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1.
Microvasc Res ; 152: 104627, 2024 03.
Artigo em Inglês | MEDLINE | ID: mdl-37963515

RESUMO

AIMS: Protein kinase D (PKD), once considered an effector of protein kinase C (PKC), now plays many pathophysiological roles in various tissues. However, little is known about role of PKD in vascular function. We investigated the role of PKD in contraction of rat aorta and human aortic smooth muscle cells (HASMCs) and in haemodynamics in rats. METHODS AND RESULTS: Isometric tension of rat aortic was measured to examine norepinephrine-induced contraction in the presence of PKD, PKC and Rho-kinase inhibitors. Phosphorylation of PKD1, myosin targeting subunit-1 (MYPT1), myosin light chain (MLC), CPI-17 and heat-shock protein 27 (HSP27), and actin polymerization were measured in the aorta. Phosphorylation of MYPT1 and MLC was also measured in HASMCs knocked down with specific siRNAs of PKD 1, 2 and 3. Intracellular calcium concentrations and cell shortening were measured in HASMCs. Norepinephrine-induced aortic contraction was accompanied by increased phosphorylation of PKD1, MYPT1 and MLC and actin polymerization, all of which were attenuated with PKD inhibitor CRT0066101. PKD1 phosphorylation was not inhibited by PKC inhibitor, chelerythrine or Rho kinase inhibitor, fasudil. In HASMCs, the phosphorylation of MYPT1 and MLC was attenuated by PKD1, but not PKD2, 3 knockdown. In HASMCs, CRT0066101 inhibited norepinephrine-induced cell shortening without affecting calcium concentration. Administration of CRT0066101 decreased systemic vascular resistance and blood pressure without affecting cardiac output in rats. CONCLUSIONS: PKD1 may play roles in aorta contraction and haemodynamics via phosphorylation of MYPT1 and actin polymerization in a calcium-independent manner.


Assuntos
Actinas , Vasoconstrição , Animais , Humanos , Ratos , Actinas/metabolismo , Cálcio/metabolismo , Contração Muscular , Músculo Liso Vascular/metabolismo , Cadeias Leves de Miosina/metabolismo , Norepinefrina/farmacologia , Norepinefrina/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/metabolismo
2.
J Biol Chem ; 298(9): 102296, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35872014

RESUMO

Cardiac muscle myosin regulatory light chain (RLC) is constitutively phosphorylated at ∼0.4 mol phosphate/mol RLC in normal hearts, and phosphorylation is maintained by balanced activities of dedicated cardiac muscle-specific myosin light chain kinase and myosin light chain phosphatase (MLCP). Previously, the identity of the cardiac-MLCP was biochemically shown to be similar to the smooth muscle MLCP, which is a well-characterized trimeric protein comprising the regulatory subunit (MYPT1), catalytic subunit PP1cß, and accessory subunit M20. In smooth muscles in vivo, MYPT1 and PP1cß co-stabilize each other and are both necessary for normal smooth muscle contractions. In the cardiac muscle, MYPT1 and MYPT2 are both expressed, but contributions to physiological regulation of cardiac myosin dephosphorylation are unclear. We hypothesized that the main catalytic subunit for cardiac-MLCP is PP1cß, and maintenance of RLC phosphorylation in vivo is dependent on regulation by striated muscle-specific MYPT2. Here, we used PP1cß conditional knockout mice to biochemically define cardiac-MLCP proteins and developed a cardiac myofibrillar phosphatase assay to measure the direct contribution of MYPT-regulated and MYPT-independent phosphatase activities toward phosphorylated cardiac myosin. We report that (1) PP1cß is the main isoform expressed in the cardiac myocyte, (2) cardiac muscle pathogenesis in PP1cß knockout animals involve upregulation of total PP1cα in myocytes and non-muscle cells, (3) the stability of cardiac MYPT1 and MYPT2 proteins in vivo is not dependent on the PP1cß expression, and (4) phosphorylated myofibrillar cardiac myosin is dephosphorylated by both myosin-targeted and soluble MYPT-independent PP1cß activities. These results contribute to our understanding of the cardiac-MLCP in vivo.


Assuntos
Miosinas Cardíacas , Fosfatase de Miosina-de-Cadeia-Leve , Proteína Fosfatase 1 , Animais , Miosinas Cardíacas/metabolismo , Camundongos , Camundongos Knockout , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Fosfatos/metabolismo , Fosforilação , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo
3.
Am J Hum Genet ; 106(1): 121-128, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31883643

RESUMO

In two independent ongoing next-generation sequencing projects for individuals with holoprosencephaly and individuals with disorders of sex development, and through international research collaboration, we identified twelve individuals with de novo loss-of-function (LoF) variants in protein phosphatase 1, regulatory subunit 12a (PPP1R12A), an important developmental gene involved in cell migration, adhesion, and morphogenesis. This gene has not been previously reported in association with human disease, and it has intolerance to LoF as illustrated by a very low observed-to-expected ratio of LoF variants in gnomAD. Of the twelve individuals, midline brain malformations were found in five, urogenital anomalies in nine, and a combination of both phenotypes in two. Other congenital anomalies identified included omphalocele, jejunal, and ileal atresia with aberrant mesenteric blood supply, and syndactyly. Six individuals had stop gain variants, five had a deletion or duplication resulting in a frameshift, and one had a canonical splice acceptor site loss. Murine and human in situ hybridization and immunostaining revealed PPP1R12A expression in the prosencephalic neural folds and protein localization in the lower urinary tract at critical periods for forebrain division and urogenital development. Based on these clinical and molecular findings, we propose the association of PPP1R12A pathogenic variants with a congenital malformations syndrome affecting the embryogenesis of the brain and genitourinary systems and including disorders of sex development.


