Smooth muscle caldesmon (CaD) binds F-actin and inhibits actomyosin ATPase activity. The inhibition is reversed by Ca2+/calmodulin (CaM). CaD is also phosphorylated upon stimulation at sites specific for mitogen-activated protein kinases (MAPKs). Because of these properties, CaD is thought to be involved in the regulation of smooth muscle contraction. The molecular mechanism of the reversal of inhibition is not well understood. We have expressed His6-tagged fragments containing the sequence of the C-terminal region of human (from M563 to V793) and chicken (from M563 to P771) CaD as well as a variant of the chicken isoform with a Q766C point mutation. By cleavages with proteases, followed by high-speed cosedimentation with F-actin and mass spectrometry, we found that within the C-terminal region of CaD there are multiple actin contact points forming two clusters. Intramolecular fluorescence resonance energy transfer between probes attached to cysteine residues (the endogenous C595 and the engineered C766) located in these two clusters revealed that the C-terminal region of CaD is elongated, but it becomes more compact when bound to actin. Binding of CaM restores the elongated conformation and facilitates dissociation of the C-terminal CaD fragment from F-actin. When the CaD fragment was phosphorylated with a MAPK, only one of the two actin-binding clusters dissociated from F-actin, whereas the other remained bound. Taken together, these results demonstrate that while both Ca2+/CaM and MAPK phosphorylation govern CaD's function via a conformational change, the regulatory mechanisms are different.

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

Heat shock markedly inhibited centrosome staining by antisera raised against the two centrosome-specific proteins, pericentrin and gamma tubulin. The inhibition of anti-pericentrin binding was measured by fluorescence imaging. Heat had the greatest effect on intact cells, followed in sensitivity by centrosomes attached to their companion nucleus, with purified centrosomes being least sensitive. The centrosomal content of pericentrin was measured by immunoprecipitation followed by western blotting. Heat caused the amount of pericentrin in the centrosomal fraction to increase, suggesting that pericentrin did not leave the centrosome during heat shock. Furthermore, the pericentrin of the centrosomal fraction became less soluble after heat shock, and could only be solubilized by the most denaturing condition of boiling in 0.1% SDS. Immunoelectron microscopy revealed a heat-induced increase in the electron-dense material comprising the pericentriolar material (PCM), consistent with protein aggregation. Lastly, in heated cells immunoelectron microscopy demonstrated an increase in the binding of heat shock protein 70 (HSP70) to numerous locations throughout the cytoplasm. These data suggest that heat shock reduces the solubility of centrosomal and other cytoplasmic proteins, most likely through protein aggregation.

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.