Diacylglycerol kinase eta augments C-Raf activity and B-Raf/C-Raf heterodimerization.
The Ras/B-Raf/C-Raf/MEK/ERK signaling cascade is critical for the control of many fundamental cellular processes, including proliferation, survival, and differentiation. This study demonstrated that small interfering RNA-dependent knockdown of diacylglycerol kinase eta (DGKeta) impaired the Ras/B-Raf/C-Raf/MEK/ERK pathway activated by epidermal growth factor (EGF) in HeLa cells. Conversely, the overexpression of DGKeta1 could activate the Ras/B-Raf/C-Raf/MEK/ERK pathway in a DGK activity-independent manner, suggesting that DGKeta serves as a scaffold/adaptor protein. By determining the activity of all the components of the pathway in DGKeta-silenced HeLa cells, this study revealed that DGKeta activated C-Raf but not B-Raf. Moreover, this study demonstrated that DGKeta enhanced EGF-induced heterodimerization of C-Raf with B-Raf, which transmits the signal to C-Raf. DGKeta physically interacted with B-Raf and C-Raf, regulating EGF-induced recruitment of B-Raf and C-Raf from the cytosol to membranes. The DGKeta-dependent activation of C-Raf occurred downstream or independently of the already known C-Raf modifications, such as dephosphorylation at Ser-259, phosphorylation at Ser-338, and interaction with 14-3-3 protein. Taken together, the results obtained strongly support that DGKeta acts as a novel critical regulatory component of the Ras/B-Raf/C-Raf/MEK/ERK signaling cascade via a previously unidentified mechanism.
Yasuda
Satoshi
S
Department of Biochemistry, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556.
Kai
Masahiro
M
Imai
Shin-Ichi
S
Takeishi
Kazuki
K
Taketomi
Akinobu
A
Toyota
Minoru
M
Kanoh
Hideo
H
Sakane
Fumio
F
D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000201:enzymology / Q000235:genetics; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000201:enzymology / Q000235:genetics; D019852:Diacylglycerol Kinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology*; D010766:Phosphorylation / Q000502:physiology; D011485:Protein Binding / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics
D034741:RNA, Small Interfering; D019852:Diacylglycerol Kinase; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D019908:Proto-Oncogene Proteins c-raf