Kinase-dependent adhesion to fibronectin: regulation by calreticulin.
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.
Papp
Sylvia
S
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.
Szabo
Eva
E
Kim
Hugh
H
McCulloch
Christopher A
CA
Opas
Michal
M
D000199:Actins / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D037282:Calreticulin / Q000172:deficiency / Q000378:metabolism*; D002448:Cell Adhesion / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004622:Embryo, Mammalian / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D005353:Fibronectins / Q000378:metabolism*; D022001:Focal Adhesions / Q000187:drug effects / Q000201:enzymology; D018942:Macrolides / Q000494:pharmacology; D051379:Mice; D008954:Models, Biological; D051419:Paxillin / Q000378:metabolism; D010641:Phenotype; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology; D016596:Vinculin / Q000378:metabolism
D000199:Actins; D002147:Calmodulin; D037282:Calreticulin; D005353:Fibronectins; D018942:Macrolides; D051419:Paxillin; D047428:Protein Kinase Inhibitors; D016596:Vinculin; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D019284:Thapsigargin; D016392:Proto-Oncogene Proteins pp60(c-src); D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; C035359:monorden; D002118:Calcium