Evidence for modulation of smooth muscle force by the p38 MAP kinase/HSP27 pathway.

Mitogen-activated protein (MAP) kinases signal to proteins that could modify smooth muscle contraction. Caldesmon is a substrate for extracellular signal-related kinases (ERK) and p38 MAP kinases in vitro and has been suggested to modulate actin-myosin interaction and contraction. Heat shock protein 27 (HSP27) is downstream of p38 MAP kinases presumably participating in the sustained phase of muscle contraction. We tested the role of caldesmon and HSP27 phosphorylation in the contractile response of vascular smooth muscle by using inhibitors of both MAP kinase pathways. In intact smooth muscle, PD-098059 abolished endothelin-1 (ET-1)-stimulated phosphorylation of ERK MAP kinases and caldesmon, but p38 MAP kinase activation and contractile response remained unaffected. SB-203580 reduced muscle contraction and inhibited p38 MAP kinase and HSP27 phosphorylation but had no effect on ERK MAP kinase and caldesmon phosphorylation. In permeabilized muscle fibers, SB-203580 and a polyclonal anti-HSP27 antibody attenuated ET-1-dependent contraction, whereas PD-098059 had no effect. These results suggest that ERK MAP kinases phosphorylate caldesmon in vivo but that activation of this pathway is unnecessary for force development. The generation of maximal force may be modulated by the p38 MAP kinase/HSP27 pathway.

Phosphorylation of caldesmon by ERK MAP kinases in smooth muscle.

Phosphorylation of h-caldesmon has been proposed to regulate airway smooth muscle contraction. Both extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinases phosphorylate h-caldesmon in vitro. To determine whether both enzymes phosphorylate caldesmon in vivo, phosphorylation-site-selective antibodies were used to assay phosphorylation of MAP kinase consensus sites. Stimulation of cultured tracheal smooth muscle cells with ACh or platelet-derived growth factor increased caldesmon phosphorylation at Ser789 by about twofold. Inhibiting ERK MAP kinase activation with 50 microM PD-98059 blocked agonist-induced caldesmon phosphorylation completely. Inhibiting p38 MAP kinases with 25 microM SB-203580 had no effect on ACh-induced caldesmon phosphorylation. Carbachol stimulation increased caldesmon phosphorylation at Ser789 in intact tracheal smooth muscle, which was blocked by the M(2) antagonist AF-DX 116 (1 microM). AF-DX 116 inhibited carbachol-induced isometric contraction by 15 +/- 1.4%, thus dissociating caldesmon phosphorylation from contraction. Activation of M(2) receptors leads to activation of ERK MAP kinases and phosphorylation of caldesmon with little or no functional effect on isometric force. P38 MAP kinases are also activated by muscarinic agonists, but they do not phosphorylate caldesmon in vivo.

Mammal-specific, ERK-dependent, caldesmon phosphorylation in smooth muscle. Quantitation using novel anti-phosphopeptide antibodies.

Extracellular signal-regulated kinases (ERKs) phosphorylate the high molecular mass isoform of the actin-binding protein caldesmon (h-CaD) at two sites (Ser(759) and Ser(789)) during smooth muscle stimulation. To investigate the role of phosphorylation at these sites, antibodies were generated against phosphopeptides analogous to the sequences around Ser(759) and Ser(789). Affinity-purified antibodies were phosho- and sequence-specific. The major site of phosphorylation in h-CaD in porcine carotid arterial muscle strips was at Ser(789); however, the amount of phosphate did not vary appreciably with either KCl or phorbol ester stimulation. Phosphorylation at Ser(759) of h-CaD was almost undetectable (<0.005 mol of phosphate/mol of protein). Moreover, phosphorylation of the low molecular mass isoform of the protein (l-CaD) at the site analogous to Ser(789) was greater in serum-stimulated cultured smooth muscle cells than in serum-starved cells. Serum-stimulated l-CaD phosphorylation was attenuated by the protein kinase inhibitor PD98059. These data 1) identify Ser(789) of h-CaD as the major site of ERK-dependent phosphorylation in carotid arteries; 2) show that the level of phosphorylation at Ser(789) is relatively constant following carotid arterial muscle stimulation, despite an increase in total protein phosphate content; and 3) suggest a functional role for ERK-dependent l-CaD phosphorylation in cell division.

p38 mitogen-activated protein kinase expression and activation in smooth muscle.

