RESUMEN
NEW FINDINGS: What is the central question of this study? DAPK3 contributes to the Ca2+ -sensitization of vascular smooth muscle contraction: does this protein kinase participate in the myogenic response of cerebral arteries? What is the main finding and its importance? Small molecule inhibitors of DAPK3 effectively block the myogenic responses of cerebral arteries. HS38-dependent changes to vessel constriction occur independent of LC20 phosphorylation, and therefore DAPK3 appears to operate via the actin cytoskeleton. A role for DAPK3 in the myogenic response was not previously reported, and the results support a potential new therapeutic target in the cerebrovascular system. ABSTRACT: The vascular smooth muscle (VSM) of resistance blood vessels is a target of intrinsic autoregulatory responses to increased intraluminal pressure, the myogenic response. In the brain, the myogenic reactivity of cerebral arteries is critical to homeostatic blood flow regulation. Here we provide the first evidence to link the death-associated protein kinase 3 (DAPK3) to the myogenic response of rat and human cerebral arteries. DAPK3 is a Ser/Thr kinase involved in Ca2+ -sensitization mechanisms of smooth muscle contraction. Ex vivo administration of a specific DAPK3 inhibitor (i.e., HS38) could attenuate vessel constrictions invoked by serotonin as well as intraluminal pressure elevation. The HS38-dependent dilatation was not associated with any change in myosin light chain (LC20) phosphorylation. The results suggest that DAPK3 does not regulate Ca2+ sensitization pathways during the myogenic response of cerebral vessels but rather operates to control the actin cytoskeleton. A slow return of myogenic tone was observed during the sustained ex vivo exposure of cerebral arteries to HS38. Recovery of tone was associated with greater LC20 phosphorylation that suggests intrinsic signalling compensation in response to attenuation of DAPK3 activity. Additional experiments with VSM cells revealed HS38- and siDAPK-dependent effects on the actin cytoskeleton and focal adhesion kinase phosphorylation status. The translational importance of DAPK3 to the human cerebral vasculature was noted, with robust expression of the protein kinase and significant HS38-dependent attenuation of myogenic reactivity found for human pial vessels.
Asunto(s)
Arterias Cerebrales , Vasoconstricción , Animales , Humanos , Ratas , Arterias Cerebrales/metabolismo , Proteínas Quinasas Asociadas a Muerte Celular/metabolismo , Proteínas Quinasas , Resistencia Vascular , Vasoconstricción/fisiologíaRESUMEN
Vascular smooth muscle cells of the renal afferent arteriole are unusual in that they must be able to contract very rapidly in response to a sudden increase in systemic blood pressure in order to protect the downstream glomerular capillaries from catastrophic damage. We showed that this could be accounted for, in part, by exclusive expression, at the protein level, of the "fast" (B) isoforms of smooth muscle myosin II heavy chains in the afferent arteriole, in contrast to other vascular smooth muscle cells such as the rat aorta and efferent arteriole which express exclusively the "slow" (A) isoforms (Shiraishi et al. (2003) FASEB. J. 17, 2284-2286). As contraction of the more rapidly contracting striated (skeletal and cardiac) muscles is regulated by the thin filament-associated troponin (Tn) system, we hypothesized that Tn or a Tn-like system may exist in afferent arteriolar cells and contribute to the unusually rapid contraction of this tissue in response to increased intraluminal pressure. We examined the expression of TnC (Ca2+ -binding subunit), TnI (inhibitory subunit), and TnT (tropomyosin-binding subunit) in vascular smooth muscle cells of the rat renal afferent arteriole at the mRNA level. Fast-twitch skeletal muscle and slow-twitch skeletal muscle/cardiac TnC isoforms and slow-twitch skeletal muscle and cardiac TnI isoforms were detected by reverse transcription-polymerase chain reaction (RT-PCR) and confirmed by cDNA sequencing. Furthermore, cardiac and slow-twitch skeletal muscle TnI isoforms, but not fast-twitch skeletal muscle TnI, were detected in isolated afferent arterioles at the protein level by proximity ligation assay. Finally, striated muscle myosin II heavy chain expression was identified in isolated rat afferent arterioles by RT-PCR. We conclude that, in addition to Ca2+ -mediated phosphorylation of myosin II regulatory light chains, contraction of the afferent arteriole may be regulated by a mechanism normally associated with the much more rapidly contracting cardiac and skeletal muscles, which involves Ca2+ binding to TnC, leading to alleviation of inhibition of the actomyosin MgATPase by TnI and tropomyosin and rapid contraction of the vessel.
Asunto(s)
Arteriolas/metabolismo , Riñón/metabolismo , Contracción Muscular/genética , Troponina/genética , Citoesqueleto de Actina/genética , Adenosina Trifosfatasas/genética , Animales , Calcio/metabolismo , Regulación de la Expresión Génica/genética , Humanos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Miosina Tipo II/genética , Fosforilación/genética , Isoformas de Proteínas/genética , Ratas , Tropomiosina/genéticaRESUMEN
The 20-kDa regulatory light chain of myosin II plays an important role in regulating smooth muscle contractile force. LC20 is phosphorylated canonically by myosin light chain kinase in a Ca2+/calmodulin-dependent manner at S19. The diphosphorylation of LC20 at T18 and S19 has been observed in smooth muscle tissues. Given that the phosphorylation of LC20 is positively correlated with tension development, the molar stoichiometry of LC20 phosphorylation is commonly profiled as a measure of smooth muscle contractility. Herein, we describe a novel multiple reaction monitoring (MRM)-mass spectrometry (MS) approach for the quantification of LC20 phosphorylation at T18 and S19. Unique precursor as well as y- and b-ion transitions were identified for unphosphorylated LC20-(TS), monophosphorylated LC20-(TpS) and diphosphorylated LC20-(pTpS) peptides. The MRM-MS assay could accurately define molar phosphorylation stoichiometries of S19 and T18 over a broad range (i.e., 0-2â¯molâ¯P/mol LC20). Correlations of the results for two quantification techniques indicate that the MRM-MS assay performs equally to Phos-tag SDS-PAGE for the determination of LC20 phosphorylation stoichiometry in arterial tissue samples. The MRM-MS technique provides a robust alternative to antibody-based detection systems for the quantification of LC20 phosphorylation.
Asunto(s)
Espectrometría de Masas/métodos , Músculo Liso Vascular/enzimología , Cadenas Ligeras de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Fragmentos de Péptidos/metabolismo , Procesamiento Proteico-Postraduccional , Cola (estructura animal)/irrigación sanguínea , Vasoconstricción , Animales , Arterias/enzimología , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/farmacología , Masculino , Toxinas Marinas , Músculo Liso Vascular/efectos de los fármacos , Oxazoles/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Proteolisis , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/farmacologíaRESUMEN
Phosphorylation of the myosin-targeting subunit 1 of myosin light chain phosphatase (MYPT1) plays an important role in the regulation of smooth muscle contraction, and several sites of phosphorylation by different protein Ser/Thr kinases have been identified. Furthermore, in some instances, phosphorylation at specific sites affects phosphorylation at neighboring sites, with functional consequences. Characterization of the complex phosphorylation of MYPT1 in tissue samples at rest and in response to contractile and relaxant stimuli is, therefore, challenging. We have exploited Phos-tag SDS-PAGE in combination with Western blotting using antibodies to MYPT1, including phosphospecific antibodies, to separate multiple phosphorylated MYPT1 species and quantify MYPT1 phosphorylation stoichiometry using purified, full-length recombinant MYPT1 phosphorylated by Rho-associated coiled-coil kinase (ROCK) and cAMP-dependent protein kinase (PKA). This approach confirmed that phosphorylation of MYPT1 by ROCK occurs at Thr(697)and Thr(855), PKA phosphorylates these two sites and the neighboring Ser(696)and Ser(854), and prior phosphorylation at Thr(697)and Thr(855)by ROCK precludes phosphorylation at Ser(696)and Ser(854)by PKA. Furthermore, phosphorylation at Thr(697)and Thr(855)by ROCK exposes two other sites of phosphorylation by PKA. Treatment of Triton-skinned rat caudal arterial smooth muscle strips with the membrane-impermeant phosphatase inhibitor microcystin or treatment of intact tissue with the membrane-permeant phosphatase inhibitor calyculin A induced slow, sustained contractions that correlated with phosphorylation of MYPT1 at 7 to ≥10 sites. Phos-tag SDS-PAGE thus provides a suitable and convenient method for analysis of the complex, multisite MYPT1 phosphorylation events involved in the regulation of myosin light chain phosphatase activity and smooth muscle contraction.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/química , Electroforesis en Gel de Poliacrilamida/métodos , Fosfatasa de Miosina de Cadena Ligera/química , Mapeo de Interacción de Proteínas/métodos , Quinasas Asociadas a rho/química , Animales , Sitios de Unión , Activación Enzimática , Masculino , Fosforilación , Unión Proteica , Subunidades de Proteína/química , Ratas , Ratas Sprague-DawleyRESUMEN
A novel inhibitor of zipper-interacting protein kinase (ZIPK) was used to examine the involvement of ZIPK in the regulation of smooth muscle contraction. Pretreatment of de-endothelialized rat caudal arterial smooth muscle strips with the pyrazolo[3,4-d]pyrimidinone inhibitor 2-((1-(3-chlorophenyl)-4-oxo-4,5-dihydro-1H-pyrazolo [3,4-d]-pyrimidin-6-yl)thio)propanamide (HS38) decreased the velocity of contraction (time to reach half-maximal force) induced by the phosphatase inhibitor calyculin A in the presence of Ca(2+) without affecting maximal force development. This effect was reversed following washout of HS38 and correlated with a reduction in the rate of phosphorylation of myosin 20-kDa regulatory light chains (LC20) but not of protein kinase C-potentiated inhibitory protein for myosin phosphatase of 17 kDa (CPI-17), prostate apoptosis response-4, or myosin phosphatase-targeting subunit 1 (MYPT1), all of which have been implicated in the regulation of vascular contractility. A structural analog of HS38, with inhibitory activity toward proviral integrations of Moloney (PIM) virus 3 kinase but not ZIPK, had no effect on calyculin A-induced contraction or protein phosphorylations. We conclude that a pool of constitutively active ZIPK is involved in regulation of vascular smooth muscle contraction through direct phosphorylation of LC20 upon inhibition of myosin light chain phosphatase activity. HS38 also significantly attenuated both phasic and tonic contractile responses elicited by phenylephrine, angiotensin II, endothelin-1, U46619, and K(+)-induced membrane depolarization in the presence of Ca(2+), which correlated with inhibition of phosphorylation of LC20, MYPT1, and CPI-17. These effects of HS38 suggest that ZIPK also lies downstream from G protein-coupled receptors that signal through both Gα12/13 and Gαq/11.
