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 smooth muscle myosin heavy and light chains. Effects of phorbol dibutyrate and agonists

A number of different protein kinases phosphorylate purified heavy chains or the 20-kDa light chain of smooth muscle myosin. The physiological significance of these phosphorylation reactions has been examined in intact smooth muscle. Myosin heavy chain was slightly phosphorylated (0.08 mol of phosphate/mol) under control conditions in bovine tracheal tissue. Treatment with carbachol, isoproterenol, or phorbol 12,13-dibutyrate resulted in no significant change. In contrast, heavy chain was phosphorylated to 0.30 mol of phosphate/mol of heavy chain in tracheal smooth muscle cells in culture. This value increased significantly with ionomycin treatment. In control tissues, 9% of the light chain was monophosphorylated with 32P in the serine site phosphorylated by myosin light chain kinase. Carbachol (0.1 microM) alone resulted in contraction and 42% monophosphorylated light chain with 32P only in the serine site phosphorylated by myosin light chain kinase. Similarly, stimulation with histamine, 5-hydroxytryptamine, or KCl resulted in 32P incorporation into only the myosin light chain kinase serine site. Phorbol 12,13-dibutyrate (1 microM) alone resulted in 22% monophosphorylated light chain. However, only 25% of the 32P was in the myosin light chain kinase serine site, whereas 75% was in a serine site phosphorylated by protein kinase C. Phorbol 12,13-dibutyrate plus carbachol resulted in 27% monophosphorylated light chain; 75% of the 32P was in the myosin light chain kinase serine site, with the remainder in the protein kinase C serine site. These results indicate that phorbol esters act to increase phosphorylation of myosin light chain by protein kinase C. However, receptor-mediated stimulation or depolarization leading to tracheal smooth muscle contraction results in phosphorylation of myosin light chain by myosin light chain kinase alone.     

Sites phosphorylated in myosin light chain in contracting smooth muscle

Purified smooth muscle myosin light chain can be phosphorylated at multiple sites by myosin light chain kinase and protein kinase C. We have determined the sites phosphorylated on myosin light chain in intact bovine tracheal smooth muscle. Stimulation with 10 microM carbachol resulted in 66 +/- 5% monophosphorylated and 11 +/- 2% diphosphorylated myosin light chain after 1 min, and 47 +/- 4% monophosphorylated and 5 +/- 2% diphosphorylated myosin light chain after 30 min. Myosin heavy chain contained 0.06 +/- 0.01 mol of phosphate/mol of protein which did not change with carbachol. At both 1 and 30 min the monophosphorylated myosin light chain contained only phosphoserine whereas the diphosphorylated myosin light chain contained both phosphoserine and phosphothreonine. Two-dimensional peptide mapping of tryptic digests of monophosphorylated and diphosphorylated myosin light chain obtained from carbachol-stimulated tissue was similar to the peptide maps of purified light chain monophosphorylated and diphosphorylated, respectively, by myosin light chain kinase; these maps were distinct from the map obtained with tracheal light chain phosphorylated by protein kinase C. Phosphorylation of tracheal smooth muscle myosin light chain by myosin light chain kinase yields the tryptic phosphopeptide ATSNVFAMFDQSQIQEFK with S the phosphoserine in the monophosphorylated myosin light chain and TS the phosphotreonine and phosphoserine in the diphosphorylated myosin light chain. Thus, stimulation of tracheal smooth muscle with a high concentration of carbachol results in formation of both monophosphorylated and diphosphorylated myosin light chain although the amount of diphosphorylated light chain is substantially less than monophosphorylated light chain. In the intact muscle, myosin light chain is phosphorylated at sites corresponding to myosin light chain kinase phosphorylation.     

Phosphorylation of brush border myosin by brush border calmodulin-dependent myosin heavy and light chain kinases

Calmodulin-dependent myosin light chain kinase isolated from chicken intestinal brush border phosphorylates brush border myosin at an apparently single serine identical to that phosphorylated by smooth muscle myosin light chain kinase. Phosphorylation to 1.8 mol phosphate/mol myosin activated the myosin actin-activated ATPase about 10-fold, to about 50 nmol/min per mg. Myosin phosphorylated on its light chains could then be further phosphorylated to a total of 3.2 mol phosphate per mol by brush border calmodulin-dependent heavy chain kinase. Heavy chain phosphorylation did not alter the actin-activated ATPase of either myosin prephosphorylated on its light chains or of unphosphorylated myosin.     