There is relatively little known about expression and activation of p38 mitogen-activated protein kinases (MAPKs) through G protein-linked, seven-transmembrane-spanning (STM) receptors in mammalian smooth muscle. To investigate the role of p38 MAPK in smooth muscle, we cloned and sequenced the p38 MAPK expressed in canine smooth muscles. A full-length clone of the canine p38 MAPK expressed in colonic smooth muscle was obtained by RT-PCR. The deduced amino acid sequence revealed 99% identity to the human p38 MAPK and differed from the human enzyme in only two conservative substitutions. The deduced molecular mass of the canine p38 MAPK is 41.2 kDa, with a calculated isoelectric point of 5.41. Canine p38 MAPK was found to be expressed in colonic, tracheal, and vascular smooth muscles and underwent increased tyrosine phosphorylation in response to motor neurotransmitters, acetylcholine (ACh) and neurokinin A (NKA), in colonic smooth muscle. There was an eightfold increase in p38 MAPK phosphorylation after a 10-min incubation with ACh and a threefold increase with NKA. We also identified a p38 immunoreactive kinase activity isolated from colonic smooth muscle homogenate by Mono Q chromatography. Partially purified p38 MAPK and activated recombinant p38 MAPK (Mpk2) phosphorylated both the known p38 MAPK substrate ATF2, as well as porcine stomach h-caldesmon in vitro. The results suggest that elements of the "stress-response" pathway may be coupled to transcriptional control as well as to cytoskeletal and possibly contractile protein phosphorylation in mammalian smooth muscle.

Activation of MAP kinase in vivo follows balloon overstretch injury of porcine coronary and carotid arteries.

Vascular restenosis involves contraction, proliferation, and remodeling of the arterial wall in response to overstretch injury. Mitogen-activated protein kinases (MAPKs) are implicated in both contraction and proliferation of vascular smooth muscle (VSM), and studies of porcine carotid arterial muscle strips have shown that mechanical stretch leads to the activation of the extracellular signal-regulated kinase (ERK) family of MAPKs in vivo. We, therefore, analyzed the acute effect of mechanical overstretch injury on ERK-MAPK (herein referred to simply as MAPK) activity in porcine coronary and carotid arteries in vivo. Balloon angioplasty catheters were inflated to 6 atm three times over 5 minutes at a balloon-artery ratio of 1.2:1 in either porcine coronary or carotid arteries. The arteries were snap-frozen after angioplasty, and MAPK activity was measured. Angioplasty of the left anterior descending (LAD, n = 5), left circumflex (LCx, n = 5), and carotid (n = 5) arteries effected an increase in MAPK activity compared with the activity in uninstrumented right coronary arteries (RCAs) or carotid arteries from the same animals used for controls. Balloon angioplasty of carotid arteries led to an increase in MAPK activity that was 7.7-fold over the activity in control arteries and comparable to the activity in stretched carotid arterial muscle strips in vivo. The increase in coronary artery kinase activity on angioplasty was variable from animal to animal. The increase in MAPK activity over that in control arteries ranged from 4.5- to 31.7-fold (mean +/- SEM, 10.7 +/- 5.3) in the LAD and 1.8- to 31.3-fold (mean +/- SEM, 9.7 +/- 5.7) in the LCx. There were no apparent inherent differences in the levels of MAPK activity in the three different types of coronary arteries (RCA, LAD, and LCx) without instrumentation. MAPK activation occurs rapidly during angioplasty, suggesting that this kinase may play an early role in initiating the injury response in both porcine coronary and carotid arteries. MAPKs may be key enzymes targeted to treat or prevent restenosis.

