STIM1 negatively regulates Ca2+ release from the sarcoplasmic reticulum in skeletal myotubes

STIM1 (stromal interaction molecule 1) mediates SOCE (store-operated Ca2+ entry) in skeletal muscle. However, the direct role(s) of STIM1 in skeletal muscle, such as Ca2+ release from the SR (sarcoplasmic reticulum) for muscle contraction, have not been identified. The times required for the maximal expression of endogenous STIM1 or Orai1, or for the appearance of puncta during the differentiation of mouse primary skeletal myoblasts to myotubes, were all different, and the formation of puncta was detected with no stimulus during differentiation, suggesting that, in skeletal muscle, the formation of puncta is a part of the differentiation. Wild-type STIM1 and two STIM1 mutants (Triple mutant, missing Ca2+-sensing residues but possessing the intact C-terminus; and E136X, missing the C-terminus) were overexpressed in the myotubes. The wild-type STIM1 increased SOCE, whereas neither mutant had an effect on SOCE. It was interesting that increases in the formation of puncta were observed in the Triple mutant as well as in wild-type STIM1, suggesting that SOCE-irrelevant puncta could exist in skeletal muscle. On the other hand, overexpression of wild-type or Triple mutant, but not E136X, attenuated Ca2+ releases from the SR in response to KCl [evoking ECC (excitation–contraction coupling) via activating DHPR (dihydropyridine receptor)] in a dominant-negative manner. The attenuation was removed by STIM1 knockdown, and STIM1 was co-immunoprecipitated with DHRP in a Ca2+-independent manner. These results suggest that STIM1 negatively regulates Ca2+ release from the SR through the direct interaction of the STIM1 C-terminus with DHPR, and that STIM1 is involved in both ECC and SOCE in skeletal muscle.

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Rem uncouples excitation–contraction coupling in adult skeletal muscle fibers

The Journal of General Physiology ◽

10.1085/jgp.201411314 ◽

2015 ◽

Vol 146 (1) ◽

pp. 97-108 ◽

Cited By ~ 7

Author(s):

Donald Beqollari ◽

Christin F. Romberg ◽

Dilyana Filipova ◽

Ulises Meza ◽

Symeon Papadopoulos ◽

...

Keyword(s):

Skeletal Muscle ◽

Direct Interaction ◽

Charge Movement ◽

Triple Mutant ◽

Membrane Expression ◽

Direct Role ◽

Release Channel ◽

Adult Skeletal Muscle ◽

Ec Coupling ◽

Conformational Coupling

In skeletal muscle, excitation–contraction (EC) coupling requires depolarization-induced conformational rearrangements in L-type Ca2+ channel (CaV1.1) to be communicated to the type 1 ryanodine-sensitive Ca2+ release channel (RYR1) of the sarcoplasmic reticulum (SR) via transient protein–protein interactions. Although the molecular mechanism that underlies conformational coupling between CaV1.1 and RYR1 has been investigated intensely for more than 25 years, the question of whether such signaling occurs via a direct interaction between the principal, voltage-sensing α1S subunit of CaV1.1 and RYR1 or through an intermediary protein persists. A substantial body of evidence supports the idea that the auxiliary β1a subunit of CaV1.1 is a conduit for this intermolecular communication. However, a direct role for β1a has been difficult to test because β1a serves two other functions that are prerequisite for conformational coupling between CaV1.1 and RYR1. Specifically, β1a promotes efficient membrane expression of CaV1.1 and facilitates the tetradic ultrastructural arrangement of CaV1.1 channels within plasma membrane–SR junctions. In this paper, we demonstrate that overexpression of the RGK protein Rem, an established β subunit–interacting protein, in adult mouse flexor digitorum brevis fibers markedly reduces voltage-induced myoplasmic Ca2+ transients without greatly affecting CaV1.1 targeting, intramembrane gating charge movement, or releasable SR Ca2+ store content. In contrast, a β1a-binding–deficient Rem triple mutant (R200A/L227A/H229A) has little effect on myoplasmic Ca2+ release in response to membrane depolarization. Thus, Rem effectively uncouples the voltage sensors of CaV1.1 from RYR1-mediated SR Ca2+ release via its ability to interact with β1a. Our findings reveal Rem-expressing adult muscle as an experimental system that may prove useful in the definition of the precise role of the β1a subunit in skeletal-type EC coupling.