Assuntos
Anormalidades Múltiplas/patologia , Transtornos do Desenvolvimento Sexual/patologia , Holoprosencefalia/patologia , Mutação , Fosfatase de Miosina-de-Cadeia-Leve/genética , Anormalidades Urogenitais/patologia , Anormalidades Múltiplas/genética , Adolescente , Criança , Pré-Escolar , Transtornos do Desenvolvimento Sexual/genética , Feminino , Idade Gestacional , Holoprosencefalia/genética , Humanos , Masculino , Fenótipo , Gravidez , Anormalidades Urogenitais/genética
4.
Pharmacol Res ; 187: 106589, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36462727

RESUMO

Copy-number variations in the ARHGAP10 gene encoding Rho GTPase-activating protein 10 are associated with schizophrenia. Model mice (Arhgap10 S490P/NHEJ mice) that carry "double-hit" mutations in the Arhgap10 gene mimic the schizophrenia in a Japanese patient, exhibiting altered spine density, methamphetamine-induced cognitive dysfunction, and activation of RhoA/Rho-kinase signaling. However, it remains unclear whether the activation of RhoA/Rho-kinase signaling due to schizophrenia-associated Arhgap10 mutations causes the phenotypes of these model mice. Here, we investigated the effects of fasudil, a brain permeable Rho-kinase inhibitor, on altered spine density in the medial prefrontal cortex (mPFC) and on methamphetamine-induced cognitive impairment in a touchscreen­based visual discrimination task in Arhgap10 S490P/NHEJ mice. Fasudil (20 mg/kg, intraperitoneal) suppressed the increased phosphorylation of myosin phosphatase-targeting subunit 1, a substrate of Rho-kinase, in the striatum and mPFC of Arhgap10 S490P/NHEJ mice. In addition, daily oral administration of fasudil (20 mg/kg/day) for 7 days ameliorated the reduced spine density of layer 2/3 pyramidal neurons in the mPFC. Moreover, fasudil (3-20 mg/kg, intraperitoneal) rescued the methamphetamine (0.3 mg/kg)-induced cognitive impairment of visual discrimination in Arhgap10 S490P/NHEJ mice. Our results suggest that Rho-kinase plays significant roles in the neuropathological changes in spine morphology and in the vulnerability of cognition to methamphetamine in mice with schizophrenia-associated Arhgap10 mutations.


Assuntos
Disfunção Cognitiva , Esquizofrenia , Animais , Camundongos , Disfunção Cognitiva/induzido quimicamente , Disfunção Cognitiva/tratamento farmacológico , Disfunção Cognitiva/genética , Mutação , Córtex Pré-Frontal/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/metabolismo , Esquizofrenia/induzido quimicamente , Esquizofrenia/tratamento farmacológico , Esquizofrenia/genética
5.
Pharmacol Res ; 194: 106838, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37390993

RESUMO

Schizophrenia (SCZ) is a severe psychiatric disorder characterized by positive symptoms, negative symptoms, and cognitive deficits. Current antipsychotic treatment in SCZ improves positive symptoms but has major side effects and little impact on negative symptoms and cognitive impairment. The pathoetiology of SCZ remains unclear, but is known to involve small GTPase signaling. Rho kinase, an effector of small GTPase Rho, is highly expressed in the brain and plays a major role in neurite elongation and neuronal architecture. This study used a touchscreen-based visual discrimination (VD) task to investigate the effects of Rho kinase inhibitors on cognitive impairment in a methamphetamine (METH)-treated male mouse model of SCZ. Systemic injection of the Rho kinase inhibitor fasudil dose-dependently ameliorated METH-induced VD impairment. Fasudil also significantly suppressed the increase in the number of c-Fos-positive cells in the infralimbic medial prefrontal cortex (infralimbic mPFC) and dorsomedial striatum (DMS) following METH treatment. Bilateral microinjections of Y-27632, another Rho kinase inhibitor, into the infralimbic mPFC or DMS significantly ameliorated METH-induced VD impairment. Two proteins downstream of Rho kinase, myosin phosphatase-targeting subunit 1 (MYPT1; Thr696) and myosin light chain kinase 2 (MLC2; Thr18/Ser19), exhibited increased phosphorylation in the infralimbic mPFC and DMS, respectively, after METH treatment, and fasudil inhibited these increases. Oral administration of haloperidol and fasudil ameliorated METH-induced VD impairment, while clozapine had little effect. Oral administration of haloperidol and clozapine suppressed METH-induced hyperactivity, but fasudil had no effect. These results suggest that METH activates Rho kinase in the infralimbic mPFC and DMS, which leads to cognitive impairment in male mice. Rho kinase inhibitors ameliorate METH-induced cognitive impairment, perhaps via the cortico-striatal circuit.


