Phosphorylated Peptide Derived from the Myosin Phosphatase Target Subunit Is a Novel Inhibitor of Protein Phosphatase-1.

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.

The Structural Effects of Phosphorylation of Protein Arginine Methyltransferase 5 on Its Binding to Histone H4.

The protein arginine methyltransferase 5 (PRMT5) enzyme is responsible for arginine methylation on various proteins, including histone H4. PRMT5 is a promising drug target, playing a role in the pathomechanism of several diseases, especially in the progression of certain types of cancer. It was recently proved that the phosphorylation of PRMT5 on T80 residue increases its methyltransferase activity; furthermore, elevated levels of the enzyme were measured in the case of human hepatocellular carcinoma and other types of tumours. In this study, we constructed the complexes of the unmodified human PRMT5-methylosome protein 50 (MEP50) structure and its T80-phosphorylated variant in complex with the full-length histone H4 peptide. The full-length histone H4 was built in situ into the human PRMT5-MEP50 enzyme using experimental H4 fragments. Extensive molecular dynamic simulations and structure and energy analyses were performed for the complexed and apo protein partners, as well. Our results provided an atomic level explanation for two important experimental findings: (1) the increased methyltransferase activity of the phosphorylated PRMT5 when compared to the unmodified type; (2) the PRMT5 methylates only the free form of histone H4 not bound in the nucleosome. The atomic level complex structure H4-PRMT5-MEP50 will help the design of new inhibitors and in uncovering further structure-function relationships of PRMT enzymes.

Smoothelin-Like Protein 1 Regulates the Thyroid Hormone-Induced Homeostasis and Remodeling of C2C12 Cells via the Modulation of Myosin Phosphatase.

The pathological elevation of the active thyroid hormone (T3) level results in the manifestation of hyperthyroidism, which is associated with alterations in the differentiation and contractile function of skeletal muscle (SKM). Myosin phosphatase (MP) is a major cellular regulator that hydrolyzes the phosphoserine of phosphorylated myosin II light chain. MP consists of an MYPT1/2 regulatory and a protein phosphatase 1 catalytic subunit. Smoothelin-like protein 1 (SMTNL1) is known to inhibit MP by directly binding to MP as well as by suppressing the expression of MYPT1 at the transcriptional level. Supraphysiological vs. physiological concentration of T3 were applied on C2C12 myoblasts and differentiated myotubes in combination with the overexpression of SMTNL1 to assess the role and regulation of MP under these conditions. In non-differentiated myoblasts, MP included MYPT1 in the holoenzyme complex and its expression and activity was regulated by SMTNL1, affecting the phosphorylation level of MLC20 assessed using semi-quantitative Western blot analysis. SMTNL1 negatively influenced the migration and cytoskeletal remodeling of myoblasts measured by high content screening. In contrast, in myotubes, the expression of MYPT2 but not MYPT1 increased in a T3-dependent and SMTNL1-independent manner. T3 treatment combined with SMTNL1 overexpression impeded the activity of MP. In addition, MP interacted with Na+/K+-ATPase and dephosphorylated its inhibitory phosphorylation sites, identifying this protein as a novel MP substrate. These findings may help us gain a better understanding of myopathy, muscle weakness and the disorder of muscle regeneration in hyperthyroid patients.

How Do Post-Translational Modifications Influence the Pathomechanistic Landscape of Huntington's Disease? A Comprehensive Review.

Huntington's disease (HD) is an autosomal dominant inherited neurodegenerative disorder characterized by the loss of motor control and cognitive ability, which eventually leads to death. The mutant huntingtin protein (HTT) exhibits an expansion of a polyglutamine repeat. The mechanism of pathogenesis is still not fully characterized; however, evidence suggests that post-translational modifications (PTMs) of HTT and upstream and downstream proteins of neuronal signaling pathways are involved. The determination and characterization of PTMs are essential to understand the mechanisms at work in HD, to define possible therapeutic targets better, and to challenge the scientific community to develop new approaches and methods. The discovery and characterization of a panoply of PTMs in HTT aggregation and cellular events in HD will bring us closer to understanding how the expression of mutant polyglutamine-containing HTT affects cellular homeostasis that leads to the perturbation of cell functions, neurotoxicity, and finally, cell death. Hence, here we review the current knowledge on recently identified PTMs of HD-related proteins and their pathophysiological relevance in the formation of abnormal protein aggregates, proteolytic dysfunction, and alterations of mitochondrial and metabolic pathways, neuroinflammatory regulation, excitotoxicity, and abnormal regulation of gene expression.