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AMP kinase is not required for the GLUT4 response to exercise and denervation in skeletal muscle

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00080.2004 ◽

2004 ◽

Vol 287 (4) ◽

pp. E739-E743 ◽

Cited By ~ 46

Author(s):

Burton F. Holmes ◽

David B. Lang ◽

Morris J. Birnbaum ◽

James Mu ◽

G. Lynis Dohm

Keyword(s):

Skeletal Muscle ◽

Dominant Negative ◽

Treadmill Running ◽

Wild Type ◽

Α Subunit ◽

Ampk Activity ◽

Denervated Muscles ◽

Amp Activated Protein Kinase ◽

Response To Exercise

An acute bout of exercise increases muscle GLUT4 mRNA in mice, and denervation decreases GLUT4 mRNA. AMP-activated protein kinase (AMPK) activity in skeletal muscle is also increased by exercise, and GLUT4 mRNA is increased in mouse skeletal muscle after treatment with AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside(AICAR). These findings suggest that AMPK activation might be responsible for the increase in GLUT4 mRNA expression in response to exercise. To investigate the role of AMPK in GLUT4 regulation in response to exercise and denervation, transgenic mice with a mutated AMPK α-subunit (dominant negative; AMPK-DN) were studied. GLUT4 did not increase in AMPK-DN mice that were treated with AICAR, demonstrating that muscle AMPK is inactive. Exercise (two 3-h bouts of treadmill running separated by 1 h of rest) increased GLUT4 mRNA in both wild-type and AMPK-DN mice. Likewise, denervation decreased GLUT4 mRNA in both wild-type and AMPK-DN mice. GLUT4 mRNA was also increased by AICAR treatment in both the innervated and denervated muscles. These data demonstrate that AMPK is not required for the response of GLUT4 mRNA to exercise and denervation.

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RSR1, a ras-like gene homologous to Krev-1 (smg21A/rap1A): role in the development of cell polarity and interactions with the Ras pathway in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.12.2.758 ◽

1992 ◽

Vol 12 (2) ◽

pp. 758-766 ◽

Cited By ~ 60

Author(s):

R Ruggieri ◽

A Bender ◽

Y Matsui ◽

S Powers ◽

Y Takai ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Polarity ◽

Copy Number ◽

Mammalian Cells ◽

Direct Interaction ◽

Dominant Negative ◽

Yeast Cells ◽

Wild Type ◽

Ras Gene ◽

Ras Pathway

The Saccharomyces cerevisiae ras-like gene RSR1 is particularly closely related to the mammalian gene Krev-1 (also known as smg21A and rap1A). RSR1 was originally isolated as a multicopy suppressor of a cdc24 mutation, which causes an inability to bud or establish cell polarity. Deletion of RSR1 itself does not affect growth but causes a randomization of bud position. We have now constructed mutant alleles of RSR1 encoding proteins with substitutions of Val for Gly at position 12 (analogous to constitutively activated Ras proteins) or Asn for Lys at position 16 (analogous to a dominant-negative Ras protein). rsr1Val-12 could not restore a normal budding pattern to an rsr1 deletion strain but could suppress a cdc24 mutation when overexpressed. rsr1Asn-16 could randomize the budding pattern of a wild-type strain even in low copy number but was not lethal even in high copy number. These and other results suggest that Rsr1p functions only in bud site selection and not in subsequent events of polarity establishment and bud formation, that this function involves a cycling between GTP-bound and GDP-bound forms of the protein, and that the suppression of cdc24 involves direct interaction between Rsr1p[GTP] and Cdc24p. Functional homology between Rsr1p and Krev-1 p21 was suggested by the observations that expression of the latter protein in yeast cells could both suppress a cdc24 mutation and randomize the budding pattern of wild-type cells. As Krev-1 overexpression can suppress ras-induced transformation of mammalian cells, we looked for effects of RSR1 on the S. cerevisiae Ras pathway. Although no suppression of the activated RAS2Val-19 allele was observed, overexpression of rsr1Val-12 suppressed the lethality of strains lacking RAS gene function, apparently through a direct activation of adenyl cyclase. This interaction of Rsr1p with the effector of Ras in S. cerevisiae suggests that Krev-1 may revert ras-induced transformation of mammalian cells by affecting the interaction of ras p21 with its effector.

