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Cerebral small vessel disease (CSVD) is the most common progressive vascular disease that causes vascular dementia. Aging and hypertension are major contributors to CSVD, but the pathophysiological mechanism remains unclear, mainly due to the lack of an ideal animal model. Our previous study revealed that vascular smooth muscle cell (VSMC)-specific myosin phosphatase target subunit 1 (MYPT1) knockout (MYPT1SMKO) leads to constant hypertension, prompting us to explore whether hypertensive MYPT1SMKO mice can be considered a novel CSVD animal model. Here, we found that MYPT1SMKO mice displayed age-dependent CSVD-like neurobehaviors, including decreased motion speed, anxiety, and cognitive decline. MYPT1SMKO mice exhibited remarkable white matter injury compared with control mice, as shown by the more prominent loss of myelin at 12 months of age. Additionally, MYPT1SMKO mice were found to exhibit CSVD-like small vessel impairment, including intravascular hyalinization, perivascular space enlargement, and microbleed and blood-brain barrier (BBB) disruption. Last, our results revealed that the brain of MYPT1SMKO mice was characterized by an exacerbated inflammatory microenvironment, which is similar to patients with CSVD. In light of the above structural and functional phenotypes that closely mimic the conditions of human CSVD, we suggest that MYPT1SMKO mice are a novel age- and hypertension-dependent animal model of CSVD. [ABSTRACT FROM AUTHOR]

Introduction: Vasodilatation in response to NO is a fundamental response of the vasculature, and during aging, the vasculature is characterized by an increase in stiffness and decrease in sensitivity to NO mediated vasodilatation. Vascular tone is regulated by the activation of smooth muscle and nonmuscle (NM) myosin, which are regulated by the activities of myosin light chain kinase (MLCK) and MLC phosphatase. MLC phosphatase is a trimeric enzyme with a catalytic subunit, myosin targeting subunit (MYPT1) and 20 kDa subunit of unknown function. Alternative mRNA splicing produces LZ+/LZ- MYPT1 isoforms and the relative expression of LZ+/LZ- MYPT1 determines the sensitivity to NO mediated vasodilatation. This study tested the hypothesis that aging is associated with changes in LZ+ MYPT1 and NM myosin expression, which alter vascular reactivity. Methods: We determined MYPT1 and NM myosin expression, force and the sensitivity of both endothelial dependent and endothelial independent relaxation in tertiary mesenteric arteries of young (6mo) and elderly (24mo) Fischer344 rats. Results: The data demonstrate that aging is associated with a decrease in both the expression of NM myosin and force, but LZ+ MYPT expression and the sensitivity to both endothelial dependent and independent vasodilatation did not change. Further, smooth muscle cell hypertrophy increases the thickness of the medial layer of smooth muscle with aging. Discussion: The reduction of NM myosin may represent an aging associated compensatory mechanism to normalize the stiffness of resistance vessels in response to the increase in media thickness observed during aging. [ABSTRACT FROM AUTHOR]

The AMP‐activated protein kinase (AMPK)‐related kinase NUAK1 (NUAK family SNF1‐like kinase 1) has emerged as a potential vulnerability in MYC‐dependent cancer but the biological roles of NUAK1 in different settings are poorly characterised, and the spectrum of cancer types that exhibit a requirement for NUAK1 is unknown. Unlike canonical oncogenes, NUAK1 is rarely mutated in cancer and appears to function as an obligate facilitator rather than a cancer driver per se. Although numerous groups have developed small‐molecule NUAK inhibitors, the circumstances that would trigger their use and the unwanted toxicities that may arise as a consequence of on‐target activity are thus undetermined. Reasoning that MYC is a key effector of RAS pathway signalling and the GTPase KRAS is almost uniformly mutated in pancreatic ductal adenocarcinoma (PDAC), we investigated whether this cancer type exhibits a functional requirement for NUAK1. Here, we show that high NUAK1 expression is associated with reduced overall survival in PDAC and that inhibition or depletion of NUAK1 suppresses growth of PDAC cells in culture. We identify a previously unknown role for NUAK1 in regulating accurate centrosome duplication and show that loss of NUAK1 triggers genomic instability. The latter activity is conserved in primary fibroblasts, raising the possibility of undesirable genotoxic effects of NUAK1 inhibition.

