Caldesmon inhibits the cooperative turning-on of the smooth muscle heavy meromyosin by tropomyosin-actin

Horiuchi, Kazuo; Chacko, Samuel

doi:10.1021/bi00449a023

Cited by 67 publications

(56 citation statements)

References 33 publications

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“…Smooth muscle actin, CaD, myosin (fully phosphorylated), and tropomyosin were purified from chicken gizzard (31)(32)(33). Bovine brain calmodulin was prepared according to Dedman and Kaetzel (34).…”

Section: Construction Of Recombinant Baculovirus Transfermentioning

confidence: 99%

Mutagenesis Analysis of Functionally Important Domains within the C-terminal End of Smooth Muscle Caldesmon

Wang

Chacko

1996

Journal of Biological Chemistry

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The ability of chicken gizzard smooth muscle caldesmon (CaD) to inhibit actomyosin ATPase activity is due mainly to an inhibitory domain that resides within the C-terminal 67 amino acid residues of the CaD molecule. In the present study, a series of C-terminal truncation and internal deletion mutants of chicken gizzard smooth muscle CaD were systematically designed using a site-directed mutagenesis approach, and these mutant proteins were overexpressed in a baculovirus expression system. Analysis of actin binding and inhibition of actomyosin ATPase activity using these mutants identified a strong actin-binding motif of 6 amino acid residues (from Lys 718 to Glu 723 ), which also form the core sequence for CaD-induced inhibition of actomyosin ATPase. However, maximal inhibition by CaD requires the presence of residues 728 -731, which are not associated with actin binding. Our data provide direct evidence for the requirement of actin binding to a specific region in CaD for CaD-induced inhibition of actin activation of smooth muscle myosin ATPase. Furthermore, our findings also show that the region between residues 690 and 717 is responsible for the weak inhibition of actomyosin ATPase and reveal that the inhibitory determinants located in the regions between residues 690 and 717 and residues 718 and 756 can function independently.

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Section: Construction Of Recombinant Baculovirus Transfermentioning

confidence: 99%

Mutagenesis Analysis of Functionally Important Domains within the C-terminal End of Smooth Muscle Caldesmon

Wang

Chacko

1996

Journal of Biological Chemistry

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“…There is strong in vitro evidence implicating caldesmon in the regulation of smooth muscle contraction and cell motility. Caldesmon inhibits the actin-activated myosin ATPase (Dabrowska et al, 1985;Smith et al, 1987;Horiuchi and Chacko, 1989;Chalovich et al, 1998) by blocking the interaction of actin and myosin (Chalovich et al, 1998;Sen et al, 2001) and/or inhibiting a kinetic step of the actomyosin ATPase cycle Marston et al, 1998). Inhibition of contraction by caldesmon can be released by binding of caldesmon to Ca 2+ /calmodulin and/or posttranslational phosphorylation.…”

Section: Introductionmentioning

confidence: 99%

Caldesmon is an integral component of podosomes in smooth muscle cells

Eves

Webb

Zhou

et al. 2006

Journal of Cell Science

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Podosomes are highly dynamic actin-based structures commonly found in motile and invasive cells such as macrophages, osteoclasts and vascular smooth muscle cells. Here, we have investigated the role of caldesmon, an actinbinding protein, in the formation of podosomes in aortic smooth muscle A7r5 cells induced by the phorbol ester PDBu. We found that endogenous low molecular weight caldesmon (l-caldesmon), which was normally localised to actin-stress fibres and membrane ruffles, was recruited to the actin cores of PDBu-induced podosomes. Overexpression of l-caldesmon in A7r5 cells caused dissociation of actin-stress fibres and disruption of focal adhesion complexes, and significantly reduced the ability of PDBu to induce podosome formation. By contrast, siRNA interference of caldesmon expression enhanced PDBuinduced formation of podosomes. The N-terminal fragment of l-caldesmon, CaD40, which contains the myosin-binding site, did not label stress fibres and was not translocated to PDBu-induced podosomes. Cad39, the C-terminal fragment housing the binding sites for actin, tropomyosin and calmodulin, was localised to stress fibres and was translocated to podosomes induced by PDBu. The caldesmon mutant, CadCamAB, which does not interact with Ca 2+ /calmodulin, was not recruited to PDBu-induced podosomes. These results show that (1) l-caldesmon is an integral part of the actin-rich core of the podosome; (2) overexpression of l-caldesmon suppresses podosome formation, whereas siRNA knock-down of l-caldesmon facilitates its formation; and (3) the actin-binding and calmodulin-binding sites on l-caldesmon are essential for the translocation of l-caldesmon to the podosomes. In summary, this data suggests that caldesmon may play a role in the regulation of the dynamics of podosome assembly and that Ca 2+ /calmodulin may be part of a regulatory mechanism in podosome formation.

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“…We and others have produced indirect evidence for a troponin-like cooperative allosteric mechanism in smooth muscle thin filaments (1,(11)(12)(13)(14)(15)41), however this article reports for the first time direct and unambiguous evidence for this mechanism. We demonstrate that smooth muscle thin filaments are a cooperative allosteric regulated system in which actintropomyosin exist in two activity states, ON and OFF that are linked by a concerted cooperative equilibrium.…”

Section: Discussionmentioning

confidence: 72%

Role of Caldesmon in the Ca2+ Regulation of Smooth Muscle Thin Filaments

Ansari

Alahyan

Marston

et al. 2008

Journal of Biological Chemistry

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Smooth muscle thin filaments are made up of actin, tropomyosin, caldesmon, and a Ca 2؉ -binding protein and their interaction with myosin is Ca 2؉ -regulated. We suggested that Ca 2؉ regulation by caldesmon and Ca 2؉ -calmodulin is achieved by controlling the state of thin filament through a cooperative-allosteric mechanism homologous to troponin-tropomyosin in striated muscles. In the present work, we have tested this hypothesis. We monitored directly the thin filament transition between the ON and OFF state using the excimer fluorescence of pyrene iodoacetamide (PIA)-labeled smooth muscle ␣␣-tropomyosin homodimers. In steady state fluorescence measurements, myosin subfragment 1 (S1) cooperatively switches the thin filaments to the ON state, and this is exhibited as an increase in the excimer fluorescence. In contrast, caldesmon decreases the excimer fluorescence, indicating a switch of the thin filament to the OFF state. Addition of Ca 2؉ -calmodulin increases the excimer fluorescence, indicating a switch of the thin filament to the ON state. The excimer fluorescence was also used to monitor the kinetics of the ON-OFF transition in a stopped-flow apparatus. When ATP induces S1 dissociation from actin-PIA-tropomyosin, the transition to the OFF state is delayed until all S1 molecules are dissociated actin. In contrast, caldesmon switches the thin filament to the OFF state in a cooperative way, and no lag is displayed in the time course of the caldesmon-induced fluorescence decrease. We have also studied caldesmon and Ca 2؉ -calmodulin-caldesmon binding to actin-tropomyosin in the ON and OFF states. The results are used to discuss both caldesmon inhibition and Ca 2؉ -calmodulin-caldesmon activation of actin-tropomyosin.

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Caldesmon inhibits the cooperative turning-on of the smooth muscle heavy meromyosin by tropomyosin-actin

Cited by 67 publications

References 33 publications

Mutagenesis Analysis of Functionally Important Domains within the C-terminal End of Smooth Muscle Caldesmon

Mutagenesis Analysis of Functionally Important Domains within the C-terminal End of Smooth Muscle Caldesmon

Caldesmon is an integral component of podosomes in smooth muscle cells

Role of Caldesmon in the Ca2+ Regulation of Smooth Muscle Thin Filaments

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