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p53 domains: structure, oligomerization, and transformation

Molecular and Cellular Biology ◽

10.1128/mcb.14.8.5182-5191.1994 ◽

1994 ◽

Vol 14 (8) ◽

pp. 5182-5191

Author(s):

P Wang ◽

M Reed ◽

Y Wang ◽

G Mayr ◽

J E Stenger ◽

...

Keyword(s):

Amino Acids ◽

Dna Binding ◽

Gel Filtration ◽

Dominant Negative ◽

Wild Type ◽

C Terminus ◽

Tetramerization Domain ◽

Wild Type P53 ◽

Negative Phenotype

Wild-type p53 forms tetramers and multiples of tetramers. Friedman et al. (P. N. Friedman, X. B. Chen, J. Bargonetti, and C. Prives, Proc. Natl. Acad. Sci. USA 90:3319-3323, 1993) have reported that human p53 behaves as a larger molecule during gel filtration than it does during sucrose gradient sedimentation. These differences argue that wild-type p53 has a nonglobular shape. To identify structural and oligomerization domains in p53, we have investigated the physical properties of purified segments of p53. The central, specific DNA-binding domain within murine amino acids 80 to 320 and human amino acids 83 to 323 behaves predominantly as monomers during analysis by sedimentation, gel filtration, and gel electrophoresis. This consistent behavior argues that the central region of p53 is globular in shape. Under appropriate conditions, however, this segment can form transient oligomers without apparent preference for a single oligomeric structure. This region does not enhance transformation by other oncogenes. The biological implications of transient oligomerization by this central segment, therefore, remain to be demonstrated. Like wild-type p53, the C terminus, consisting of murine amino acids 280 to 390 and human amino acids 283 to 393, behaves anomalously during gel filtration and apparently has a nonglobular shape. Within this region, murine amino acids 315 to 350 and human amino acids 323 to 355 are sufficient for assembly of stable tetramers. The finding that murine amino acids 315 to 360 enhance transformation by other oncogenes strongly supports the role of p53 tetramerization in oncogenesis. Amino acids 330 to 390 of murine p53 and amino acids 340 to 393 of human p53, which have been implicated by Sturzbecher et al. in tetramerization (H.-W. Sturzbecher, R. Brain, C. Addison, K. Rudge, M. Remm, M. Grimaldi, E. Keenan, and J. R. Jenkins, Oncogene 7:1513-1523, 1992), do not form stable tetramers under our conditions. Our findings indicate that p53 has at least two autonomous oligomerization domains: a strong tetramerization domain in its C-terminal region and a weaker oligomerization domain in the central DNA binding region of p53. Together, these domains account for the formation of tetramers and multiples of tetramers by wild-type p53. The tetramerization domain is the major determinant of the dominant negative phenotype leading to transformation by mutant p53s.

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The Naturally Occurring Variant of Estrogen Receptor (ER) ERΔE7 Suppresses Estrogen-Dependent Transcriptional Activation by Both Wild-Type ERα and ERβ

Endocrinology ◽

10.1210/en.2002-0027 ◽

2003 ◽

Vol 144 (7) ◽

pp. 2967-2976 ◽

Cited By ~ 41

Author(s):

Juana M. García Pedrero ◽

Pedro Zuazua ◽

Carlos Martínez-Campa ◽

Pedro S. Lazo ◽

Sofía Ramos

Keyword(s):