The 204-kDa smooth muscle myosin heavy chain is phosphorylated in intact cells by casein kinase II on a serine near the carboxyl terminus

The heavy chain of smooth muscle myosin was found to be phosphorylated following immunoprecipitation from cultured bovine aortic smooth muscle cells. Of a variety of serine/threonine kinases assayed, only casein kinase II and calcium/calmodulin-dependent protein kinase II phosphorylated the smooth muscle myosin heavy chain to a significant extent in vitro. Two-dimensional maps of tryptic peptides derived from heavy chains phosphorylated in cultured cells revealed one major and one minor phosphopeptide. Identical tryptic peptide maps were obtained from heavy chains phosphorylated in vitro with casein kinase II but not with calcium/calmodulin-dependent protein kinase II. Of note, the 204-kDa smooth muscle myosin heavy chain but not the 200-kDa heavy chain isoform was phosphorylated by casein kinase II. Partial sequence of the tryptic phosphopeptides generated following phosphorylation by casein kinase II yielded Val-Ile-Glu-Asn-Ala-Asp-Gly-Ser*-Glu-Glu-Glu-Val. The Ser* represents the Ser(PO4) which is in an acidic environment, as is typical for casein kinase II phosphorylation sites. By comparison with the deduced amino acid sequence for rabbit uterine smooth muscle myosin (Nagai, R., Kuro-o, M., Babij, P., and Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737), we have localized the phosphorylated serine residue to the non-helical tail of the 204-kDa isoform of the smooth muscle myosin heavy chain. The ability of the 204-kDa isoform, but not the 200-kDa isoform, to serve as a substrate for casein kinase II suggests that these two isoforms can be regulated differentially.     

The heavy chain of macrophage myosin is phosphorylated at the tip of the tail

Myosin purified from rabbit alveolar macrophages has been shown previously to be phosphorylated on the rod portion of the heavy chain and on the 20-kDa light chains (Trotter, J.A. (1982) Biochem Biophys. Res. Commun. 106, 1071-1077). Phosphorylation of the 20-kDa light chains by endogenous kinase activity is associated with a significant enhancement of the actin-activated MgATPase activity (Trotter, J.A., and Adelstein, R.S. (1979) J. Biol. Chem. 254, 8781-8785), whereas the function of heavy-chain phosphorylation is unknown. The isolated heavy chains of myosin purified from freshly harvested cells contain between 0.4 and 1.5 mol of PO4/mol of heavy chain, all esterified to serine residues. Using myosin phosphorylated by incubating living unstimulated macrophages in the presence of 32Pi, two-dimensional thin-layer mapping of tryptic peptides derived from heavy chains yields four phosphopeptides, which are phosphorylated to different extents. Limited trypsin digestion of similar radioactive myosin removes all radioactivity from the heavy chain while reducing its apparent molecular mass by less than 10 kDa. It is concluded that the heavy chain of macrophage myosin is phosphorylated on as many as four serines within 10 kDa of the tip of the tail.     

Phosphorylation of smooth muscle heavy meromyosin by calcium-activated, phospholipid-dependent protein kinase. The effect on actin-activated MgATPase activity

Smooth muscle heavy meromyosin (HMM) can serve as a substrate for the Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) as well as for the Ca2+/calmodulin-dependent kinase, myosin light chain kinase. When turkey gizzard HMM is incubated with protein kinase C, 1.7-2.2 mol of phosphate are incorporated per mol of HMM, all of it into the 20,000-Da light chain of HMM. Two-dimensional peptide mapping following tryptic hydrolysis revealed that protein kinase C phosphorylated a different site on the 20,000-Da HMM light chain than did myosin light chain kinase. Moreover, sequential phosphorylation of HMM by myosin light chain kinase and protein kinase C resulted in the incorporation of 4 mol of phosphate/mol of HMM, i.e. 2 mol of phosphate into each 20,000-Da light chain. When unphosphorylated HMM was phosphorylated by myosin light chain kinase, its actin-activated MgATPase activity increased from 4 nmol to 156 nmol of phosphate released/mg of HMM/min. Subsequent phosphorylation of this phosphorylated HMM by protein kinase C decreased the actin-activated MgATPase activity of HMM to 75 nmol of phosphate released/mg of HMM/min.     

