Search

Non-muscle caldesmon (l-CaD) is involved in the regulation of actin cytoskeletal remodeling in the podosome formation, but its function in osteoclastogenesis remains to be determined. In this study, RANKL-induced differentiation of RAW264.7 murine macrophages to osteoclast-like cells (OCs) was used as a model to determine the physiological role of l-CaD and its phosphorylation in osteoclastogenesis. Upon RANKL treatment, RAW264.7 cells undergo cell-cell fusion into multinucleate, and TRAP-positive large OCs with a concomitant increase of l-CaD expression. Using gain- and loss-of-function in OC precursor cells followed by RANKL induction, we showed that the expression of l-CaD in response to RANKL activation is an important event for osteoclastogenesis, and bone resorption. To determine the effect of l-CaD phosphorylation in osteoclastogenesis, three decoy peptides of l-CaD were used with, respectively, Ser-to-Ala mutations at the Erk- and Pak1-mediated phosphorylation sites, and Ser-to-Asp mutation at the Erk-mediated phosphorylation sites. Both the former two peptides competed with the C-terminal segment of l-CaD for F-actin binding and accelerated formation of podosome-like structures in RANKL-induced OCs, while the third peptide did not significantly affect the F-actin binding of l-CaD, and decreased the formation of podosome-like structures in OCs. With the experiments using dephosphorylated and phosphorylated l-CaD mutants, we further showed that dephosphorylated l-CaD mutant facilitated RANKL-induced TRAP activity with an increased cell fusion index, whereas phosphorylated l-CaD decreased the TRAP activity and cell fusion. Our findings suggested that both the level of l-CaD expression and the extent of l-CaD phosphorylation play a role in RANKL-induced osteoclast differentiation.

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 an early phase of polymerization, interferes with this process (Huang et al. J Biol Chem 2010; 285:71). The pre-transition filament is characterized by relatively low pyrene-fluorescence intensity when pyrene-labeled actin is used as a reporter of subunit assembly into filaments, whereas the mature filament emits a characteristic enhanced fluorescence. Previously reported co-sedimentation experiments suggest that filament formation is not inhibited by the presence of CaD, despite blocking the transition associated with filament maturation. In this study we visualized structural effects of CaD on the assembly of actin filaments by TIRF and electron microscopy. CaD-free actin forms “rough” filaments with irregular edges and indistinct subunit organization during the initial phase (∼20 min under our conditions) of polymerization as reported previously by others (Steinmetz et al. J Cell Biol 1997; 138:559; Galinska-Rakoczy et al. J Mol Biol 2009; 387:869), which most likely correspond to the pre-transition state preceding the maturation step. Later during the polymerization process “mature” filaments exhibit a smoother F-actin appearance with easily detectible double helically arranged actin subunits. While the inclusion of the actin-binding domain of CaD during actin polymerization does not affect the elongation rate, it is associated with a prolonged pre-transition phase, characterized by a delayed alteration (rough to smooth) of the appearance of filaments, consistent with a later onset of the maturation process.

Actin polymerization and depolymerization plays a central role in controlling a wide spectrum of cellular processes. There are many actin-binding proteins in eukaryotic cells. Their roles in the remodeling of the actin architecture and whether they work cooperatively await further study. Caldesmon (CaD) is an actin-binding protein present in nearly all mammalian cells. Cortactin is another actin-binding protein found mainly in the cell cortex. There have been no reports suggesting that CaD and cortactin interact with each other or work as partners. Here, we present evidence that CaD binds cortactin directly by overlay, pull-down assays, ELISA, and by column chromatography. The interaction involves the N-terminal region of cortactin and the C-terminal region of CaD, and appears to be enhanced by divalent metal ions. Cortactin competes with both full-length CaD and its C-terminal fragment for actin binding. Binding of cortactin partially alleviates the inhibitory effect of CaD on the actomyosin ATPase activity. Not only can binding be demonstrated in vitro, the two proteins also co-localize in activated cells at the cortex. Whether such interactions bear any functional significance awaits further investigation.