Assuntos
Disfunção Cognitiva , Metanfetamina , Proteínas Monoméricas de Ligação ao GTP , Inibidores de Proteínas Quinases , Esquizofrenia , Animais , Masculino , Camundongos , Clozapina , Disfunção Cognitiva/tratamento farmacológico , Haloperidol/farmacologia , Haloperidol/uso terapêutico , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Esquizofrenia/induzido quimicamente , Esquizofrenia/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico
6.
Biochemistry (Mosc) ; 88(8): 1126-1138, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37758312

RESUMO

Hyperglycemia is a hallmark of type 2 diabetes implicated in vascular endothelial dysfunction and cardiovascular complications. Many in vitro studies identified endothelial apoptosis as an early outcome of experimentally modeled hyperglycemia emphasizing cell demise as a significant factor of vascular injury. However, endothelial apoptosis has not been observed in vivo until the late stages of type 2 diabetes. Here, we studied the long-term (up to 4 weeks) effects of high glucose (HG, 30 mM) on human umbilical vein endothelial cells (HUVEC) in vitro. HG did not alter HUVEC monolayer morphology, ROS levels, NO production, and exerted minor effects on the HUVEC apoptosis markers. The barrier responses to various clues were indistinguishable from those by cells cultured in physiological glucose (5 mM). Tackling the key regulators of cytoskeletal contractility and endothelial barrier revealed no differences in the histamine-induced intracellular Ca2+ responses, nor in phosphorylation of myosin regulatory light chain or myosin light chain phosphatase. Altogether, these findings suggest that vascular endothelial cells may well tolerate HG for relatively long exposures and warrant further studies to explore mechanisms involved in vascular damage in advanced type 2 diabetes.

7.
Int J Mol Sci ; 24(5)2023 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-36902219

RESUMO

Identification of specific protein phosphatase-1 (PP1) inhibitors is of special importance regarding the study of its cellular functions and may have therapeutic values in diseases coupled to signaling processes. In this study, we prove that a phosphorylated peptide of the inhibitory region of myosin phosphatase (MP) target subunit (MYPT1), R690QSRRS(pT696)QGVTL701 (P-Thr696-MYPT1690-701), interacts with and inhibits the PP1 catalytic subunit (PP1c, IC50 = 3.84 µM) and the MP holoenzyme (Flag-MYPT1-PP1c, IC50 = 3.84 µM). Saturation transfer difference NMR measurements established binding of hydrophobic and basic regions of P-Thr696-MYPT1690-701 to PP1c, suggesting interactions with the hydrophobic and acidic substrate binding grooves. P-Thr696-MYPT1690-701 was dephosphorylated by PP1c slowly (t1/2 = 81.6-87.9 min), which was further impeded (t1/2 = 103 min) in the presence of the phosphorylated 20 kDa myosin light chain (P-MLC20). In contrast, P-Thr696-MYPT1690-701 (10-500 µM) slowed down the dephosphorylation of P-MLC20 (t1/2 = 1.69 min) significantly (t1/2 = 2.49-10.06 min). These data are compatible with an unfair competition mechanism between the inhibitory phosphopeptide and the phosphosubstrate. Docking simulations of the PP1c-P-MYPT1690-701 complexes with phosphothreonine (PP1c-P-Thr696-MYPT1690-701) or phosphoserine (PP1c-P-Ser696-MYPT1690-701) suggested their distinct poses on the surface of PP1c. In addition, the arrangements and distances of the surrounding coordinating residues of PP1c around the phosphothreonine or phosphoserine at the active site were distinct, which may account for their different hydrolysis rate. It is presumed that P-Thr696-MYPT1690-701 binds tightly at the active center but the phosphoester hydrolysis is less preferable compared to P-Ser696-MYPT1690-701 or phosphoserine substrates. Moreover, the inhibitory phosphopeptide may serve as a template to synthesize cell permeable PP1-specific peptide inhibitors.


Assuntos
Inibidores Enzimáticos , Fosfopeptídeos , Proteína Fosfatase 1 , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Fosfopeptídeos/química , Fosfopeptídeos/farmacologia , Fosforilação , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Proteína Fosfatase 1/antagonistas & inibidores , Proteína Fosfatase 1/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia
8.
Int J Mol Sci ; 23(6)2022 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-35328517

RESUMO

Somatostatin is an inhibitory peptide, which regulates the release of several hormones, and affects neurotransmission and cell proliferation via its five Gi protein-coupled receptors (SST1-5). Although its endocrine regulatory and anti-tumour effects have been thoroughly studied, little is known about its effect on the vascular system. The aim of the present study was to analyse the effects and potential mechanisms of somatostatin on endothelial barrier function. Cultured human umbilical vein endothelial cells (HUVECs) express mainly SST1 and SST5 receptors. Somatostatin did not affect the basal HUVEC permeability, but primed HUVEC monolayers for thrombin-induced hyperpermeability. Western blot data demonstrated that somatostatin activated the phosphoinositide 3-kinases (PI3K)/protein kinase B (Akt) and p42/44 mitogen-activated protein kinase (MAPK) pathways by phosphorylation. The HUVEC barrier destabilizing effects were abrogated by pre-treating HUVECs with mitogen-activated protein kinase kinase/extracellular signal regulated kinase (MEK/ERK), but not the Akt inhibitor. Moreover, somatostatin pre-treatment amplified vascular endothelial growth factor (VEGF)-induced angiogenesis (3D spheroid formation) in HUVECs. In conclusion, the data demonstrate that HUVECs under quiescence conditions express SST1 and SST5 receptors. Moreover, somatostatin primes HUVECs for thrombin-induced hyperpermeability mainly via the activation of MEK/ERK signalling and promotes HUVEC proliferation and angiogenesis in vitro.


