Phosphorylated l-caldesmon is involved in disassembly of actin stress fibers and postmitotic spreading |
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Authors: | Kordowska Jolanta Hetrick Tracy Adam Leonard P Wang C-L Albert |
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Affiliation: | Muscle and Motility Group, Boston Biomedical Research Institute, Watertown, MA 02472, USA. |
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Abstract: | 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. |
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Keywords: | BSA, bovine serum albumin CaD, caldesmon h-CaD, smooth muscle caldesmon l-CaD, non-muscle caldesmon CaM, calmodulin DAPI, 4′,6-diamidino-2-phenylindole ERK, extracellular signal-regulated kinase FITC, fluorescein isothiocyanate GFP, green fluorescent protein GST, glutathione-S-transferase HASM, human aorta smooth muscle cells HPLC, high pressure liquid chromatography MAPK, mitogen-activated protein kinase MDCK, Madin-Darby canine kidney cells PAK, p21-activated protein kinase PBS, phosphate buffered saline pCaD, phospho-CaD RAF, rat aorta fibroblast cells Tm, tropomyosin |
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