Differential involvement of H- and K-Ras in Raf-1 activation determines the role of calmodulin in MAPK signaling.
We have previously demonstrated that inhibition of calmodulin (CaM) and the concomitant reduction of PI3K interfere with H-Ras-mediated activation of Raf-1 [1]. In the present study, we show that CaM has completely opposite effects on K-Ras-mediated Raf-1 activation. The differential contribution of CaM in the regulation of Raf-1 kinase activity via K- or H-Ras correlates with the stimulatory or inhibitory effect of CaM on MAPK phosphorylation depending on the cell type analyzed. FRET microscopy and biochemical analysis show that inhibition of CaM increases K-Ras-GTP levels and consequently its association with Raf-1. Though inhibition of CaM, using the CaM antagonist W-13, significantly increased Raf-1 activation by K-Ras-GTP, MAPK activation downstream K-Ras/Raf-1 was strongly reduced in COS-1 and several other cell lines. In contrast, in other cell lines such as NIH3T3-wt8, W-13-mediated inhibition of CaM increased Raf-1 activity, but resulted in an increase in MAPK phosphorylation. These findings suggest that modulation of K-Ras activity via CaM regulates MAPK signaling only in certain cell types. In support of this hypothesis, the comparison of H- and K-Ras expression, GTP loading and Raf-1 interaction in COS-1 and NIH3T3-wt8 suggests that the overall role of CaM in MAPK signal output is determined by the ratio of activated H- and K-Ras and the cell-specific contribution of each isoform in Raf-1 activation.
Moretó
Jemina
J
Departament de Biologia Cel.lular, Immunologia i Neurociències, Facultat de Medicina, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Casanova 143, 08036-Barcelona, Spain.
Vidal-Quadras
Maite
M
Pol
Albert
A
Santos
Eugenio
E
Grewal
Thomas
T
Enrich
Carlos
C
Tebar
Francesc
F
D000818:Animals; D019556:COS Cells; D002147:Calmodulin / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D020935:MAP Kinase Signaling System; D051379:Mice; D041681:NIH 3T3 Cells; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism*
D002147:Calmodulin; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins