scholarly journals Enhancement of mDia2 activity by Rho-kinase-dependent phosphorylation of the diaphanous autoregulatory domain

2011 ◽  
Vol 439 (1) ◽  
pp. 57-65 ◽  
Author(s):  
Dean P. Staus ◽  
Joan M. Taylor ◽  
Christopher P. Mack
Keyword(s):  
Smooth Muscle ◽  
Actin Polymerization ◽  
Serum Response Factor ◽  
Rho Kinase ◽  
Specific Gene ◽  
Related Transcription Factor ◽  
Inhibitory Domain ◽  
Serum Response ◽  
Acidic Region ◽  
Basic Region

It is clear that RhoA activates the DRF (diaphanous-related formin) mDia2 by disrupting the molecular interaction between the DAD (diaphanous autoregulatory domain) and the DID (diaphanous inhibitory domain). Previous studies indicate that a basic motif within the DAD contributes to mDia2 auto-inhibition, and results shown in the present study suggest these residues bind a conserved acidic region within the DID. Furthermore, we demonstrate that mDia2 is phosphorylated by ROCK (Rho-kinase) at two conserved residues (Thr1061 and Ser1070) just C-terminal to the DAD basic region. Phosphomimetic mutations to these residues in the context of the full-length molecule enhanced mDia2 activity as measured by increased actin polymerization, SRF (serum response factor)-dependent smooth muscle-specific gene transcription, and nuclear localization of myocardin-related transcription factor B. Biochemical and functional data indicate that the T1061E/S1070E mutation significantly inhibited the ability of DAD to interact with DID and enhanced mDia2 activation by RhoA. Taken together, the results of the present study indicate that ROCK-dependent phosphorylation of the mDia2 DAD is an important determinant of mDia2 activity and that this signalling mechanism affects actin polymerization and smooth muscle cell-specific gene expression.

scholarly journals Regulation of myocardin factor protein stability by the LIM-only protein FHL2

2008 ◽  
Vol 295 (3) ◽  
pp. H1067-H1075 ◽  
Author(s):  
Jeremiah S. Hinson ◽  
Matt D. Medlin ◽  
Joan M. Taylor ◽  
Christopher P. Mack
Keyword(s):  
Smooth Muscle ◽  
Protein Stability ◽  
Serum Response Factor ◽  
Proteasome Inhibitors ◽  
Specific Gene ◽  
Lim Domain ◽  
Protein Levels ◽  
Binding Partners ◽  
Related Transcription Factor ◽  
Serum Response

Extensive evidence indicates that serum response factor (SRF) regulates muscle-specific gene expression and that myocardin family SRF cofactors are critical for smooth muscle cell differentiation. In a yeast two hybrid screen for novel SRF binding partners expressed in aortic SMC, we identified four and a half LIM domain protein 2 (FHL2) and confirmed this interaction by GST pull-down and coimmunoprecipitation assays. FHL2 also interacted with all three myocardin factors and enhanced myocardin and myocardin-related transcription factor (MRTF)-A-dependent transactivation of smooth muscle α-actin, SM22, and cardiac atrial natriuretic factor promoters in 10T1/2 cells. The expression of FHL2 increased myocardin and MRTF-A protein levels, and, importantly, this effect was due to an increase in protein stability not due to an increase in myocardin factor mRNA expression. Treatment of cells with proteasome inhibitors MG-132 and lactacystin strongly upregulated endogenous MRTF-A protein levels and resulted in a substantial increase in ubiquitin immunoreactivity in MRTF-A immunoprecipitants. Interestingly, the expression of FHL2 attenuated the effects of RhoA and MRTF-B on promoter activity, perhaps through decreased MRTF-B nuclear localization or decreased SRF-CArG binding. Taken together, these data indicate that myocardin factors are regulated by proteasome-mediated degradation and that FHL2 regulates SRF-dependent transcription by multiple mechanisms, including stabilization of myocardin and MRTF-A.

scholarly journals Four isoforms of serum response factor that increase or inhibit smooth-muscle-specific promoter activity

2000 ◽  
Vol 345 (3) ◽  
pp. 445-451 ◽  
Author(s):  
Paul R. KEMP ◽  
James C. METCALFE
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Serum Response Factor ◽  
Muscle Cells ◽  
Dominant Negative ◽  
C2c12 Cells ◽  
Specific Gene ◽  
Response Factor ◽  
Transactivation Domain ◽  
Serum Response

Serum response factor (SRF) is a key transcriptional activator of the c-fos gene and of muscle-specific gene expression. We have identified four forms of the SRF coding sequence, SRF-L (the previously identified form), SRF-M, SRF-S and SRF-I, that are produced by alternative splicing. The new forms of SRF lack regions of the C-terminal transactivation domain by splicing out of exon 5 (SRF-M), exons 4 and 5 (SRF-S) and exons 3, 4 and 5 (SRF-I). SRF-M is expressed at similar levels to SRF-L in differentiated vascular smooth-muscle cells and skeletal-muscle cells, whereas SRF-L is the predominant form in many other tissues. SRF-S expression is restricted to vascular smooth muscle and SRF-I expression is restricted to the embryo. Transfection of SRF-L and SRF-M into C2C12 cells showed that both forms are transactivators of the promoter of the smooth-muscle-specific gene SM22α, whereas SRF-I acted as a dominant negative form of SRF.

scholarly journals Physiological Control of Smooth Muscle-specific Gene Expression through Regulated Nuclear Translocation of Serum Response Factor