Calponin and mitogen-activated protein kinase signaling in differentiated vascular smooth muscle.

Contraction of smooth muscle cells is generally assumed to require Ca2+/calmodulin-dependent phosphorylation of the 20-kDa myosin light chains. However, we report here that in the absence of extracellular calcium, phenylephrine induces a contraction of freshly isolated ferret aorta cells in the absence of increases in intracellular ionized calcium or light chain phosphorylation levels but in the presence of activation of mitogen-activated protein kinase. A protein at 36 kDa co-immunoprecipitated with the mitogen-activated protein kinase and was identified as the actin-binding protein, calponin, by immunoblot. An overlay assay further confirmed an interaction between the kinase and calponin, even though the kinase did not phosphorylate calponin in vitro. Calponin also co-immunoprecipitated from smooth muscle cells with protein kinase C-epsilon. High resolution digital confocal studies indicated that calponin redistributes to the cell membrane during phenylephrine stimulation at a time when mitogen-activated protein kinase and protein kinase C-epsilon are targeted to the plasmalemma. These results suggest a role for calponin as a signaling molecule, possibly an adapter protein, linking the targeting of mitogen-activated protein kinase and protein kinase C-epsilon to the surface membrane.

Detection of osteopontin in calcified human aortic valves.

Cardiac valve calcification often results in obstruction of blood flow, which eventually leads to valve replacement. The molecular mechanisms resulting in valve calcification are unknown. Collagen and specific bone matrix proteins are thought to provide the framework for ectopic tissue calcification. This investigation was performed to determine whether the bone matrix protein osteopontin was present in calcified human aortic valves. Proteins extracted from human aortic valve tissue were subjected to polyacrylamide gel electrophoresis followed by Western blotting, using polyclonal antibodies directed against osteopontin. Fresh frozen tissue sections were also screened for osteopontin and macrophages using immunohistochemical techniques. Osteopontin was present in both heavily and minimally calcified aortic valves and absent in noncalcified purely regurgitant or normal aortic valves by both radioimmunoassay (n = 16) and immunohistochemical techniques (n = 8). Osteopontin colocalized with valvular calcific deposits, and macrophages were identified in the vicinity of osteopontin. These results, in addition to showing that osteopontin is present in calcified human aortic valves, suggest that osteopontin is a regulatory protein in pathological calcification. Identification of the cells producing osteopontin in abnormal cardiac valves and of proximate stimuli for its secretion may lead to novel therapeutic strategies to prevent and/or reverse calcific valve disease.

Stretch-dependent activation and desensitization of mitogen-activated protein kinase in carotid arteries.

Arterial smooth muscle stretch is an important physiological modulator of vascular function. To identify intracellular processes altered during muscle stretch, we found previously that extracellular signal-regulated kinase-mitogen-activated protein kinase (MAPK) activity increased in response to the application of mechanical loads. In the present study, stretch-dependent activation of MAPK in porcine carotid arteries was investigated as was the phosphorylation of the thin filament-binding protein caldesmon, which is known to be a substrate for the kinase in fully differentiated smooth muscle. MAPK activity was 67 pmol.min-1.mg protein-1 in unloaded muscle strips immediately after attachment to force transducers and 139 pmol.min-1.mg protein-1 within 30 s of muscle stretch. When muscle strips were continually stretched, MAPK activity remained elevated for approximately 2 h and then decreased over 16 h to 16 pmol.min-1.mg protein-1. When muscle strips were stretched and then unloaded, MAPK activity decreased within 1 h to the level present in the muscle before the stretch. These effects of muscle stretch on MAPK activity were additive to the effects of KCl or phorbol ester stimulation and were partially inhibited by reducing extracellular Ca2+. Eliminating extracellular Ca2+ had no effect on phorbol 12,13-dibutyrate (PDBu)-dependent contractions or MAPK activity; however, KCl-dependent contractions and MAPK activity were completely abolished by this procedure. An antibody specific for detecting caldesmon phosphorylated by MAPK, vs. protein kinase C (PKC), was developed and used to assess relative caldesmon phosphorylation in unstimulated and PDBu-stimulated muscle strips. In all cases investigated, the level of MAPK activity correlated with phosphocaldesmon immunoreactivity. Because arterial MAPK activity is regulated by PKC- and stretch-dependent mechanisms, these data are consistent with a role for MAPK and the subsequent phosphorylation of caldesmon as mediators in the stretch activation of vascular smooth muscle.