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 muscle caldesmon binds actin and inhibits actomyosin ATPase activity. Phosphorylation of caldesmon by extracellular signal-regulated kinase (ERK) reverses this inhibitory effect and weakens actin binding. To better understand this function, we have examined the phosphorylation-dependent contact sites of caldesmon on actin by low dose electron microscopy and three-dimensional reconstruction of actin filaments decorated with a C-terminal fragment, hH32K, of human caldesmon containing the principal actin-binding domains. Helical reconstruction of negatively stained filaments demonstrated that hH32K is located on the inner portion of actin subdomain 1, traversing its upper surface toward the C-terminal segment of actin, and forms a bridge to the neighboring actin monomer of the adjacent long pitch helical strand by connecting to its subdomain 3. Such lateral binding was supported by cross-linking experiments using a mutant isoform, which was capable of cross-linking actin subunits. Upon ERK phosphorylation, however, the mutant no longer cross-linked actin to polymers. Three-dimensional reconstruction of ERK-phosphorylated hH32K indeed indicated loss of the interstrand connectivity. These results, together with fluorescence quenching data, are consistent with a phosphorylation-dependent conformational change that moves the C-terminal end segment of caldesmon near the phosphorylation site but not the upstream region around Cys(595), away from F-actin, thus neutralizing its inhibitory effect on actomyosin interactions. The binding pattern of hH32K suggests a mechanism by which unphosphorylated, but not ERK-phosphorylated, caldesmon could stabilize actin filaments and resist F-actin severing or depolymerization in both smooth muscle and nonmuscle cells.

The biological functions of the myosin light chain 1 (LC1) have not been clearly elucidated yet. In this work we cloned and expressed N- and C- terminal fragments of human ventricular LC1 (HVLC1) containing amino acid residues 1–98 and 99–195 and two parts, NN and NC of N fragment in GST-fusion forms, respectively. Using GST pull-down assay, the direct binding experiments of LC1 with rat cardiac G -actin, F -actin and thin filaments, as well as rat cardiac myosin heavy chain (RCMHC) have been performed. Furthermore, the recombinant complexes of rat myosin S1 with N- and C-fragments, as well as the whole molecular of HVLC1 were generated. The results suggested that both binding sites of HVLC1 for actin and myosin heavy chain are positioned in its N-terminal fragment, which may contain several actin-binding sites in tandem. The polymerization of G -actin, the tropomyosin and troponin molecules located in the thin filaments do not hinder the binding of N-terminal fragment of HVLC1 with actin and thin filaments in vitro. The recombinant complex of rat cardiac myosin S1 (RCMS1) with N fragment of HVLC1 greatly decreased actin-actived Mg 2+ -ATPase activity for lack of C fragment. We conclude that the N-fragment is the binding domain of human ventricular LC1, whereas the C-fragment serves as a functional domain, which may be more involved in the modulation of the actin-activated ATPase activity of myosin.

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 up-regulation 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 cross-bridges. 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 cross-bridges. Our finding that h-CaD modulates the rate of smooth muscle relaxation clearly supports a role in the control of vascular tone.

Actin is a highly ubiquitous protein in eukaryotic cells that plays a crucial role in cell mechanics and motility. Cell motility is driven by assembling actin as polymerizing actin drives cell protrusions in a process closely involving a host of other actin-binding proteins, notably the actin-related protein 2/3 (Arp2/3) complex, which nucleates actin and forms branched filamentous structures. The Arp2/3 complex preferentially binds specific actin networks at the cell leading edge and forms branched filamentous structures, which drive cell protrusions, but the exact regulatory mechanism behind this process is not well understood. Here we show using in vitro imaging and binding assays that a fragment of the actin-binding protein caldesmon added to polymerizing actin increases the Arp2/3-mediated branching activity, whereas it has no effect on branch formation when binding to aged actin filaments. Because this caldesmon effect is shown to be independent of nucleotide hydrolysis and phosphate release from actin, our results suggest a mechanism by which caldesmon maintains newly polymerized actin in a distinct state that has a higher affinity for the Arp2/3 complex. Our data show that this new state does not affect the level of cooperativity of binding by Arp2/3 complex or its distribution on actin. This presents a novel regulatory mechanism by which caldesmon, and potentially other actin-binding proteins, regulates the interactions of actin with its binding partners.

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.

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.

Smooth muscle caldesmon binds actin and inhibits actomyosin ATPase activity. Phosphorylation of caldesmon by extracellular signal-regulated kinase (ERK) reverses this inhibitory effect and weakens actin binding. To better understand this function, we have examined the phosphorylation-dependent contact sites of caldesmon on actin by low dose electron microscopy and three-dimensional reconstruction of actin filaments decorated with a C-terminal fragment, hH32K, of human caldesmon containing the principal actin-binding domains. Helical reconstruction of negatively stained filaments demonstrated that hH32K is located on the inner portion of actin subdomain 1, traversing its upper surface toward the C-terminal segment of actin, and forms a bridge to the neighboring actin monomer of the adjacent long pitch helical strand by connecting to its subdomain 3. Such lateral binding was supported by cross-linking experiments using a mutant isoform, which was capable of cross-linking actin subunits. Upon ERK phosphorylation, however, the mutant no longer cross-linked actin to polymers. Three-dimensional reconstruction of ERK-phosphorylated hH32K indeed indicated loss of the interstrand connectivity. These results, together with fluorescence quenching data, are consistent with a phosphorylation-dependent conformational change that moves the C-terminal end segment of caldesmon near the phosphorylation site but not the upstream region around Cys(595), away from F-actin, thus neutralizing its inhibitory effect on actomyosin interactions. The binding pattern of hH32K suggests a mechanism by which unphosphorylated, but not ERK-phosphorylated, caldesmon could stabilize actin filaments and resist F-actin severing or depolymerization in both smooth muscle and nonmuscle cells.