2000 ◽  
Vol 275 (39) ◽  
pp. 30387-30393 ◽  
Author(s):  
Blanca Camoretti-Mercado ◽  
Hong-W. Liu ◽  
Andrew J. Halayko ◽  
Sean M. Forsythe ◽  
John W. Kyle ◽  
...  
Keyword(s):  
Gene Expression ◽  
Smooth Muscle ◽  
Serum Response Factor ◽  
Nuclear Translocation ◽  
Specific Gene ◽  
Response Factor ◽  
Physiological Control ◽  
Specific Gene Expression ◽  
Serum Response

scholarly journals The actin-MRTF-SRF transcriptional circuit controls tubulin acetylation via α-TAT1 gene expression

2018 ◽  
Vol 217 (3) ◽  
pp. 929-944 ◽  
Author(s):  
Jaime Fernández-Barrera ◽  
Miguel Bernabé-Rubio ◽  
Javier Casares-Arias ◽  
Laura Rangel ◽  
Laura Fernández-Martín ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Actin Polymerization ◽  
Serum Response Factor ◽  
Cell Types ◽  
Mrna Levels ◽  
Tubulin Acetylation ◽  
Hereditary Disorders ◽  
Related Transcription Factor ◽  
Transcriptional Complex ◽  
Serum Response

The role of formins in microtubules is not well understood. In this study, we have investigated the mechanism by which INF2, a formin mutated in degenerative renal and neurological hereditary disorders, controls microtubule acetylation. We found that silencing of INF2 in epithelial RPE-1 cells produced a dramatic drop in tubulin acetylation, increased the G-actin/F-actin ratio, and impaired myocardin-related transcription factor (MRTF)/serum response factor (SRF)–dependent transcription, which is known to be repressed by increased levels of G-actin. The effect on tubulin acetylation was caused by the almost complete absence of α-tubulin acetyltransferase 1 (α-TAT1) messenger RNA (mRNA). Activation of the MRTF-SRF transcriptional complex restored α-TAT1 mRNA levels and tubulin acetylation. Several functional MRTF-SRF–responsive elements were consistently identified in the α-TAT1 gene. The effect of INF2 silencing on microtubule acetylation was also observed in epithelial ECV304 cells, but not in Jurkat T cells. Therefore, the actin-MRTF-SRF circuit controls α-TAT1 transcription. INF2 regulates the circuit, and hence microtubule acetylation, in cell types where it has a prominent role in actin polymerization.

scholarly journals LIM kinase and Diaphanous cooperate to regulate serum response factor and actin dynamics

2002 ◽  
Vol 157 (5) ◽  
pp. 831-838 ◽  
Author(s):  
Olivier Geneste ◽  
John W. Copeland ◽  
Richard Treisman
Keyword(s):  
Actin Polymerization ◽  
Serum Response Factor ◽  
Rho Kinase ◽  
Small Gtpase ◽  
Actin Dynamics ◽  
Response Factor ◽  
Lim Kinase ◽  
Serum Stimulation ◽  
Effector Pathways ◽  
Serum Response

The small GTPase RhoA controls activity of serum response factor (SRF) by inducing changes in actin dynamics. We show that in PC12 cells, activation of SRF after serum stimulation is RhoA dependent, requiring both actin polymerization and the Rho kinase (ROCK)–LIM kinase (LIMK)–cofilin signaling pathway, previously shown to control F-actin turnover. Activation of SRF by overexpression of wild-type LIMK or ROCK-insensitive LIMK mutants also requires functional RhoA, indicating that a second RhoA-dependent signal is involved. This is provided by the RhoA effector mDia: dominant interfering mDia1 derivatives inhibit both serum- and LIMK-induced SRF activation and reduce the ability of LIMK to induce F-actin accumulation. These results demonstrate a role for LIMK in SRF activation, and functional cooperation between RhoA-controlled LIMK and mDia effector pathways.

scholarly journals Cooperative signaling by TGF-β1 and WNT-11 drives sm-α-actin expression in smooth muscle via Rho kinase-actin-MRTF-A signaling

2016 ◽  
Vol 311 (3) ◽  
pp. L529-L537 ◽  
Author(s):  
Kuldeep Kumawat ◽  
Tim Koopmans ◽  
Mark H. Menzen ◽  
Alita Prins ◽  
Marieke Smit ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Nuclear Translocation ◽  
Rho Kinase ◽  
Specific Gene ◽  
Cytoskeletal Remodeling ◽  
Gene And Protein Expression ◽  
Signaling Axis ◽  
Related Transcription Factor ◽  
Actin Expression ◽  
Tgf Β1

Airway smooth muscle (ASM) remodeling is a key feature in asthma and includes changes in smooth muscle-specific gene and protein expression. Despite this being a major contributor to asthma pathobiology, our understanding of the mechanisms governing ASM remodeling remains poor. Here, we studied the functional interaction between WNT-11 and TGF-β1 in ASM cells. We demonstrate that WNT-11 is preferentially expressed in contractile myocytes and is strongly upregulated following TGF-β1-induced myocyte maturation. Knock-down of WNT-11 attenuated TGF-β1-induced smooth muscle (sm)-α-actin expression in ASM cells. We demonstrate that TGF-β1-induced sm-α-actin expression is mediated by WNT-11 via RhoA activation and subsequent actin cytoskeletal remodeling, as pharmacological inhibition of either Rho kinase by Y27632 or actin remodeling by latrunculin A attenuated sm-α-actin induction. Moreover, we show that TGF-β1 regulates the nuclear expression of myocardin-related transcription factor-A (MRTF-A) in a Rho kinase-dependent fashion, which in turn mediates sm-α-actin expression. Finally, we demonstrate that TGF-β1-induced MRTF-A nuclear translocation is dependent on endogenous WNT-11. The present study thus demonstrates a WNT-11-dependent Rho kinase-actin-MRTF-A signaling axis that regulates the expression of sm-α-actin in ASM cells.

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