Asunto(s)
Calcio/metabolismo , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Quinasas Quinasa Quinasa PAM/metabolismo , Músculo Liso Vascular/enzimología , Pirazoles/farmacología , Pirimidinas/farmacología , Animales , Masculino , Músculo Liso Vascular/efectos de los fármacos , Técnicas de Cultivo de Órganos , Pirazoles/química , Pirimidinas/química , Pirimidinonas/química , Pirimidinonas/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Depolarization of the vascular smooth muscle cell membrane evokes a rapid (phasic) contractile response followed by a sustained (tonic) contraction. We showed previously that the sustained contraction involves genistein-sensitive tyrosine phosphorylation upstream of the RhoA/Rho-associated kinase (ROK) pathway leading to phosphorylation of MYPT1 (the myosin-targeting subunit of myosin light chain phosphatase (MLCP)) and myosin regulatory light chains (LC20). In this study, we addressed the hypothesis that membrane depolarization elicits activation of the Ca(2+)-dependent tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2). Pyk2 was identified as the major tyrosine-phosphorylated protein in response to membrane depolarization. The tonic phase of K(+)-induced contraction was inhibited by the Pyk2 inhibitor sodium salicylate, which abolished the sustained elevation of LC20 phosphorylation. Membrane depolarization induced autophosphorylation (activation) of Pyk2 with a time course that correlated with the sustained contractile response. The Pyk2/focal adhesion kinase (FAK) inhibitor PF-431396 inhibited both phasic and tonic components of the contractile response to K(+), Pyk2 autophosphorylation, and LC20 phosphorylation but had no effect on the calyculin A (MLCP inhibitor)-induced contraction. Ionomycin, in the presence of extracellular Ca(2+), elicited a slow, sustained contraction and Pyk2 autophosphorylation, which were blocked by pre-treatment with PF-431396. Furthermore, the Ca(2+) channel blocker nifedipine inhibited peak and sustained K(+)-induced force and Pyk2 autophosphorylation. Inhibition of Pyk2 abolished the K(+)-induced translocation of RhoA to the particulate fraction and the phosphorylation of MYPT1 at Thr-697 and Thr-855. We conclude that depolarization-induced entry of Ca(2+) activates Pyk2 upstream of the RhoA/ROK pathway, leading to MYPT1 phosphorylation and MLCP inhibition. The resulting sustained elevation of LC20 phosphorylation then accounts for the tonic contractile response to membrane depolarization.
Asunto(s)
Quinasa 2 de Adhesión Focal/metabolismo , Músculo Liso Vascular/fisiología , Animales , Electroforesis en Gel de Poliacrilamida , Masculino , Contracción Muscular/fisiología , Músculo Liso Vascular/enzimología , Fosforilación , Ratas , Ratas WistarRESUMEN
OsCaM61 is one of five calmodulins known to be present in Oryza sativa that relays the increase of cytosolic [Ca(2+)] to downstream targets. OsCaM61 bears a unique C-terminal extension with a prenylation site. Using nuclear magnetic resonance (NMR) spectroscopy we studied the behavior of the calmodulin (CaM) domain and the C-terminal extension of OsCaM61 in the absence and presence of Ca(2+). NMR dynamics data for OsCaM61 indicate that the two lobes of the CaM domain act together unlike the independent behavior of the lobes seen in mammalian CaM and soybean CaM4. Also, data demonstrate that the positively charged nuclear localization signal region in the tail in apo-OsCaM61 is helical, whereas it becomes flexible in the Ca(2+)-saturated protein. The extra helix in apo-OsCaM61 provides additional interactions in the C-lobe and increases the structural stability of the closed apo conformation. This leads to a decrease in the Ca(2+) binding affinity of EF-hands III and IV in OsCaM61. In Ca(2+)-OsCaM61, the basic nuclear localization signal cluster adopts an extended conformation, exposing the C-terminal extension for prenylation or enabling OsCaM61 to be transferred to the nucleus. Moreover, Ser(172) and Ala(173), residues in the tail, interact with different regions of the protein. These interactions affect the ability of OsCaM61 to activate different target proteins. Altogether, our data show that the tail is not simply a linker between the prenyl group and the protein but that it also provides a new regulatory mechanism that some plants have developed to fine-tune Ca(2+) signaling events.
Asunto(s)
Señalización del Calcio/fisiología , Calmodulina/química , Oryza/química , Proteínas de Plantas/química , Prenilación de Proteína/fisiología , Animales , Calmodulina/genética , Calmodulina/metabolismo , Señales de Localización Nuclear , Resonancia Magnética Nuclear Biomolecular , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Glycine max/química , Glycine max/genética , Glycine max/metabolismoRESUMEN
During activation of smooth muscle contraction, one myosin light chain kinase (MLCK) molecule rapidly phosphorylates many smooth muscle myosin (SMM) molecules, suggesting that muscle activation rates are influenced by the kinetics of MLCK-SMM interactions. To determine the rate-limiting step underlying activation of SMM by MLCK, we measured the kinetics of calcium-calmodulin (Ca²âºCaM)-MLCK-mediated SMM phosphorylation and the corresponding initiation of SMM-based F-actin motility in an in vitro system with SMM attached to a coverslip surface. Fitting the time course of SMM phosphorylation to a kinetic model gave an initial phosphorylation rate, kp(o), of ~1.17 heads s⻹ MLCK⻹. Also, we measured the dwell time of single streptavidin-coated quantum dot-labeled MLCK molecules interacting with surface-attached SMM and phosphorylated SMM using total internal reflection fluorescence microscopy. From these data, the dissociation rate constant from phosphorylated SMM was 0.80 s⻹, which was similar to the kp(o) mentioned above and with rates measured in solution. This dissociation rate was essentially independent of the phosphorylation state of SMM. From calculations using our measured dissociation rates and Kd values, and estimates of SMM and MLCK concentrations in muscle, we predict that the dissociation of MLCK from phosphorylated SMM is rate-limiting and that the rate of the phosphorylation step is faster than this dissociation rate. Also, association with SMM (11-46 s⻹) would be much faster than with pSMM (<0.1-0.2 s⻹). This suggests that the probability of MLCK interacting with unphosphorylated versus phosphorylated SMM is 55-460 times greater. This would avoid sequestering MLCK to unproductive interactions with previously phosphorylated SMM, potentially leading to faster rates of phosphorylation in muscle.
