Ras and calcium signaling pathways converge at Raf1 via the Shoc2 scaffold protein.
Situated downstream of Ras is a key signaling molecule, Raf1. Increase in Ca(2+) concentration has been shown to modulate the Ras-dependent activation of Raf1; however, the mechanism underlying this effect remains elusive. Here, to characterize the role of Ca(2+) in Ras signaling to Raf1, we used a synthetic guanine nucleotide exchange factor (GEF) for Ras, eGRF. In HeLa cells expressing eGRF, Ras was activated by the cAMP analogue 007 as efficiently as by epidermal growth factor (EGF), whereas the activation of Raf1, MEK, and ERK by 007 was about half of that by EGF. Using a biosensor based on fluorescence resonance energy transfer, it was found that activation of Raf1 at the plasma membrane required not only Ras activation but also an increase in Ca(2+) concentration or inhibition of calmodulin. Furthermore, the Ca(2+)-dependent activation of Raf1 was found to be abrogated by knockdown of Shoc2, a scaffold protein that binds both Ras and Raf1. These observations indicated that the Shoc2 scaffold protein modulates Ras-dependent Raf1 activation in a Ca(2+)- and calmodulin-dependent manner.
Yoshiki
Sayaka
S
Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
Matsunaga-Udagawa
Rie
R
Aoki
Kazuhiro
K
Kamioka
Yuji
Y
Kiyokawa
Etsuko
E
Matsuda
Michiyuki
M
D000818:Animals; D020013:Calcium Signaling / Q000502:physiology*; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000031:analogs & derivatives; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D048369:MAP Kinase Kinase 1 / Q000235:genetics / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
D002147:Calmodulin; D020662:Guanine Nucleotide Exchange Factors; D047908:Intracellular Signaling Peptides and Proteins; C112913:SHOC2 protein, human; D000242:Cyclic AMP; D019908:Proto-Oncogene Proteins c-raf; D048049:Extracellular Signal-Regulated MAP Kinases; D048369:MAP Kinase Kinase 1; D018631:ras Proteins