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.

Actin undergoes a conformational transition during polymerization, known as maturation. Caldesmon (CaD) or its C-terminal fragment, H32K, when present before the transition, inhibits this process and arrests the actin filament at the pre-transitional “young” state, which is otherwise transient (Huang et al., J Biol Chem 285, 71-79, 2010). However, CaD in cells usually binds to stable actin filaments, except when it is phosphorylated and translocated to the cell leading edge where actin is being actively assembled (Kordowska et al., Exp Cell Res 312, 95-110, 2006); this may be the only occasion that CaD could interfere with actin maturation in vivo. We hypothesized that the young actin filaments stabilized by phosphorylated CaD are rendered more amenable to remodeling. Electron microscopic images indeed show that these young filaments have a rough and fuzzy morphology (Collins et al., BioArchitecture 1, 127-133, 2011). The question then arises: Could such an irregular appearance in fact provide more available docking sites for other actin-binding proteins, and thereby facilitate actin dynamics? To test this idea, we have performed binding experiments between Arp2/3 and actin filaments formed under the condition favoring either the young or the mature state. Actin filaments polymerized in the presence and absence of the CaD fragment H32K were incubated with the Arp2/3 complex and centrifuged; the amount of actin-bound Arp2/3 in the pellet was then analyzed by immuno-reactivity toward anti-Arp2 after serial dilutions. We found that the level of Arp2/3 cosedimented with actin filaments polymerized in the presence of H32K was higher than that bound to CaD-free F-actin, indicating stronger binding of Arp2/3 to the young actin filaments than to the mature filaments. These results appear to support our hypothesis. Supported by a grant from NIH.

Our preliminary experiments suggest that actin undergoes a conformational transition during polymerization. Both caldesmon (CaD) and nonmuscle tropomyosin (Tm) arrest actin filaments at an intermediate state if added initially, but stabilize “matured” filaments when added after the transition. We have tested this hypothesis by making use of the calmodulin (CaM)-dependent dissociation of a CaD fragment, H32K, from F-actin. When CaM was added to H32K-arrested, pyrene-labeled F-actin in the presence of Ca2+, the pyrene-actin emission increased, reflecting the maturation of actin filaments upon dissociation of H32K. When free Ca2+ was removed by EGTA and H32K became re-associated with F-actin, we observed an accelerated increase in pyrene fluorescence. These results are consistent with the hypothesized conformational transition and a differential effect of CaD on the two states of actin filaments. The combined effect of CaD and Tm on the actin conformational transition was also tested during polymerization. H32K and Tm5a were first incubated with actin before polymerization was initiated. Shortly after polymerization started, CaM was added to dissociate H32K. Since Tm5a alone was sufficient to inhibit the maturation process, the transition corresponding to the conformational change was not observed, and the pyrene-actin emission jumped to the level of F-actin•Tm5a. At a later time when CaD fragment re-associated with the addition of EGTA, we saw an instant decrease, instead of a further increase, in pyrene fluorescence to the level of actin with both H32K and Tm5a, indicating that F-actin was still kept at the intermediate state by Tm5a. Since the actin cytoskeleton at the cell leading edge is extremely dynamic, it is expected that actin filaments there are kept at a less stable configuration. CaD and Tm5a may therefore function to maintain such a configuration by binding to nascent actin filaments as they are assembled.

Caldesmon (CaD) is thought to regulate smooth muscle contraction, because it binds actin and inhibits actomyosin interactions. A synthetic actin-binding peptide (GS17C) corresponding to Gly666–Ser682 of chicken gizzard CaD has been shown to induce force development in permeabilized smooth muscle cells. The mechanism of GS17C’s action remains unclear, although a structural effect was postulated. By photo-crosslinking and fluorescence quenching experiments with a gizzard CaD fragment (H32K; Met563-Pro771) and its mutants, we showed that GS17C indeed dissociated the C-terminal region of H32K from actin, in a manner similar to extracellular signal-regulated kinase-mediated phosphorylation, thereby reversing the CaD-imposed inhibition and enabling the actomyosin interaction.

