A role for serine-175 in modulating the molecular conformation of calponin

Jin, Jian‐Ping; Walsh, Michael P.; Sutherland, Cindy; Chen, Wenhua

doi:10.1042/bj3500579

Cited by 25 publications

(18 citation statements)

References 32 publications

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“…Although the fusion protein contained aa 92-298, it included the putative phosphorylation site (Ser 175 ) (21). The fusion protein could also be identified by anticalponin monoclonal antibody, indicating that the expressed fusion protein was functional.…”

Section: Discussionmentioning

confidence: 99%

Direct association and translocation of PKC-α with calponin

Patil

Pawar

Bitar

2004

American Journal of Physiology-Gastrointestinal and Liver Physi

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Calponin has been implicated in the regulation of smooth muscle contraction through its interaction with F-actin and inhibition of the actin-activated MgATPase activity of phosphorylated myosin. Calponin has also been shown to interact with PKC. We have studied the interaction of calponin with PKC-α and with the low molecular weight heat-shock protein (HSP)27 in contraction of colonic smooth muscle cells. Particulate fractions from isolated smooth muscle cells were immunoprecipitated with antibodies to calponin and Western blot analyzed with antibodies to HSP27 and to PKC-α. Acetylcholine induced a sustained increase in the immunocomplexing of calponin with HSP27 and of calponin with PKC-α in the particulate fraction, indicating an association of the translocated proteins in the membrane. To examine whether the observed interaction in vivo is due to a direct interaction of calponin with PKC-α, a cDNA of 1.3 kb of human calponin gene was PCR amplified. PCR product encoding 622 nt of calponin cDNA (nt 351–972 corresponding to amino acids 92–229) was expressed as fusion glutathione S-transferase (GST) protein in the vector pGEX -KT. We have studied the direct association of GST-calponin fusion protein with recombinant PKC-α in vitro. Western blot analysis of the fractions collected after elution with reduced glutathione buffer (pH 8.0) show a coelution of GST-calponin with PKC-α, indicating a direct association of GST-calponin with PKC-α. These data suggest that there is a direct association of translocated calponin and PKC-α in the membrane and a role for the complex calponin-PKC-α-HSP27, in contraction of colonic smooth muscle cells.

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Section: Discussionmentioning

confidence: 99%

Direct association and translocation of PKC-α with calponin

Patil

Pawar

Bitar

2004

American Journal of Physiology-Gastrointestinal and Liver Physi

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“…mAbs provide reagents for recognizing specific epitopes and can be used in monitoring protein conformation, structural dynamics, and folding (18). We have applied antibody probes against epitopes in allosteric proteins to detect conformational differences and changes using high throughput microtiter plate ELISA under native conditions (23,24,27,39).…”

Section: Methodsmentioning

confidence: 99%

The heart-specific NH₂-terminal extension regulates the molecular conformation and function of cardiac troponin I

Akhter

Zhang

Jin

2012

American Journal of Physiology-Heart and Circulatory Physiology

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Akhter S, Zhang Z, Jin JP. The heart-specific NH2-terminal extension regulates the molecular conformation and function of cardiac troponin I. Am J Physiol Heart Circ Physiol 302: H923-H933, 2012. First published December 2, 2011; doi:10.1152/ajpheart.00637.2011.-In addition to the core structure conserved in all troponin I isoforms, cardiac troponin I (cTnI) has an ϳ30 amino acids NH 2-terminal extension. This peptide segment is a heart-specific regulatory structure containing two Ser residues that are substrates of PKA. Under ␤-adrenergic regulation, phosphorylation of cTnI in the NH 2-terminal extension increases the rate of myocardial relaxation. The NH 2-terminal extension of cTnI is also removable by restrictive proteolysis to produce functional adaptation to hemodynamic stresses. The molecular mechanism for the NH 2-terminal modifications to regulate the function of cTnI is not fully understood. In the present study, we tested a hypothesis that the NH 2-terminal extension functions by modulating the conformation of other regions of cTnI. Monoclonal antibody epitope analysis and protein binding experiments demonstrated that deletion of the NH 2-terminal segment altered epitopic conformation in the middle, but not COOH-terminal, region of cTnI. PKA phosphorylation produced similar effects. This targeted longrange conformational modulation corresponded to changes in the binding affinities of cTnI for troponin T and for troponin C in a Ca 2ϩ -dependent manner. The data suggest that the NH2-terminal extension of cTnI regulates cardiac muscle function through modulating molecular conformation and function of the core structure of cTnI.troponin I phosphorylation; proteolytic NH 2-terminal truncation; TnITnT interface; cardiac muscle regulation; protein conformation analysis THE CONTRACTION OF CARDIAC muscle is regulated by binding of cytosolic Ca 2ϩ to troponin, which activates cross bridge cycling between sarcomeric myosin and actin filaments. The troponin complex consists of three protein subunits: the Ca 2ϩ -binding subunit troponin C (TnC), the tropomyosin-binding subunit troponin T (TnT), and the inhibitory subunit troponin I (TnI) (19). The function of TnI is essential to cardiac muscle contraction (34).Three homologous genes are present in vertebrate species encoding the fast skeletal muscle, slow skeletal muscle, and cardiac isoforms of TnI (20,29). Cardiac TnI (cTnI) is the newest member evolved in the family of TnI isoform genes (7). In addition to the ϳ180 amino acids core structure that is highly conserved in the three TnI isoforms in all vertebrates, cTnI has a unique NH 2 -terminal extension of ϳ30 amino acids, which is not found in the two skeletal muscle TnI isoforms.Showing functional conservation and exchangeability among the TnI isoforms, embryonic cardiac muscle utilizes solely slow skeletal muscle TnI. During development, it is completely replaced by cTnI in the adult heart (22, 36). Therefore, the NH 2 -terminal extension of cTnI is not an essential structure for the basic contractility of c...