Asunto(s)
Quinasa de Cadena Ligera de Miosina/metabolismo , Miosinas del Músculo Liso/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinas/química , Actinas/metabolismo , Animales , Señalización del Calcio , Calmodulina/metabolismo , Pollos , Activación Enzimática , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/metabolismo , Cinética , Quimografía , Metilcelulosa/química , Metilcelulosa/metabolismo , Microscopía Fluorescente , Quinasa de Cadena Ligera de Miosina/química , Fosforilación , Procesamiento Proteico-Postraduccional , Miosinas del Músculo Liso/química , Propiedades de SuperficieRESUMEN
The principal signal to activate smooth muscle contraction is phosphorylation of the regulatory light chains of myosin (LC(20)) at Ser(19) by Ca(2+)/calmodulin-dependent myosin light chain kinase. Inhibition of myosin light chain phosphatase leads to Ca(2+)-independent phosphorylation at both Ser(19) and Thr(18) by integrin-linked kinase and/or zipper-interacting protein kinase. The functional effects of phosphorylation at Thr(18) on steady-state isometric force and relaxation rate were investigated in Triton-skinned rat caudal arterial smooth muscle strips. Sequential phosphorylation at Ser(19) and Thr(18) was achieved by treatment with adenosine 5'-O-(3-thiotriphosphate) in the presence of Ca(2+), which induced stoichiometric thiophosphorylation at Ser(19), followed by microcystin (phosphatase inhibitor) in the absence of Ca(2+), which induced phosphorylation at Thr(18). Phosphorylation at Thr(18) had no effect on steady-state force induced by Ser(19) thiophosphorylation. However, phosphorylation of Ser(19) or both Ser(19) and Thr(18) to comparable stoichiometries (0.5 mol of P(i)/mol of LC(20)) and similar levels of isometric force revealed differences in the rates of dephosphorylation and relaxation following removal of the stimulus: t(½) values for dephosphorylation were 83.3 and 560 s, and for relaxation were 560 and 1293 s, for monophosphorylated (Ser(19)) and diphosphorylated LC(20), respectively. We conclude that phosphorylation at Thr(18) decreases the rates of LC(20) dephosphorylation and smooth muscle relaxation compared with LC(20) phosphorylated exclusively at Ser(19). These effects of LC(20) diphosphorylation, combined with increased Ser(19) phosphorylation (Ca(2+)-independent), may underlie the hypercontractility that is observed in response to certain physiological contractile stimuli, and under pathological conditions such as cerebral and coronary arterial vasospasm, intimal hyperplasia, and hypertension.
Asunto(s)
Arterias/metabolismo , Arterias/fisiología , Relajación Muscular/fisiología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiología , Cadenas Ligeras de Miosina/metabolismo , Fosforilación/fisiología , Animales , Técnicas In Vitro , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Ca(2+) sensitization of smooth muscle contraction depends upon the activities of protein kinases, including Rho-associated kinase, that phosphorylate the myosin phosphatase targeting subunit (MYPT1) at Thr(697) and/or Thr(855) (rat sequence numbering) to inhibit phosphatase activity and increase contractile force. Both Thr residues are preceded by the sequence RRS, and it has been suggested that phosphorylation at Ser(696) prevents phosphorylation at Thr(697). However, the effects of Ser(854) and dual Ser(696)-Thr(697) and Ser(854)-Thr(855) phosphorylations on myosin phosphatase activity and contraction are unknown. We characterized a suite of MYPT1 proteins and phosphospecific antibodies for specificity toward monophosphorylation events (Ser(696), Thr(697), Ser(854), and Thr(855)), Ser phosphorylation events (Ser(696)/Ser(854)) and dual Ser/Thr phosphorylation events (Ser(696)-Thr(697) and Ser(854)-Thr(855)). Dual phosphorylation at Ser(696)-Thr(697) and Ser(854)-Thr(855) by cyclic nucleotide-dependent protein kinases had no effect on myosin phosphatase activity, whereas phosphorylation at Thr(697) and Thr(855) by Rho-associated kinase inhibited phosphatase activity and prevented phosphorylation by cAMP-dependent protein kinase at the neighboring Ser residues. Forskolin induced phosphorylation at Ser(696), Thr(697), Ser(854), and Thr(855) in rat caudal artery, whereas U46619 induced Thr(697) and Thr(855) phosphorylation and prevented the Ser phosphorylation induced by forskolin. Furthermore, pretreatment with forskolin prevented U46619-induced Thr phosphorylations. We conclude that cross-talk between cyclic nucleotide and RhoA signaling pathways dictates the phosphorylation status of the Ser(696)-Thr(697) and Ser(854)-Thr(855) inhibitory regions of MYPT1 in situ, thereby regulating the activity of myosin phosphatase and contraction.