Strong interaction between caldesmon and calponin.

Caldesmon was labeled at either Cys-153 in the NH2-terminal domain or Cys-580 in the COOH-terminal domain with a 6-acryloyl-2-dimethylaminonaphthalene (acrylodan) fluorescence probe. The addition of smooth muscle calponin to Cys-580-labeled caldesmon resulted in an 18% drop in fluorescence intensity, which titrated with a stoichiometry of 0.9 and a binding constant of 9.5 x 10(7) M-1. For Cys-153-labeled caldesmon, there was no change in fluorescence upon adding calponin. These findings indicate strong binding between calponin and the COOH-domain of caldesmon. The association was sensitive to ionic strength, suggesting that ionic interactions between calponin, a basic protein, and caldesmon, an acidic protein, contribute to the stabilization of the protein complex. That non-muscle acidic calponin interacts with caldesmon with a much reduced association constant of 3.5 x 10(6) M-1 supports such a model. The binding between acidic calponin and caldesmon is strengthened to 1.8 x 10(7) M-1 in the presence of Ca2+, which might bind to acidic residues of the calponin and partially neutralize its negative charge. The strong, specific binding between calponin and caldesmon suggests that this interaction occurs within smooth muscle cells and possibly plays a role in the regulation of contraction.

Intracellular signaling by 8-epi-prostaglandin F2 alpha is mediated by thromboxane A2/prostaglandin endoperoxide receptors in porcine carotid arteries.

To investigate the mechanisms for intracellular signaling and increased vascular tone by 8-epi-prostaglandin F2 alpha (8-epi-PGF2 alpha), we measured mitogen-activated protein kinase (MAPK) activity and myosin regulatory light chain (LC20) phosphorylation in porcine carotid arteries incubated with 8-epi-PGF2 alpha or PGF2 alpha. With stimulation by either 8-epi-PGF2 alpha or PGF2 alpha. MAPK activity and the force of contraction rose in parallel and were maintained during the time of exposure to agonist (2 hours). LC20 phosphorylation levels rose and then partially declined during stimulation with either agonist. The effects of 8-epi-PGF2 alpha on contraction, MAPK activity, and myosin light chain phosphorylation were completely inhibited by the receptor antagonists, SQ-29548 and BMS-180291; the effects of PGF2 alpha were only partially inhibited by these compounds. Thus, intracellular signaling by 8-epi-PGF2 alpha in fully differentiated vascular smooth muscle, resulting in MAPK activation and increased myosin phosphorylation, is specifically mediated by an activation of thromboxane A2/prostaglandin endoperoxide receptors. Lipid peroxidation and 8-epi-PGF2 alpha production, resulting from such vascular pathological processes as atherosclerosis, lead to an activation of two intracellular signaling pathways in smooth muscle: one pathway results in the activation of MAPK, while the other results in myosin light chain phosphorylation.

Calponin is not phosphorylated during contractions of porcine carotid arteries.

