Chih-Lueh Albert Wang

Smooth muscle caldesmon (h-CaD) is an actin- and myosin-binding protein that reversibly inhibits the actomyosin ATPase activity in vitro. To test the function of h-CaD in vivo, we eliminated its expression in mice. The h-CaD-null animals appeared normal and fertile, although the litter size was smaller. Tissues from the homozygotes lacked h-CaD and exhibited upregulation of the non-muscle isoform, l-CaD, in visceral, but not vascular tonic smooth muscles. While the Ca²⁺ sensitivity of force generation of h-CaD-deficient smooth muscle remained largely unchanged, the kinetic behavior during relaxation in arteries was different. Both intact and permeabilized arterial smooth muscle tissues from the knockout animals relaxed more slowly than those of the wild type. Since this difference occurred after myosin dephosphorylation was complete, the kinetic effect most likely resulted from slower detachment of unphosphorylated crossbridges. Detailed analyses revealed that the apparently slower relaxation of h-CaD-null smooth muscle was due to an increase in the amplitude of a slower component of the biphasic tension decay. While the identity of this slower process has not been unequivocally determined, we propose it reflects a thin filament state that elicits fewer re-attached crossbridges. Our finding that h-CaD modulates the rate of smooth muscle relaxation clearly supports a role in the control of vascular tone.

We have previously reported that actin undergoes a conformational transition (which we named "maturation") during polymerization, and that the actin-binding protein, caldesmon (CaD), when added at ...

Background: Migration of vascular smooth muscle cells (SMCs) from the media to intima constitutes a critical step in the development of proliferative vascular diseases. To elucidate the regulatory mechanism of vacular SMC motility, the roles of caldesmon (CaD) and its phosphorylation were investigated. Methods: We have performed Transwell migration assays, immunofluorescence microscopy, traction microscopy and cell rounding assays using A7r5 cells transfected with EGFP (control), EGFP-wtCaD or phosphomimetic CaD mutants, including EGFP-A1A2 (the two PAK sites Ser452 and Ser482 converted to Ala), EGFP-A3A4 (the two Erk sites Ser497 and Ser527 converted to Ala), EGFP-A1234 (both PAK- and Erk-sites converted to Ala) and EGFP-D1234 (both PAK- and Erk-sites converted to Asp). Results: We found that cells transfected with wtCaD, A1A2 or A3A4 mutants of CaD migrated at a rate approximately 50% more slowly than those EGFP-transfected cells. The migration activity for A1234 cells was only about 13% of control cells. Thus it seems both MAPK and PAK contribute to the motility of A7r5 cells and the effects are comparable and additive. The A1234 mutant also gave rise to highest strain energy and lowest rate of cell rounding. The migratory and contractile properties of these cells are consistent with stabilized actin cytoskeletal structures. Indeed, the A1234 mutant cells exhibited most robust stress fibers, whereas cells transfected with wtCaD or A3A4 (and A1A2) had moderately reinforced actin cytoskeleton. The control cells (transfected with EGFP alone) exhibited actin cytoskeleton that was similar to that in untransfected cells, and also migrated at about the same speed as the untransfected cells. Conclusions: These results suggest that both the expression level and the level of MAPK- and/or PAK-mediated phosphorylation of CaD play key roles in regulating the cell motility by modulating the actin cytoskeleton stability in dedifferentiated vascular SMCs such as A7r5.

Caldesmon (CaD) is an extraordinary actin-binding protein, because in addition to actin, it also binds myosin, calmodulin and tropomyosin. As a component of the smooth muscle and nonmuscle contractile apparatus CaD inhibits the actomyosin ATPase activity and its inhibitory action is modulated by both Ca²⁺ and phosphorylation. The multiplicity of binding partners and diverse biochemical properties suggest CaD is a potent and versatile regulatory protein both in contractility and cell motility. However, after decades of investigation in numerous laboratories, hard evidence is still lacking to unequivocally identify its in vivo functions, although indirect evidence is mounting to support an important role in connection with the actin cytoskeleton. This chapter reviews the highlights of the past findings and summarizes the current views on this protein, with emphasis of its interaction with tropomyosin.

The actin-binding protein caldesmon (CaD) exists both in smooth muscle (the heavy isoform, h-CaD) and non-muscle cells (the light isoform, l-CaD). In smooth muscles h-CaD binds to myosin and actin simultaneously and modulates the actomyosin interaction. In non-muscle cells l-CaD binds to actin and stabilizes␣the actin stress fibers; it may also mediate the interaction between actin and non-muscle myosins. Both h- and l-CaD are phosphorylated in vivo upon stimulation. The major phosphorylation sites of h-CaD when activated by phorbol ester are the Erk-specific sites, modification of which is attenuated by the MEK inhibitor PD98059. The same sites in l-CaD are also phosphorylated when cells are stimulated to migrate, whereas in dividing cells l-CaD is phosphorylated more extensively, presumably by cdc2 kinase. Both Erk and cdc2 are members of the MAPK family. Thus it appears that CaD is a downstream effector of the Ras signaling pathways. Significantly, the phosphorylatable serine residues shared by both CaD isoforms are in the C-terminal region that also contains the actin-binding sites. Biochemical and structural studies indicated that phosphorylation of CaD at the Erk sites is accompanied by a conformational change that partially dissociates CaD from actin. Such a structural change in h-CaD exposes the myosin-binding sites on the actin surface and allows actomyosin interactions in smooth muscles. In the case of non-muscle cells, the change in l-CaD weakens the stability of the actin filament and facilitates its disassembly. Indeed, the level of l-CaD modification correlates very well in a reciprocal manner with the level of actin stress fibers. Since both cell migration and cell division require dynamic remodeling of actin cytoskeleton that leads to cell shape changes, phosphorylation of CaD may therefore serve as a plausible means to regulate these processes. Thus CaD not only links the smooth muscle contractility and non-muscle motility, but also provides a common mechanism for the regulation of cell migration and cell proliferation.

Smooth muscles exist in the wall of hollow organs in our body and are responsible for controlling the flow of vital fluids that are essential for the normal function of the cardiovascular,...