Inhibition of protein phosphatase-1 and -2A by ellagitannins: structure-inhibitory potency relationships and influences on cellular systems.

Several ellagitannins inhibited the activity of protein phosphatase-1 (PP1) and -2 A (PP2A) catalytic subunits (PP1c and PP2Ac) with preferential suppression of PP1c over PP2Ac. The inhibitory potency for PP1c followed the order of tellimagrandin I > mahtabin A > praecoxin B > 1.2-Di-O-galloyl-4.6-(S)-HHDP-ß-D-glucopyranose > pedunculagin with IC50 values ranging from 0.20 µM to 2.47 µM. The interaction of PP1c and tellimagrandin I was assessed by NMR saturation transfer difference, surface plasmon resonance, isothermal titration calorimetry, and microscale thermophoresis based binding techniques. Tellimagrandin I suppressed viability and phosphatase activity of HeLa cells, while mahtabin A was without effect. Conversely, mahtabin A increased the phosphorylation level of SNAP-25Thr138 and suppressed exocytosis of cortical synaptosomes, whereas tellimagrandin I was without influence. Our results establish ellagitannins as partially selective inhibitors of PP1 and indicate that these polyphenols may act distinctly in cellular systems depending on their membrane permeability and/or their actions on cell membranes.

Aralkyl selenoglycosides and related selenosugars in acetylated form activate protein phosphatase-1 and -2A.

Aralkyl and aryl selenoglycosides as well as glycosyl selenocarboxylate derivatives were assayed on the activity of protein phosphatase-1 (PP1) and -2A (PP2A) catalytic subunits (PP1c and PP2Ac) in search of compounds for PP1c and PP2Ac effectors. The majority of tested selenoglycosides activated both PP1c and PP2Ac by ∼2-4-fold in a phosphatase assay with phosphorylated myosin light chain substrate when the hydroxyl groups of the glycosyl moiety were acetylated, but they were without any effects in the non-acetylated forms. A peptide from the myosin phosphatase target subunit-1 (MYPT123-38) that included an RVxF PP1c-binding motif attenuated activation of PP1c by 2-Trifluoromethylbenzyl 2,3,4,6-tetra-O-acetyl-1-seleno-ß-d-glucopyranoside (TFM-BASG) and 4-Bromobenzyl 2,3,4,6-tetra-O-acetyl-1-seleno-ß-d-glucopyranoside (Br-BASG). MYPT123-38 stimulated PP2Ac and contributed to PP2Ac activation exerted by either Br-BASG or TFM-BASG. Br-BASG and TFM-BASG suppressed partially binding of PP1c to MYPT1 in surface plasmon resonance based binding experiments. Molecular docking predicted that the hydrophobic binding surfaces in PP1c for interaction with either the RVxF residues of PP1c-interactors or selenoglycosides are partially overlapped. Br-BASG and TFM-BASG caused a moderate increase in the phosphatase activity of HeLa cells in 1 h, and suppressed cell viability in 24 h incubations. In conclusion, our present study identified selenoglycosides as novel activators of PP1 and PP2A as well as provided insights into the structural background of their interactions establishing a molecular model for future design of more efficient phosphatase activator molecules.

Pregnancy and Smoothelin-like Protein 1 (SMTNL1) Deletion Promote the Switching of Skeletal Muscle to a Glycolytic Phenotype in Human and Mice.

Pregnancy promotes physiological adaptations throughout the body, mediated by the female sex hormones progesterone and estrogen. Changes in the metabolic properties of skeletal muscle enable the female body to cope with the physiological challenges of pregnancy and may also be linked to the development of insulin resistance. We conducted global microarray, proteomic, and metabolic analyses to study the role of the progesterone receptor and its transcriptional regulator, smoothelin-like protein 1 (SMTNL1) in the adaptation of skeletal muscle to pregnancy. We demonstrate that pregnancy promotes fiber-type changes from an oxidative to glycolytic isoform in skeletal muscle. This phenomenon is regulated through an interaction between SMTNL1 and progesterone receptor, which alters the expression of contractile and metabolic proteins. smtnl1(-/-) mice are metabolically less efficient and show impaired glucose tolerance. Pregnancy antagonizes these effects by inducing metabolic activity and increasing glucose tolerance. Our results suggest that SMTNL1 has a role in mediating the actions of steroid hormones to promote fiber switching in skeletal muscle during pregnancy. Our findings also bear on the management of gestational diabetes that develops as a complication of pregnancy in ~4% of women.

