Kinase-dependent adhesion to fibronectin: regulation by calreticulin |
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Authors: | Papp Sylvia Szabo Eva Kim Hugh McCulloch Christopher A Opas Michal |
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Affiliation: | Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada. |
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Abstract: | We studied the phosphorylation (activation status) of c-Src and CaMKII in MEFs either wild type for calreticulin, calreticulin-null, or rescued with full-length calreticulin. We found that calreticulin-null cells were poorly spread on the substratum and formed few, if any, focal contacts. Fibronectin expression and deposition were lower in calreticulin-null MEFs compared to calreticulin-expressing cells, which also exhibited increased c-Src and CaMKII phosphorylation (activity). Plating MEFs on preformed fibronectin rescued the poor adhesive phenotype of calreticulin-null cells, and caused a decrease in c-Src Y418 phosphorylation (activity). c-Src inhibition caused the calreticulin-null MEFs to become well spread on the substratum and to make many prominent focal contacts. Calmodulin and CaMKII inhibition caused similar results, along with a notable increase in paxillin phosphorylation (activation). To test if the calcium storage function of calreticulin was responsible for these effects, we manipulated intracellular [Ca(2+)]. Lowering [Ca(2+)](ER) caused an increase in c-Src phosphorylation and a decrease in fibronectin abundance. Conversely, increasing [Ca(2+)] caused opposite effects. These results suggest that calreticulin regulates both the c-Src and calmodulin/CaMKII pathways, enabling cells to be better spread on the substratum by allowing greater fibronectin deposition and increased focal contact formation. |
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Keywords: | [Ca2+]c, cytosolic Ca2+ concentration CaMKII, Ca2+/calmodulin-dependent protein kinase II ECM, extracellular matrix ER, endoplasmic reticulum FAK, focal adhesion kinase K41, wild type cells K42, calreticulin-null cells K42CRT, calreticulin-null cells rescued with full-length calreticulin MEF, mouse embryonic fibroblast |
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