Background: Genitourinary and/or brain malformation syndrome (GUBS) is a recently discovered syndrome involving abnormalities of the neurological or urogenital system. PPP1R12A may be the pathological gene causing this syndrome. Currently, to our knowledge, there is only one study related to GUBS in the world. Here, we report a clinical case of a Chinese newborn with congenital micropenis caused by a non‐coding sequence pathogenic variant of PPP1R12A, providing additional evidence on genetic causes of genital malformation. Methods: The genetic cause of the patient's malformation was detected using trio‐whole exome sequencing and Sanger sequencing, and reverse transcription‐PCR analysis was performed by constructing the minigene mutant plasmid in vitro. Results: Genetic testing revealed a novel heterozygous variant, c.2666+3A>G, of the PPP1R12A gene of the patient. The parents at this site were wild‐type, indicating that this might be a de novo variant. The minigene experiment showed that the c.2666+3A>G plasmid led to the deletion of 17 bp in exon 20, and a new mRNA product c.2650_2666del (p.Thr884IleTer2) with skipping of exon 20 was produced. This may lead to PPP1R12A haploinsufficiency and cause biological harm. Conclusions: To our knowledge, this is the first clinical study on a rare variant of PPP1R12A in the Chinese population. The c.2666+3A>G may lead to external genitalia malformation, such as congenital micropenis in male neonates. The results of this study further verified the correlation between GUBS and PPP1R12A haploinsufficiency and revealed the important role of a non‐coding sequence variant in the pathogenesis of the disease. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Myosin phosphatase target subunit 1 (MYPT1) is a subunit of myosin phosphatase that is capable of regulating smooth muscle contraction. MYPT1 has been reported to be involved in a wide variety of tumours, but its expression and biological functions in renal clear cell carcinoma (ccRCC) remain obscure. Herein, we analysed the relationship between patient clinicopathological characteristics and MYPT1 expression levels in ccRCC patients using a tissue microarray (TMA) and data retrieved from the TCGA‐KIRC dataset. MYPT1 was overexpressed or depleted using siRNA in ccRCC cells to assess the effects on migration and invasion in vitro and in vivo. Additionally, RNA‐sequencing and bioinformatics analysis were performed to investigate the precise mechanism. MYPT1 expression in ccRCC tissues was observed to be lower than that in nonmalignant tissues (P

The AMP‐activated protein kinase (AMPK)‐related kinase NUAK1 (NUAK family SNF1‐like kinase 1) has emerged as a potential vulnerability in MYC‐dependent cancer but the biological roles of NUAK1 in different settings are poorly characterised, and the spectrum of cancer types that exhibit a requirement for NUAK1 is unknown. Unlike canonical oncogenes, NUAK1 is rarely mutated in cancer and appears to function as an obligate facilitator rather than a cancer driver per se. Although numerous groups have developed small‐molecule NUAK inhibitors, the circumstances that would trigger their use and the unwanted toxicities that may arise as a consequence of on‐target activity are thus undetermined. Reasoning that MYC is a key effector of RAS pathway signalling and the GTPase KRAS is almost uniformly mutated in pancreatic ductal adenocarcinoma (PDAC), we investigated whether this cancer type exhibits a functional requirement for NUAK1. Here, we show that high NUAK1 expression is associated with reduced overall survival in PDAC and that inhibition or depletion of NUAK1 suppresses growth of PDAC cells in culture. We identify a previously unknown role for NUAK1 in regulating accurate centrosome duplication and show that loss of NUAK1 triggers genomic instability. The latter activity is conserved in primary fibroblasts, raising the possibility of undesirable genotoxic effects of NUAK1 inhibition. [ABSTRACT FROM AUTHOR]

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.

Myosin phosphatase target subunit 1 (MYPT1) is a subunit of myosin phosphatase that is capable of regulating smooth muscle contraction. MYPT1 has been reported to be involved in a wide variety of tumours, but its expression and biological functions in renal clear cell carcinoma (ccRCC) remain obscure. Herein, we analysed the relationship between patient clinicopathological characteristics and MYPT1 expression levels in ccRCC patients using a tissue microarray (TMA) and data retrieved from the TCGA‐KIRC dataset. MYPT1 was overexpressed or depleted using siRNA in ccRCC cells to assess the effects on migration and invasion in vitro and in vivo. Additionally, RNA‐sequencing and bioinformatics analysis were performed to investigate the precise mechanism. MYPT1 expression in ccRCC tissues was observed to be lower than that in nonmalignant tissues (P < 0.05). In addition, MYPT1 downregulation was closely linked to advanced pathological stage (P < 0.05), and poor OS (overall survival; P < 0.05). Functionally, increased expression of MYPT1 suppressed ccRCC migration and invasion in vitro, and inhibited tumour metastasis in vivo. In addition, MYPT1 overexpression exerted its suppressive effects via the MAPK8/N‐cadherin pathway in ccRCC. [ABSTRACT FROM AUTHOR]

The myosin light chain phosphatase target subunit 1 (MYPT1), encoded by the PPP1R12A gene, is a key component of the myosin light chain phosphatase (MLCP) protein complex. MYPT1 isoforms have been described as products of the cassette-type alternative splicing of exons E13, E14, E22, and E24. Through in silico analysis of the publicly available EST and mRNA databases, we established that PPP1R12A contains 32 exons (6 more than the 26 previously reported), of which 29 are used in 11 protein-coding transcripts. An in silico analysis of publicly available RNAseq data combined with validation by reverse transcription (RT)-PCR allowed us to determine the relative abundance of each transcript in three cell types of the circulatory system where MYPT1 plays important roles: human umbilical vein endothelial cells (HUVEC), human saphenous vein smooth muscle cells (HSVSMC), and platelets. All three cell types express up to 10 transcripts at variable frequencies. HUVECs and HSVSMCs predominantly express the full-length variant (58.3% and 64.3%, respectively) followed by the variant skipping E13 (33.7% and 23.1%, respectively), whereas in platelets the predominant variants are those skipping E14 (51.4%) and E13 (19.9%), followed by the full-length variant (14.4%). Variants including E24 account for 5.4% of transcripts in platelets but are rare (<1%) in HUVECs and HSVSMCs. Complex transcriptional profiles were also found across organs using in silico analysis of RNAseq data from the GTEx project. Our findings provide a platform for future studies investigating the specific (patho)physiological roles of understudied MYPT1 isoforms. [ABSTRACT FROM AUTHOR]