Assuntos
Proteínas Proto-Oncogênicas c-akt , Fator A de Crescimento do Endotélio Vascular , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neovascularização Patológica/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Somatostatina/metabolismo , Somatostatina/farmacologia , Trombina/metabolismo , Trombina/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo
9.
J Biol Chem ; 295(21): 7341-7349, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32295844

RESUMO

The role of O-linked N-acetylglucosamine (O-GlcNAc) modification in the cell cycle has been enigmatic. Previously, both O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) disruptions have been shown to derail the mitotic centrosome numbers, suggesting that mitotic O-GlcNAc oscillation needs to be in concert with mitotic progression to account for centrosome integrity. Here, using both chemical approaches and biological assays with HeLa cells, we attempted to address the underlying molecular mechanism and observed that incubation of the cells with the OGA inhibitor Thiamet-G strikingly elevates centrosomal distances, suggestive of premature centrosome disjunction. These aberrations could be overcome by inhibiting Polo-like kinase 1 (PLK1), a mitotic master kinase. PLK1 inactivation is modulated by the myosin phosphatase targeting subunit 1 (MYPT1)-protein phosphatase 1cß (PP1cß) complex. Interestingly, MYPT1 has been shown to be abundantly O-GlcNAcylated, and the modified residues have been detected in a recent O-GlcNAc-profiling screen utilizing chemoenzymatic labeling and bioorthogonal conjugation. We demonstrate here that MYPT1 is O-GlcNAcylated at Thr-577, Ser-585, Ser-589, and Ser-601, which antagonizes CDK1-dependent phosphorylation at Ser-473 and attenuates the association between MYPT1 and PLK1, thereby promoting PLK1 activity. We conclude that under high O-GlcNAc levels, PLK1 is untimely activated, conducive to inopportune centrosome separation and disruption of the cell cycle. We propose that too much O-GlcNAc is equally deleterious as too little O-GlcNAc, and a fine balance between the OGT/OGA duo is indispensable for successful mitotic divisions.


Assuntos
Centrossomo/metabolismo , Mitose , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Glicosilação , Humanos , Fosfatase de Miosina-de-Cadeia-Leve/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Quinase 1 Polo-Like
10.
Int J Mol Sci ; 22(5)2021 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-33802280

RESUMO

Monocyte to macrophage differentiation is characterized by the activation of various signal transduction pathways, which may be modulated by protein phosphorylation; however, the impact of protein kinases and phosphatases is not well understood yet. It has been demonstrated that actomyosin rearrangement during macrophage differentiation is dependent on Rho-associated protein kinase (ROCK). Myosin phosphatase (MP) target subunit-1 (MYPT1) is one of the major cellular substrates of ROCK, and MP is often a counter enzyme of ROCK; therefore, MP may also control macrophage differentiation. Changes in MP activity and the effects of MP activation were studied on PMA or l,25(OH)2D3-induced differentiation of monocytic THP-1 cells. During macrophage differentiation, phosphorylation of MYPT1 at Thr696 and Thr853 increased significantly, resulting in inhibition of MP. The ROCK inhibitor H1152 and the MP activator epigallocatechin-3-gallate (EGCG) attenuated MYPT1 phosphorylation and concomitantly decreased the extent of phosphorylation of 20 kDa myosin light chain. H1152 and EGCG pretreatment also suppressed the expression of CD11b and weakened the PMA-induced adherence of the cells. Our results indicate that MP activation/inhibition contributes to the efficacy of monocyte to macrophage differentiation, and this enzyme may be a target for pharmacological interventions in the control of disease states that are affected by excessive macrophage differentiation.


Assuntos
Diferenciação Celular/fisiologia , Macrófagos/metabolismo , Monócitos/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Células THP-1/metabolismo , Células Cultivadas , Humanos , Macrófagos/fisiologia , Monócitos/fisiologia , Fosforilação/fisiologia , Transdução de Sinais/fisiologia , Células THP-1/fisiologia , Quinases Associadas a rho/metabolismo
11.
Genesis ; 58(2): e23345, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31705616

RESUMO

Myosin phosphatase targeting subunit 1 (Mypt1) is the regulatory subunit of myosin phosphatase which dephosphorylates the light chain of myosin II to inhibit its contraction. Although biochemical properties of Mypt1 have been characterized in detail, its biological functions in organisms are not well understood. The zebrafish mypt1 sq181 allele was found defective in the ventral pancreatic bud and extrapancreatic duct development, resulting in dysplasia of exocrine pancreas. In mypt1 sq181 mutant, the early growth of the ventral pancreatic bud was initiated but failed to expand due to impaired cell proliferation and increased cell apoptosis. As Mypt1 is essential for cell migration, the loss-of-function of Mypt1 in the mutant disrupted the lateral plate mesoderm migration during gut looping, therefore, altering the Bmp2a expression pattern within it, and eventually leading to impaired Bmp signaling in the adjacent exocrine pancreas. Overexpression of bmp2a could rescue the development of exocrine pancreas, suggesting that the impaired Bmp2a signaling is responsible for the pancreatic development defects. Bmp2a has been reported to promote the early specification of the ventral pancreatic bud, and our study reveals that it continues to serve as a cell proliferation/survival signal to ensure pancreatic bud growth properly in zebrafish.


Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Pâncreas Exócrino/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Apoptose , Proteína Morfogenética Óssea 2/genética , Regulação da Expressão Gênica no Desenvolvimento , Mutação com Perda de Função , Fosfatase de Miosina-de-Cadeia-Leve/genética , Pâncreas Exócrino/embriologia , Transdução de Sinais , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
12.
J Cell Mol Med ; 24(2): 1413-1427, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31778027

RESUMO

BACKGROUND: Increased Rho-kinase activity in circulating leucocytes is observed in heart failure with reduced ejection fraction (HFrEF). However, there is little information in HFrEF regarding other Rho-kinase pathway components an on the relationship between Rho-kinase and apoptosis. Here, Rho-kinase activation levels and phosphorylation of major downstream molecules and apoptosis levels were measured for the first time both in HFrEF patients and healthy individuals. METHODS: Cross-sectional study comparing HFrEF patients (n = 20) and healthy controls (n = 19). Rho-kinase activity in circulating leucocytes (peripheral blood mononuclear cells, PBMCs) was determined by myosin light chain phosphatase 1 (MYPT1) and ezrin-radixin-moesin (ERM) phosphorylation. Rho-kinase cascade proteins phosphorylation p38-MAPK, myosin light chain-2, JAK and JNK were also analysed along with apoptosis. RESULTS: MYPT1 and ERM phosphorylation were significantly elevated in HFrEF patients, (3.9- and 4.8-fold higher than in controls, respectively). JAK phosphorylation was significantly increased by 300% over controls. Phosphorylation of downstream molecules p38-MAPK and myosin light chain-2 was significantly higher by 360% and 490%, respectively, while JNK phosphorylation was reduced by 60%. Catecholamine and angiotensin II levels were significantly higher in HFrEF patients, while angiotensin-(1-9) levels were lower. Apoptosis in circulating leucocytes was significantly increased in HFrEF patients by 2.8-fold compared with controls and significantly correlated with Rho-kinase activation. CONCLUSION: Rho-kinase pathway is activated in PMBCs from HFrEF patients despite optimal treatment, and it is closely associated with neurohormonal activation and with apoptosis. ROCK cascade inhibition might induce clinical benefits in HFrEF patients, and its assessment in PMBCs could be useful to evaluate reverse remodelling and disease regression.


Assuntos
Apoptose , Insuficiência Cardíaca/sangue , Insuficiência Cardíaca/fisiopatologia , Leucócitos Mononucleares/enzimologia , Leucócitos Mononucleares/patologia , Transdução de Sinais , Volume Sistólico , Quinases Associadas a rho/metabolismo , Angiotensinas/sangue , Animais , Antígenos CD/metabolismo , Catecolaminas/sangue , Citocinas/sangue , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Ativação Enzimática , Feminino , Insuficiência Cardíaca/diagnóstico por imagem , Humanos , Janus Quinase 2/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Pessoa de Meia-Idade , Miocárdio/patologia , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Peptídeo Natriurético Encefálico/sangue , Fosforilação , Ratos , Sístole , Remodelação Ventricular
13.
J Biol Chem ; 294(36): 13280-13291, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31315927

RESUMO

Transforming growth factor-ß membrane associated protein (TIMAP) is an endothelial cell (EC)-predominant PP1 regulatory subunit and a member of the myosin phosphatase target (MYPT) protein family. The MYPTs preferentially bind the catalytic protein phosphatase 1 subunit PP1cß, forming myosin phosphatase holoenzymes. We investigated whether TIMAP/PP1cß could also function as a myosin phosphatase. Endogenous PP1cß, myosin light chain 2 (MLC2), and myosin IIA heavy chain coimmunoprecipitated from EC lysates with endogenous TIMAP, and endogenous MLC2 colocalized with TIMAP in EC projections. Purified recombinant GST-TIMAP interacted directly with purified recombinant His-MLC2. However, TIMAP overexpression in EC enhanced MLC2 phosphorylation, an effect not observed with a TIMAP mutant that does not bind PP1cß. Conversely, MLC2 phosphorylation was reduced in lung lysates from TIMAP-deficient mice and upon silencing of endogenous TIMAP expression in ECs. Ectopically expressed TIMAP slowed the rate of MLC2 dephosphorylation, an effect requiring TIMAP-PP1cß interaction. The association of MYPT1 with PP1cß was profoundly reduced in the presence of excess TIMAP, leading to proteasomal MYPT1 degradation. In the absence of TIMAP, MYPT1-associated PP1cß readily bound immobilized microcystin-LR, an active-site inhibitor of PP1c. By contrast, TIMAP-associated PP1cß did not interact with microcystin-LR, indicating that the active site of PP1cß is blocked when it is bound to TIMAP. Thus, TIMAP inhibits myosin phosphatase activity in ECs by competing with MYPT1 for PP1cß and blocking the PP1cß active site.


