Functional and Structural Relationship between the Calmodulin-binding, Actin-binding, and Actomyosin-ATPase Inhibitory Domains on the C Terminus of Smooth Muscle Caldesmon

Wang, Ze; Yang, Zhaopeng; Chacko, Samuel

doi:10.1074/jbc.272.27.16896

Cited by 21 publications

(24 citation statements)

References 50 publications

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“…In the 'flip-flop' model (Sobue et al, 1983), Ca 2+ -CaM competes with actin for the binding sites on CaD and therefore reverses the inhibitory effects of CaD on actomyosin ATPase by dissociating it from actin filaments. In favor of this model, the Ca 2+ -CaM binding domain has been shown to be juxtaposed to the actin-binding domain (Fraser et al, 1997;Huber et al, 1998a;Wang et al, 1997). By contrast, the 'four state' model proposes that Ca 2+ -CaM can reverse the inhibitory effects of CaD by two means: either by dissociating CaD from the actin filaments or by forming an uninhibited ternary complex of Ca 2+ -CaM-CaDactin-tropomyosin.…”

Section: Discussionmentioning

confidence: 93%

Caldesmon mutant defective in Ca2+-calmodulin binding interferes with assembly of stress fibers and affects cell morphology, growth and motility

Lin

Reiter

et al. 2004

Journal of Cell Science

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Despite intensive in vitro studies, little is known about the regulation of caldesmon (CaD) by Ca2+-calmodulin (Ca2+-CaM) in vivo. To investigate this regulation, a mutant was generated of the C-terminal fragment of human fibroblast CaD, termed CaD39-AB, in which two crucial tryptophan residues involved in Ca2+-CaM binding were each replaced with alanine. The mutation abolished most CaD39-AB binding to Ca2+-CaM in vitro but had little effect on in vitro binding to actin filaments and the ability to inhibit actin/tropomyosin-activated heavy meromyosin ATPase. To study the functional consequences of these mutations in vivo, we transfected an expression plasmid carrying CaD39-AB cDNA into Chinese hamster ovary (CHO) cells and isolated several clones expressing various amounts of CaD39-AB. Immunofluorescence microscopy revealed that mutant CaD39-AB was distributed diffusely throughout the cytoplasm but also concentrated at membrane ruffle regions. Stable expression of CaD39-AB in CHO cells disrupted assembly of stress fibers and focal adhesions, altered cell morphology, and slowed cell cycle progression. Moreover, CaD39-AB-expressing cells exhibited motility defects in a wound-healing assay, in both velocity and the persistence of translocation, suggesting a role for CaD regulation by Ca2+-CaM in cell migration. Together, these results demonstrate that CaD plays a crucial role in mediating the effects of Ca2+-CaM on the dynamics of the actin cytoskeleton during cell migration.

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Section: Discussionmentioning

confidence: 93%

Caldesmon mutant defective in Ca2+-calmodulin binding interferes with assembly of stress fibers and affects cell morphology, growth and motility

Lin

Reiter

et al. 2004

Journal of Cell Science

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“…Domain analysis of CaD and CaD fragments produced from limited proteolysis, chemical cleavage, and expression systems has led to the precise localization of various functional domains within the CaD molecule (33)(34)(35)(36)(37)(38)(39). For example, the myosinbinding site was first localized to the amino-terminal region of CaD (42,43) and was recently mapped to the sequence between residues 24 and 53 (33).…”

Section: Resultsmentioning

confidence: 99%

Tyrosine Phosphorylation of Caldesmon Is Required for Binding to the Shc·Grb2 Complex

Wang

Danielsen

Maihle

et al. 1999

Journal of Biological Chemistry

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“…As we were unable to achieve total knockdown of caldesmon using any of the siRNAs, it is not clear to what extent the structure of stress fibres would be affected in (Velaz et al, 1990;Bogatcheva et al, 1993;Huber et al, 1995) and a tropomyosin-binding site (black) (Smith et al, 1987;. The C-terminal fragment of lcaldesmon (Cad39; amino acids 236-532) contains two tropomyosin-binding sites, two actin-interacting regions (diamonds) (Wang et al, 1997b;Marston et al, 1998), and the two Ca 2+ /calmodulin binding sites A and B, as indicated (dark-grey boxes) Wang et al, 1996). Both Ca 2+ /calmodulin-binding sites contain a key tryptophan residue (W454 and W487) that is essential for this interaction (Graether et al, 1997); replacement of these tryptophan residues with alanine (W454A and W487A) generates an l-caldesmon mutant that is unable to interact with Ca 2+ /calmodulin (CadCamAB).…”

Section: Effects Of Expression Of Egfp-caldesmon On Pdbuinduced Podosmentioning

confidence: 99%

“…We generated an EGFP-caldesmon construct (CadCamAB), in which the two tryptophan residues, W454 and W487, known to be essential for calmodulin-binding were mutated to Ala. The W454A and W487A mutations have been shown to abolish interaction between caldesmon and calmodulin, but have little effect on binding of caldesmon to actin (Graether et al, 1997;Wang et al, 1997b;Li et al, 2004). Depending on the expression level, CadCamAB caused various degrees of stress fibre disassembly.…”

Section: Intact Calmodulin-binding Sites Are Required For Targeting Cmentioning

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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Functional and Structural Relationship between the Calmodulin-binding, Actin-binding, and Actomyosin-ATPase Inhibitory Domains on the C Terminus of Smooth Muscle Caldesmon

Cited by 21 publications

References 50 publications

Caldesmon mutant defective in Ca2+-calmodulin binding interferes with assembly of stress fibers and affects cell morphology, growth and motility

Caldesmon mutant defective in Ca2+-calmodulin binding interferes with assembly of stress fibers and affects cell morphology, growth and motility

Tyrosine Phosphorylation of Caldesmon Is Required for Binding to the Shc·Grb2 Complex

Caldesmon is an integral component of podosomes in smooth muscle cells

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