R-Ras regulates exocytosis by Rgl2/Rlf-mediated activation of RalA on endosomes.
R-Ras is a Ras-family small GTPase that regulates various cellular functions such as apoptosis and cell adhesion. Here, we demonstrate a role of R-Ras in exocytosis. By the use of specific anti-R-Ras antibody, we found that R-Ras was enriched on both early and recycling endosomes in a wide range of cell lines. Using a fluorescence resonance energy transfer-based probe for R-Ras activity, R-Ras activity was found to be higher on endosomes than on the plasma membrane. This high R-Ras activity on the endosomes correlated with the accumulation of an R-Ras effector, the Rgl2/Rlf guanine nucleotide exchange factor for RalA, and also with high RalA activity. The essential role played by R-Ras in inducing high levels of RalA activity on the endosomes was evidenced by the short hairpin RNA (shRNA)-mediated suppression of R-Ras and by the expression of R-Ras GAP. In agreement with the reported role of RalA in exocytosis, the shRNA of either R-Ras or RalA was found to suppress calcium-triggered exocytosis in PC12 pheochromocytoma cells. These data revealed that R-Ras activates RalA on endosomes and that it thereby positively regulates exocytosis.
Takaya
Akiyuki
A
Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, Yamadaoka, Osaka 565-0871, Japan.
Kamio
Takahiro
T
Masuda
Michitaka
M
Mochizuki
Naoki
N
Sawa
Hirofumi
H
Sato
Mami
M
Nagashima
Kazuo
K
Mizutani
Akiko
A
Matsuno
Akira
A
Kiyokawa
Etsuko
E
Matsuda
Michiyuki
M
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D004285:Dogs; D011992:Endosomes / Q000378:metabolism*; D005089:Exocytosis / Q000502:physiology*; D006801:Humans; D051379:Mice; D016716:PC12 Cells; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020725:ral GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018631:ras Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D034741:RNA, Small Interfering; D014157:Transcription Factors; D020691:rab GTP-Binding Proteins; D020725:ral GTP-Binding Proteins; D018631:ras Proteins