Assuntos
Proteínas de Membrana/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/antagonistas & inibidores , Proteína Fosfatase 1/metabolismo , Animais , Biocatálise , Linhagem Celular , Células Endoteliais/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo
14.
J Biol Chem ; 294(28): 10846-10862, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31138649

RESUMO

Cell migration is essential to embryonic development, wound healing, and cancer cell dissemination. Cells move via leading-edge protrusion, substrate adhesion, and retraction of the cell's rear. The molecular mechanisms by which extracellular cues signal to the actomyosin cytoskeleton to control these motility mechanics are poorly understood. The growth factor-responsive and oncogenically activated protein extracellular signal-regulated kinase (ERK) promotes motility by signaling in actin polymerization-mediated edge protrusion. Using a combination of immunoblotting, co-immunoprecipitation, and myosin-binding experiments and cell migration assays, we show here that ERK also signals to the contractile machinery through its substrate, p90 ribosomal S6 kinase (RSK). We probed the signaling and migration dynamics of multiple mammalian cell lines and found that RSK phosphorylates myosin phosphatase-targeting subunit 1 (MYPT1) at Ser-507, which promotes an interaction of Rho kinase (ROCK) with MYPT1 and inhibits myosin targeting. We find that by inhibiting the myosin phosphatase, ERK and RSK promote myosin II-mediated tension for lamella expansion and optimal edge dynamics for cell migration. These findings suggest that ERK activity can coordinately amplify both protrusive and contractile forces for optimal cell motility.


Assuntos
Movimento Celular/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Citoesqueleto de Actina/metabolismo , Actomiosina/metabolismo , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Citoesqueleto/metabolismo , Citoesqueleto/fisiologia , Humanos , Contração Muscular , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/fisiologia , Miosinas/metabolismo , Fosforilação , Ligação Proteica , Proteínas Quinases S6 Ribossômicas 90-kDa/fisiologia , Transdução de Sinais , Quinases Associadas a rho/metabolismo
15.
Mol Cancer ; 19(1): 7, 2020 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-31926547

RESUMO

BACKGROUND: Ovarian cancer is one of the most common and malignant cancers, partly due to its late diagnosis and high recurrence. Chemotherapy resistance has been linked to poor prognosis and is believed to be linked to the cancer stem cell (CSC) pool. Therefore, elucidating the molecular mechanisms mediating therapy resistance is essential to finding new targets for therapy-resistant tumors. METHODS: shRNA depletion of MYPT1 in ovarian cancer cell lines, miRNA overexpression, RT-qPCR analysis, patient tumor samples, cell line- and tumorsphere-derived xenografts, in vitro and in vivo treatments, analysis of data from ovarian tumors in public transcriptomic patient databases and in-house patient cohorts. RESULTS: We show that MYPT1 (PPP1R12A), encoding myosin phosphatase target subunit 1, is downregulated in ovarian tumors, leading to reduced survival and increased tumorigenesis, as well as resistance to platinum-based therapy. Similarly, overexpression of miR-30b targeting MYPT1 results in enhanced CSC-like properties in ovarian tumor cells and is connected to the activation of the Hippo pathway. Inhibition of the Hippo pathway transcriptional co-activator YAP suppresses the resistance to platinum-based therapy induced by either low MYPT1 expression or miR-30b overexpression, both in vitro and in vivo. CONCLUSIONS: Our work provides a functional link between the resistance to chemotherapy in ovarian tumors and the increase in the CSC pool that results from the activation of the Hippo pathway target genes upon MYPT1 downregulation. Combination therapy with cisplatin and YAP inhibitors suppresses MYPT1-induced resistance, demonstrating the possibility of using this treatment in patients with low MYPT1 expression, who are likely to be resistant to platinum-based therapy.


Assuntos
Biomarcadores Tumorais/metabolismo , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , MicroRNAs/genética , Células-Tronco Neoplásicas/patologia , Neoplasias Ovarianas/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Via de Sinalização Hippo , Humanos , Camundongos , Camundongos Nus , Invasividade Neoplásica , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Prognóstico , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Cardiovasc Diabetol ; 19(1): 56, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32375786

