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.

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.

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 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.

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.

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.

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.

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.

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.

The Short-Chain Fatty Acid Propionate Inhibits Adipogenic Differentiation of Human Chorion-Derived Mesenchymal Stem Cells Through the Free Fatty Acid Receptor 2.

Free fatty acid receptor 2 (FFAR2, also known as GPR43) is a G-protein-coupled receptor activated by short-chain fatty acids that are produced by gut microbiota through fermentation of nondigestible carbohydrates. FFAR2 functions as a metabolic sensor and is expressed in metabolically active tissues, such as adipose tissue. Earlier studies proved the connection between FFAR2 and adipocyte differentiation in mice. The aim of this study was to investigate the implication of FFAR2 receptor in adipogenesis in human chorion-derived mesenchymal stem cells (cMSCs). The short-chain fatty acid, propionate, and phenylacetamide a selective FFAR2 agonist resulted in a marked suppression of lipid droplet accumulation during the adipogenic differentiation of cMSCs. Western blot studies revealed that FFAR2 was detectable at any time point of the differentiation period. The direct involvement of FFAR2 in the differentiation into adipocytes was proven by the downregulation of its gene expression in cMSCs by lentiviral messenger RNA (mRNA) silencing transduction particles. Our results showed that a significant suppression in lipid accumulation upon FFAR2 agonist treatments was elicited by FFAR2-silencing. Based on these results we suggest that propionate inhibits the formation of adipocytes from MSCs and acts on adipogenesis predominantly via FFAR2.

Correction: Myosin phosphatase and RhoA-activated kinase modulate neurotransmitter release by regulating SNAP-25 of SNARE complex.

[This corrects the article DOI: 10.1371/journal.pone.0177046.].

Myosin phosphatase and RhoA-activated kinase modulate neurotransmitter release by regulating SNAP-25 of SNARE complex.

Reversible phosphorylation of neuronal proteins plays an important role in the regulation of neurotransmitter release. Myosin phosphatase holoenzyme (MP) consists of a protein phosphatase-1 (PP1) catalytic subunit (PP1c) and a regulatory subunit, termed myosin phosphatase targeting subunit (MYPT1). The primary function of MP is to regulate the phosphorylation level of contractile proteins; however, recent studies have shown that MP is localized to neurons, and is also involved in the mediation of neuronal processes. Our goal was to investigate the effect of RhoA-activated kinase (ROK) and MP on the phosphorylation of one potential neuronal substrate, the synaptosomal-associated protein of 25 kDa (SNAP-25). SNAP-25 is a member of the SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex, along with synaptobrevin and syntaxin, and the primary role of SNAP25 is to mediate vesicle fusion. We showed that MYPT1 interacts with SNAP-25, as revealed by immunoprecipitation and surface plasmon resonance based binding studies. Mass spectrometry analysis and in vitro phosphorylation/dephosphorylation assays demonstrated that ROK phosphorylates, while MP dephosphorylates, SNAP-25 at Thr138. Silencing MYPT1 in B50 neuroblastoma cells increased phosphorylation of SNAP-25 at Thr138. Inhibition of PP1 with tautomycetin increased, whereas inhibition of ROK by H1152, decreased the phosphorylation of SNAP-25 at Thr138 in B50 cells, in cortical synaptosomes, and in brain slices. In response to the transduction of the MP inhibitor, kinase-enhanced PP1 inhibitor (KEPI), into synaptosomes, an increase in phosphorylation of SNAP-25 and a decrease in the extent of neurotransmitter release were detected. The interaction between SNAP-25 and syntaxin increased with decreasing phosphorylation of SNAP-25 at Thr138, upon inhibition of ROK. Our data suggest that ROK/MP play a crucial role in vesicle trafficking, fusion, and neurotransmitter release by oppositely regulating the phosphorylation of SNAP-25 at Thr138.

Interplay of myosin phosphatase and protein phosphatase-2A in the regulation of endothelial nitric-oxide synthase phosphorylation and nitric oxide production.

