Modes of Caldesmon Binding to Actin

Abstract

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 crosslinking actin subunits. Upon ERK phosphorylation, however, the mutant no longer cross-linked actin to polymers. Three-dimensional reconstruction of ERKphosphorylated 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.

Regulation of Microfilament Organization by Kaposi Sarcoma-associated Herpes Virus-cyclin·CDK6 Phosphorylation of Caldesmon

“…In contrast, mitotic CDK1 and MAPK/ERK both modify CALD1 within the carboxyl-terminal actin-binding region and affect its association with actin, although the impact of these kinases on CALD1 function appears to differ to some extend. Recent evidence suggests that in vitro MAPK/ ERK phosphorylation does not abolish the association of CALD1 with F-actin, although it disables the trans-filament linkage and thus filament bundling (70). In contrast, mitotic CDK1 leads to full dissociation of CALD1 from F-actin filaments (48).…”

Caldesmon regulates the motility of vascular smooth muscle cells by modulating the actin cytoskeleton stability

“…In light of the findings that unphosphorylated and phosphorylated CaD display quite different actin-binding properties [11,39] and intracellular distributions [16,21], variations in CaD phosphorylation may have contributed to this apparent discrepancy. To better understand the role of CaD phosphorylation in vascular SMCs, we have force-expressed phosphomimetic mutants of CaD in A7r5 cells, a model for remodelled or diseased vascular SMCs, and examined the resulting cells in terms of their morphology, migration activity, contractility and detachment kinetics.…”

Caldesmon as a Therapeutic Target for Proliferative Vascular Diseases

“…Dephosphorylation of caldesmon is catalyzed by protein phosphatase(s), but relatively little has been published on caldesmon phosphatases, except that one caldesmon phosphatase has been identified as a type 2A protein phosphatase [51]. As shown in Table 1 , majority of the caldesmon kinases phosphorylate sites at the actin-binding carboxyl terminus of caldesmon, which may lead to the detachment of caldesmon from F-actin, and removing the inhibitory effect of caldesmon [52, 53]. Among the six caldesmon kinases shown in Table 1 , cdc2 kinase, Erk1/2 MAPK, and PAK have been well documented as regulators of caldesmon function on the actin cytoskeleton.…”