RESUMO

BACKGROUND: The intracellular ROCK signaling pathway is an important modulator of blood pressure and of cardiovascular and renal remodeling when Rho-kinase activity is increased. Besides, in preclinical models of diabetes, ROCK activation has also a role in abnormal glucose metabolism as well as in subsequent vascular and myocardial dysfunction. In humans, there are a few data assessing ROCK activation in patients with type 2 diabetes mellitus (T2D) and no studies assessing upstream/downstream components of the ROCK pathway. We assessed here levels of ROCK activation and some of the RhoA/ROCK cascade molecules in peripheral blood mononuclear cells (PBMCs) in T2D patients under current treatment. METHODS: Cross-sectional observational study comparing 28 T2D patients under current antidiabetic treatment with 31 consecutive healthy subjects, matched by age and gender. Circulating levels of malondialdehyde, angiotensin II and inflammatory cytokines IL-6 and IL-8 were determined in all subjects. ROCK activation in PMBCs, upstream and downstream cascade proteins, and levels of the proinflammatory molecules VCAM, ICAM-1 and IL-8 were determined in their PMBCs by Western blot. RESULTS: Compared to healthy controls, ROCK activation in T2D patients measured by 2 direct ROCK targets in PBMCs was increased by 420 and 570% (p < 0001) and it correlated significantly with serum glucose levels. p38 MAPK phosphorylation (downstream from ROCK) and JAK-2 (upstream from ROCK) were significantly higher in the T2D patients by 580% and 220%, respectively. In T2D patients, significantly increased PBMC levels of the proinflammatory molecules VCAM-1, ICAM-1 and IL-8 were observed compared to control subjects (by 180%, 360% and 260%, respectively). Circulating levels of Ang II and MDA were significantly higher in T2D patients by 29 and 63%, respectively. CONCLUSIONS: T2D patients under treatment with glucose-lowering drugs, antihypertensive treatment as well as with statins have significantly increased ROCK activation in their circulating leukocytes along with higher phosphorylation of downstream cascade proteins despite pharmacologic treatment, along with increased plasma angiotensin II and MDA levels. ROCK inhibition might have an additional role in the prevention and treatment of T2D.


Assuntos
Diabetes Mellitus Tipo 2/enzimologia , Leucócitos Mononucleares/enzimologia , Quinases Associadas a rho/sangue , Idoso , Angiotensina II/sangue , Anti-Hipertensivos/uso terapêutico , Biomarcadores/sangue , Estudos de Casos e Controles , Estudos Transversais , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Ativação Enzimática , Feminino , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Hipoglicemiantes/uso terapêutico , Molécula 1 de Adesão Intercelular/sangue , Interleucina-8/sangue , Janus Quinase 2/sangue , Masculino , Malondialdeído/sangue , Pessoa de Meia-Idade , Transdução de Sinais , Molécula 1 de Adesão de Célula Vascular/sangue , Proteínas Quinases p38 Ativadas por Mitógeno/sangue
17.
Int J Mol Sci ; 21(16)2020 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-32784920

RESUMO

The Cpi-17 (ppp1r14) gene family is an evolutionarily conserved, vertebrate specific group of protein phosphatase 1 (PP1) inhibitors. When phosphorylated, Cpi-17 is a potent inhibitor of myosin phosphatase (MP), a holoenzyme complex of the regulatory subunit Mypt1 and the catalytic subunit PP1. Myosin phosphatase dephosphorylates the regulatory myosin light chain (Mlc2) and promotes actomyosin relaxation, which in turn, regulates numerous cellular processes including smooth muscle contraction, cytokinesis, cell motility, and tumor cell invasion. We analyzed zebrafish homologs of the Cpi-17 family, to better understand the mechanisms of myosin phosphatase regulation. We found single homologs of both Kepi (ppp1r14c) and Gbpi (ppp1r14d) in silico, but we detected no expression of these genes during early embryonic development. Cpi-17 (ppp1r14a) and Phi-1 (ppp1r14b) each had two duplicate paralogs, (ppp1r14aa and ppp1r14ab) and (ppp1r14ba and ppp1r14bb), which were each expressed during early development. The spatial expression pattern of these genes has diverged, with ppp1r14aa and ppp1r14bb expressed primarily in smooth muscle and skeletal muscle, respectively, while ppp1r14ab and ppp1r14ba are primarily expressed in neural tissue. We observed that, in in vitro and heterologous cellular systems, the Cpi-17 paralogs both acted as potent myosin phosphatase inhibitors, and were indistinguishable from one another. In contrast, the two Phi-1 paralogs displayed weak myosin phosphatase inhibitory activity in vitro, and did not alter myosin phosphorylation in cells. Through deletion and chimeric analysis, we identified that the difference in specificity for myosin phosphatase between Cpi-17 and Phi-1 was encoded by the highly conserved PHIN (phosphatase holoenzyme inhibitory) domain, and not the more divergent N- and C- termini. We also showed that either Cpi-17 paralog can rescue the knockdown phenotype, but neither Phi-1 paralog could do so. Thus, we provide new evidence about the biochemical and developmental distinctions of the zebrafish Cpi-17 protein family.


Assuntos
Proteínas de Peixes/genética , Genes Duplicados/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Musculares/genética , Proteínas/genética , Sequência de Aminoácidos , Animais , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Evolução Molecular , Proteínas de Peixes/classificação , Proteínas de Peixes/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/classificação , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Musculares/classificação , Proteínas Musculares/metabolismo , Fosfoproteínas Fosfatases/classificação , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Filogenia , Proteínas/classificação , Proteínas/metabolismo , Homologia de Sequência de Aminoácidos , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
18.
J Biol Chem ; 293(43): 16677-16686, 2018 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-30185619

