The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Avril V. Somlyo - One of the best experts on this subject based on the ideXlab platform.
-
RESEARCH ARTICLE Role of Telokin in Regulating Murine Gastric Fundus Smooth Muscle Tension
2016Co-Authors: Bhupal P. Bhetwal¤b, Avril V. Somlyo, Kenton M. S, A. PerrinoAbstract:Telokin phosphorylation by cyclic GMP-dependent protein kinase facilitates smooth muscle relaxation. In this study we examined the relaxation of gastric fundus smooth muscles from basal tone, or pre-contracted with KCl or carbachol (CCh), and the phosphorylation of telo-kin S13, myosin light chain (MLC) S19, MYPT1 T853, T696, and CPI-17 T38 in response to 8-Bromo-cGMP, the NO donor sodium nitroprusside (SNP), or nitrergic neurotransmission. We compared MLC phosphorylation and the contraction and relaxation responses of gastric fundus smooth muscles from Telokin-/- mice and their wild-type littermates to KCl or CCh, and 8-Bromo-cGMP, SNP, or nitrergic neurotransmission, respectively. We compared the relaxation responses and Telokin phosphorylation of gastric fundus smooth muscles from wild-type mice andW/WVmice which lack ICC-IM, to 8-Bromo-cGMP, SNP, or nitrergic neu-rotransmission. We found that Telokin S13 is basally phosphorylated and that 8-Bromo-cGMP and SNP increased basal Telokin phosphorylation. In muscles pre-contracted with KCl or CCh, 8-Bromo-cGMP and SNP had no effect on CPI-17 or MYPT1 phosphorylation, but increased Telokin phosphorylation and reduced MLC phosphorylation. In Telokin-/- gas
-
Role of Telokin in Regulating Murine Gastric Fundus Smooth Muscle Tension.
PLOS ONE, 2015Co-Authors: Bhupal P. Bhetwal, Kenton M. Sanders, Avril V. Somlyo, Brian A. PerrinoAbstract:Telokin phosphorylation by cyclic GMP-dependent protein kinase facilitates smooth muscle relaxation. In this study we examined the relaxation of gastric fundus smooth muscles from basal tone, or pre-contracted with KCl or carbachol (CCh), and the phosphorylation of Telokin S13, myosin light chain (MLC) S19, MYPT1 T853, T696, and CPI-17 T38 in response to 8-Bromo-cGMP, the NO donor sodium nitroprusside (SNP), or nitrergic neurotransmission. We compared MLC phosphorylation and the contraction and relaxation responses of gastric fundus smooth muscles from Telokin-/- mice and their wild-type littermates to KCl or CCh, and 8-Bromo-cGMP, SNP, or nitrergic neurotransmission, respectively. We compared the relaxation responses and Telokin phosphorylation of gastric fundus smooth muscles from wild-type mice and W/WV mice which lack ICC-IM, to 8-Bromo-cGMP, SNP, or nitrergic neurotransmission. We found that Telokin S13 is basally phosphorylated and that 8-Bromo-cGMP and SNP increased basal Telokin phosphorylation. In muscles pre-contracted with KCl or CCh, 8-Bromo-cGMP and SNP had no effect on CPI-17 or MYPT1 phosphorylation, but increased Telokin phosphorylation and reduced MLC phosphorylation. In Telokin-/- gastric fundus smooth muscles, basal tone and constitutive MLC S19 phosphorylation were increased. Pre-contracted Telokin-/- gastric fundus smooth muscles have increased contractile responses to KCl, CCh, or cholinergic neurotransmission and reduced relaxation to 8-Bromo-cGMP, SNP, and nitrergic neurotransmission. However, basal Telokin phosphorylation was not increased when muscles were stimulated with lower concentrations of SNP or when the muscles were stimulated by nitrergic neurotransmission. SNP, but not nitrergic neurotransmission, increased Telokin Ser13 phosphorylation in both wild-type and W/WV gastric fundus smooth muscles. Our findings indicate that Telokin may play a role in attenuating constitutive MLC phosphorylation and provide an additional mechanism to augment gastric fundus mechanical responses to inhibitory neurotransmission.
-
The contractile responses of Telokin-/- gastric fundus smooth muscles are increased compared to wild-type controls.