Asunto(s)
AMP Cíclico/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteína Fosfatasa 1/metabolismo , Sistemas de Mensajero Secundario/fisiología , Miosinas del Músculo Liso/metabolismo , Quinasas Asociadas a rho/metabolismo , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Sustitución de Aminoácidos , Animales , Colforsina/farmacología , AMP Cíclico/genética , Masculino , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Mutación Missense , Miocitos del Músculo Liso/citología , Fosforilación/efectos de los fármacos , Proteína Fosfatasa 1/genética , Ratas , Ratas Sprague-Dawley , Sistemas de Mensajero Secundario/efectos de los fármacos , Miosinas del Músculo Liso/genética , Vasoconstrictores/farmacología , Quinasas Asociadas a rho/genética , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
The protein prostate-apoptosis response (Par)-4 has been implicated in the regulation of smooth muscle contraction, based largely on studies with the A7r5 cell line. A mechanism has been proposed whereby Par-4 binding to MYPT1 (the myosin-targeting subunit of myosin light chain phosphatase, MLCP) blocks access of zipper-interacting protein kinase (ZIPK) to Thr697 and Thr855 of MYPT1, whose phosphorylation is associated with MLCP inhibition. Phosphorylation of Par-4 at Thr155 disrupts its interaction with MYPT1, exposing the sites of phosphorylation in MYPT1 and leading to MLCP inhibition and contraction. We tested this "padlock" hypothesis in a well-characterized vascular smooth muscle system, the rat caudal artery. Par-4 was retained in Triton-skinned tissue, suggesting a tight association with the contractile machinery, and indeed Par-4 co-immunoprecipitated with MYPT1. Treatment of Triton-skinned tissue with the phosphatase inhibitor microcystin (MC) evoked phosphorylation of Par-4 at Thr155, but did not induce its dissociation from the contractile machinery. Furthermore, analysis of the time courses of MC-induced phosphorylation of MYPT1 and Par-4 revealed that MYPT1 phosphorylation at Thr697 or Thr855 preceded Par-4 phosphorylation. Par-4 phosphorylation was inhibited by the non-selective kinase inhibitor staurosporine, but not by inhibitors of ZIPK, Rho-associated kinase or protein kinase C. In addition, Par-4 phosphorylation did not occur upon addition of constitutively-active ZIPK to skinned tissue. We conclude that phosphorylation of Par-4 does not regulate contraction of this vascular smooth muscle tissue by inducing dissociation of Par-4 from MYPT1 to allow phosphorylation of MYPT1 and inhibition of MLCP.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Músculo Liso Vascular/metabolismo , Proteína Fosfatasa 1/metabolismo , Amidas/farmacología , Animales , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Arterias/efectos de los fármacos , Arterias/metabolismo , Western Blotting , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Asociadas a Muerte Celular , Inmunoprecipitación , Indoles/farmacología , Masculino , Maleimidas/farmacología , Microcistinas/farmacología , Contracción Muscular , Músculo Liso Vascular/efectos de los fármacos , Octoxinol/metabolismo , Fosforilación , Unión Proteica , Proteína Fosfatasa 1/genética , Piridinas/farmacología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/farmacología , Estaurosporina/farmacología , Treonina/metabolismo , Factores de Tiempo , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
Myosin regulatory light chain (LC20) phosphorylation plays an important role in vascular smooth muscle contraction and cell migration. Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates LC20 (its only known substrate) exclusively at S19. Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in the regulation of LC20 phosphorylation via direct phosphorylation of LC20 at T18 and S19 and indirectly via phosphorylation of MYPT1 (the myosin targeting subunit of myosin light chain phosphatase, MLCP) and Par-4 (prostate-apoptosis response-4). Phosphorylation of MYPT1 at T696 and T853 inhibits MLCP activity whereas phosphorylation of Par-4 at T163 disrupts its interaction with MYPT1, exposing the sites of phosphorylation in MYPT1 and leading to MLCP inhibition. To evaluate the roles of MLCK, ROCK and ZIPK in these phosphorylation events, we investigated the time courses of phosphorylation of LC20, MYPT1 and Par-4 in serum-stimulated human vascular smooth muscle cells (from coronary and umbilical arteries), and examined the effects of siRNA-mediated MLCK, ROCK and ZIPK knockdown and pharmacological inhibition on these phosphorylation events. Serum stimulation induced rapid phosphorylation of LC20 at T18 and S19, MYPT1 at T696 and T853, and Par-4 at T163, peaking within 30-120 s. MLCK knockdown or inhibition, or Ca2+ chelation with EGTA, had no effect on serum-induced LC20 phosphorylation. ROCK knockdown decreased the levels of phosphorylation of LC20 at T18 and S19, of MYPT1 at T696 and T853, and of Par-4 at T163, whereas ZIPK knockdown decreased LC20 diphosphorylation, but increased phosphorylation of MYPT1 at T696 and T853 and of Par-4 at T163. ROCK inhibition with GSK429286A reduced serum-induced phosphorylation of LC20 at T18 and S19, MYPT1 at T853 and Par-4 at T163, while ZIPK inhibition by HS38 reduced only LC20 diphosphorylation. We also demonstrated that serum stimulation induced phosphorylation (activation) of ZIPK, which was inhibited by ROCK and ZIPK down-regulation and inhibition. Finally, basal phosphorylation of LC20 in the absence of serum stimulation was unaffected by MLCK, ROCK or ZIPK knockdown or inhibition. We conclude that: (i) serum stimulation of cultured human arterial smooth muscle cells results in rapid phosphorylation of LC20, MYPT1, Par-4 and ZIPK, in contrast to the slower phosphorylation of kinases and other proteins involved in other signaling pathways (Akt, ERK1/2, p38 MAPK and HSP27), (ii) ROCK and ZIPK, but not MLCK, are involved in serum-induced phosphorylation of LC20, (iii) ROCK, but not ZIPK, directly phosphorylates MYPT1 at T853 and Par-4 at T163 in response to serum stimulation, (iv) ZIPK phosphorylation is enhanced by serum stimulation and involves phosphorylation by ROCK and autophosphorylation, and (v) basal phosphorylation of LC20 under serum-free conditions is not attributable to MLCK, ROCK or ZIPK.