Protein phosphatase-1 is involved in the maintenance of normal homeostasis and in UVA irradiation-induced pathological alterations in HaCaT cells and in mouse skin.

The number of ultraviolet (UV) radiation-induced skin diseases such as melanomas is on the rise. The altered behavior of keratinocytes is often coupled with signaling events in which Ser/Thr specific protein kinases and phosphatases regulate various cellular functions. In the present study the role of protein phosphatase-1 (PP1) was investigated in the response of human keratinocyte (HaCaT) cells and mouse skin to UV radiation. PP1 catalytic subunit (PP1c) isoforms, PP1cα/γ and PP1cδ, are all localized to the cytoskeleton and cytosol of keratinocytes, but PP1cδ was found to be dominant over PP1α/γ in the nucleus. PP1c-silencing in HaCaT cells decreased the phosphatase activity and suppressed the viability of the cells. Exposure to a 10 J/cm(2) UVA dose induced HaCaT cell death and resulted in a 30% decrease of phosphatase activity. PP1c-silencing and UVA irradiation altered the gene expression profile of HaCaT cells and suggested that the expression of 19 genes was regulated by the combined treatments with many of these genes being involved in malignant transformation. Microarray analysis detected altered expression levels of genes coding for melanoma-associated proteins such as keratin 1/10, calcium binding protein S100A8 and histone 1b. Treatment of Balb/c mice with the PP1-specific inhibitor tautomycin (TM) exhibited increased levels of keratin 1/10 and S100A8, and a decreased level of histone 1b proteins following UVA irradiation. Moreover, TM treatment increased pigmentation of the skin which was even more apparent when TM was followed by UVA irradiation. Our data identify PP1 as a regulator of the normal homeostasis of keratinocytes and the UV-response.

Smoothelin-like protein 1 promotes insulin sensitivity and modulates the contractile properties of endometrial epithelial cells with insulin resistance.

Introduction: The incidence of infertility is significantly higher in women with diseases linked to impaired glucose homeostasis, such as insulin resistance. Defective glucose metabolism interferes with fertilization; however, the molecular mechanism underlying this interference is unclear. Smoothelin-like protein 1 (SMTNL1) was isolated from muscle and steroid hormone-responsive tissues and regulates the contractile functions of various cell types through the inhibition of myosin phosphatase (MP) holoenzyme. In addition, SMTNL-1 after phosphorylation at Ser301 by protein kinase A translocates to the nucleus and functions as a transcriptional co-activator of the progesterone receptor-B. SMTNL1 null mice exhibit reduced reproductive fitness and are more prone to type 2 diabetes mellitus. However, the role of SMTNL1 in endometrial epithelial cells is not known. Methods: The effect of SMTNL1 overexpression was investigated in pregnancy and in gestational diabetic endometrial epithelial cell models by immunofluorescent staining, cell migration, and semi quantitative Western blot analysis and glucose uptake assay. Results: We show that SMTNL1 promotes the differentiation of endometrial epithelial cells in a progesterone-dependent manner to attenuate insulin resistance. Furthermore, SMTNL1 hampers the migration capacity of epithelial cells in a gestational diabetes model by inhibiting the expression of MYPT1, the regulatory subunit of MP, and the activity of the holoenzyme, resulting in increased phosphorylation of the 20 kDa regulatory myosin light chain. SMTNL1 also acts as an insulin-sensitizing agent by increasing the gene expression of PP2A and DUPS9 protein phosphatases, resulting in decreased ERK1/2 activity and, hence, decreasing the phosphorylation of IRS-1 at Ser612 under gestational diabetes conditions. Conclusion: SMTNL1 may have therapeutic relevance to the progesterone-dependent inhibition of endometrial epithelial cell migration under hyperglycemic conditions and insulin sensitivity in the endometrium in gestational diabetes or other metabolic disorders.

Smoothelin-like 1 protein is a bifunctional regulator of the progesterone receptor during pregnancy.