2015Co-Authors: Bhupal P. Bhetwal, Kenton M. Sanders, Avril V. Somlyo, Brian A. PerrinoAbstract:A. Representative recordings of the contractile responses of wild-type and Telokin-/- gastric fundus smooth muscles to 1μM CCh (n = 4). B. Representative recordings of the contractile responses of wild-type and Telokin-/- gastric fundus smooth muscles to 15mM, 30mM, and 60mM KCl. C. Average integrals and peak amplitudes of contraction ± SD of wild-type and Telokin-/- gastric fundus smooth muscles to 15mM, 30mM, and 60mM KCl (#P
-
molecular mechanism of Telokin mediated disinhibition of myosin light chain phosphatase and camp cgmp induced relaxation of gastrointestinal smooth muscle
Journal of Biological Chemistry, 2012Co-Authors: Alexander S Khromov, Ko Momotani, Mykhaylo V Artamonov, John D Shannon, Avril V. SomlyoAbstract:Phospho-Telokin is a target of elevated cyclic nucleotide concentrations that lead to relaxation of gastrointestinal and some vascular smooth muscles (SM). Here, we demonstrate that in Telokin-null SM, both Ca2+-activated contraction and Ca2+ sensitization of force induced by a GST-MYPT1(654–880) fragment inhibiting myosin light chain phosphatase were antagonized by the addition of recombinant S13D Telokin, without changing the inhibitory phosphorylation status of endogenous MYPT1 (the regulatory subunit of myosin light chain phosphatase) at Thr-696/Thr-853 or activity of Rho kinase. Cyclic nucleotide-induced relaxation of force in Telokin-null ileum muscle was reduced but not correlated with a change in MYPT1 phosphorylation. The 40% inhibited activity of phosphorylated MYPT1 in Telokin-null ileum homogenates was restored to nonphosphorylated MYPT1 levels by addition of S13D Telokin. Using the GST-MYPT1 fragment as a ligand and SM homogenates from WT and Telokin KO mice as a source of endogenous proteins, we found that only in the presence of endogenous Telokin, thiophospho-GST-MYPT1 co-precipitated with phospho-20-kDa myosin regulatory light chain 20 and PP1. Surface plasmon resonance studies showed that S13D Telokin bound to full-length phospho-MYPT1. Results of a protein ligation assay also supported interaction of endogenous phosphorylated MYPT1 with Telokin in SM cells. We conclude that the mechanism of action of phospho-Telokin is not through modulation of the MYPT1 phosphorylation status but rather it contributes to cyclic nucleotide-induced relaxation of SM by interacting with and activating the inhibited full-length phospho-MYPT1/PP1 through facilitating its binding to phosphomyosin and thus accelerating 20-kDa myosin regulatory light chain dephosphorylation.
-
Molecular Mechanism of Telokin-mediated Disinhibition of Myosin Light Chain Phosphatase and cAMP/cGMP-induced Relaxation of Gastrointestinal Smooth Muscle
The Journal of biological chemistry, 2012Co-Authors: Alexander S Khromov, Ko Momotani, Mykhaylo V Artamonov, John D Shannon, Li Jin, Masumi Eto, Avril V. SomlyoAbstract:Phospho-Telokin is a target of elevated cyclic nucleotide concentrations that lead to relaxation of gastrointestinal and some vascular smooth muscles (SM). Here, we demonstrate that in Telokin-null SM, both Ca(2+)-activated contraction and Ca(2+) sensitization of force induced by a GST-MYPT1(654-880) fragment inhibiting myosin light chain phosphatase were antagonized by the addition of recombinant S13D Telokin, without changing the inhibitory phosphorylation status of endogenous MYPT1 (the regulatory subunit of myosin light chain phosphatase) at Thr-696/Thr-853 or activity of Rho kinase. Cyclic nucleotide-induced relaxation of force in Telokin-null ileum muscle was reduced but not correlated with a change in MYPT1 phosphorylation. The 40% inhibited activity of phosphorylated MYPT1 in Telokin-null ileum homogenates was restored to nonphosphorylated MYPT1 levels by addition of S13D Telokin. Using the GST-MYPT1 fragment as a ligand and SM homogenates from WT and Telokin KO mice as a source of endogenous proteins, we found that only in the presence of endogenous Telokin, thiophospho-GST-MYPT1 co-precipitated with phospho-20-kDa myosin regulatory light chain 20 and PP1. Surface plasmon resonance studies showed that S13D Telokin bound to full-length phospho-MYPT1. Results of a protein ligation assay also supported interaction of endogenous phosphorylated MYPT1 with Telokin in SM cells. We conclude that the mechanism of action of phospho-Telokin is not through modulation of the MYPT1 phosphorylation status but rather it contributes to cyclic nucleotide-induced relaxation of SM by interacting with and activating the inhibited full-length phospho-MYPT1/PP1 through facilitating its binding to phosphomyosin and thus accelerating 20-kDa myosin regulatory light chain dephosphorylation.