The methyltransferase EZH2 plays an important role in regulating chromatin conformation and gene transcription. Phosphorylation of EZH2 at S21 by AKT kinase suppresses its function. However, protein phosphatases responsible for the dephosphorylation of EZH2-S21 remain elusive. Here, it is demonstrated that EZH2 is highly expressed in the ocular lens, and AKT-EZH2 axis is important in TGFβ-induced epithelial-mesenchymal transition (EMT). More importantly, it is identified that MYPT1/PP1 dephosphorylates EZH2-S21 and thus modulates its functions. MYPT1 knockout accelerates EMT, but expression of the EZH2-S21A mutant suppresses EMT through control of multiple families of genes. Furthermore, the phosphorylation status and gene expression modulation of EZH2 are implicated in control of anterior subcapsular cataracts (ASC) in human and mouse eyes. Together, the results identify the specific phosphatase for EZH2-S21 and reveal EZH2 dephosphorylation control of several families of genes implicated in lens EMT and ASC pathogenesis. These results provide important novel information in EZH2 function and regulation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Wei-Ping Xiao,1,* Li-Li-Qiang Ding,2,* You-Jiang Min,1,3 Hua-Yuan Yang,4 Hai-Hua Yao,3 Jie Sun,1 Xuan Zhou,1 Xue-Bo Zeng,1 Wan Yu1 1Spinal Department of Orthopedics and Department of Acupuncture, Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine, Nanchang, Jiangxi, People’s Republic of China; 2Department of Hypertension, Shanghai Institute of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China; 3Department of Traditional Chinese Medicine, Shanghai Eighth People’s Hospital, Shanghai, People’s Republic of China; 4Institute of Traditional Chinese Medicine Engineering, Shanghai University of Traditional Chinese Medicine, Shanghai, People’s Republic of China*These authors contributed equally to this workCorrespondence: You-Jiang MinShanghai Eighth People’s Hospital, Caobao Road 8, Shanghai 200235, People’s Republic of ChinaTel +86 18221786381Email myj2002@126.com Hua-Yuan YangShanghai University of Traditional Chinese Medicine, Cairen Road 1200, Shanghai 201203, People’s Republic of ChinaTel +86 13651968830Email yhyabcd@sina.comPurpose: To observe the changes of Nogo/NgR and Rho/ROCK signaling pathway-related gene and protein expression in rats with spinal cord injury (SCI) treated with electroacupuncture (EA) and to further investigate the possible mechanism of EA for treating SCI.Methods: Allen’s method was used to create the SCI rat model. Sixty-four model rats were further subdivided into four subgroups, namely, the SCI model group (SCI), EA treatment group (EA), blocking agent Y27632 treatment group (Y27632) and EA+blocking agent Y27632 treatment group (EA+Y), according to the treatment received. The rats were subjected to EA and/or blocking agent Y27632 treatment. After 14 days, injured spinal cord tissue was extracted for analysis. The mRNA and protein expression levels were determined by real-time fluorescence quantitative PCR and Western blotting, respectively. Cell apoptosis changes in the spinal cord were evaluated by in situ hybridization. Hindlimb motor function in the rats was evaluated by Basso-Beattie-Bresnahan assessment methods.Results: Except for RhoA protein expression, compared with the SCI model group, EA, blocking agent Y27632 and EA+blocking agent Y27632 treatment groups had significantly reduced mRNA and protein expression of Nogo-A, NgR, LINGO-1, RhoA and ROCK II in spinal cord tissues, increased mRNA and protein expression of MLCP, decreased p-MYPT1 protein expression and p-MYPT1/MYPT1 ratio, and caspase3 expression, and improved lower limb movement function after treatment for 14 days (P

In this study, we investigated the efficacy of kaempferol (a flavonoid found in plants and plant-derived foods such as kale, beans, tea, spinach and broccoli) on vascular contractibility and aimed to clarify the detailed mechanism underlying the relaxation. Isometric contractions of divested muscles were stored and linked with western blot analysis which was carried out to estimate the phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and phosphorylation-dependent inhibitory protein for myosin phosphatase (CPI-17) and to estimate the effect of kaempferol on the RhoA/ROCK/CPI-17 pathway. Kaempferol conspicuously impeded phorbol ester-, fluoride- and a thromboxane mimetic-derived contractions regardless of endothelial nitric oxide synthesis, indicating its direct effect on smooth muscles. It also conspicuously impeded the fluoride-derived elevation in phospho-MYPT1 rather than phospho-CPI-17 levels and phorbol 12,13-dibutyrate-derived increase in phospho-CPI-17 and phospho-ERK1/2 levels, suggesting the depression of PKC and MEK activities and subsequent phosphorylation of CPI-17 and ERK1/2. Taken together, these outcomes suggest that kaempferol-derived relaxation incorporates myosin phosphatase retrieval and calcium desensitization, which appear to be modulated by CPI-17 dephosphorylation mainly through PKC inactivation.

