SummaryThe 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.Abbreviations: BPM -benzophenone maleimide; CaD -caldesmon; CaM -calmodulin; EM -electron microscopy; Erk -extracellular signal-regulated kinase; FRET -fluorescence resonance energy transfer; GFP -green fluorescent protein; h-CaD -smooth muscle caldesmon; IAEDANS -5-(iodoacetamidoethyl) aminonaphthalene-1-sulfonic acid; l-CaD -non-muscle caldesmon; MAPK -mitogen activated protein kinase; MLCK -myosin light chain kinase; MS -mass spectrometry; Pak -p21-activated protein kinase; PMA -phorbol 12-myristate 13-acetate; Raf -rat aorta fibroblast cells; Tm -tropomyosin Remodeling of actin cytoskeleton plays a central role in a variety of cellular processes that involve shape change and movement. Malfunction of these processes could lead to pathological consequences, but how actin-mediated motility is regulated is only beginning to be understood. With recent

The actin-binding protein caldesmon (CaD) reversibly inhibits smooth muscle contraction. In non-muscle cells, a shorter CaD isoform co-exists with microfilaments in the stress fibers at the quiescent state, but the phosphorylated CaD is found at the leading edge of migrating cells where dynamic actin filament remodeling occurs. We have studied the effect of a C-terminal fragment of CaD (H32K) on the kinetics of the in vitro actin polymerization by monitoring the fluorescence of pyrene-labeled actin. Addition of H32K or its phosphorylated form either attenuated or accelerated the pyrene emission enhancement, depending on whether it was added at the early or the late phase of actin polymerization. However, the CaD fragment had no effect on the yield of sedimentable actin, nor did it affect the actin ATPase activity. Our findings can be explained by a model in which nascent actin filaments undergo a maturation process that involves at least two intermediate conformational states. If present at early stages of actin polymerization, CaD stabilizes one of the intermediate states and blocks the subsequent filament maturation. Addition of CaD at a later phase accelerates F-actin formation. The fact that CaD is capable of inhibiting actin filament maturation provides a novel function for CaD and suggests an active role in the dynamic reorganization of the actin cytoskeleton.

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BACKGROUND A previous comparative tissue proteomics study by the authors of the current study led to the identification of caldesmon (CaD) as one of the proteins associated with cervical metastasis of oral cavity squamous cell carcinoma (OSCC). In the current investigation, the authors focused on the potential functions of CaD in patients with OSCC. METHODS CaD expression was examined in tissue samples from 155 patients using immunohistochemical analysis. The expression of CaD variants was determined by Western blot analysis and reverse transcriptase‐polymerase chain reaction. In addition, the specific effects of CaD gene overexpression and silence were determined in OSCC cell lines. RESULTS CaD expression was found to be significantly higher in tumor cells from metastatic lymph nodes compared with primary tumor cells, and was nearly absent in normal oral epithelia. Higher CaD expression was found to be correlated with positive N classification, poor differentiation, perineural invasion, and tumor depth (P = .001, P = .029, P = .001, and P = .031, respectively). In survival analyses, OSCC patients with higher CaD expression were found to have poorer prognosis with regard to disease‐specific survival and disease‐free survival (P = .003 and P = .014, respectively). Multivariate analyses further indicated that higher CaD expression was an independent predictor of disease‐specific survival (P = .043). Serum CaD levels were found to be significantly higher in patients with OSCC, but this finding was not associated with clinicopathological manifestations. Data obtained from in vitro suppression, rescue, and overexpression of CaD in OEC‐M1 cells indicated that CaD promotes migration and invasive processes in OSCC cells. CONCLUSIONS The findings of the current study collectively suggest that the low‐molecular‐weight CaD expression in OSCC tumors is associated with tumor metastasis and patient survival. Cancer 2013;119:4003–4011. © 2013 American Cancer Society.

The motor function of smooth muscle myosin (SmM) is regulated by phosphorylation of the regulatory light chain (RLC) bound to the neck region of the SmM heavy chain. It is generally accepted that unphosphorylated RLC induces interactions between the two heads and between the head and the tail, thus inhibiting the motor activity of SmM, whereas phosphorylation of RLC interrupts those interactions, thus reversing the inhibition and restoring the motor activity to the maximal value. One assumption of this model is that single-headed SmM is fully active regardless of phosphorylation. To re-evaluate this model, we produced a number of SmM constructs with coiled coils of various lengths and examined their structure and regulation. With these constructs we identified the segment in the coiled-coil key for the formation of a stable double-headed structure. In agreement with the current model, we found that the actin-activated ATPase activity of unphosphorylated SmM increased with shortening of the coiled-coil. However, contrary to the current model, we found that the actin-activated ATPase activity of phosphorylated SmM decreased with shortening coiled-coil and only the stable double-headed SmM was fully activated by phosphorylation. These results indicate that single-headed SmM is neither fully active nor fully inhibited. Based on our findings, we propose that cooperation between the two heads is essential, not only for the inhibition of unphosphorylated SmM, but also for the activation of phosphorylated SmM.