B. Paul Herring - One of the best experts on this subject based on the ideXlab platform.
-
Regulation of 130-kDa Smooth Muscle Myosin Light Chain Kinase Expression by an Intronic CArG Element
The Journal of biological chemistry, 2013Co-Authors: Meng Chen, April M Hoggatt, Wenwu Zhang, Susan J. Gunst, Ghassan S. Kassab, Johnathan D. Tune, B. Paul HerringAbstract:The mylk1 gene encodes a 220-kDa nonmuscle myosin light chain kinase (MLCK), a 130-kDa smooth muscle MLCK (smMLCK), as well as the non-catalytic product Telokin. Together, these proteins play critical roles in regulating smooth muscle contractility. Changes in their expression are associated with many pathological conditions; thus, it is important to understand the mechanisms regulating expression of mylk1 gene transcripts. Previously, we reported a highly conserved CArG box, which binds serum response factor, in intron 15 of mylk1. Because this CArG element is near the promoter that drives transcription of the 130-kDa smMLCK, we examined its role in regulating expression of this transcript. Results show that deletion of the intronic CArG region from a β-galactosidase reporter gene abolished transgene expression in mice in vivo. Deletion of the CArG region from the endogenous mylk1 gene, specifically in smooth muscle cells, decreased expression of the 130-kDa smMLCK by 40% without affecting expression of the 220-kDa MLCK or Telokin. This reduction in 130-kDa smMLCK expression resulted in decreased phosphorylation of myosin light chains, attenuated smooth muscle contractility, and a 24% decrease in small intestine length that was associated with a significant reduction of Ki67-positive smooth muscle cells. Overall, these data show that the CArG element in intron 15 of the mylk1 gene is necessary for maximal expression of the 130-kDa smMLCK and that the 130-kDa smMLCK isoform is specifically required to regulate smooth muscle contractility and small intestine smooth muscle cell proliferation.
-
Hprt-targeted transgenes provide new insights into smooth muscle-restricted promoter activity
American journal of physiology. Cell physiology, 2006Co-Authors: Ketrija Touw, April M Hoggatt, Gina Simon, B. Paul HerringAbstract:Mouse Telokin and SM22α promoters have previously been shown to direct smooth muscle cell-specific expression of transgenes in vivo in adult mice. However, the activity of these promoters is highly...
-
Regulation of myosin light chain kinase and Telokin expression in smooth muscle tissues
American journal of physiology. Cell physiology, 2006Co-Authors: B. Paul Herring, Patricia J. Gallagher, Feng Yin, Omar El-mounayri, Jiliang ZhouAbstract:The mylk1 gene is a large gene spanning ∼250 kb and comprising at least 31 exons. The mylk1 gene encodes at least four protein products: two isoforms of the 220-kDa myosin light chain kinase (MLCK)...
-
Smooth muscle-specific genes are differentially sensitive to inhibition by Elk-1
Molecular and cellular biology, 2005Co-Authors: Jiliang Zhou, B. Paul HerringAbstract:Understanding the mechanism of smooth muscle cell (SMC) differentiation will provide the foundation for elucidating SMC-related diseases, such as atherosclerosis, restenosis, and asthma. In the current study, overexpression of Elk-1 in SMCs down-regulated expression of several endogenous smooth muscle-restricted proteins, including Telokin, SM22alpha, and smooth muscle alpha-actin. In contrast, down-regulation of endogenous Elk-1 in smooth muscle cells increased the expression of only Telokin and SM22alpha, suggesting that smooth muscle-specific promoters are differentially sensitive to the inhibitory effects of Elk-1. Consistent with this, overexpression of the DNA binding domain of Elk-1, which acts as a dominant-negative protein by displacing endogenous Elk-1, enhanced the expression of Telokin and SM22alpha without affecting expression of smooth muscle alpha-actin. Elk-1 suppressed the activity of smooth muscle-restricted promoters, including the Telokin promoter that does not contain a consensus Elk-1 binding site, through its ability to block myocardin-induced activation of the promoters. Gel mobility shift and chromatin immunoprecipitation assays revealed that Elk-1 binds to a nonconsensus binding site in the Telokin promoter and Elk-1 binding is dependent on serum response factor (SRF) binding to a nearby CArG box. Although overexpression of the SRF-binding B-box domain of Elk-1 is sufficient to repress the myocardin activation of the Telokin promoter, this repression is not as complete as that seen with an Elk-1 fragment that includes the DNA binding domain. In addition, reporter gene assays demonstrate that an intact Elk-1 binding site in the Telokin promoter is required for Elk-1 to maximally inhibit promoter activity. Together, these data suggest that the differential sensitivity of smooth muscle-specific genes to inhibition by Elk-1 may play a role in the complex changes in smooth muscle-specific protein expression that are observed under pathological conditions.
-
Regulation of smooth muscle-specific gene expression by homeodomain proteins, Hoxa10 and Hoxb8.