Estrogen Receptor ◽

Transcriptional Activation ◽

Mammalian Cells ◽

Activation Function ◽

Inhibitory Effect ◽

Dominant Negative ◽

Wild Type ◽

Independent Manner ◽

Naturally Occurring ◽

Estrogen Response

Abstract We have isolated and functionally characterized the exon 7-skipped variant (ERΔE7) of estrogen receptor (ER)α, which has emerged as the predominant variant expressed in multiple normal and tumoral tissues. However, to date no function has been established for this variant in mammalian cells. ERΔE7 exhibits a negligible ability to bind ligands, insensitivity to allosteric modulation by estrogen and antiestrogens, and loss of estrogen-dependent interaction with p160 coactivators such as SRC-1 and AIB1. ERΔE7 is able to form heterodimers with both ERα and ERβ in a ligand-independent manner. Transient expression experiments in HeLa cells show that increasing amounts of ERΔE7 result in a progressive inhibition of the estrogen-dependent transcriptional activation by both wild-type ERα and ERβ on estrogen response element-driven promoters. The inhibitory effect of ERΔE7 is due to the inhibition of binding of wild-type receptors to their responsive elements. Surprisingly, the activation function (AF)-1-dependent transactivation triggered by epithelial growth factor and phorbol-12-myristate-13-acetate is also abolished in ERΔE7 despite AF1 integrity, suggesting a cross-talk between AF1 and AF2 regions of the receptor. These results indicate that the naturally occurring variant ERΔE7 is a dominant negative receptor that, when expressed at high levels relative to wild-type ERs, might have profound effects on several estrogen-dependent functions.

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Functional characterization of FlgM in the regulation of flagellar synthesis and motility in Yersinia pseudotuberculosis

Microbiology ◽

10.1099/mic.0.026294-0 ◽

2009 ◽

Vol 155 (6) ◽

pp. 1890-1900 ◽

Cited By ~ 18

Author(s):

Lisha Ding ◽

Yao Wang ◽

Yangbo Hu ◽

Steve Atkinson ◽

Paul Williams ◽

...

Keyword(s):

Yersinia Pseudotuberculosis ◽

Functional Characterization ◽

Direct Interaction ◽

Site Directed Mutagenesis ◽

Wild Type ◽

C Terminus ◽

In Trans ◽

Differential Gene ◽

Flagellar Biogenesis

We describe here the functional characterization of the flgM gene in Yersinia pseudotuberculosis. Direct interaction of FlgM with the alternative sigma factor σ 28 (FliA) was first confirmed. A conserved region in the C-terminus of FlgM was found which included the σ 28 binding domain. By site-directed mutagenesis, bacterial two-hybrid analysis and Western blotting, the primary FlgM binding sites with σ 28 were shown to be Ile85, Ala86 and Leu89. A role for FlgM in swimming motility was demonstrated by inactivation of flgM and subsequent complementation in trans. Transcriptional fusion analyses showed differential gene expression of flhDC, fliA, flgM and fliC in the fliA and flgM mutants compared with the wild-type. flhDC expression was not influenced by σ 28 or FlgM while fliA expression was abolished in the fliA mutant and considerably reduced in the flgM mutant when compared to the wild-type, indicating that both FliA and FlgM can activate fliA transcription. Conversely, flgM transcription was higher in the fliA mutant when compared to the wild-type, suggesting that flgM transcription was repressed by σ 28. Interestingly, fliC expression was markedly increased in the flgM mutant, suggesting a negative regulatory role for FlgM in fliC expression. The transcription of other σ-dependent genes (cheW, flgD, flaA, csrA and fliZ) was also examined in fliA and flgM mutant backgrounds and this revealed that other σ-factors apart from σ 28 may be involved in flagellar biogenesis in Y. pseudotuberculosis. Taking together the motility phenotypes and effects of flgM mutation on the regulation of these key motility genes, we propose that the mechanisms regulating flagellar biogenesis in Y. pseudotuberculosis may differ from those described for other bacteria.

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Hyperphosphorylation of Histone Deacetylase 2 by Alphaherpesvirus US3 Kinases

Journal of Virology ◽

10.1128/jvi.00981-10 ◽

2010 ◽

Vol 84 (19) ◽

pp. 9666-9676 ◽

Cited By ~ 33

Author(s):

Matthew S. Walters ◽

Paul R. Kinchington ◽

Bruce W. Banfield ◽

Saul Silverstein

Keyword(s):