Tropomyosin (Tm) isoforms are known to target different subpopulations of actin filaments in cells. For example, Tm2 is typically co-localized with stress fibers, whereas Tm5a/b are localized to cell peripheries where the actin cytoskeleton is more dynamically remodeled. We have reported previously that actin undergoes an obligatory conformational transition during polymerization, called maturation. Both Tm5a and caldesmon (CaD) interfere with this process and maintain actin filaments in the pre-transition (or “young”) state if present at early stages of polymerization, but stabilize the matured actin filaments when binding occurs after this transition. When Tm5a and CaD are present together, they synergistically modulate actin maturation. The commonality between Tm5a and CaD apparently is not shared by all actin-binding proteins. Here we found that Tm2, when included in the F-buffer used to initiate actin polymerization, the pyrene-actin fluorescence enhancement was only slightly suppressed. When Tm2 and CaD were added together at the beginning of actin polymerization, the pyene-actin emission enhancement was greatly suppressed, although not as much as that caused by CaD alone. When calmodulin was added to dissociate CaD from actin in the presence of Tm2, the suppressed pyrene-actin fluorescence recovered quickly, with the final emission intensity approaching the level of Tm2•actin, which is about the same as that of actin alone. Upon addition of EGTA, on the other hand, the pyrene fluorescence only changed slightly to the level of CaD-bound mature F-actin. Clearly, actin had already passed the critical transition even Tm2 was still present. Our observations thus indicated that the maturation process can be blocked by Tm5a, but not by Tm2. Whether such a difference is related to their subcellular localizations and whether this finding holds true for other Tm isoforms await future investigations.

Caldesmon is an actin-binding protein found in nearly all vertebrate cells. The heavy caldesmon isoform, which is specific to smooth muscle cells, is known to inhibit actomyosin interactions in vitro in a phosphorylation-dependent manner. However, possible roles of caldesmon as a regulator of actin mechanics and turnover are being explored, and the effects of caldesmon on actin dynamics and structure are not well understood.We have recently demonstrated that polymerizing actin undergoes an irreversible structural transition (termed “maturation”), and that the caldesmon C-terminal fragment, H32K, if added during the early stage of actin polymerization, prevents this maturation process (Huang et al., 2010 J Biol Chem 285(1):71-79). Actin filaments stabilized in this “nascent” state by H32K appear rough under electron microscopy and exhibits attenuated pyrene fluorescence enhancement compared to normal, mature F-actin, but H32K does not otherwise affect the polymerization kinetics of actin (Collins et al., 2011 Bioarchitechture 1(3):127-133).Both phosphorylated caldesmon and the actin-branching protein complex Arp2/3 are present at the leading edge of motile cells, where actin assembly provides the force needed to protrude the plasma membrane. Here, we study the interaction of H32K-stabilized nascent F-actin with Arp2/3 complex. By direct visualization of actin assembly in the presence of Arp2/3 complex using confocal fluorescence microscopy, we show that actin polymerized in the presence of H32K exhibits increased branching activity. We propose that the observed change is a result of the structural state of the young actin filament, which is stabilized by the early added H32K. This effect suggests a role of caldesmon as a regulator of actin structure, in turn dictating the interaction of other actin-binding proteins with actin.

Migration of differentiated smooth muscle cells is usually pathogenic. It contributes to diseases such as atherosclerosis. The low molecular weight isoform of caldesmon (l-CaD) binds actin filaments in mammalian cells and modulates the assembly of the actin cytoskeleton. To determine the effect of phosphorylation of l-CaD on the mobility of cultured vascular smooth muscle cells, we have performed Transwell migration assays on A7r5 cells expressing l-CaD variants with mutations at the phosphorylation sites mediated by PAK (Ser452 and Ser482) and/or ERK (Ser497 and Ser527). The chemotactic migration activity of transfected cells compared to the normal, untransfected cells was evaluated. Among all constructs the A1234 mutant (Ser residues at all 4 positions changed to Ala) resulted in most hindered mobility. The relative migration activity for the A1234-transfected cells was about 13% of the vehicle-transfected cells. Transfection with wild-type l-CaD decreased the rate of cell migration to a lesser extent (∼50% of the control cells). Cells transfected with l-CaD mutated only at the PAK sites (A1A2) or the ERK sites (A3A4) also migrated approximately 50% slower than those control cells. Apparently, both ERK and PAK contribute to the mobility of A7r5 cells and the effects are comparable and additive. Phosphorylation was indeed found at the ERK sites of ectopically expressed A1A2 and l-CaD, but not that of A3A4 and A1234. The mobility of cells transfected with D1234 (all 4 Ser changed to Asp) was higher than that of cells transfected with all other CaD variants, but still about 20% lower than the control cells. Taken together, these results suggest l-CaD plays a role in controlling the migration activity of smooth muscle cells, and reversible phosphorylation of l-CaD facilitates this activity. Supported by NIH (HL-92252).