During pregnancy, uterine smooth muscle (USM) coordinately adapts its contractile phenotype in order to accommodate the developing fetus and then prepare for delivery. Herein we show that SMTNL1 plays a major role in pregnancy to promote adaptive responses in USM and that this process is specifically mediated through interactions of SMTNL1 with the steroid hormone receptor PR-B. In vitro and in vivo SMTNL1 selectively binds PR and not other steroid hormone receptors. The physiological relationship between the two proteins was also established in global gene expression and transcriptional reporter studies in pregnant smtnl1(-/-) mice and by RNA interference in progesterone-sensitive cell lines. We show that the contraction-associated and progestin-sensitive genes (oxytocin receptor, connexin 43, and cyclooxygenase-2) and prolactins are down-regulated in pregnant smtnl1(-/-) mice. We suggest that SMTNL1 is a bifunctional co-regulator of PR-B signaling and thus provides a molecular mechanism whereby PR-B is targeted to alter gene expression patterns within USM cells to coordinately promote alterations in USM function during pregnancy.

Protein phosphatase-1M and Rho-kinase affect exocytosis from cortical synaptosomes and influence neurotransmission at a glutamatergic giant synapse of the rat auditory system.

Protein phosphatase-1M (PP1M, myosin phosphatase) consists of a PP1 catalytic subunit (PP1c) and the myosin phosphatase target subunit-1 (MYPT1). RhoA-activated kinase (ROK) regulates PP1M via inhibitory phosphorylation of MYPT1. Using multidisciplinary approaches, we have studied the roles of PP1M and ROK in neurotransmission. Electron microscopy demonstrated the presence of MYPT1 and ROK in both pre- and post-synaptic terminals. Tautomycetin (TMC), a PP1-specific inhibitor, decreased the depolarization-induced exocytosis from cortical synaptosomes. trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride, a ROK-specific inhibitor, had the opposite effect. Mass spectrometry analysis identified several MYPT1-bound synaptosomal proteins, of which interactions of synapsin-I, syntaxin-1, calcineurin-A subunit, and Ca(2+) /calmodulin-dependent kinase II with MYPT1 were confirmed. In intact synaptosomes, TMC increased, whereas Y27632 decreased the phosphorylation levels of MYPT1(Thr696) , myosin-II light chain(Ser19) , synapsin-I(Ser9) , and syntaxin-1(Ser14) , indicating that PP1M and ROK influence their phosphorylation status. Confocal microscopy indicated that MYPT1 and ROK are present in the rat ventral cochlear nucleus both pre- and post-synaptically. Analysis of the neurotransmission in an auditory glutamatergic giant synapse demonstrated that PP1M and ROK affect neurotransmission via both pre- and post-synaptic mechanisms. Our data suggest that both PP1M and ROK influence synaptic transmission, but further studies are needed to give a full account of their mechanism of action.

Mechanisms by which smoothelin-like protein 1 reverses insulin resistance in myotubules and mice.

Insulin resistance (InR) is manifested in skeletal muscle by decreased insulin-stimulated glucose uptake due to impaired insulin signaling and multiple post-receptor intracellular defects. Chronic glucose-induced insulin resistance leads to the activation of Ser/Thr kinases and elevated phosphorylation of insulin receptor substrate 1 (IRS1) on Ser residues. Phosphorylation of IRS1 triggers the dissociation of IRS1 and its downstream effector, phosphatidylinositol 3-kinase. In the present study, we provide evidence for the insulin-sensitizing role of smoothelin-like protein 1 (SMTNL1) that is a ligand-dependent co-regulator of steroid receptors, predominantly the progesterone receptor. SMTNL1 was transiently overexpressed in insulin-resistant C2C12 myotubes. A proteome profiler array revealed that mTOR and Ser/Thr kinases were SMTNL1-dependent signaling pathways. In the presence of progesterone, overexpression was coupled to decreased Ser phosphorylation of IRS1 at Ser307, Ser318, and Ser612 residues. SMTNL1 also induced the expression and activity of the p85 subunit of PI3K. SMTNL1 regulated the expression of PKCε, which phosphorylates IRS1 at Ser318 residue. SMTNL1 also regulated ERK1/2 and JNK, which phosphorylate IRS1 at Ser612 and Ser307, respectively. Real-time metabolic measurements of oxygen consumption rate and extracellular acidification rate revealed that SMTNL1 improved glycolysis and promoted the utilization of alternative carbon fuels. SMTNL1 also rescued the mitochondrial respiration defect induced by chronic insulin exposure. Collectively, SMTNL1 plays a crucial role in maintaining the physiological ratio of Tyr/Ser IRS1 phosphorylation and attenuates the insulin-signaling cascade that contributes to impaired glucose disposal, which makes it a potential therapeutic target for improving InR.