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. 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. PKD1 may play roles in aorta contraction and haemodynamics via phosphorylation of MYPT1 and actin polymerization in a calcium-independent manner. • Little is known about role of Protein kinase D (PKD) in vascular function. • Norepinephrine increased phosphorylation of PKD1 during aortic contraction. • The phosphorylation of MYPT1 and MLC were attenuated with PKD-inhibitor and SiRNA. • PKD-inhibitor decreased cell shortening without change of calcium concentrations. • PKD1 might play roles in hemodynamics in a calcium-independent manner. [ABSTRACT FROM AUTHOR]

Protein kinases exert physiological functions through phosphorylating their specific substrates; however, the mode of kinase–substrate recognition is not fully understood. Rho-kinase is a Ser/Thr protein kinase that regulates cytoskeletal reorganization through phosphorylating myosin light chain (MLC) and the myosin phosphatase targeting subunit 1 (MYPT1) of MLC phosphatase (MLCP) and is involved in various diseases, due to its aberrant cellular contraction, morphology, and movement. Despite the importance of the prediction and identification of substrates and phosphorylation sites, understanding of the precise regularity in phosphorylation preference of Rho-kinase remains far from satisfactory. Here we analyzed the Rho-kinase–MYPT1 interaction, to understand the mode of Rho-kinase substrate recognition and found that the three short regions of MYPT1 close to phosphorylation sites (referred to as docking motifs (DMs); DM1 (DLQEAEKTIGRS), DM2 (KSQPKSIRERRRPR), and DM3 (RKARSRQAR)) are important for interactions with Rho-kinase. The phosphorylation levels of MYPT1 without DMs were reduced, and the effects were limited to the neighboring phosphorylation sites. We further demonstrated that the combination of pseudosubstrate (PS) and DM of MYPT1 (PS1 + DM3 and PS2 + DM2) serves as a potent inhibitor of Rho-kinase. The present information will be useful in identifying new substrates and developing selective Rho-kinase inhibitors.

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. [ABSTRACT FROM AUTHOR]

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. miR‐124 was a promoter of necrotizing enterocolitis (NEC), which promotes the intestine cell apoptosis and inflammatory cell infiltration through the inhibition of Toll‐like receptor 9 (TLR9) expression by targeting rho‐associated coiled‐coil‐containing protein kinase 1 (ROCK1). [ABSTRACT FROM AUTHOR]

Abstract The miR-143/145 cluster is down-regulated in cervical tumor cells suggesting a role in tumorigenesis including cytoskeleton remodeling, a key event for tumor progression. The aim of the present work was to determine the role of miR-143/145 in the modulation of the myosin regulator phospho-myosin light chain (pMLC). HeLa monolayer and tridimensional cultures were transfected with miR-143 or miR-145 mimics inhibiting cell viability, proliferation, migration and invasion, mainly through miR-145. MiR-145 transfection increased pMLC levels by targeting the MYPT1 subunit of the regulatory myosin phosphatase. MYPT1 knockdown by siRNAs reproduced miR-145 effects suggesting miR-145 as a tumor suppressor through MYPT1 targeting, leading to a subsequent increase of pMLC levels with implications for cervical cell viability, migration and invasion. Graphical abstract Image 1 Highlights • miR-143/145 affect viability, migration and invasion on monolayer and spheroid cultures. • miR-145 produces a stronger inhibitory effect and increases pMLC levels through MYPT1 targeting. • MYPT1 silencing by siRNAs reproduces miR-145 effects. [ABSTRACT FROM AUTHOR]

Abstract Myosin phosphatase is an evolutionarily conserved regulator of actomyosin contractility, comprised of a regulatory subunit (Mypt1), and a catalytic subunit (PP1). Zebrafish has become an ideal model organism for the study of the genetic and cell physiological role of the myosin phosphatase in morphogenesis and embryonic development. We identified and characterized a novel splice variant of Mypt1 ( ppp1r12a-tv202 ) from zebrafish, which is widely expressed during early embryonic development. Importantly, mutant alleles and antisense morpholinos that have been used to demonstrate the important role of Mypt1 in early development, not only disrupt the longer splice variants, but also tv202. The protein product of ppp1r12a-tv202 (Mypt1-202) contains the PP1-binding N-terminus, but lacks the regulatory C-terminus, which contains two highly conserved inhibitory phosphorylation sites. We observed that the protein product of tv202 assembled a constitutively active myosin phosphatase uninhibited by kinases such as Zipk. Thus, we propose that Mypt1-202 plays an important role in maintaining baseline Mlc2 dephosphorylation and actomyosin relaxation during early zebrafish development. Highlights • Zebrafish broadly express a novel splice variant of Mypt1, ppp1r12a-tv202. • Mypt1-202 lacks critical inhibitory phosphorylation sites. • Mypt1-202 forms a constitutively active myosin phosphatase. [ABSTRACT FROM AUTHOR]