Calponin and caldesmon were purified from porcine carotid arteries that were preincubated with [32P]orthophosphate, and the stoichiometry of phosphorylation was measured. In resting arteries, caldesmon was phosphorylated to a level of 0.41 mol PO4/mol protein, while calponin was phosphorylated to levels < 0.01 mol PO4/mol protein. Stimulation by histamine (1 or 5 min), KCl (1, 5 or 60 min), or phorbol 12,13-dibutyrate (PDBu; 1 microM for 15 or 60 min) did not lead to measurable increases in the PO4 content of calponin. Because dephosphorylation of calponin during the purification procedure could account for these results, we also determined stoichiometries after firat denaturing endogenous phosphatases with trichloroacetic acid. In these experiments, calponin was determined to be phosphorylated to the same low levels as in the first set of experiments. Collectively, these data show that calponin is not phosphorylated to significant levels during contractions of carotid arteries under conditions where caldesmon phosphorylation is apparent. The circumstances under which calponin may be phosphorylated in intact smooth muscle, and the purpose that may be served by this potential regulatory process, remain to be determined.

Phosphorylation of dense-plaque proteins talin and paxillin during tracheal smooth muscle contraction.

Reorganization of cytoskeletal-membrane interactions during contractile stimulation may contribute to the regulation of airway smooth muscle contraction. We investigated the effect of contractile stimulation on the phosphorylation of the actin-membrane attachment proteins talin, vinculin, and paxillin. Stimulation of 32P-labeled canine tracheal smooth muscle strips with acetylcholine (ACh; 10(-3) M) resulted in a rapid 2.6-fold increase in phosphorylation of serine and/or threonine residues, compared with resting levels of 0.22 mol PO4(3-)/mol talin. After stimulation with ACh, phosphorylation of tyrosine residues on paxillin increased approximately threefold. Two-dimensional phosphopeptide mapping of in vivo labeled talin and paxillin indicated phosphorylation on a limited number of sites. Vinculin phosphorylation was undetectable in either resting or ACh-stimulated muscle. We conclude that phosphorylation of talin and paxillin occurs during ACh-stimulated contraction of tracheal smooth muscle and that distinct signaling pathways activate a serine/threonine kinase that phosphorylates talin and a tyrosine kinase that phosphorylates paxillin. The pharmacological activation of airway smooth muscle cells might involve the anchoring of contractile filaments to the membrane.

Activation of mitogen-activated protein kinase in porcine carotid arteries.

The thin-filament protein h-caldesmon (the high molecular weight isoform of caldesmon) is phosphorylated in resting and contracted porcine carotid arteries. Phosphorylation of h-caldesmon in intact tissue occurs at sites that are covalently modified by mitogen-activated protein kinase (MAPK) in vitro. In this study, we have evaluated MAPK activation in arteries in response to mechanical load and pharmacological stimulation. MAPK was extracted from resting and stimulated porcine carotid arteries and then partially purified by anion-exchange fast-performance liquid chromatography. MAPK activity was separated into two peaks corresponding to the tyrosine-phosphorylated 42- and 44-kD isoforms of MAPK (p42MAPK and p44MAPK, respectively). Of the total MAPK activity, 42% was associated with p42MAPK, and 58% was associated with p44MAPK, this percentage was not altered by stimulation of the muscles with either KCl (110 mmol/L) or phorbol 12,13-dibutyrate (PDBu, 1 mumol/L). Both p42MAPK and p44MAPK, purified from porcine carotid arteries, phosphorylated h-caldesmon at the same sites and to levels approaching or > 1 mol phosphate per mole protein. In unloaded muscle strips, MAPK activity was 39 pmol.min-1.mg protein-1 when assayed with the peptide substrate APRTPG-GRR. MAPK activity increased in response to incremental mechanical loading to a maximum of 99 pmol.min-1.mg protein-1 at 16 x 10(3) N/m2. MAPK activity could be further increased in loaded muscles by pharmacological stimulation. With KCl stimulation, MAPK activities rose to a peak of 205 pmol.min-1.mg protein-1 at 10 minutes and then declined to basal values at 30 and 60 minutes.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of isotonic shortening velocity by second messengers in tracheal smooth muscle.