The Journal of biological chemistry, 2005Co-Authors: Omar El-mounayri, Jason W. Triplett, Charles W. Yates, B. Paul HerringAbstract:Abstract Smooth muscle cells arise from different populations of precursor cells during embryonic development. The mechanisms that specify the smooth muscle cell phenotype in each of these populations of cells are largely unknown. In many tissues and organs, homeodomain transcription factors play a key role in directing cell specification. However, little is known about how these proteins regulate smooth muscle differentiation. Using degenerate reverse transcription-PCR coupled to cDNA library screening we identified two homeodomain proteins, Hoxa10 and Hoxb8, which are expressed in adult mouse smooth muscle tissues. All three of the previously described transcripts of the Hoxa10 gene, Hoxa10-1, Hoxa10-2, and Hoxa10-3, were identified. Hoxa10-1 directly activated the smooth muscle-specific Telokin promoter but did not activate the SM22α, smooth muscle α-actin, or smooth muscle myosin heavy chain promoters. Small interfering RNA-mediated knock-down of Hoxa10-1 demonstrated that Hoxa10-1 is required for high levels of Telokin expression in smooth muscle cells from uterus and colon. On the other hand, Hoxb8 inhibited the activity of the Telokin, SM22α, and smooth muscle α-actin promoters. Cotransfection of Hoxa10-1 together with Hoxa10-2 or Hoxb8 suggested that Hoxa10-2 and Hoxb8 act as competitive inhibitors of Hoxa10-1. Results from gel mobility shift assays demonstrated that Hoxa10-1, Hoxa10-2, and Hoxb8 bind directly to multiple sites in the Telokin promoter. Mutational analysis of Telokin promoter reporter genes demonstrated that the three homeodomain protein binding sites located between -80 and -75, +2 and +6, and +14 and +17 were required for maximal promoter activation by Hoxa10-1 and maximal inhibition by Hoxb8. Together these data demonstrate that the genes encoding smooth muscle-restricted proteins are direct transcriptional targets of clustered homeodomain proteins and that different homeodomain proteins have distinct effects on the promoters of these genes.
Gabriele Pfitzer - One of the best experts on this subject based on the ideXlab platform.
-
Neonatal mouse ileum: functional properties and protein composition of the contractile machinery
Pediatric Research, 2014Co-Authors: Frank Eifinger, Gabriele Pfitzer, Lubomir T. Lubomirov, Elena Dercks, Borislav Genchev, Bernhard Roth, Wolfram F. Neiss, Mechthild M SchroeterAbstract:Background: Immature motility of the ileum may contribute to life-threatening diseases. Little is known about the normal biomechanics of the neonatal ileum in relation to the protein composition of its contractile machinery. Methods: We analyzed the tissue architecture, the biomechanics in intact and β-escin-permeabilized preparations, and the protein composition in neonatal (P0) and adult murine ileum. Results: Muscle thickness of the P0 ileum was −50% of the adult ileum and passive compliance was higher. Carbachol- and KCl-elicited contractions were tonic rather than phasic as in the adult. Ca^2+ sensitivity was higher and relaxation rate was slower in β-escin-permeabilized P0 compared with adult ileum. The expression level of β-actin relative to α-actin was higher, and those of total actin, myosin, myosin light chain kinase, the catalytic subunit of myosin phosphatase and Telokin were lower compared with the adult. The expression level of MYPT1 was similar, but P0 ileum expressed only the M133; the adult ileum also expressed the M130 isoform. Conclusion: The mechanical features and protein composition of the P0 ileum are similar to those of adult tonic smooth muscles. We propose that this is highly adaptive during fetal life allowing the small intestine to act predominantly as a container.
-
kinase related protein Telokin inhibits ca2 independent contraction in triton skinned guinea pig taenia coli
Biochemical Journal, 2010Co-Authors: Olga V Shcherbakova, Vladimir P. Shirinsky, Alexander V. Vorotnikov, Daria Serebryanaya, Alexander Postnikov, Mechthild M Schroeter, Stefan Zittrich, Angelika A Noegel, Gabriele PfitzerAbstract:KRP (kinase-related protein), also known as Telokin, has been proposed to inhibit smooth muscle contractility by inhibiting the phosphorylation of the rMLC (regulatory myosin light chain) by the Ca2+-activated MLCK (myosin light chain kinase). Using the phosphatase inhibitor microcystin, we show in the present study that KRP also inhibits Ca2+-independent rMLC phosphorylation and smooth muscle contraction mediated by novel Ca2+-independent rMLC kinases. Incubating KRP-depleted Triton-skinned taenia coli with microcystin at p Ca>8 induced a slow contraction reaching 90% of maximal force ( F max) at p Ca 4.5 after ~25 min. Loading the fibres with KRP significantly slowed down the force development, i.e. the time to reach 50% of F max was increased from 8 min to 35 min. KRP similarly inhibited rMLC phosphorylation of HMM (heavy meromyosin) in vitro by MLCK or by the constitutively active MLCK fragment (61K-MLCK) lacking