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. 2007 Jul 16;178(2):185-91.
doi: 10.1083/jcb.200611138.

G protein-independent Ras/PI3K/F-actin circuit regulates basic cell motility

Atsuo T Sasaki  1 , Chris Janetopoulos, Susan Lee, Pascale G Charest, Kosuke Takeda, Lauren W Sundheimer, Ruedi Meili, Peter N Devreotes, Richard A Firtel
Affiliations

G protein-independent Ras/PI3K/F-actin circuit regulates basic cell motility

Atsuo T Sasaki et al. J Cell Biol. .

Abstract

Phosphoinositide 3-kinase (PI3K)gamma and Dictyostelium PI3K are activated via G protein-coupled receptors through binding to the Gbetagamma subunit and Ras. However, the mechanistic role(s) of Gbetagamma and Ras in PI3K activation remains elusive. Furthermore, the dynamics and function of PI3K activation in the absence of extracellular stimuli have not been fully investigated. We report that gbeta null cells display PI3K and Ras activation, as well as the reciprocal localization of PI3K and PTEN, which lead to local accumulation of PI(3,4,5)P(3). Simultaneous imaging analysis reveals that in the absence of extracellular stimuli, autonomous PI3K and Ras activation occur, concurrently, at the same sites where F-actin projection emerges. The loss of PI3K binding to Ras-guanosine triphosphate abolishes this PI3K activation, whereas prevention of PI3K activity suppresses autonomous Ras activation, suggesting that PI3K and Ras form a positive feedback circuit. This circuit is associated with both random cell migration and cytokinesis and may have initially evolved to control stochastic changes in the cytoskeleton.

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Figures

Figure 1.
Figure 1.
G protein–independent PI3K activation in the absence of extracellular stimuli. (A) The vegetative state cells were plated in Na/KPO4 buffer. Fluorescent images of GFP-PHcrac in wild-type and gβ− cells before or after the addition of 50 μM LY294002 (left). The cells were exposed to a uniform concentration of chemoattractant (50 μM folic acid; right). The folic-induced PH translocation is weak compared with the spontaneous PH accumulation at the plasma membrane. (B) Spontaneous activation of Akt/PKB is shown. (C) Fluorescent images of GFP-PHcrac in wild-type and gβ− cells before or after the addition of 5 μM LatB and after the removal of LatB. The LatB-treated cells increased GFP intensity due to the loss of membrane PH, and the cell became round and shrank. (D) Fluorescent images of GFP-tagged PI3K (left) and PTEN (right) in wild-type cells. The arrows indicate PI3K accumulation sites (left) and PTEN dissociation sites (right). Bars, 5 μm.
Figure 2.
Figure 2.
G protein–independent, PI3K-dependent Ras activation without extracellular stimuli. (A) Fluorescent images of GFP-RBD in wild-type and gβ− cells before or after the addition of 50 μM LY294002 and after the removal of LY294002. (B and C) Spontaneous activation of Ras in indicated cells is shown. Cells were treated or not treated with 50 μM LY294002 (LY) or DMSO for 5 min. (D) Fluorescent images of GFP-RBD in wild-type and gβ− cells before or after the addition of 5 μM LatB and after the removal of LatB. (E) Translocation of GFP-RBD in a wild-type cell treated with 50 μM LY294002 for 10 min or 5 μM LatB for 20 min before folic acid stimulation. (F) Ras activation level in response to folic acid. (G) Translocation of GFP-RBD in wild-type and gβ null cells in response to folic acid. Bars, 5 μm.
Figure 3.
Figure 3.
Tight correlation of the sites and timing of autonomous Ras and PI3K activation. (A and B) Fluorescent images of GFP-RBD and RFP-PHcrac in a wild-type cell. Simultaneous imaging of spontaneous RBD and PH domain accumulation (A) and disappearance (B) are shown. (C) Translocation of GFP-RBD and PHcrac in developed wild-type cells by cAMP is depicted. (D) Translocation of GFP-RBD and PHcrac in vegetative (left) and developed pten null cells by folic acid and cAMP is depicted.
Figure 4.
Figure 4.
Spontaneous PI3K activation requires Ras binding and regulates random cell movement. (A) Time-lapse recording of random movement of the indicated strains or wild-type cells treated with LY294002. The vegetative state cells and the nonaxenic NC4 cells grown on bacteria were placed in Na/KPO4 buffer. (B) Fluorescent images of GFP-RBD and RFP-PHcrac in the indicated strains. We note that the spontaneous RBD accumulation in the pi3k mutant cells was completely blocked by LY294002. (C) Time sequence of GFP-RBD in wild-type and gβ− cells during cytokinesis.
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