Smoothelin-like 1 protein regulates myosin phosphatase-targeting subunit 1 expression during sexual development and pregnancy.

Pregnancy coordinately alters the contractile properties of both vascular and uterine smooth muscles reducing systemic blood pressure and maintaining uterine relaxation. The precise molecular mechanisms underlying these pregnancy-induced adaptations have yet to be fully defined but are likely to involve changes in the expression of proteins regulating myosin phosphorylation. Here we show that smoothelin like protein 1 (SMTNL1) is a key factor governing sexual development and pregnancy induced adaptations in smooth and striated muscle. A primary target gene of SMTNL1 in these muscles is myosin phosphatase-targeting subunit 1 (MYPT1). Deletion of SMTNL1 increases expression of MYPT1 30-40-fold in neonates and during development expression of both SMTNL1 and MYPT1 increases over 20-fold. Pregnancy also regulates SMTNL1 and MYPT1 expression, and deletion SMTNL1 greatly exaggerates expression of MYPT1 in vascular smooth muscle, producing a profound reduction in force development in response to phenylephrine as well as sensitizing the muscle to acetylcholine. We also show that MYPT1 is expressed in Type2a muscle fibers in mice and humans and its expression is regulated during pregnancy, suggesting unrecognized roles in mediating skeletal muscle plasticity in both species. Our findings define a new conserved pathway in which sexual development and pregnancy mediate smooth and striated muscle adaptations through SMTNL1 and MYPT1.

Src mediates cytokine-stimulated gene expression in airway myocytes through ERK MAPK.

The p38 and extracellular signal-regulated kinases (ERK) mitogen-activated protein kinases (MAPK) participate in cytokine-stimulated inflammatory gene expression in airway smooth muscle cells. The following study was undertaken to determine whether Src tyrosine kinases are signaling intermediaries upstream of cytokine-stimulated MAPK activation and gene expression. Treating human airway myocytes with interleukin (IL)-1ß, tumor necrosis factor (TNF) α and interferon (IFN) γ caused a rapid 1.8-fold increase in Src family tyrosine kinase activity within 1 minute that remained 2.3 to 2.7 fold above basal conditions for 15 minutes. This activity was blocked by addition of 30 µM PP1, a pyrimidine inhibitor specific for Src family tyrosine kinases, in immune-complex assays to confirm that this stimulus activates Src tyrosine kinase. Addition of PP1 also blocked cytokine-stimulated expression of IL-1ß, IL-6 and IL-8, while decreasing phosphorylation of ERK, but not p38 MAPK. Since this inflammatory stimulus may activate additional inflammatory signaling pathways downstream of Src, we tested the effects of PP1 on phosphorylation of signal transducers and activators of transcription (STAT). PP1 had no effect on cytokine-stimulated STAT 1 or STAT 3 phosphorylation. These results demonstrate that Src tyrosine kinases participate in the regulation of IL-1ß, IL-6 and IL-8 expression and that these effects of Src are mediated through activation of ERK MAPK and not p38 MAPK or STAT1/STAT3 phosphorylation.

Smoothelin-Like Protein 1 Regulates Development and Metabolic Transformation of Skeletal Muscle in Hyperthyroidism.