the myosin-docking KRP domain. A C-terminally truncated KRP defective in myosin binding inhibited neither force nor HMM phosphorylation. Phosphorylated KRP inhibited the rMLC phosphorylation of HMM in vitro and Ca2+-insensitive contractions in fibres similar to unphosphorylated KRP, whereby the phosphorylation state of KRP was not altered in the fibres. We conclude that (i) KRP inhibits not only MLCK-induced contractions, but also those elicited by Ca2+-independent rMLC kinases; (ii) phosphorylation of KRP does not modulate this effect; (iii) binding of KRP to myosin is essential for this inhibition; and (iv) KRP inhibition of rMLC phosphorylation is most probably due to the shielding of the phosphorylation site on the rMLC. Abbreviations: aa, amino acids; DAPI, 4′,6-diamidino-2-phenylindole; DTT, dithiothreitol; ECL, enhanced chemiluminescence; F-actin, filamentous actin; GST, glutathione transferase; HMM, heavy meromyosin; ILK, integrin-linked kinase; KRP, kinase-related protein; MAPK, mitogen-activated protein kinase; MEK1, MAPK/ERK (extracellular-signal-regulated kinase) kinase 1; MHC, myosin heavy chain; MLC, myosin light chain; MLCK, MLC kinase; MLCP, MLC phosphatase; PKA, protein kinase A; PKG, protein kinase G; rMLC, regulatory MLC; ROK, Rho kinase; TBS, Tris-buffered saline; TCA, trichloroacetic acid; wt, wild-type; WME, whole muscle extract; ZIP kinase, MYPT1-associated zipper-interacting kinase
-
Kinase related protein / Telokin inhibits Ca2+-independent contraction in triton skinned guinea pig taenia coli
Biochemical Journal, 2010Co-Authors: Olga Shcherbakova, Daria Serebryanaya, Alexander Postnikov, Mechthild M Schroeter, Stefan Zittrich, Angelika A Noegel, Vladimir Shirinsky, Alexander Vorotnikov, Gabriele PfitzerAbstract:The kinase-related protein, KRP, also known as Telokin, has been proposed to inhibit smooth muscle contractility by inhibiting the phosphorylation of the regulatory light chains of myosin (rMLC) by the Ca2+-activated myosin light chain kinase (MLCK). Using the phosphatase inhibitor, microcystin, we now show that KRP also inhibits Ca2+-independent rMLC phosphorylation and smooth muscle contraction mediated by novel Ca2+-independent rMLC kinases. Incubating KRP depleted triton skinned taenia coli with microcystin at pCa
-
Kinase-related protein/Telokin inhibits Ca2+-independent contraction in Triton-skinned guinea pig taenia coli
The Biochemical journal, 2010Co-Authors: Olga V Shcherbakova, Vladimir P. Shirinsky, Alexander V. Vorotnikov, Daria Serebryanaya, Alexander Postnikov, Mechthild M Schroeter, Stefan Zittrich, Angelika A Noegel, Gabriele PfitzerAbstract:KRP (kinase-related protein), also known as Telokin, has been proposed to inhibit smooth muscle contractility by inhibiting the phosphorylation of the rMLC (regulatory myosin light chain) by the Ca2+-activated MLCK (myosin light chain kinase). Using the phosphatase inhibitor microcystin, we show in the present study that KRP also inhibits Ca2+-independent rMLC phosphorylation and smooth muscle contraction mediated by novel Ca2+-independent rMLC kinases. Incubating KRP-depleted Triton-skinned taenia coli with microcystin at p Ca>8 induced a slow contraction reaching 90% of maximal force ( F max) at p Ca 4.5 after ~25 min. Loading the fibres with KRP significantly slowed down the force development, i.e. the time to reach 50% of F max was increased from 8 min to 35 min. KRP similarly inhibited rMLC phosphorylation of HMM (heavy meromyosin) in vitro by MLCK or by the constitutively active MLCK fragment (61K-MLCK) lacking the myosin-docking KRP domain. A C-terminally truncated KRP defective in myosin binding inhibited neither force nor HMM phosphorylation. Phosphorylated KRP inhibited the rMLC phosphorylation of HMM in vitro and Ca2+-insensitive contractions in fibres similar to unphosphorylated KRP, whereby the phosphorylation state of KRP was not altered in the fibres. We conclude that (i) KRP inhibits not only MLCK-induced contractions, but also those elicited by Ca2+-independent rMLC kinases; (ii) phosphorylation of KRP does not modulate this effect; (iii) binding of KRP to myosin is essential for this inhibition; and (iv) KRP inhibition of rMLC phosphorylation is most probably due to the shielding of the phosphorylation site on the rMLC. Abbreviations: aa, amino acids; DAPI, 4′,6-diamidino-2-phenylindole; DTT, dithiothreitol; ECL, enhanced chemiluminescence; F-actin, filamentous actin; GST, glutathione transferase; HMM, heavy meromyosin; ILK, integrin-linked kinase; KRP, kinase-related protein; MAPK, mitogen-activated protein kinase; MEK1, MAPK/ERK (extracellular-signal-regulated kinase) kinase 1; MHC, myosin heavy chain; MLC, myosin light chain; MLCK, MLC kinase; MLCP, MLC phosphatase; PKA, protein kinase A; PKG, protein kinase G; rMLC, regulatory MLC; ROK, Rho kinase; TBS, Tris-buffered saline; TCA, trichloroacetic acid; wt, wild-type; WME, whole muscle extract; ZIP kinase, MYPT1-associated zipper-interacting kinase
Brian A. Perrino - One of the best experts on this subject based on the ideXlab platform.
