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C-L Albert Wang

Publications and source records attributed to C-L Albert Wang.

9 recordsLinked to original sources
PubMed2006

Direct interaction between caldesmon and cortactin.

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.

Animals↗
PubMed2006

Genetic ablation of zyxin causes Mena/VASP mislocalization, increased motility, and deficits in actin remodeling.

Focal adhesions are specialized regions of the cell surface where integrin receptors and associated proteins link the extracellular matrix to the actin cytoskeleton. To define the cellular role of the focal adhesion protein zyxin, we characterized the phenotype of fibroblasts in which the zyxin gene was deleted by homologous recombination. Zyxin-null fibroblasts display enhanced integrin-dependent adhesion and are more migratory than wild-type fibroblasts, displaying reduced dependence on extracellular matrix cues. We identified differences in the profiles of 75- and 80-kD tyrosine-phosphorylated proteins in the zyxin-null cells. Tandem array mass spectrometry identified both modified proteins as isoforms of the actomyosin regulator caldesmon, a protein known to influence contractility, stress fiber formation, and motility. Zyxin-null fibroblasts also show deficits in actin stress fiber remodeling and exhibit changes in the molecular composition of focal adhesions, most notably by severely reduced accumulation of Ena/VASP proteins. We postulate that zyxin cooperates with Ena/VASP proteins and caldesmon to influence integrin-dependent cell motility and actin stress fiber remodeling.

Actins↗
PubMed2005

A caldesmon peptide activates smooth muscle via a mechanism similar to ERK-mediated phosphorylation.

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.

Actomyosin↗
PubMed2005

Phosphorylated l-caldesmon is involved in disassembly of actin stress fibers and postmitotic spreading.

The function of the ubiquitous actin-binding protein, caldesmon (l-CaD) in mammalian non-muscle cells remains elusive. During mitosis, l-CaD becomes markedly phosphorylated at Ser497 and Ser527 (in the rat sequence), therefore, it has been suggested that l-CaD is involved in cytokinesis by inhibiting the actomyosin interaction until it is phosphorylated, although direct in vivo evidence is still missing. In the present study, we used F-actin staining and specific antibodies against these two phosphorylation sites of l-CaD to simultaneously monitor actin assembly and l-CaD phosphorylation. Our observations demonstrated that the level of l-CaD phosphorylation undergoes dynamic changes during the cell cycle. The spatial and temporal distributions of phospho-CaD do not correlate with cytokinesis per se, but rather, with the level of actin bundles in a reciprocal manner. The highest l-CaD phosphorylation level coincides with the disassembly of actin cytoskeleton during mitotic cell rounding. Ser-to-Ala mutations at these two positions prevent stress fibers from disassembly upon migratory stimulation. In addition, phospho-CaD appears to colocalize with nascent focal adhesion complexes during postmitotic spreading. These findings suggest that l-CaD phosphorylation plays an important role not only in cytoskeleton remodeling during cell shape changes, but also in cell spreading and migration.

Actins↗
PubMed2005

Specific disruption of smooth muscle caldesmon expression in mice.

Caldesmon (CaD) is an actin-binding protein that is capable of inhibiting the actomyosin ATPase activity in vitro. CaD has a single gene that is alternatively spliced to generate the smooth muscle-specific form, h-CaD, and a shorter isoform, l-CaD, that is present only in non-muscle cells. The difference between h- and l-CaD is a highly charged repeating sequence, corresponding to a 35 nm-long single helical region that separates the N-terminal domain from the C-terminal domain of h-CaD. To test whether such an elongated h-CaD is essential for smooth muscles to function properly, we have specifically abrogated its expression in the mouse by targeting h-CaD without affecting the expression of l-CaD. After genotyping, we have obtained homozygous knockout mice that indeed lack h-CaD, but nevertheless express varying amounts of l-CaD in a tissue-dependent fashion. The contractility of smooth muscles isolated from the knockout animals is currently under investigation.

Alternative Splicing↗
PubMed2004

Modes of caldesmon binding to actin: sites of caldesmon contact and modulation of interactions by phosphorylation.

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.

Acrylamide↗
PubMed2003

Caldesmon binding to actin is regulated by calmodulin and phosphorylation via different mechanisms.

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.

Actins↗
PubMed2002

Crystal structures of S100A6 in the Ca(2+)-free and Ca(2+)-bound states: the calcium sensor mechanism of S100 proteins revealed at atomic resolution.

S100A6 is a member of the S100 family of Ca(2+) binding proteins, which have come to play an important role in the diagnosis of cancer due to their overexpression in various tumor cells. We have determined the crystal structures of human S100A6 in the Ca(2+)-free and Ca(2+)-bound states to resolutions of 1.15 A and 1.44 A, respectively. Ca(2+) binding is responsible for a dramatic change in the global shape and charge distribution of the S100A6 dimer, leading to the exposure of two symmetrically positioned target binding sites. The results are consistent with S100A6, and most likely other S100 proteins, functioning as Ca(2+) sensors in a way analogous to the prototypical sensors calmodulin and troponin C. The structures have important implications for our understanding of target binding and cooperativity of Ca(2+) binding in the S100 family.

Amino Acid Sequence↗