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“…We previously showed that phosphoryltation of calponin decreases the binding affinity for F-actin (39). On the other hand, the observation that phosphorylated calponin was hardly detectable in living cells (40) may implicate that phosphorylation-induced calponin dissociation from the actin filaments is coupled with rapid degradation.…”

Section: Mechanical Tension Regulates H2-calponin By Controlling Bothmentioning

confidence: 99%

Cytoskeletal Tension Regulates Both Expression and Degradation of h2-Calponin in Lung Alveolar Cells

Hossain

Smith

et al. 2006

Biochemistry

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Calponin is an actin filament-associated regulatory protein and its h2 isoform is expressed in lung alveolar epithelial cells under postnatal up-regulation during lung development corresponding to the commencement of respiratory expansion. Consistent with this correlation to mechanical tension, the expression of h2-calponin in alveolar cells is dependent on substrate stiffness and cytoskeleton tension. The function of h2-calponin in the stability of actin cytoskeleton implicates a role in balancing the strength and compliance of alveoli. An interesting finding is a rapid degradation of h2-calponin in lung after prolonged deflation, which is prevented by inflation of the lung to the in situ expanded volume. Decreasing mechanical tension in cultured alveolar cells by reducing the dimension of culture matrix reproduced the degradation of h2-calponin. Inhibition of myosin II ATPase also resulted in the degradation of h2-calponin in alveolar cells, showing a determining role of the tension in the actin cytoskeleton. Alveolar cells statically cultured on silicon rubber membrane build high tension in the cytoskeleton corresponding to a high expression of h2-calponin. Chronic cyclic stretching of cells on the membrane did not increase but decreased the expression of h2-calponin. This finding suggests that when cellular structure adapted to the stretched dimension, cyclic relaxations periodically release cytoskeleton tension and lower the total amount of tension that the cell senses over time. Therefore, the isometric tension, other than tension dynamics, determines the expression of h2-calponin. The tension regulation of h2-calponin synthesis and degradation demonstrate a novel mechanical regulation of cellular biochemistry.Living cells respond to mechanical forces by changes in cellular structure and function through gene regulation and posttranslational protein modification (1)(2)(3)(4)(5). Reorganization of the cytoskeleton is a key component of the cellular response to mechanical stimuli (3,6). The actin cytoskeleton is a dynamic filamentous network that determines cell shape and strength and plays an important role in cellular response to mechanical tension (7). Calponin is an actin filament-associated regulatory protein (8) and has been extensively studied for its role in the contractility of smooth muscle (9,10). Biochemical activities of calponin have been documented in details mainly from experiments using the h1 isoform in smooth muscles (11). Through high affinity binding to F-actin, calponin inhibits the actin-activated smooth muscle myosin MgATPase and the production of force (12). Despite the extensive investigations, the physiological function of calponin in living cells remains to be established.The h2 isoform of calponin (11) is found in smooth muscle and non-muscle cells. Calponin's association with actin stress fibers and function in regulating actin-myosin interaction suggest † This study was supported by grants from the National Institutes of Health (AR048816 and HL078773).*To whom correspon...

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A role for serine-175 in modulating the molecular conformation of calponin

Cited by 25 publications

References 32 publications

Direct association and translocation of PKC-α with calponin

Direct association and translocation of PKC-α with calponin

The heart-specific NH₂-terminal extension regulates the molecular conformation and function of cardiac troponin I

Cytoskeletal Tension Regulates Both Expression and Degradation of h2-Calponin in Lung Alveolar Cells

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