Abstract: Aim: During early post‐natal development, arterial contraction depends less on Ca2+‐signalling pathways but more on changes in Ca2+‐sensitivity compared to adult animals. Whether this difference is related to Rho‐kinase, one of the major players affecting Ca2+‐sensitivity, is unknown for intact vessels. Thus, we tested the hypothesis that Rho‐kinase critically contributes to the higher Ca2+‐sensitivity of contraction in intact arteries of 1‐week‐old rats. Methods: We studied 1‐week‐old, 4‐ to 5‐week‐old and 10‐ to 12‐week‐old rats performing isometric myography, Ca2+‐fluorimetry and Western blotting using intact saphenous arteries and arterial pressure measurements under urethane anaesthesia. Results: In 10‐ to 12‐week‐old rats, methoxamine (MX) produced vasoconstriction associated with an increase in [Ca2+]i and Ca2+‐sensitivity. In contrast, in 1‐week‐old rats these contractions were accompanied only by an increase in Ca2+‐sensitivity. All MX‐induced effects were reduced by the Rho‐kinase inhibitor Y‐27632; this reduction was complete only in 1‐week‐old rats. The Rho‐kinase specific site Thr855 on MYPT1 was increasingly phosphorylated by MX in vessels of 1‐week‐old, but not 10‐ to 12‐week‐old rats; this effect was also inhibited completely by Y‐27632. The Rho‐kinase inhibitor fasudil in a dose not affecting the pressor response to MX in 4‐ to 5‐week‐old rats reduced it considerably in 1‐week‐old rats. Conclusion: Our results suggest that the higher Ca2+‐sensitivity of arterial contraction in 1‐week‐old compared to 10‐ to 12‐week‐old rats is due to a greater Rho‐kinase activity. Constitutively active Rho‐kinase contributes to MX‐induced contraction in 10‐ to 12‐week‐old rats. In 1‐week‐old rats, additional Rho‐kinase activation is involved. This remodelling of the Rho‐kinase pathway is associated with its increased contribution to adrenergic arterial pressure responses. [ABSTRACT FROM AUTHOR]

PKC-potentiated phosphorylation-dependent inhibitory protein of protein phosphatase 1 (CPI-17), an endogenous myosin phosphatase inhibitory protein, is considered a key molecule for Ca2+ sensitization of the contractile apparatus. Here, we have used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 to generate CPI-17-deficient [knockout (KO)] and threonine 38 (T38)-phospho-resistant mice [threonine mutant into alanine (TA)], and then effects of CPI-17 on vascular contractility in vitro and mean blood pressure (MBP) in vivo were investigated. In isolated thoracic aorta, phorbol 12, 13-dibutyrate induced a sustained contraction of wild-type (WT) mice, whereas no contraction showed from TA or KO mice. A high concentration of KCl solution-induced contraction was not different between transgenic and WT mice. In contrast, phenylephrine (PE)-induced contractions in both mutant strains were significantly smaller than those of WT mice in association with a low level of myosin phosphorylation, suggesting that at least part of PE-induced contraction is regulated by phosphorylation of CPI-17 at T38. Finally, the physiologic role of CPI-17 in the regulation of blood pressure was investigated using radio telemetry. MBP was decreased significantly in both transgenic mice, even with a compensatory increase in heart rate. In summary, we generated KO and constitutively phospho-resistant mouse models of CPI-17 for the first time. p-CPI-17 at T38, possibly by PKC, could be important to maintain vascular contractility and blood pressure in vivo. [ABSTRACT FROM AUTHOR]

Polo-like kinase 1 (Plk1) is an instrumental kinase that modulates many aspects of the cell cycle. Previous investigations have indicated that Plk1 is a target of the DNA damage response, and Plk1 inhibition is dependent on ATM/ATR and Chk1. But the exact mechanism remains elusive. In a proteomic screen to identify Chk1-interacting proteins, we found that myosin phosphatase targeting protein 1 (MYPT1) was present in the immunocomplex. MYPT1 is phosphorylated by CDK1, thus recruiting protein phosphatase 1β (PP1cβ) to dephosphorylate and inactivate Plk1. Here we identified that Chk1 directly interacts with MYPT1 and preferentially phosphorylates MYPT1 at Ser20, which is essential for MYPT1-PP1cβ interaction and subsequent Plk1 dephosphorylation. Phosphorylation of Ser20 is abolished during mitotic damage when Chk1 is inhibited. The degradation of MYPT1 is also regulated by Chk1 phosphorylation. Our results thus unveil the underlying machinery that attenuates Plk1 activity during mitotic damage through Chk1-induced phosphorylation of MYPT1. [ABSTRACT FROM AUTHOR]