-
Role of Telokin in Regulating Murine Gastric Fundus Smooth Muscle Tension.
PLOS ONE, 2015Co-Authors: Bhupal P. Bhetwal, Kenton M. Sanders, Avril V. Somlyo, Brian A. PerrinoAbstract:Telokin phosphorylation by cyclic GMP-dependent protein kinase facilitates smooth muscle relaxation. In this study we examined the relaxation of gastric fundus smooth muscles from basal tone, or pre-contracted with KCl or carbachol (CCh), and the phosphorylation of Telokin S13, myosin light chain (MLC) S19, MYPT1 T853, T696, and CPI-17 T38 in response to 8-Bromo-cGMP, the NO donor sodium nitroprusside (SNP), or nitrergic neurotransmission. We compared MLC phosphorylation and the contraction and relaxation responses of gastric fundus smooth muscles from Telokin-/- mice and their wild-type littermates to KCl or CCh, and 8-Bromo-cGMP, SNP, or nitrergic neurotransmission, respectively. We compared the relaxation responses and Telokin phosphorylation of gastric fundus smooth muscles from wild-type mice and W/WV mice which lack ICC-IM, to 8-Bromo-cGMP, SNP, or nitrergic neurotransmission. We found that Telokin S13 is basally phosphorylated and that 8-Bromo-cGMP and SNP increased basal Telokin phosphorylation. In muscles pre-contracted with KCl or CCh, 8-Bromo-cGMP and SNP had no effect on CPI-17 or MYPT1 phosphorylation, but increased Telokin phosphorylation and reduced MLC phosphorylation. In Telokin-/- gastric fundus smooth muscles, basal tone and constitutive MLC S19 phosphorylation were increased. Pre-contracted Telokin-/- gastric fundus smooth muscles have increased contractile responses to KCl, CCh, or cholinergic neurotransmission and reduced relaxation to 8-Bromo-cGMP, SNP, and nitrergic neurotransmission. However, basal Telokin phosphorylation was not increased when muscles were stimulated with lower concentrations of SNP or when the muscles were stimulated by nitrergic neurotransmission. SNP, but not nitrergic neurotransmission, increased Telokin Ser13 phosphorylation in both wild-type and W/WV gastric fundus smooth muscles. Our findings indicate that Telokin may play a role in attenuating constitutive MLC phosphorylation and provide an additional mechanism to augment gastric fundus mechanical responses to inhibitory neurotransmission.
-
The contractile responses of Telokin-/- gastric fundus smooth muscles are increased compared to wild-type controls.
2015Co-Authors: Bhupal P. Bhetwal, Kenton M. Sanders, Avril V. Somlyo, Brian A. PerrinoAbstract:A. Representative recordings of the contractile responses of wild-type and Telokin-/- gastric fundus smooth muscles to 1μM CCh (n = 4). B. Representative recordings of the contractile responses of wild-type and Telokin-/- gastric fundus smooth muscles to 15mM, 30mM, and 60mM KCl. C. Average integrals and peak amplitudes of contraction ± SD of wild-type and Telokin-/- gastric fundus smooth muscles to 15mM, 30mM, and 60mM KCl (#P
-
Telokin S13 phosphorylation is not increased by nitrergic neurotransmission in W/WV gastric fundus smooth muscles.
2015Co-Authors: Bhupal P. Bhetwal, Kenton M. Sanders, Avril V. Somlyo, Brian A. PerrinoAbstract:Representative recordings of the relaxation responses evoked by 30 sec of 5Hz EFS and 10Hz EFS (A), or 10μM SNP (B) from wild-type and W/WV fundus muscles. B. Representative western blot of phosphorylated and non-phosphorylated MLC from wild-type and W/WV fundus muscles treated with 10μM SNP. C. Average ratios ± SD of pS13:Telokin and representative western blots of phosphorylated Telokin S13 and Telokin from wild-type and W/WV muscles stimulated by 10Hz/30 sec EFS, or incubated with 10μM SNP. *P
-
The relaxation responses of pre-contracted Telokin-/- gastric fundus smooth muscles are reduced compared to wild-type controls.
2015Co-Authors: Bhupal P. Bhetwal, Kenton M. Sanders, Avril V. Somlyo, Brian A. PerrinoAbstract:Representative recordings of the relaxation responses of wild-type and Telokin-/- gastric fundus smooth muscles to (A) 10μM SNP, (B) 50μM 8Br-cGMP, and (C) 30 sec of 5Hz or 10Hz EFS. Representative recordings of the relaxation responses of wild-type and Telokin-/- gastric fundus smooth muscles evoked by 10μM SNP in the presence of (D) 1μM CCh or (E) 30mM KCl. F. Representative recording of the relaxation responses evoked by 30 sec of 5Hz or 10Hz EFS in wild-type and Telokin-/- gastric fundus smooth muscles incubated with 30mM KCl and 1μM atropine, 1μM phentolamine, and 1μM propranolol.