RESUMO

Contractile force development of smooth muscle is controlled by balanced kinase and phosphatase activities toward the myosin regulatory light chain (RLC). Numerous biochemical and pharmacological studies have investigated the specificity and regulatory activity of smooth muscle myosin light-chain phosphatase (MLCP) bound to myosin filaments and comprised of the regulatory myosin phosphatase target subunit 1 (MYPT1) and catalytic protein phosphatase 1cß (PP1cß) subunits. Recent physiological and biochemical evidence obtained with smooth muscle tissues from a conditional MYPT1 knockout suggests that a soluble, MYPT1-unbound form of PP1cß may additionally contribute to myosin RLC dephosphorylation and relaxation of smooth muscle. Using a combination of isoelectric focusing and isoform-specific immunoblotting, we found here that more than 90% of the total PP1c in mouse smooth muscles is the ß isoform. Moreover, conditional knockout of PP1cα or PP1cγ in adult smooth muscles did not result in an apparent phenotype in mice up to 6 months of age and did not affect smooth muscle contractions ex vivo In contrast, smooth muscle-specific conditional PP1cß knockout decreased contractile force development in bladder, ileal, and aortic tissues and reduced mouse survival. Bladder smooth muscle tissue from WT mice was selectively permeabilized to remove soluble PP1cß to measure contributions of total (α-toxin treatment) and myosin-bound (Triton X-100 treatment) phosphatase activities toward phosphorylated RLC in myofilaments. Triton X-100 reduced PP1cß content by 60% and the rate of RLC dephosphorylation by 2-fold. These results are consistent with the selective dephosphorylation of RLC by both MYPT1-bound and -unbound PP1cß forms in smooth muscle.


Assuntos
Músculo Liso/enzimologia , Proteína Fosfatase 1/metabolismo , Animais , Íleo/enzimologia , Íleo/fisiologia , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Camundongos , Camundongos Knockout , Contração Muscular , Músculo Liso/fisiologia , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/fisiologia , Fosforilação , Proteína Fosfatase 1/genética , Bexiga Urinária/enzimologia , Bexiga Urinária/fisiologia
19.
J Biol Chem ; 293(39): 15152-15162, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-30115685

RESUMO

The protein Ser/Thr phosphatase PP1 catalyzes an important fraction of protein dephosphorylation events and forms highly specific holoenzymes through an association with regulatory interactors of protein phosphatase one (RIPPOs). The functional characterization of individual PP1 holoenzymes is hampered by the lack of straightforward strategies for substrate mapping. Because efficient substrate recruitment often involves binding to both PP1 and its associated RIPPO, here we examined whether PP1-RIPPO fusions can be used to trap substrates for further analysis. Fusions of an hypoactive point mutant of PP1 and either of four tested RIPPOs accumulated in HEK293T cells with their associated substrates and were co-immunoprecipitated for subsequent identification of the substrates by immunoblotting or MS analysis. Hypoactive fusions were also used to study RIPPOs themselves as substrates for associated PP1. In contrast, substrate trapping was barely detected with active PP1-RIPPO fusions or with nonfused PP1 or RIPPO subunits. Our results suggest that hypoactive fusions of PP1 subunits represent an easy-to-use tool for substrate identification of individual holoenzymes.


Assuntos
Núcleo Celular/química , Holoenzimas/química , Proteína Fosfatase 1/química , Receptores de Neuropeptídeo Y/química , Animais , Sítios de Ligação , Células COS , Núcleo Celular/genética , Chlorocebus aethiops/genética , Células HEK293 , Holoenzimas/genética , Humanos , Imunoprecipitação , Fosforilação , Ligação Proteica , Proteína Fosfatase 1/genética , Receptores de Neuropeptídeo Y/genética , Especificidade por Substrato
20.
J Cell Physiol ; 234(7): 10218-10224, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30480807

RESUMO

BACKGROUNDS: Necrotizing enterocolitis (NEC) was one of the main causes of morbidity and mortality in neonates. Our objective was to detect the mechanism of miR-124 in small bowel tissues of NEC. METHODS: Hematoxylin and eosin (H&E) staining was used to detect the repair of the damaged tissues in rat NEC model. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was used to evaluate the cell apoptosis level in intestinal tissue. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the messenger RNA (mRNA) expression level of miR-124, Rho-associated coiled-coil-containing protein kinase 1 (ROCK1), myosin phosphatase target subunit 1 (MYPT1), and Toll-like receptor 9 (TLR9) in NEC tissues and IEC-6 cells. Luciferase reporter assay was used to verify whether ROCK1 is a direct target of miR-124. RESULTS: miR-124 was overexpressed in the NEC tissues, while ROCK1 and MYPT1 was downregulated in the NEC tissues. Inhibition of miR-124, suppressed the intestinal cell apoptosis and promoted the expression of ROCK1 and MYPT1. What is more, overexpression of miR-124 could inhibit the expression of ROCK1, TLR9, and MYPT1. Luciferase assay confirmed that miR-124 can regulate the transcriptional activity of ROCK1 through binding its 3'-UTR region. CONCLUSION: miR-124 was a promoter of NEC, which promotes the intestine cell apoptosis and inflammatory cell infiltration through the inhibition of TLR9 expression by targeting ROCK1.


Assuntos
Enterocolite Necrosante/metabolismo , MicroRNAs/metabolismo , Proteína Fosfatase 1/metabolismo , Transdução de Sinais/fisiologia , Receptor Toll-Like 9/metabolismo , Animais , Animais Recém-Nascidos , Enterocolite Necrosante/genética , Regulação da Expressão Gênica/fisiologia , MicroRNAs/genética , Proteína Fosfatase 1/genética , Ratos , Receptor Toll-Like 9/genética , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo
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