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Quinasas Asociadas a Muerte Celular/metabolismo , Músculo Liso Vascular/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Miosinas/metabolismo , Quinasas Asociadas a rho/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Arterias/citología , Arterias/metabolismo , Células Cultivadas , Proteínas Quinasas Asociadas a Muerte Celular/antagonistas & inhibidores , Proteínas Quinasas Asociadas a Muerte Celular/genética , Humanos , Músculo Liso Vascular/citología , Quinasa de Cadena Ligera de Miosina/antagonistas & inhibidores , Quinasa de Cadena Ligera de Miosina/genética , Fosfatasa de Miosina de Cadena Ligera/genética , Fosforilación , ARN Interferente Pequeño/genética , Suero/metabolismo , Transducción de Señal , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/genéticaRESUMEN
Sustained vascular smooth muscle hypercontractility promotes hypertension and cardiovascular disease. The etiology of hypercontractility is not completely understood. New therapeutic targets remain vitally important for drug discovery. Here we report that Pim kinases, in combination with DAPK3, regulate contractility and control hypertension. Using a co-crystal structure of lead molecule (HS38) in complex with DAPK3, a dual Pim/DAPK3 inhibitor (HS56) and selective DAPK3 inhibitors (HS94 and HS148) were developed to provide mechanistic insight into the polypharmacology of hypertension. In vitro and ex vivo studies indicated that Pim kinases directly phosphorylate smooth muscle targets and that Pim/DAPK3 inhibition, unlike selective DAPK3 inhibition, significantly reduces contractility. In vivo, HS56 decreased blood pressure in spontaneously hypertensive mice in a dose-dependent manner without affecting heart rate. These findings suggest including Pim kinase inhibition within a multi-target engagement strategy for hypertension management. HS56 represents a significant step in the development of molecularly targeted antihypertensive medications.
Asunto(s)
Proteínas Quinasas Asociadas a Muerte Celular/antagonistas & inhibidores , Hipertensión/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Presión Sanguínea/efectos de los fármacos , Cristalografía por Rayos X , Proteínas Quinasas Asociadas a Muerte Celular/química , Proteínas Quinasas Asociadas a Muerte Celular/metabolismo , Humanos , Hipertensión/metabolismo , Hipertensión/fisiopatología , Masculino , Ratones , Modelos Moleculares , Terapia Molecular Dirigida , Contracción Muscular/efectos de los fármacos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-pim-1/química , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Ratas Sprague-Dawley , Alineación de SecuenciaRESUMEN
Hyperphosphorylation of the cardiac Ca2+ release channel (ryanodine receptor, RyR2) by protein kinase A (PKA) at serine-2808 has been proposed to be a key mechanism responsible for cardiac dysfunction in heart failure (HF). However, the sites of PKA phosphorylation in RyR2 and their phosphorylation status in HF are not well defined. Here we used various approaches to investigate the phosphorylation of RyR2 by PKA. Mutating serine-2808, which was thought to be the only PKA phosphorylation site in RyR2, did not abolish the phosphorylation of RyR2 by PKA. Two-dimensional phosphopeptide mapping revealed two major PKA phosphopeptides, one of which corresponded to the known serine-2808 site. Another, novel, PKA phosphorylation site, serine 2030, was identified by Edman sequencing. Using phospho-specific antibodies, we showed that the novel serine-2030 site was phosphorylated in rat cardiac myocytes stimulated with isoproterenol, but not in unstimulated cells, whereas serine-2808 was considerably phosphorylated before and after isoproterenol treatment. We further showed that serine-2030 was stoichiometrically phosphorylated by PKA, but not by CaMKII, and that mutations of serine-2030 altered neither the FKBP12.6-RyR2 interaction nor the Ca2+ dependence of [3H]ryanodine binding. Moreover, the levels of phosphorylation of RyR2 at serine-2030 and serine-2808 in both failing and non-failing canine hearts were similar. Together, our data indicate that serine-2030 is a major PKA phosphorylation site in RyR2 responding to acute beta-adrenergic stimulation, and that RyR2 is not hyperphosphorylated by PKA in canine HF.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Insuficiencia Cardíaca/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Animales , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Perros , Isoproterenol/farmacología , Miocitos Cardíacos/metabolismo , Fosforilación , Serina/metabolismoRESUMEN
CPI-17 is a cytosolic protein of 17 kDa that becomes a potent inhibitor of certain type 1 protein serine/threonine phosphatases, including smooth muscle myosin light-chain phosphatase (MLCP), when phosphorylated at Thr38. Several protein kinases are capable of phosphorylating CPI-17 at this site in vitro; however, in intact tissue, compelling evidence only exists for phosphorylation by protein kinase C (PKC). Agonist-induced activation of heterotrimeric G proteins of the Gq/11 family via seven-transmembrane domain-containing, G protein-coupled receptors results in phospholipase Cbeta-mediated hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG). IP3 triggers Ca2+ release from the sarcoplasmic reticulum. DAG and Ca2+ together activate classical isoforms of PKC, and DAG activates novel PKC isoforms without a requirement for Ca2+. Activated PKC phosphorylates CPI-17 at Thr38, enhancing its potency of inhibition of MLCP approx 1000-fold. The myosin light-chain kinase (MLCK):MLCP activity ratio is thereby increased at the prevailing cytosolic free-Ca2+ concentration ([Ca2+]i), resulting in an increase in phosphorylation of the 20-kDa light chains of myosin II (LC20) catalyzed by Ca2+- and calmodulin-dependent MLCK and contraction of the smooth muscle. Physiologically, this mechanism can account for some instances of Ca2+ sensitization of smooth muscle contraction (i.e., an increase in force in response to agonist stimulation without a change in [Ca2+]i).