-
Telokin phosphorylation is increased by incubation of non-contracted gastric fundus smooth muscles with compounds that elevate cGMP.
2015Co-Authors: Bhupal P. Bhetwal, Kenton M. Sanders, Avril V. Somlyo, Brian A. PerrinoAbstract:A. Representative recordings of the relaxation responses evoked by 10μM SNP in the absence or presence of 1μM ODQ (n = 6, each), or 50μM 8Br-cGMP (n = 6). B. Representative western blots of Telokin S13 phosphorylation in untreated control muscles and muscles incubated with 50μM 8Br-cGMP for 5 min (n = 6) or 10μM SNP (n = 6) for 1 min. C. Average ratios ± SD of pS13:Telokin in the absence or presence of 50μM 8Br-cGMP (n = 6) or 10μM SNP. *P
Alexander V. Vorotnikov - One of the best experts on this subject based on the ideXlab platform.
-
kinase related protein Telokin inhibits ca2 independent contraction in triton skinned guinea pig taenia coli
Biochemical Journal, 2010Co-Authors: Olga V Shcherbakova, Vladimir P. Shirinsky, Alexander V. Vorotnikov, Daria Serebryanaya, Alexander Postnikov, Mechthild M Schroeter, Stefan Zittrich, Angelika A Noegel, Gabriele PfitzerAbstract:KRP (kinase-related protein), also known as Telokin, has been proposed to inhibit smooth muscle contractility by inhibiting the phosphorylation of the rMLC (regulatory myosin light chain) by the Ca2+-activated MLCK (myosin light chain kinase). Using the phosphatase inhibitor microcystin, we show in the present study that KRP also inhibits Ca2+-independent rMLC phosphorylation and smooth muscle contraction mediated by novel Ca2+-independent rMLC kinases. Incubating KRP-depleted Triton-skinned taenia coli with microcystin at p Ca>8 induced a slow contraction reaching 90% of maximal force ( F max) at p Ca 4.5 after ~25 min. Loading the fibres with KRP significantly slowed down the force development, i.e. the time to reach 50% of F max was increased from 8 min to 35 min. KRP similarly inhibited rMLC phosphorylation of HMM (heavy meromyosin) in vitro by MLCK or by the constitutively active MLCK fragment (61K-MLCK) lacking the myosin-docking KRP domain. A C-terminally truncated KRP defective in myosin binding inhibited neither force nor HMM phosphorylation. Phosphorylated KRP inhibited the rMLC phosphorylation of HMM in vitro and Ca2+-insensitive contractions in fibres similar to unphosphorylated KRP, whereby the phosphorylation state of KRP was not altered in the fibres. We conclude that (i) KRP inhibits not only MLCK-induced contractions, but also those elicited by Ca2+-independent rMLC kinases; (ii) phosphorylation of KRP does not modulate this effect; (iii) binding of KRP to myosin is essential for this inhibition; and (iv) KRP inhibition of rMLC phosphorylation is most probably due to the shielding of the phosphorylation site on the rMLC. Abbreviations: aa, amino acids; DAPI, 4′,6-diamidino-2-phenylindole; DTT, dithiothreitol; ECL, enhanced chemiluminescence; F-actin, filamentous actin; GST, glutathione transferase; HMM, heavy meromyosin; ILK, integrin-linked kinase; KRP, kinase-related protein; MAPK, mitogen-activated protein kinase; MEK1, MAPK/ERK (extracellular-signal-regulated kinase) kinase 1; MHC, myosin heavy chain; MLC, myosin light chain; MLCK, MLC kinase; MLCP, MLC phosphatase; PKA, protein kinase A; PKG, protein kinase G; rMLC, regulatory MLC; ROK, Rho kinase; TBS, Tris-buffered saline; TCA, trichloroacetic acid; wt, wild-type; WME, whole muscle extract; ZIP kinase, MYPT1-associated zipper-interacting kinase
-
Kinase-related protein/Telokin inhibits Ca2+-independent contraction in Triton-skinned guinea pig taenia coli
The Biochemical journal, 2010Co-Authors: Olga V Shcherbakova, Vladimir P. Shirinsky, Alexander V. Vorotnikov, Daria Serebryanaya, Alexander Postnikov, Mechthild M Schroeter, Stefan Zittrich, Angelika A Noegel, Gabriele PfitzerAbstract:KRP (kinase-related protein), also known as Telokin, has been proposed to inhibit smooth muscle contractility by inhibiting the phosphorylation of the rMLC (regulatory myosin light chain) by the Ca2+-activated MLCK (myosin light chain kinase). Using the phosphatase inhibitor microcystin, we show in the present study that KRP also inhibits Ca2+-independent rMLC phosphorylation and smooth muscle contraction mediated by novel Ca2+-independent rMLC kinases. Incubating KRP-depleted Triton-skinned taenia coli with microcystin at p Ca>8 induced a slow contraction reaching 90% of maximal force ( F max) at p Ca 4.5 after ~25 min. Loading the fibres with KRP significantly slowed down