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 vertebrate nonmuscle and smooth muscle myosin heavy chains and light chains

In this article we review the various amino acids present in vertebrate nonmuscle and smooth muscle myosin that can undergo phosphorylation. The sites for phosphorylation in the 20 kD myosin light chain include serine-19 and threonine-18 which are substrates for myosin light chain kinase and serine-1 and/or-2 and threonine-9 which are substrates for protein kinase C. The sites in vertebrate smooth muscle and nonmuscle myosin heavy chains that can be phosphorylated by protein kinase C and casein kinase II are also summarized.Original data indicating that treatment of human T-lymphocytes (Jurkat cell line) with phorbol 12-myristate 13-acetate results in phosphorylation of both the 20 kD myosin light chain as well as the 200 kD myosin heavy chain is presented. We identified the amino acids phosphorylated in the human T-lymphocytes myosin light chains as serine-1 or serine-2 and in the myosin heavy chains as serine-1917 by 1-dimensional isoelectric focusing of tryptic phosphopeptides. Untreated T-lymphocytes contain phosphate in the serine-19 residue of teh myosin light chain and in a residue tentatively identified as serine-1944 in the myosin heavy chain.Abbreviations MLC myosin light chain - MHC myosin heavy chain - Tris tris(hydroxymethyl)aminomethane - EGTA [ethylenebis(oxyethylenenitrilo)]tetraacetic acid - EDTA ethylenediaminetetraacetate - TPCK N-tosyl-L-phenylalanine chloromethyl ketone - PMA phorbol 12-myristate 13-acetate     

Identification of the serine residue phosphorylated by protein kinase C in vertebrate nonmuscle myosin heavy chains

Two-dimensional mapping of the tryptic phosphopeptides generated following in vitro protein kinase C phosphorylation of the myosin heavy chain isolated from human platelets and chicken intestinal epithelial cells shows a single radioactive peptide. These peptides were found to comigrate, suggesting that they were identical, and amino acid sequence analysis of the human platelet tryptic peptide yielded the sequence -Glu-Val-Ser-Ser(PO4)-Leu-Lys-. Inspection of the amino acid sequence for the chicken intestinal epithelial cell myosin heavy chain (196 kDa) derived from cDNA cloning showed that this peptide was identical with a tryptic peptide present near the carboxyl terminal of the predicted alpha-helix of the myosin rod. Although other vertebrate nonmuscle myosin heavy chains retain neighboring amino acid sequences as well as the serine residue phosphorylated by protein kinase C, this residue is notably absent in all vertebrate smooth muscle myosin heavy chains (both 204 and 200 kDa) sequenced to date.     

Protein kinase C modulates in vitro phosphorylation of the smooth muscle heavy meromyosin by myosin light chain kinase

Protein kinase C phosphorylates different sites on the 20,000-Da light chain of smooth muscle heavy meromyosin (HMM) than did myosin light chain kinase (Nishikawa, M., Hidaka, H., and Adelstein, R. S. (1983) J. Biol. Chem. 258, 14069-14072). Although protein kinase C incorporates 1 mol of phosphate into 1 mol of 20,000-Da light chain when either HMM or the whole myosin molecule is used as a substrate, it catalyzes the incorporation of up to 3 mol of phosphate/mol of 20,000-Da light chain when the isolated light chains are used as a substrate. Threonine is the major phosphoamino acid resulting from phosphorylation of HMM by protein kinase C. Prephosphorylation of HMM by protein kinase C decreases the rate of phosphorylation of HMM by myosin light chain kinase due to a 9-fold increase of the Km for prephosphorylated HMM compared to that of unphosphorylated HMM. Prephosphorylation of HMM by myosin light chain kinase also results in a decrease of the rate of phosphorylation by protein kinase C due to a 2-fold increase of the Km for HMM. Both prephosphorylations have little or no effect on the maximum rate of phosphorylation. The sequential phosphorylation of HMM by myosin light chain kinase and protein kinase C results in a decrease in actin-activated MgATPase activity due to a 7-fold increase of the Km for actin over that observed with phosphorylated HMM by myosin light chain kinase but has little effect on the maximum rate of the actin-activated MgATPase activity. The decrease of the actin-activated MgATPase activity correlates well with the extent of the additional phosphorylation of HMM by protein kinase C following initial phosphorylation by myosin light chain kinase.     