The inhibitory phosphorylation of endothelial nitric oxide (NO) synthase (eNOS) at Thr497 (eNOSpThr497) by protein kinase C or RhoA-activated kinase is a major regulatory determinant of eNOS activity. The signalling mechanisms involved in the dephosphorylation of eNOSpThr497 have not yet been clarified. This study identifies myosin phosphatase (MP) holoenzyme consisting of protein phosphatase-1 catalytic subunit (PP1c) and MP target subunit-1 (MYPT1) as an eNOSpThr497 phosphatase. In support of this finding are: (i) eNOS and MYPT1 interacts in various endothelial cells (ECs) and in in vitro binding assays (ii) MYPT1 targets and stimulates PP1c toward eNOSpThr497 substrate (iii) phosphorylation of MYPT1 at Thr696 (MYPT1pThr696) controls the activity of MP on eNOSpThr497. Phosphatase inhibition suppresses both NO production and transendothelial resistance (TER) of ECs. In contrast, epigallocatechin-3-gallate (EGCG) signals ECs via the 67 kDa laminin-receptor (67LR) resulting in protein kinase A dependent activation of protein phosphatase-2A (PP2A). PP2A dephosphorylates MYPT1pThr696 and thereby stimulates MP activity inducing dephosphorylation of eNOSpThr497 and the 20 kDa myosin II light chains. Thus an interplay of MP and PP2A is involved in the physiological regulation of EC functions implying that an EGCG dependent activation of these phosphatases leads to enhanced NO production and EC barrier improvement.

Myosin phosphatase and RhoA-activated kinase modulate arginine methylation by the regulation of protein arginine methyltransferase 5 in hepatocellular carcinoma cells.

Myosin phosphatase (MP) holoenzyme is a protein phosphatase-1 (PP1) type Ser/Thr specific enzyme that consists of a PP1 catalytic (PP1c) and a myosin phosphatase target subunit-1 (MYPT1). MYPT1 is an ubiquitously expressed isoform and it targets PP1c to its substrates. We identified the protein arginine methyltransferase 5 (PRMT5) enzyme of the methylosome complex as a MYPT1-binding protein uncovering the nuclear MYPT1-interactome of hepatocellular carcinoma cells. It is shown that PRMT5 is regulated by phosphorylation at Thr80 by RhoA-associated protein kinase and MP. Silencing of MYPT1 increased the level of the PRMT5-specific symmetric dimethylation on arginine residues of histone 2 A/4, a repressing gene expression mark, and it resulted in a global change in the expression of genes affecting cellular processes like growth, proliferation and cell death, also affecting the expression of the retinoblastoma protein and c-Myc. The phosphorylation of the MP inhibitory MYPT1T850 and the regulatory PRMT5T80 residues as well as the symmetric dimethylation of H2A/4 were elevated in human hepatocellular carcinoma and in other types of cancers. These changes correlated positively with the grade and state of the tumors. Our results suggest the tumor suppressor role of MP via inhibition of PRMT5 thereby regulating gene expression through histone arginine dimethylation.

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.

Identification of protein phosphatase interacting proteins from normal and UVA-irradiated HaCaT cell lysates by surface plasmon resonance based binding technique using biotin-microcystin-LR as phosphatase capturing molecule.

Identification of the interacting proteins of protein phosphatases is crucial to understand the cellular roles of these enzymes. Microcystin-LR (MC-LR), a potent inhibitor of protein phosphatase-1 (PP1), -2A (PP2A), PP4, PP5 and PP6, was biotinylated, immobilized to streptavidin-coupled sensorchip surface and used in surface plasmon resonance (SPR) based binding experiments to isolate phosphatase binding proteins. Biotin-MC-LR captured PP1 catalytic subunit (PP1c) stably and the biotin-MC-LR-PP1c complex was able to further interact with the regulatory subunit (MYPT1) of myosin phosphatase. Increased biotin-MC-LR coated sensorchip surface in the Surface Prep unit of Biacore 3000 captured PP1c, PP2Ac and their regulatory proteins including MYPT1, MYPT family TIMAP, inhibitor-2 as well as PP2A-A and -Bα-subunits from normal and UVA-irradiated HaCaT cell lysates as revealed by dot blot analysis of the recovered proteins. Biotin-MC-LR was used for the subcellular localization of protein phosphatases in HaCaT cells by identification of phosphatase-bound biotin-MC-LR with fluorescent streptavidin conjugates. Partial colocalization of the biotin-MC-LR signals with those obtained using anti-PP1c and anti-PP2Ac antibodies was apparent as judged by confocal microscopy. Our results imply that biotin-MC-LR is a suitable capture molecule in SPR for isolation of protein phosphatase interacting proteins from cell lysates in sufficient amounts for immunological detection.