the force development, i.e. the time to reach 50% of F max was increased from 8 min to 35 min. KRP similarly inhibited rMLC phosphorylation of HMM (heavy meromyosin) in vitro by MLCK or by the constitutively active MLCK fragment (61K-MLCK) lacking the myosin-docking KRP domain. A C-terminally truncated KRP defective in myosin binding inhibited neither force nor HMM phosphorylation. Phosphorylated KRP inhibited the rMLC phosphorylation of HMM in vitro and Ca2+-insensitive contractions in fibres similar to unphosphorylated KRP, whereby the phosphorylation state of KRP was not altered in the fibres. We conclude that (i) KRP inhibits not only MLCK-induced contractions, but also those elicited by Ca2+-independent rMLC kinases; (ii) phosphorylation of KRP does not modulate this effect; (iii) binding of KRP to myosin is essential for this inhibition; and (iv) KRP inhibition of rMLC phosphorylation is most probably due to the shielding of the phosphorylation site on the rMLC. Abbreviations: aa, amino acids; DAPI, 4′,6-diamidino-2-phenylindole; DTT, dithiothreitol; ECL, enhanced chemiluminescence; F-actin, filamentous actin; GST, glutathione transferase; HMM, heavy meromyosin; ILK, integrin-linked kinase; KRP, kinase-related protein; MAPK, mitogen-activated protein kinase; MEK1, MAPK/ERK (extracellular-signal-regulated kinase) kinase 1; MHC, myosin heavy chain; MLC, myosin light chain; MLCK, MLC kinase; MLCP, MLC phosphatase; PKA, protein kinase A; PKG, protein kinase G; rMLC, regulatory MLC; ROK, Rho kinase; TBS, Tris-buffered saline; TCA, trichloroacetic acid; wt, wild-type; WME, whole muscle extract; ZIP kinase, MYPT1-associated zipper-interacting kinase
-
Telokin/KRP differentially modulates myosin II filament assembly and regulatory light chain phosphorylation in fibroblasts
Biophysics, 2006Co-Authors: D. V. Serebryanaya, Vladimir P. Shirinsky, O. V. Shcherbakova, T. V. Dudnakova, Alexander V. VorotnikovAbstract:Transgenic 3T3 fibroblasts were made to express either wild-type Telokin (KRP) or its truncated version lacking the C-terminal domain essential for binding to myosin. The content of myosin II filaments was markedly increased while regulatory light chain phosphorylation was decreased in the cells expressing KRP but not the C-truncated version. It could be concluded that (i) KRP promotes polymerization of nonmuscle myosin but reduces its RLC phosphorylation, (ii) these effects involve direct KRP binding to myosin, and (iii) KRP-expressing fibroblasts are a convenient model for assessing the role of myosin structural dynamics in cell motility.
-
Telokin krp differentially modulates myosin ii filament assembly and regulatory light chain phosphorylation in fibroblasts
Biophysics, 2006Co-Authors: D. V. Serebryanaya, Vladimir P. Shirinsky, O. V. Shcherbakova, T. V. Dudnakova, Alexander V. VorotnikovAbstract:Transgenic 3T3 fibroblasts were made to express either wild-type Telokin (KRP) or its truncated version lacking the C-terminal domain essential for binding to myosin. The content of myosin II filaments was markedly increased while regulatory light chain phosphorylation was decreased in the cells expressing KRP but not the C-truncated version. It could be concluded that (i) KRP promotes polymerization of nonmuscle myosin but reduces its RLC phosphorylation, (ii) these effects involve direct KRP binding to myosin, and (iii) KRP-expressing fibroblasts are a convenient model for assessing the role of myosin structural dynamics in cell motility.
-
KRP/Telokin differentially regulates filament assembly and phosphorylation of light chains of non-muscle myosin II in fibroblasts
Biofizika, 2006Co-Authors: O. V. Shcherbakova, T. V. Dudnakova, Alexander V. VorotnikovAbstract:Transgenic 3T3 fibroblasts have been generated that express either the wild-type KRP or its truncated mutant lacking the C-terminal domain, which primarily contributes to myosin binding of KRP. It was found that KRP-expressing cells display a significantly increased content of myosin filaments and a reduced level of rMLC phosphorylation, whereas the mock transfected cells or cells expressing the C-terminally truncated KRP do not. Our results suggest that (1) KRP promotes the polymerization of myosin II and reduces the rMLC phosphorylation level in cells, (2) KRP acts through direct binding to myosin II, and (3) transgenic 3T3 fibroblasts stably expressing KRP represent a useful and versatile model to study the role of myosin II filament dynamics in cell motility.