Identification, phosphorylation, and dephosphorylation of a second site for myosin light chain kinase on the 20,000-dalton light chain of smooth muscle myosin

At relatively high concentrations of myosin light chain kinase, a second site on the 20,000-dalton light chain of smooth muscle myosin is phosphorylated (Ikebe, M., and Hartshorne, D. J. (1985) J. Biol. Chem. 260, 10027-10031). In this communication the site is identified and kinetics associated with its phosphorylation and dephosphorylation are described. The doubly phosphorylated 20,000-dalton light chain from turkey gizzard myosin was hydrolyzed with alpha-chymotrypsin and the phosphorylated peptide was isolated by reverse phase chromatography. Following amino acid analyses and partial sequence determinations the second site of phosphorylation is shown to be threonine 18. This site is distinct from the threonine residue phosphorylated by protein kinase C. The time courses of phosphorylation of serine 19 and threonine 18 in isolated light chains follow a single exponential indicating a random process, although the phosphorylation rates differ considerably. The values of kcat/Km for serine 19 and threonine 18 for isolated light chains are 550 and 0.2 min-1 microM-1, respectively. With intact myosin, phosphorylation of serine 19 is biphasic; kcat/Km values are 22.5 and 7.5 min-1 microM-1 for the fast and slow phases, respectively. In contrast, phosphorylation of threonine 18 in intact myosin is a random, but markedly slower process, kcat/Km = 0.44 min-1 microM-1. Dephosphorylation of doubly phosphorylated myosin (approximately 4 mol of phosphate/mol of myosin) and isolated light chains (approximately 2 mol of phosphate/mol of light chain) follows a random process and dephosphorylation of the serine 19 and threonine 18 sites occurs at similar rates.     

Analysis of myosin light chain phosphopeptides in phorbol dibutyrate-contracted artery

The incorporation of [32P]phosphate into the 20 kDa myosin light chain of phorbol dibutyrate-contracted artery was slightly increased as compared to that of resting muscle. Addition of K+ to the 1-h phorbol dibutyrate-contracted artery immediately doubled the force and greatly increased the light chain phosphorylation. Two-dimensional phosphopeptide mapping of light chain from phorbol dibutyrate-contracted muscle showed distinct peptides phosphorylated on serine residues by myosin light chain kinase and protein kinase C. In addition, the peptide phosphorylated on threonine residue by protein kinase C was revealed for the first time in intact muscle. Upon addition of K+, the distribution of phosphopeptides shifted toward the myosin light chain kinase catalyzed pattern.     

Chemoattractant-elicited increases in Dictyostelium myosin phosphorylation are due to changes in myosin localization and increases in kinase activity

We previously reported (Berlot, C. H., Spudich, J. A., and Devreotes, P. N. (1985) Cell 43, 307-314) that cAMP stimulation of chemotactically competent Dictyostelium amoebae causes transient increases in phosphorylation of the myosin heavy chain and 18,000-dalton light chain in vivo and in vitro. In this report we investigate the mechanisms involved in these changes in phosphorylation. In the case of heavy chain phosphorylation, the amount of substrate available for phosphorylation appears to be the major factor regulating the in vitro phosphorylation rate. Almost all heavy chain kinase activity is insoluble in Triton X-100, and the increase in the heavy chain phosphorylation rate in vitro parallels an increase in Triton insolubility of myosin. Changes in heavy chain phosphatase activity are not involved in the changes in the in vitro phosphorylation rate. In the case of light chain phosphorylation, increases in the vitro phosphorylation rate occur under conditions where the amount of substrate available for phosphorylation is constant and phosphatase activity is undetectable, implicating light chain kinase activation as the means of regulation. The specificity of the myosin kinases operating in vivo and in vitro was explored using phosphoamino acid and chymotryptic phosphopeptide analysis. The light chain is phosphorylated on serine both in vivo and in vitro, and phosphopeptide maps of the light chain phosphorylated in vivo and in vitro are indistinguishable. In the case of the heavy chain, both serine and threonine are phosphorylated in vivo and in vitro, although the cAMP-stimulated increases in phosphorylation occur primarily on threonine. Phosphopeptide maps of the heavy chain show that the peptides phosphorylated in vitro represent a major subset of those phosphorylated in vivo. The kinetics of the transient increases in myosin phosphorylation rates observed in vitro can be predicted quantitatively from the in vivo myosin phosphorylation data assuming that there is a constant phosphatase activity.     

Sequence of the sites phosphorylated by protein kinase C in the smooth muscle myosin light chain

We have determined the sequence of the sites phosphorylated by protein kinase C in the turkey gizzard smooth muscle myosin light chain. In contrast to previous work (Nishikawa, M., Hidaka, H., and Adelstein, R. S. (1983) J. Biol. Chem. 258, 14069-14072), two-dimensional tryptic peptide maps of both heavy meromyosin and the isolated myosin light chain showed two major phosphopeptides, one containing phosphoserine and the other phosphothreonine. We have purified the succinylated tryptic phosphopeptides using reverse phase and DEAE high pressure liquid chromatography. The serine-containing peptide, residues 1-4 (Ac-SSKR), is the NH2-terminal peptide. The phosphorylated serine residue may be either serine 1 or serine 2. The threonine-containing peptide, residues 5-16, yielded the sequence AKAKTTKKRPQR. Analysis of the yields and radioactivity of the products from automated Edman degradation showed that threonine 9 is the phosphorylation site.     

