation was determined in resting and activated platelets by estimating the specific activity of metabolic [gamma-32P]ATP from the specific activity of phosphatidic acid. Approximately 0.01 mol of P/mol of GPIIIa was phosphorylated in resting platelets and 0.03 mol of P/mol of GPIIIa was phosphorylated in thrombin-, phorbol ester-, or U46619-treated platelets. Myosin light chain (MLC) phosphorylation served as a positive control for this method (1.2 mol of P/mol of MLC). Phosphorylation of purified GPIIb-IIIa by human platelet protein kinase C (PKC) resulted in levels of GPIIIa phosphorylation similar to that in platelets (0.05 mol of P/mol of GPIIIa). However, while GPIIIa in platelets was phosphorylated primarily on threonine, purified GPIIIa treated with PKC was phosphorylated primarily on serine. These results suggest that PKC may not directly phosphorylate GPIIIa in intact platelets. Ca2+/calmodulin-dependent kinase II phosphorylated purified GPIIIa to higher levels (0.5 mol of P/mol of GPIIIa) with phosphorylation on both threonine and serine. The limited phosphorylation of GPIIIa in intact platelets suggests that this event is unlikely to affect functions involving large populations of GPIIb-IIIa, such as its conversion to a fibrinogen receptor. However, these results may suggest the existence of a more readily phosphorylated subpopulation of GPIIb-IIIa with potentially distinct structural or functional properties.     

Determination of the phosphorylation sites of smooth muscle caldesmon by protein kinase C

Smooth muscle caldesmon was phosphorylated by protein kinase C up to 1.90 mol P/mol caldesmon. Phosphorylated caldesmon was completely digested by trypsin and the produced phosphopeptides were purified by C-8 and C-18 reverse phase chromatography. Four phosphopeptides were determined and two phosphoserines were identified. Both were localized in the C-terminal domain at serine-587 and serine-726. By following the time course of phosphorylation, serine-587 was found to be the preferred site. Effects of the phosphorylation of caldesmon by protein C on the inhibition of acto-H-meromyosin ATPase activity was also examined. While unphosphorylated caldesmon inhibited the ATPase activity by 60%, phosphorylated caldesmon hardly inhibited the ATPase activity. Therefore, it was concluded that the phosphorylation at serine-726 and serine-587 reverses the inhibitory activity of caldesmon.     

Phosphorylation of the 165-kDa dihydropyridine/phenylalkylamine receptor from skeletal muscle by protein kinase C

Dihydropyridine-sensitive Ca2+ channels exist in many different types of cells and are believed to be regulated by various protein phosphorylation and dephosphorylation reactions. The present study concerns the phosphorylation of a putative component of dihydropyridine-sensitive Ca2+ channels by the calcium and phospholipid-dependent protein kinase, protein kinase C. A skeletal muscle peptide of 165 kDa, which is known to contain receptors for dihydropyridines, phenylalkylamines, and other Ca2+ channel effectors, was found to be an efficient substrate for protein kinase C when the peptide was phosphorylated in its membrane-bound state. Protein kinase C incorporated 1.5-2.0 mol of phosphate/mol of peptide within 2 min into the 165-kDa peptide in incubations carried out at 37 degrees C. In contrast to the membrane-bound peptide, the purified 165-kDa peptide in detergent solution was phosphorylated to a markedly less extent than its membrane-bound counterpart; less than 0.1 mol of phosphate/mol of peptide was incorporated. Preincubation of the membranes with several types of drugs known to be Ca2+ channel activators or inhibitors had no specific effects on the rate and/or extent of phosphorylation of the 165-kDa peptide by protein kinase C. The phosphorylation of the membrane-bound 165-kDa peptide by protein kinase C was compared to that catalyzed by cAMP-dependent protein kinase and was found to be not additive. Prior phosphorylation of the 165-kDa peptide by cAMP-dependent protein kinase prevented subsequent phosphorylation of the peptide by protein kinase C. Phosphoamino acid analysis indicated that protein kinase C phosphorylated the 165-kDa peptide at both serine and threonine residues. Phosphopeptide mapping experiments showed that protein kinase C phosphorylated one unique site in the 165-kDa peptide, and, in addition, other sites that were phosphorylated by either cAMP-dependent protein kinase or a multifunctional Ca2+/calmodulin-dependent protein kinase. The results suggest that the 165-kDa dihydropyridine/phenylalkylamine receptor could serve as a physiological substrate of protein kinase C in intact cells. It is therefore possible that the regulation of dihydropyridine-sensitive Ca2+ channels by activators of protein kinase C may occur at the level of this peptide.