Evidence suggests that the mechanical behavior of smooth muscle tissues is regulated by Ca(2+)-dependent changes in the phosphorylation of the 20,000-Da light chain of myosin (MLC). However, alternative mechanisms activated by specific kinases may be involved in regulating the shortening velocity in some smooth muscle tissues. To determine how the activation of protein kinases A or C affects the regulation of the shortening velocity in canine tracheal smooth muscle, we evaluated the effects of forskolin (10(-5) M) and phorbol 12,13-dibutyrate (PDBu, 3 x 10(-6) M) on active stress, intracellular Ca2+ ([Ca2+]i), MLC phosphorylation, and isotonic shortening velocity during contractions elicited by 60 mM KCl. Forskolin depressed and PDBu increased active stress, [Ca2+]i, MLC phosphorylation, and shortening velocity; thus the effects of these agents on the shortening velocity may result from changes in Ca(2+)-dependent MLC phosphorylation. In contrast, the decline in velocity that occurred with time during tonic contractions elicited by K+ depolarization was not associated with significant changes in MLC phosphorylation; thus the time-dependent changes in shortening velocity may be regulated by a mechanism other than MLC phosphorylation.

Identification of a latent Ca2+/calmodulin dependent protein kinase II phosphorylation site in vascular calpain II.

The Ca(2+)-dependent protease, calpain II, isolated from vascular smooth muscle was found to be a substrate for Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in vitro. Phosphorylation was dependent upon prior autolysis of the regulatory subunit of calpain II. The stoichiometry of phosphorylation of native, unautolyzed calpain II was 0.02 +/- 0.01 mol PO4/mol enzyme while for autolyzed calpain, the stoichiometry was 1.04 +/- 0.15 mol PO4/mol enzyme. All phosphate was incorporated into the 76 kDa catalytic subunit of calpain II. A single serine residue in domain III of the catalytic subunit was identified as the phosphate acceptor: RGS*TAGGCR. Phosphorylation doubled enzyme activity measured both as proteolysis of an exogenous substrate (alpha-casein) as well as by intermolecular catalytic subunit autolysis. The effects of phosphorylation could be reversed by dephosphorylation using a type IIA phosphoprotein phosphatase. These results demonstrate that calpain II possesses a latent CaM kinase II phosphorylation site that is unmasked by autolysis.

Disulphide cross-linking of smooth-muscle and non-muscle caldesmon to the C-terminus of actin in reconstituted and native thin filaments.

It was reported that chicken gizzard smooth-muscle caldesmon Cys-580 can be disulphide-cross-linked to the C-terminal pen-ultimate residue (Cys-374) of actin, indicating that these residues are close in the protein complex [Graceffa, P. and Jancso, A. (1991) J. Biol. Chem. 266, 20305-20310]. Since the possibility that the cross-link involves a cysteine residue other than actin Cys-374 was not absolutely excluded, more direct evidence was sought for the identify of the cysteine residues involved in the cross-link. We show here that caldesmon could not be disulphide-cross-linked to actin which had Cys-374 removed by carboxypeptidase A digestion, providing direct support for the participation of actin Cys-374 in the cross-link to caldesmon. In order to assign the caldesmon cysteine residue involved in the cross-link, use was made of caldesmon from porcine stomach muscle, which is shown to contain one cysteine residue close to, or at, position 580, in contrast with chicken gizzard caldesmon, which has an additional cysteine residue at position 153. The porcine stomach caldesmon also formed a disulphide-cross-link to actin, further supporting the original conclusion that Cys-580 of the chicken gizzard caldesmon had been cross-linked to actin. Disulphide-cross-linking with similar yield was also observed in native chicken gizzard muscle thin filaments, indicating that the interaction between actin and the C-terminal domain of caldesmon is the same in native and reconstituted thin filaments. The much smaller non-muscle isoform of caldesmon, from rabbit liver, could be similarly cross-linked to actin, consistent with the sequence similarity between the C-terminal domain of muscle and non-muscle caldesmon. The ability to cross-link caldesmon Cys-580 to actin Cys-374 suggests the possibility that the Cys-580 region of caldesmon and the C-terminus of actin form part of the actin-caldesmon binding interface.