Correlation between multiple phosphorylation of gizzard myosin light chains and actin-activated myosin ATPase activity

With large amounts of gizzard Mr 135,000 calmodulin-binding protein (myosin light chain kinase), the phosphate incorporation into myosin light chains was determined to be 2 mol/mol of myosin light chain. The actin-activated ATPase activity was dramatically enhanced when myosin light chains were phosphorylated by more than 1 mol of phosphate incorporated/mol of myosin light chain.     

Stretch activates myosin light chain kinase in arterial smooth muscle

Stretching of porcine carotid arterial muscle increased the phosphorylation of the 20 kDa myosin light chain from 0.23 to 0.68 mol [32P]phosphate/mol light chain, whereas stretching of phorbol dibutyrate treated muscle increased the phosphorylation from 0.30 to 0.91 mol/mol. Two-dimensional gel electrophoresis followed by two-dimensional tryptic phosphopeptide mapping was used to identify the enzyme involved in the stretch-induced phosphorylation. Quantitation of the [32P]phosphate content of the peptides revealed considerable light chain phosphorylation by protein kinase C only in the phorbol dibutyrate treated arterial muscle, whereas most of the light chain phosphorylation was attributable to myosin light chain kinase. Upon stretch of either the untreated or treated muscle, the total increment in [32P]phosphate incorporation into the light chain could be accounted for by peptides characteristic for myosin light chain kinase catalyzed phosphorylation, demonstrating that the stretch-induced phosphorylation is caused by this enzyme exclusively.     

Phosphorylation of mammalian myosin light chain kinases by the catalytic subunit of cyclic AMP-dependent protein kinase and by cyclic GMP-dependent protein kinase

The phosphorylation of the calmodulin-dependent enzyme myosin light chain kinase, purified from bovine tracheal smooth muscle and human blood platelets, by the catalytic subunit of cAMP-dependent protein kinase and by cGMP-dependent protein kinase was investigated. When myosin light chain kinase which has calmodulin bound is phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, 1 mol of phosphate is incorporated per mol of tracheal myosin light chain kinase or platelet myosin light chain kinase, with no effect on the catalytic activity. Phosphorylation when calmodulin is not bound results in the incorporation of 2 mol of phosphate and significantly decreases the activity. The decrease in myosin light chain kinase activity is due to a 5 to 7-fold increase in the amount of calmodulin required for half-maximal activation of both tracheal and platelet myosin light chain kinase. In contrast to the results with the catalytic subunit of cAMP-dependent protein kinase, cGMP-dependent protein kinase cannot phosphorylate tracheal myosin light chain kinase in the presence of bound calmodulin. When calmodulin is not bound to tracheal myosin light chain kinase, cGMP-dependent protein kinase phosphorylates only one site, and this phosphorylation has no effect on myosin light chain kinase activity. On the other hand, cGMP-dependent protein kinase incorporates phosphate into two sites in platelet myosin light chain kinase when calmodulin is not bound. The sites phosphorylated by the two cyclic nucleotide-dependent protein kinases were compared by two-dimensional peptide mapping following extensive tryptic digestion of the phosphorylated myosin light chain kinases. With respect to the tracheal myosin light chain kinase, the single site phosphorylated by cGMP-dependent protein kinase when calmodulin is not bound appears to be the same site phosphorylated in the tracheal enzyme by the catalytic subunit of cAMP-dependent protein kinase when calmodulin is bound. With respect to the platelet myosin light chain kinase, the additional site that was phosphorylated by cGMP-dependent protein kinase when calmodulin was not bound was different from that phosphorylated by the catalytic subunit of cAMP-dependent protein kinase.     

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.     

Protein kinase C phosphorylates both the light chains and the head portion of the heavy chains of brain myosin

Protein kinase C phosphorylated both the 19/21-kDa regulatory light chains and heavy chains of bovine brain myosin. The major phosphorylation sites of the light chains were on their threonyl residues, while those for myosin light chain kinase were on their seryl residues. Whereas several non-muscle regular myosins have been reported to be phosphorylated by different types of protein kinases at the non-helical small segments at the tail ends of the heavy chains, the phosphorylation sites for protein kinase C were localized on the head portion of the heavy chains of brain myosin. The possible role of phosphorylation of brain myosin by protein kinase C in the regulation of motility of neural cells is discussed.