Calmodulin modulates H-Ras mediated Raf-1 activation.
We have previously demonstrated that, in COS-1 cells, inhibition of calmodulin increases Ras-GTP levels although it decreases Raf-1 activity and consequently MAPK. The present study analyzes the role of calmodulin in the regulation of Raf-1. First we show, using FRET microscopy, that inhibition of Raf-1 was not a consequence of a decreased interaction between H-Ras and Raf-1. Besides, the analysis of the phosphorylation state of Raf-1 showed that calmodulin, through downstream PI3K, is essential to ensure the Ser338-Raf-1 phosphorylation, critical for Raf-1 activation. We also show that the expression of a dominant negative mutant of PI3K impairs the calmodulin-mediated Raf-1 activation; in addition, both calmodulin and PI3K inhibitors decrease phospho-Ser338 and Raf-1 activity from upstream active H-Ras (H-RasG12V) and this effect is dependent on endocytosis. Importantly, in H-Ras depleted COS-1 cells, calmodulin does not modulate MAPK activation. Altogether, the results suggest that calmodulin regulation of MAPK in COS-1 cells relies upon H-Ras control of Raf-1 activity and involves PI3K.
Moretó
Jemina
J
Departament de Biologia Cel.lular, Facultat de Medicina, Universitat de Barcelona, Casanova 143, 08036-Barcelona, Spain.
Lladó
Anna
A
Vidal-Quadras
Maite
M
Calvo
Maria
M
Pol
Albert
A
Enrich
Carlos
C
Tebar
Francesc
F
D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002147:Calmodulin / Q000378:metabolism*; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D004705:Endocytosis; D004789:Enzyme Activation; D020935:MAP Kinase Signaling System; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D012694:Serine / Q000378:metabolism
D002147:Calmodulin; D019869:Phosphatidylinositol 3-Kinases; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras); D012694:Serine