Identification of mitogen-activated protein kinase phosphorylation sequences in mammalian h-Caldesmon.

h-Caldesmon in vascular smooth muscle is phosphorylated in response to pharmacologic stimulation. Although many kinases phosphorylate h-caldesmon, in vitro, the responsible kinase in intact tissue is unknown. The sites of phosphorylation in caldesmon from intact canine aortas have recently been identified and are consensus sequences for a proline-directed protein kinase. In this study, we investigated the phosphorylation of h-caldesmon by mitogen-activated protein kinase (MAPK). Purified, recombinant MAPK phosphorylated porcine stomach h-caldesmon to a stoichiometry approaching 2 mol phosphate/mol protein. Phosphorylated h-caldesmon was subjected to proteolysis and the phosphopeptides were purified by high performance liquid chromatography. Two major phosphopeptides were identified and sequenced. These two peptides, VTS*PTKV and S*PAPK, were identical to the sequences of the sites phosphorylated in intact tissue. Antibodies to several enzymes implicated in the cascade of activation of MAPK were used to evaluate vascular smooth muscle by Western blotting. All components were found to be present. These data suggest that MAPK can function as a 'caldesmon kinase' in vascular smooth muscle.

Phosphorylation sequences in h-caldesmon from phorbol ester-stimulated canine aortas.

The high molecular weight form of caldesmon (h-caldesmon) is phosphorylated in vascular smooth muscle. The stoichiometry of caldesmon phosphorylation increases in response to stimulation of the muscle by several contractile agonists; however, the responsible kinase has not been identified. In this study, we have sequenced the phosphopeptides prepared from h-caldesmon phosphorylated in vitro by protein kinase C (PKC) as well as the phosphopeptides prepared from caldesmon phosphorylated in intact canine aortas that were stimulated to contract with PDBu. PKC phosphorylated three sites located in the C terminus: GSS*LKIEE, AEFLNKS*VQK and NLWEKQS*VDK, while h-caldesmon from intact tissue was phosphorylated at two separate sites also in the C terminus: VTS*PTKV and S*PAPK. By comparison to known substrate consensus sequences for various protein kinases these data suggest that h-caldesmon is directly phosphorylated by a proline-directed protein kinase and not by PKC.

Myosin light chain and caldesmon phosphorylation in arterial muscle stimulated with endothelin-1.

Endothelin-1 contracts porcine carotid arterial smooth muscle with an ED50 of 10 nM. Contraction is associated with phosphorylation of the 20,000 dalton-regulatory light chain subunits of vascular myosin. Phosphopeptide mapping of light chains isolated from 32PO4-loaded muscle strips stimulated by endothelin-1 (5 x 10(-8) M) and comparison with maps generated from light chains phosphorylated in vitro or muscles stimulated with KCl (110 mM) or angiotensin-II (5 x 10(-8) M) indicates that Ca2(+)-calmodulin activation of myosin light chain kinase is a biochemical pathway stimulated by all three agonists. However, a small amount of phosphate (17%) was detected in a light chain peptide phosphorylated by protein kinase C. Endothelin-1 also stimulated phosphorylation of the thin filament protein, caldesmon, (from 0.35 mol PO4/mol caldesmon to 0.52 mol PO4/mol). Collectively, these results provide evidence that the effects of endothelin-1 on force generation and maintenance in vascular muscle may be dependent upon myosin light chain phosphorylation by Ca2+ calmodulin--requiring myosin light chain kinase and upon a thin filament mechanism that is modulated by phosphorylation of caldesmon.