Hyperthyroidism triggers a glycolytic shift in skeletal muscle (SKM) by altering the expression of metabolic proteins, which is often accompanied by peripheral insulin resistance. Our previous results show that smoothelin-like protein 1 (SMTNL1), a transcriptional co-regulator, promotes insulin sensitivity in SKM. Our aim was to elucidate the role of SMTNL1 in SKM under physiological and pathological 3,3',5-Triiodo-L-thyronine (T3) concentrations. Human hyper- and euthyroid SKM biopsies were used for microarray analysis and proteome profiler arrays. Expression of genes related to energy production, nucleic acid- and lipid metabolism was changed significantly in hyperthyroid samples. The phosphorylation levels and activity of AMPKα2 and JNK were increased by 15% and 23%, respectively, in the hyperthyroid samples compared to control. Moreover, SMTNL1 expression showed a 6-fold decrease in the hyperthyroid samples and in T3-treated C2C12 cells. Physiological and supraphysiological concentrations of T3 were applied on differentiated C2C12 cells upon SMTNL1 overexpression to assess the activity and expression level of the elements of thyroid hormone signaling, insulin signaling and glucose metabolism. Our results demonstrate that SMTNL1 selectively regulated TRα expression. Overexpression of SMTNL1 induced insulin sensitivity through the inhibition of JNK activity by 40% and hampered the non-genomic effects of T3 by decreasing the activity of ERK1/2 through PKCδ. SMTNL1 overexpression reduced IRS1 Ser307 and Ser612 phosphorylation by 52% and 53%, respectively, in hyperthyroid model to restore the normal responsiveness of glucose transport to insulin. SMTNL1 regulated glucose phosphorylation and balances glycolysis and glycogen synthesis via the downregulation of hexokinase II by 1.3-fold. Additionally, mitochondrial respiration and glycolysis were measured by SeaHorse analysis to determine cellular metabolic function/phenotype of our model system in real-time. T3 overload strongly increased the rate of acidification and a shift to glycolysis, while SMTNL1 overexpression antagonizes the T3 effects. These lines of evidence suggest that SMTNL1 potentially prevents hyperthyroidism-induced changes in SKM, and it holds great promise as a novel therapeutic target in insulin resistance.

MicroRNA Regulatory Pathways in the Control of the Actin-Myosin Cytoskeleton.

MicroRNAs (miRNAs) are key modulators of post-transcriptional gene regulation in a plethora of processes, including actin-myosin cytoskeleton dynamics. Recent evidence points to the widespread effects of miRNAs on actin-myosin cytoskeleton dynamics, either directly on the expression of actin and myosin genes or indirectly on the diverse signaling cascades modulating cytoskeletal arrangement. Furthermore, studies from various human models indicate that miRNAs contribute to the development of various human disorders. The potentially huge impact of miRNA-based mechanisms on cytoskeletal elements is just starting to be recognized. In this review, we summarize recent knowledge about the importance of microRNA modulation of the actin-myosin cytoskeleton affecting physiological processes, including cardiovascular function, hematopoiesis, podocyte physiology, and osteogenesis.

Myosin phosphatase interacts with and dephosphorylates the retinoblastoma protein in THP-1 leukemic cells: its inhibition is involved in the attenuation of daunorubicin-induced cell death by calyculin-A.

Reversible phosphorylation of the retinoblastoma protein (pRb) is an important regulatory mechanism in cell cycle progression. The role of protein phosphatases is less understood in this process, especially concerning the regulatory/targeting subunits involved. It is shown that pretreatment of THP-1 leukemic cells with calyculin-A (CL-A), a cell-permeable phosphatase inhibitor, attenuated daunorubicin (DNR)-induced cell death and resulted in increased pRb phosphorylation and protection against proteolytic degradation. Protein phosphatase-1 catalytic subunits (PP1c) dephosphorylated the phosphorylated C-terminal fragment of pRb (pRb-C) slightly, whereas when PP1c was complexed to myosin phosphatase target subunit-1 (MYPT1) in myosin phosphatase (MP) holoenzyme dephosphorylation was stimulated. The pRb-C phosphatase activity of MP was partially inhibited by anti-MYPT1(1-296) implicating MYPT1 in targeting PP1c to pRb. MYPT1 became phosphorylated on both inhibitory sites (Thr695 and Thr850) upon CL-A treatment of THP-1 cells resulting in the inhibition of MP activity. MYPT1 and pRb coprecipitated from cell lysates by immunoprecipitation with either anti-MYPT1 or anti-pRb antibodies implying that pRb-MYPT1 interaction occurred at cellular levels. Surface plasmon resonance-based experiments confirmed binding of pRb-C to both PP1c and MYPT1. In control and DNR-treated cells, MYPT1 and pRb were predominantly localized in the nucleus exhibiting partial colocalization as revealed by immunofluorescence using confocal microscopy. Upon CL-A treatment, nucleo-cytoplasmic shuttling of both MYPT1 and pRb, but not PP1c, was observed. The above data imply that MP, with the targeting role of MYPT1, may regulate the phosphorylation level of pRb, thereby it may be involved in the control of cell cycle progression and in the mediation of chemoresistance of leukemic cells.