Although recent studies reveal that activation of the metabolic and Ca2+ sensor AMPK strongly inhibits smooth muscle contraction, there is a paucity of information about the potential linkage between pharmacological AMPK activation and vascular smooth muscle (VSM) contraction regulation. Our aim was to test the general hypothesis that the allosteric AMPK activator A-769662 causes VSM relaxation via inhibition of contractile protein activation, and to specifically determine which activation mechanism(s) is(are) affected. The ability of A-769662 to cause endothelium-independent relaxation of contractions induced by several contractile stimuli was examined in large and small musculocutaneous and visceral rabbit arteries. For comparison, the structurally dissimilar AMPK activators MET, SIM, and BBR were assessed. A-769662 displayed artery- and agonist-dependent differential inhibitory activities that depended on artery size and location. A-769662 did not increase AMPK-pT172 levels, but did increase phosphorylation of the downstream AMPK substrate, acetyl-CoA carboxylase (ACC). A-769662 did not inhibit basal phosphorylation levels of several contractile protein regulatory proteins, and did not alter the activation state of rhoA. A-769662 did not inhibit Ca2+- and GTPγS-induced contractions in β-escin-permeabilized muscle, suggesting that A-769662 must act by inhibiting Ca2+ signaling. In intact artery, A-769662 immediately reduced basal intracellular free calcium ([Ca2+]i), inhibited a stimulus-induced increase in [Ca2+]i, and inhibited a cyclopiazonic acid (CPA)-induced contraction. MET increased AMPK-pT172, and caused neither inhibition of contraction nor inhibition of [Ca2+]i. Together, these data support the hypothesis that the differential inhibition of stimulus-induced arterial contractions by A-769662 was due to selective inhibition of a Ca2+ mobilization pathway, possibly involving CPA-dependent Ca2+ entry via an AMPK-independent pathway. That MET activated AMPK without causing arterial relaxation suggests that AMPK activation does not necessarily cause VSM relaxation.

The present study determined the changes in the expression levels of MYPT1, CPI-17 and MLC20 in the ileum of mice with neonatal induced necrotizing enterocolitis (NEC) to provide a basis for a pathogenesis model that includes smooth muscle changes during NEC. A group of 7-day-old BALB/c mice were fed with formula (40 μl/g, 5 times/day) and given hypoxia treatments (5% O2 and 95% N2 for 10 min, twice daily) for 4 days to induce NEC and establish a mouse model. A control group of 7-day-old BALB/c mice were left with their mother for the duration of the treatment. After establishing the model, the two groups of mice were sacrificed, and the terminal ileum tissue was collected and subjected to western blot analysis and immunohistochemistry. The results showed the expression levels of MYPT1 and pMYPT1 in the ileum of the mice in the NEC group were lower than those in the control group (P<0.01). The levels of CPI17 and pCPI17 were higher in the NEC group compared with those in the control group. The expression level of MLC20 in NEC group was lower than that in the control group (P<0.01), but the level of pMLC20 in the NEC group was higher (P<0.05). The results of immunohistochemistry showed that the staining intensities of MYPT1, CPI-17 and MLC20 in the NEC group were lighter than those in the control group, and the proportion of positive cells was also lower in the NEC group (P<0.01). Taken together our results suggest that establishment of NEC is accompanied by changes in the protein levels of MYPT1 and pCPI-17, which can regulate smooth muscle contraction in the ileum. [ABSTRACT FROM AUTHOR]

Smooth muscle is a major component of most hollow organ systems (e.g., airways, vasculature, bladder and gut/gastrointestine); therefore, the coordinated regulation of contraction is a key property of smooth muscle. When smooth muscle functions normally, it contributes to general health and wellness, but its dysfunction is associated with morbidity and mortality. Rho-associated protein kinase (ROCK) is central to calcium-independent, actomyosin-mediated contractile force generation in the vasculature, thereby playing a role in smooth muscle contraction, cell motility and adhesion. Recent evidence supports an important role for ROCK in the increased vasoconstriction and remodeling observed in various models of hypertension. This review will provide a commentary on the development of specific ROCK inhibitors and their clinical application. Fasudil will be discussed as an example of bench-to-bedside development of a clinical therapeutic that is used to treat conditions of vascular hypercontractility. Due to the wide spectrum of biological processes regulated by ROCK, many additional clinical indications might also benefit from ROCK inhibition. Apart from the importance of ROCK in smooth muscle contraction, a variety of other protein kinases are known to play similar roles in regulating contractile force. The zipper-interacting protein kinase (ZIPK) and integrin-linked kinase (ILK) are two well-described regulators of contraction. The relative contribution of each kinase to contraction depends on the muscle bed as well as hormonal and neuronal stimulation. Unfortunately, specific inhibitors for ZIPK and ILK are still in the development phase, but the success of fasudil suggests that inhibitors for these other kinases may also have valuable clinical applications. Notably, the directed inhibition of ZIPK with a pseudosubstrate molecule shows unexpected effects on the contractility of gastrointestinal smooth muscle.