Histone Deacetylase ◽

Pseudorabies Virus ◽

Wild Type ◽

Independent Manner ◽

Specific Chemical ◽

Varicella Zoster ◽

Histone Deacetylase 2 ◽

C Terminus ◽

In Cells ◽

Hsv 1

ABSTRACT A serine/threonine (S/T) kinase encoded by the US3 gene of herpes simplex virus type 1 (HSV-1) is conserved in varicella-zoster virus (VZV) and pseudorabies virus (PRV). Expression of US3 kinase in cells transformed with US3 expression plasmids or infected with each virus results in hyperphosphorylation of histone deacetylase 2 (HDAC2). Mapping studies revealed that each US3 kinase phosphorylates HDAC2 at the same unique conserved Ser residue in its C terminus. HDAC2 was also hyperphosphorylated in cells infected with PRV lacking US3 kinase, indicating that hyperphosphorylation of HDAC2 by PRV occurs in a US3-independent manner. Specific chemical inhibition of class I HDAC activity increases the plaquing efficiency of VZV and PRV lacking US3 or its enzymatic activity, whereas only minimal effects are observed with wild-type viruses, suggesting that VZV and PRV US3 kinase activities target HDACs to reduce viral genome silencing and allow efficient viral replication. However, no effect was observed for wild-type or US3 null HSV-1. Thus, we have demonstrated that while HDAC2 is a conserved target of alphaherpesvirus US3 kinases, the functional significance of these events is virus specific.

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Activation of AMPK is essential for AICAR-induced glucose uptake by skeletal muscle but not adipocytes

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00455.2001 ◽

2002 ◽

Vol 282 (6) ◽

pp. E1239-E1244 ◽

Cited By ~ 56

Author(s):

Hideyuki Sakoda ◽

Takehide Ogihara ◽

Motonobu Anai ◽

Midori Fujishiro ◽

Hiraku Ono ◽

...

Keyword(s):

Skeletal Muscle ◽

Glucose Uptake ◽

Glucose Transport ◽

Muscle Cells ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Ampk Activation ◽

Wild Type ◽

Rat Soleus Muscle ◽

Amp Activated Protein Kinase

5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) reportedly activates AMP-activated protein kinase (AMPK) and stimulates glucose uptake by skeletal muscle cells. In this study, we investigated the role of AMPK in AICAR-induced glucose uptake by 3T3-L1 adipocytes and rat soleus muscle cells by overexpressing wild-type and dominant negative forms of the AMPKα2 subunit by use of adenovirus-mediated gene transfer. Overexpression of the dominant negative mutant had no effect on AICAR-induced glucose transport in adipocytes, although AMPK activation was almost completely abolished. This suggests that AICAR-induced glucose uptake by 3T3-L1 adipocytes is independent of AMPK activation. By contrast, overexpression of the dominant negative AMPKα2 mutant in muscle markedly suppressed both AICAR-induced glucose uptake and AMPK activation, although insulin-induced uptake was unaffected. Overexpression of the wild-type AMPKα2 subunit significantly increased AMPK activity in muscle but did not enhance glucose uptake. Thus, although AMPK activation may not, by itself, be sufficient to increase glucose transport, it appears essential for AICAR-induced glucose uptake in muscle.

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Transmembrane orientation of the N-terminal and C-terminal ends of the ryanodine receptor in the sarcoplasmic reticulum of rabbit skeletal muscle

Biochemical Journal ◽

10.1042/bj2980743 ◽

1994 ◽

Vol 298 (3) ◽

pp. 743-749 ◽

Cited By ~ 21

Author(s):

I Marty ◽

M Villaz ◽

G Arlaud ◽

I Bally ◽

M Ronjat

Keyword(s):

Skeletal Muscle ◽

Sarcoplasmic Reticulum ◽

Ryanodine Receptor ◽

Synthetic Peptides ◽

Rabbit Skeletal Muscle ◽

Cytoplasmic Localization ◽

Carboxypeptidase A ◽

Western Blots ◽

C Terminus ◽

Cytoplasmic Side

Antibodies were raised against synthetic peptides corresponding to the N-terminal (residues 2-15) and the C-terminal (residues 5027-5037) parts of the rabbit skeletal muscle ryanodine receptor. The specificity of the antibodies generated was tested by e.l.i.s.a., Western blotting and immunofluorescence. All these tests demonstrated the specificity of the antibodies and their ability to react with both the native and the denaturated ryanodine receptor. Both the anti-N-terminus and the anti-C-terminus antibodies bound to sarcoplasmic reticulum vesicles, indicating that each end of the membrane-embedded ryanodine receptor is exposed to the cytoplasmic side of the vesicles. These immunological data were complemented with proteolysis experiments using carboxypeptidase A. Carboxypeptidase A induced degradation of the C-terminal end of the ryanodine receptor in sarcoplasmic reticulum vesicles and a concomitant loss of reactivity of the anti-C-terminus antibodies in Western blots, providing extra evidence for the cytoplasmic localization of the C-terminal end of the ryanodine receptor.

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