Myosin phosphatase: Unexpected functions of a long-known enzyme.

Myosin phosphatase (MP) holoenzyme is a Ser/Thr specific enzyme, which is the member of protein phosphatase type 1 (PP1) family and composed of a PP1 catalytic subunit (PP1c/PPP1CB) and a myosin phosphatase targeting subunit (MYPT1/PPP1R12A). PP1c is required for the catalytic activity of the holoenzyme, while MYPT1 regulates MP through targeting the holoenzyme to its substrates. Above the well-characterized function of MP, as the major regulator of smooth muscle contractility mediating the dephosphorylation of 20 kDa myosin light chain, accumulating data support its role in other, non-contractile functions. In this review, we summarize the scaffold function of MP holoenzyme and its roles in processes such as cell cycle, development, gene expression regulation and neurotransmitter release. In particular, we highlight novel interacting proteins of MYPT1 and pathophysiological functions of MP relevant to tumorigenesis, insulin resistance and neurodegenerative disorders. This article is part of a Special Issue entitled: Protein Phosphatases as Critical Regulators for Cellular Homeostasis edited by Prof. Peter Ruvolo and Dr. Veerle Janssens.

Granulocyte colony stimulating factor increases drug resistance of leukaemic blast cells to daunorubicin.

Acute leukaemia is known as the most common cancer in childhood. Febrile neutropenia is a common serious side effect of the cytostatic treatment of malignancies. The clinical use of Granulocyte Colony Stimulating Factor (G-CSF) has become widespread to minimize chemotherapy-induced myelosuppression and febrile neutropenia in childhood solid tumors, acute lymphoid leukaemia (ALL) and in several trials with AML. In case of ALL this seems to be reasonable because, due to the absence of G-CSF receptor (G-CSFR) on the surface of normal lymphoid cells, G-CSF does not have any influence on the pathways of proliferation and differentiation of lymphoid lineage cells. It has been suggested, however, that ALL blasts with B or T cell surface antigens as well as biphenotypic leukaemia cells express G-CSFR, and they are able to respond to exogenously added G-CSF with proliferation. In this study we investigated how G-CSF might influence the sensitivity of leukemic cells to daunorubicin induced cell death using MTT assay, flow cytometry and Western blot analysis. After pretreatment of KG-1 leukaemic cells with G-CSF a moderate increase in the resistance of these cells to daunorubicin could be observed. These results draw attention to the risk of G-CSF application as an adjuvant therapy of childhood ALL. In addition, adjuvant treatment of AML patients with G-CSF in order to prevent neutropenia, or its use in priming regimens might result resistance to daunorubicin.

Myosin phosphatase accelerates cutaneous wound healing by regulating migration and differentiation of epidermal keratinocytes via Akt signaling pathway in human and murine skin.

Wound healing is a complex sequence of cellular and molecular processes such as inflammation, cell migration, proliferation and differentiation. ROCK is a widely investigated Ser/Thr kinase with important roles in rearranging the actomyosin cytoskeleton. ROCK inhibitors have already been approved to improve corneal endothelial wound healing. The purpose of this study was to investigate the functions of myosin phosphatase (MP or PPP1CB), a type-1 phospho-Ser/Thr-specific protein phosphatase (PP1), one of the counter enzymes of ROCK, in skin homeostasis and wound healing. To confirm our hypotheses, we applied tautomycin (TM), a selective PP1 inhibitor, on murine skin that caused the arrest of wound closure. TM suppressed scratch closure of HaCaT human keratinocytes without having influence on the survival of the cells. Silencing of, the regulatory subunit of MP (MYPT1 or PPP1R12A), had a negative impact on the migration of keratinocytes and it influenced the cell-cell adhesion properties by decreasing the impedance of HaCaT cells. We assume that MP differentially activates migration and differentiation of keratinocytes and plays a key role in the downregulation of transglutaminase-1 in lower layers of skin where no differentiation is required. MAPK Proteome Profiler analysis on human ex vivo biopsies with MYPT1-silencing indicated that MP contributes to the mediation of wound healing by regulating the Akt signaling pathway. Our findings suggest that MP plays a role in the maintenance of normal homeostasis of skin and the process of wound healing.