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Fosfoproteínas Fosfatasas/metabolismo , Fosfoproteínas/metabolismo , Proteína Quinasa C/metabolismo , Animales , Calcio/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Fosforilación , PorcinosRESUMEN
Dissociation of FKBP12.6 from the cardiac Ca2+-release channel (RyR2) as a consequence of protein kinase A (PKA) hyperphosphorylation of RyR2 at a single amino acid residue, serine-2808, has been proposed as an important mechanism underlying cardiac dysfunction in heart failure. However, the issue of whether PKA phosphorylation of RyR2 can dissociate FKBP12.6 from RyR2 is controversial. To additionally address this issue, we investigated the effect of PKA phosphorylation and mutations at serine-2808 of RyR2 on recombinant or native FKBP12.6-RyR2 interaction. Site-specific antibodies, which recognize the serine-2808 phosphorylated or nonphosphorylated form of RyR2, were used to unambiguously correlate the phosphorylation state of RyR2 at serine-2808 with its ability to bind FKBP12.6. We found that FKBP12.6 can bind to both the serine-2808 phosphorylated and nonphosphorylated forms of RyR2. The S2808D mutant thought to mimic constitutive phosphorylation also retained the ability to bind FKBP12.6. Complete phosphorylation at serine-2808 by exogenous PKA disrupted neither the recombinant nor native FKBP12.6-RyR2 complex. Furthermore, binding of site-specific antibodies to the serine-2808 phosphorylation site did not dissociate FKBP12.6 from or prevent FKBP12.6 from binding to RyR2. Taken together, our results do not support the notion that PKA phosphorylation at serine-2808 dissociates FKBP12.6 from RyR2.
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Señalización del Calcio/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Mutación Missense , Miocitos Cardíacos/metabolismo , Mutación Puntual , Procesamiento Proteico-Postraduccional , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Proteínas de Unión a Tacrolimus/metabolismo , Sustitución de Aminoácidos , Animales , Reacciones Antígeno-Anticuerpo , Señalización del Calcio/genética , Línea Celular , Perros , Humanos , Riñón , Sustancias Macromoleculares , Ratones , Miocitos Cardíacos/efectos de los fármacos , Fosforilación , Fosfoserina/química , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/química , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/inmunología , Retículo Sarcoplasmático/metabolismo , Sirolimus/farmacología , Relación Estructura-ActividadRESUMEN
The signal transduction pathway whereby the TxA2 (thromboxane A2) mimetic U-46619 activates vascular smooth muscle contraction was investigated in de-endothelialized rat caudal artery. U-46619-evoked contraction was inhibited by the TP receptor (TxA2 receptor) antagonist SQ-29548, the ROK (Rho-associated kinase) inhibitors Y-27632 and H-1152, the MLCK (myosin light-chain kinase) inhibitors ML-7, ML-9 and wortmannin, the voltagegated Ca2+-channel blocker nicardipine, and removal of extracellular Ca2+; the protein kinase C inhibitor GF109203x had no effect. U-46619 elicited Ca2+ sensitization in a-toxin-permeabilized tissue. U-46619 induced activation of the small GTPase RhoA, consistent with the involvement of ROK. Two downstream targets of ROK were investigated: CPI-17 [protein kinase C-potentiated inhibitory protein for PP1 (protein phosphatase type 1) of 17 kDa], a myosin light-chain phosphatase inhibitor, was not phosphorylated at the functional site (Thr-38); phosphorylation of MYPT1 (myosin-targeting subunit of myosin light-chain phosphatase) was significantly increased at Thr-855, but not Thr-697. U-46619-evoked contraction correlated with phosphorylation of the 20 kDa light chains of myosin. We conclude that: (i) U-46619 induces contraction via activation of the Ca2+/calmodulin/MLCK pathway and of the RhoA/ROK pathway; (ii) Thr-855 of MYPT1 is phosphorylated by ROK at rest and in response to U-46619 stimulation; (iii) Thr-697 of MYPT1 is phosphorylated by a kinase other than ROK under resting conditions, and is not increased in response to U-46619 treatment; and (iv) neither ROK nor protein kinase C phosphorylates CPI-17 in this vascular smooth muscle in response to U-46619.
