Spatio-temporal images of growth-factor-induced activation of Ras and Rap1.
G proteins of the Ras family function as molecular switches in many signalling cascades; however, little is known about where they become activated in living cells. Here we use FRET (fluorescent resonance energy transfer)-based sensors to report on the spatio-temporal images of growth-factor-induced activation of Ras and Rap1. Epidermal growth factor activated Ras at the peripheral plasma membrane and Rap1 at the intracellular perinuclear region of COS-1 cells. In PC12 cells, nerve growth factor-induced activation of Ras was initiated at the plasma membrane and transmitted to the whole cell body. After three hours, high Ras activity was observed at the extending neurites. By using the FRAP (fluorescence recovery after photobleaching) technique, we found that Ras at the neurites turned over rapidly; therefore, the sustained Ras activity at neurites was due to high GTP/GDP exchange rate and/or low GTPase activity, but not to the retention of the active Ras. These observations may resolve long-standing questions as to how Ras and Rap1 induce different cellular responses and how the signals for differentiation and survival are distinguished by neuronal cells.
Mochizuki
N
N
Department of Structural Analysis, National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita-shi, Osaka 565-8565, Japan.
Yamashita
S
S
Kurokawa
K
K
Ohba
Y
Y
Nagai
T
T
Miyawaki
A
A
Matsuda
M
M
D000818:Animals; D019556:COS Cells; D004815:Epidermal Growth Factor / Q000378:metabolism*; D005453:Fluorescence; D020558:GTP Phosphohydrolases; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D008164:Luminescent Proteins; D008969:Molecular Sequence Data; D016501:Neurites / Q000378:metabolism; D016716:PC12 Cells; D017434:Protein Structure, Tertiary / Q000235:genetics; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins; D020827:rap1 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D008164:Luminescent Proteins; C430803:RAP1A protein, human; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D004815:Epidermal Growth Factor; D019908:Proto-Oncogene Proteins c-raf; C430804:Raichu-Ras protein; D020558:GTP Phosphohydrolases; D020827:rap1 GTP-Binding Proteins; D018631:ras Proteins