The regulation of intestinal epithelial permeability requires phosphorylation of myosin regulatory light chain (MLC). The phosphorylation status of MLC is regulated by myosin light chain phosphatase (MLCP) activities. The activity of the catalytic subunit of MLCP (PP1cδ) toward MLC depends on its regulatory subunit (MYPT1). In this study, we revealed the presence of two MYPT1 isoforms, full length and variant 2 in human intestinal (Caco-2) epithelial cells and isolated intestinal epithelial cells (IECs) from mice. In confluent Caco-2 cells, MYPT1 was distributed at cell–cell contacts and colocalized with F-actin. These results suggest that MYPT1 isoforms are expressed in intestinal epithelial cells and MYPT1 may be involved in the regulation of intestinal epithelial barrier function. [ABSTRACT FROM AUTHOR]

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 touchscreen‑based 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. [Display omitted] [ABSTRACT FROM AUTHOR]

Lysophosphatidic acid (LPA) species accumulate in the ascites of ovarian high-grade serous cancer (HGSC) and are associated with short relapse-free survival. LPA is known to support metastatic spread of cancer cells by activating a multitude of signaling pathways via G-protein-coupled receptors of the LPAR family. Systematic unbiased analyses of the LPA-regulated signal transduction network in ovarian cancer cells have, however, not been reported to date. Methods: LPA-induced signaling pathways were identified by phosphoproteomics of both patient-derived and OVCAR8 cells, RNA sequencing, measurements of intracellular Ca 2+ and cAMP as well as cell imaging. The function of LPARs and downstream signaling components in migration and entosis were analyzed by selective pharmacological inhibitors and RNA interference. Results: Phosphoproteomic analyses identified > 1100 LPA-regulated sites in > 800 proteins and revealed interconnected LPAR1, ROCK/RAC, PKC/D and ERK pathways to play a prominent role within a comprehensive signaling network. These pathways regulate essential processes, including transcriptional responses, actomyosin dynamics, cell migration and entosis. A critical component of this signaling network is MYPT1, a stimulatory subunit of protein phosphatase 1 (PP1), which in turn is a negative regulator of myosin light chain 2 (MLC2). LPA induces phosphorylation of MYPT1 through ROCK (T853) and PKC/ERK (S507), which is majorly driven by LPAR1. Inhibition of MYPT1, PKC or ERK impedes both LPA-induced cell migration and entosis, while interference with ROCK activity and MLC2 phosphorylation selectively blocks entosis, suggesting that MYPT1 figures in both ROCK/MLC2-dependent and -independent pathways. We finally show a novel pathway governed by LPAR2 and the RAC-GEF DOCK7 to be indispensable for the induction of entosis. Conclusion: We have identified a comprehensive LPA-induced signal transduction network controlling LPA-triggered cytoskeletal changes, cell migration and entosis in HGSC cells. Due to its pivotal role in this network, MYPT1 may represent a promising target for interfering with specific functions of PP1 essential for HGSC progression., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

In this investigation, we made a study of the efficacy of luteolin (a flavonoid found in plants such as vegetables, herbs and fruits) on vascular contractibility and to elucidate the mechanism underlying the relaxation. Isometric contractions of denuded muscles were stored and combined with western blot analysis which was conducted to assess the phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and phosphorylation-dependent inhibitory protein for myosin phosphatase (CPI-17) and to examine the effect of luteolin on the RhoA/ROCK/CPI-17 pathway. Luteolin significantly alleviated phorbol ester-, fluoride- and thromboxane mimetic-elicited contractions regardless of endothelial nitric oxide synthesis, implying its direct effect on smooth muscle. It also significantly alleviated the fluoride-elicited elevation in pCPI-17 and pMYPT1 levels and phorbol 12,13-dibutyrate-elicited increase in pERK1/2 level, suggesting depression of ROCK and PKC/MEK activity and ensuing phosphorylation of MYPT1, CPI-17 and ERK1/2. Taken together, these results suggest that luteolin-elicited relaxation includes myosin phosphatase reactivation and calcium desensitization, which seems to be arbitrated by CPI-17 dephosphorylation via ROCK/PKC inhibition.

Variability in myosin phosphatase (MP) subunits may provide specificity in signaling pathways that regulate muscle tone. We utilized public databases and computational algorithms to investigate the phylogenetic diversity of MP regulatory (PPP1R12A-C) and inhibitory (PPP1R14A-D) subunits. The comparison of exonic coding sequences and expression data confirmed or refuted the existence of isoforms and their tissue-specific expression in different model organisms. The comparison of intronic and exonic sequences identified potential expressional regulatory elements. As examples, smooth muscle MP regulatory subunit (PPP1R12A) is highly conserved through evolution. Its alternative exon E24 is present in fish through mammals with two invariant features: 1) a reading frame shift generating a premature termination codon and 2) a hexanucleotide sequence adjacent to the 3= splice site hypothesized to be a novel suppressor of exon splicing. A characteristic of the striated muscle MP regulatory subunit (PPP1R12B) locus is numerous and phylogenetically variable transcriptional start sites. In fish this locus only codes for the small (M21) subunit, suggesting the primordial function of this gene. Inhibitory subunits show little intragenic variability; their diversity is thought to have arisen by expansion and tissue-specific expression of different gene family members. We demonstrate differences in the regulatory landscape between smooth muscle enriched (PPP1R14A) and more ubiquitously expressed (PPP1R14B) family members and identify deeply conserved intronic sequence and predicted transcriptional cis-regulatory elements. This bioinformatic and computational study has uncovered a number of attributes of MP subunits that supports selection of ideal model organisms and testing of hypotheses regarding their physiological significance and regulated expression. [ABSTRACT FROM AUTHOR]