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Señalización del Calcio , Calcio/fisiología , Proteínas Portadoras/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Tromboxano A2/fisiología , Vasoconstricción/fisiología , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Secuencia de Aminoácidos , Animales , Arterias/fisiología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L , Proteínas Portadoras/química , Péptidos y Proteínas de Señalización Intracelular , Proteínas Musculares/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Quinasa de Cadena Ligera de Miosina/metabolismo , Fosfoproteínas Fosfatasas/química , Fosfoproteínas/metabolismo , Fosforilación , Proteína Fosfatasa 1 , Ratas , Tromboxano A2/antagonistas & inhibidores , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/farmacología , Quinasas Asociadas a rhoRESUMEN
Smooth muscle contraction is activated by phosphorylation at Ser-19 of LC20 (the 20 kDa light chains of myosin II) by Ca2+/calmodulin-dependent MLCK (myosin light-chain kinase). Diphosphorylation of LC20 at Ser-19 and Thr-18 is observed in smooth muscle tissues and cultured cells in response to various contractile stimuli, and in pathological circumstances associated with hypercontractility. MLCP (myosin light-chain phosphatase) inhibition can lead to LC20 diphosphorylation and Ca2+-independent contraction, which is not attributable to MLCK. Two kinases have emerged as candidates for Ca2+-independent LC20 diphosphorylation: ILK (integrin-linked kinase) and ZIPK (zipper-interacting protein kinase). Triton X-100-skinned rat caudal arterial smooth muscle was used to investigate the relative importance of ILK and ZIPK in Ca2+-independent, microcystin (phosphatase inhibitor)-induced LC20 diphosphorylation and contraction. Western blotting and in-gel kinase assays revealed that both kinases were retained in this preparation. Ca2+-independent contraction of calmodulin-depleted tissue in response to microcystin was resistant to MLCK inhibitors [AV25 (a 25-amino-acid peptide derived from the autoinhibitory domain of MLCK), ML-7, ML-9 and wortmannin], protein kinase C inhibitor (GF109203X) and Rho-associated kinase inhibitors (Y-27632 and H-1152), but blocked by the non-selective kinase inhibitor staurosporine. ZIPK was inhibited by AV25 (IC50 0.63+/-0.05 microM), whereas ILK was insensitive to AV25 (at concentrations as high as 100 microM). AV25 had no effect on Ca2+-independent, microcystin-induced LC20 mono- or di-phosphorylation, with a modest effect on force. We conclude that direct inhibition of MLCP in the absence of Ca2+ unmasks ILK activity, which phosphorylates LC20 at Ser-19 and Thr-18 to induce contraction. ILK is probably the kinase responsible for myosin diphosphorylation in vascular smooth muscle cells and tissues.
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Calcio/metabolismo , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/fisiología , Miosinas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Calcio/farmacología , Quinasas Quinasa Quinasa PAM , Microcistinas , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Péptidos Cíclicos/farmacología , Fosforilación/efectos de los fármacos , RatasRESUMEN
Smooth muscle contraction is activated primarily by phosphorylation at Ser19 of the regulatory light chain subunits (LC20) of myosin II, catalysed by Ca(2+)/calmodulin-dependent myosin light chain kinase. Ca(2+)-independent contraction can be induced by inhibition of myosin light chain phosphatase, which correlates with diphosphorylation of LC20 at Ser19 and Thr18, catalysed by integrin-linked kinase (ILK) and zipper-interacting protein kinase (ZIPK). LC20 diphosphorylation at Ser19 and Thr18 has been detected in mammalian vascular smooth muscle tissues in response to specific contractile stimuli (e.g. endothelin-1 stimulation of rat renal afferent arterioles) and in pathophysiological situations associated with hypercontractility (e.g. cerebral vasospasm following subarachnoid hemorrhage). Comparison of the effects of LC 20 monophosphorylation at Ser19 and diphosphorylation at Ser19 and Thr18 on contraction and relaxation of Triton-skinned rat caudal arterial smooth muscle revealed that phosphorylation at Thr18 has no effect on steady-state force induced by Ser19 phosphorylation. On the other hand, the rates of dephosphorylation and relaxation are significantly slower following diphosphorylation at Thr18 and Ser19 compared to monophosphorylation at Ser19. We propose that this diphosphorylation mechanism underlies the prolonged contractile response of particular vascular smooth muscle tissues to specific stimuli, e.g. endothelin-1 stimulation of renal afferent arterioles, and the vasospastic behavior observed in pathological conditions such as cerebral vasospasm following subarachnoid hemorrhage and coronary arterial vasospasm. ILK and ZIPK may, therefore, be useful therapeutic targets for the treatment of such conditions.
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Músculo Liso Vascular/fisiología , Miosina Tipo II/química , Miosina Tipo II/fisiología , Vasoconstricción/genética , Lesión Renal Aguda/tratamiento farmacológico , Animales , Catálisis , Vasoespasmo Coronario/tratamiento farmacológico , Proteínas Quinasas Asociadas a Muerte Celular/fisiología , Proteínas Quinasas Asociadas a Muerte Celular/uso terapéutico , Endotelina-1/farmacología , Humanos , Hipertensión/tratamiento farmacológico , Microcirculación/efectos de los fármacos , Microcirculación/genética , Terapia Molecular Dirigida , Quinasa de Cadena Ligera de Miosina/fisiología , Fosfatasa de Miosina de Cadena Ligera/fisiología , Fosforilación , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Serina-Treonina Quinasas/uso terapéutico , Ratas , Circulación Renal/efectos de los fármacos , Circulación Renal/genética , Vasoespasmo Intracraneal/tratamiento farmacológicoRESUMEN
DAPK1 and ZIPK (also called DAPK3) are closely related serine/threonine protein kinases that regulate programmed cell death and phosphorylation of non-muscle and smooth muscle myosin. We have developed a fluorescence linked enzyme chemoproteomic strategy (FLECS) for the rapid identification of inhibitors for any element of the purinome and identified a selective pyrazolo[3,4-d]pyrimidinone (HS38) that inhibits DAPK1 and ZIPK in an ATP-competitive manner at nanomolar concentrations. In cellular studies, HS38 decreased RLC20 phosphorylation. In ex vivo studies, HS38 decreased contractile force generated in mouse aorta, rabbit ileum, and calyculin A stimulated arterial muscle by decreasing RLC20 and MYPT1 phosphorylation. The inhibitor also promoted relaxation in Ca(2+)-sensitized vessels. A close structural analogue (HS43) with 5-fold lower affinity for ZIPK produced no effect on cells or tissues. These findings are consistent with a mechanism of action wherein HS38 specifically targets ZIPK in smooth muscle. The discovery of HS38 provides a lead scaffold for the development of therapeutic agents for smooth muscle related disorders and a chemical means to probe the function of DAPK1 and ZIPK across species.