Zipper-interacting protein kinase (ZIPK) is a conserved vertebrate-specific regulator of actomyosin contractility in smooth muscle and non-muscle cells. Murine ZIPK has undergone an unusual divergence in sequence and regulation compared to other ZIPK orthologs. In humans, subcellular localization is controlled by phosphorylation of threonines 299 and 300. In contrast, ZIPK subcellular localization in mouse and rat is controlled by interaction with PAR-4. We carried out a comparative biochemical characterization of the regulation of the zebrafish ortholog of ZIPK. Like the human orthologs zebrafish ZIPK undergoes nucleocytoplasmic-shuttling and is abundant in the cytoplasm, unlike the primarily nuclear rat ZIPK. Rat ZIPK, but not human or zebrafish ZIPK, interacts with zebrafish PAR-4. Mutation of the conserved residues required for activation of the mammalian orthologs abrogated activity of the zebrafish ZIPK. In contrast to the human ortholog, mutation of threonine 299 and 300 in the zebrafish ZIPK has no effect on the activity or subcellular localization. Thus, we found that zebrafish ZIPK functions in a manner most similar to the human ZIPK and quite distinct from murine orthologs, yet the regulation of subcellular localization is not conserved. [ABSTRACT FROM AUTHOR]

Vascular smooth muscle contraction and the myogenic response regulate blood flow in the resistance vascular and contribute to systemic blood pressure. Three pathways are currently known to contribute to the development of the myogenic response: (i) Ca2+-dependent phosphorylation of LC20; (ii) Ca2+ sensitization through inhibition of myosin phosphatase; and (iii) cortical actin polymerization. A number of regulatory smooth muscle proteins are integrated with these pathways to fine tune the response and facilitate adaptations to vascular (patho)physiologies. Of particular interest is the SMTN family of proteins, consisting of SMTN-A, SMTN-B, and the SMTN-like protein, SMTNL1. The SMTN-B and SMTNL1 proteins are both implicated in regulating smooth muscle contractility and contributing to vascular adaptations associated with hypertension, pregnancy, and exercise training. In the case of SMTNL1, the protein plays multiple roles in regulating contraction through functional interactions with contractile regulators as well as transcriptional control of the contractile phenotype and Ca2+-sensitizing capacity. For the first time, preliminary results suggest SMTNL1 is involved in the myogenic response of the cerebral resistance vasculature. In this regard, global SMTNL1 deletion is associated with greater myogenic reactivity of cerebral arterioles, although the precise mechanism accounting for this finding remains to be defined. [ABSTRACT FROM AUTHOR]

The dephosphorylation of myosin by the MP causes smooth muscle relaxation. MP is also a key target of signals that regulate vascular tone and thus blood flow and pressure. Here, we review studies from the past two decades that support the hypothesis that the regulated expression of MP subunits is a critical determinant of smooth muscle responses to constrictor and dilator signals. In particular, the highly regulated splicing of the regulatory subunit Mypt1 Exon 24 is proposed to tune sensitivity to NO/ cGMP-mediated relaxation. The regulated transcription of the MP inhibitory subunit CPI-17 is proposed to determine sensitivity to agonist-mediated constriction. The expression of these subunits is specific in the microcirculation and varies in developmental and disease contexts. To date, the relationship between MP subunit expression and vascular function in these different contexts is correlative; confirmation of the hypothesis will require the generation of genetically engineered mice to test the role of MP subunits and their isoforms in the specificity of vascular smooth muscle responses to constrictor and dilator signals. [ABSTRACT FROM AUTHOR]

Cancer cell migration during metastasis is mediated by a highly polarized cytoskeleton. MARK2 and its invertebrate homolog Par1B are kinases that regulate the microtubule cytoskeleton to mediate polarization of neurons in mammals and embryos in invertebrates. However, the role of MARK2 in cancer cell migration is unclear. Using osteosarcoma cells, we found that in addition to its known localizations on microtubules and the plasma membrane, MARK2 also associates with the actomyosin cytoskeleton and focal adhesions. Cells depleted of MARK proteins demonstrated that MARK2 promotes phosphorylation of both myosin II and the myosin phosphatase targeting subunit MYPT1 to synergistically drive myosin II contractility and stress fiber formation in cells. Studies with isolated proteins showed that MARK2 directly phosphorylates myosin II regulatory light chain, while its effects on MYPT1 phosphorylation are indirect. Using a mutant lacking the membrane-binding domain, we found that membrane association is required for focal adhesion targeting of MARK2, where it specifically enhances cell protrusion by promoting FAK phosphorylation and formation of focal adhesions oriented in the direction of migration to mediate directionally persistent cell motility. Together, our results define MARK2 as a master regulator of the actomyosin and microtubule cytoskeletal systems and focal adhesions to mediate directional cancer cell migration. • MARK2 associates with the actomyosin cytoskeleton • MARK2 promotes phosphorylation of MRLC and MYPT1 to drive contractility • MARK2 associates with focal adhesions via its membrane-binding domain • MARK2 promotes FAK activation and focal adhesion formation and orientation Pasapera et al. show that the microtubule regulatory and polarity protein MARK2 associates with actomyosin and promotes phosphorylation of myosin II regulatory light chain and MYPT1 to drive contractility. MARK2 also associates with focal adhesions (FAs) and promotes FAK activation and FA formation/orientation to drive directional cell migration. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]