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First published online 14 August 2007
doi: 10.1242/jcs.005298

Journal of Cell Science 120, 3138-3146 (2007)
Published by The Company of Biologists 2007
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The distinct roles of Ras and Rac in PI 3-kinase-dependent protrusion during EGF-stimulated cell migration

Shu-Chin Yip1,2,*, Mirvat El-Sibai1,*, Salvatore J. Coniglio3, Ghassan Mouneimne2, Robert J. Eddy2, Beth E. Drees3, Paul O. Neilsen3, Sumanta Goswami4, Marc Symons5, John S. Condeelis2 and Jonathan M. Backer1,{ddagger}

1 Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
2 Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
3 Echelon Biosciences, Inc., Salt Lake City, UT, USA
4 Department of Biology, Yeshiva University, NY, USA
5 Center for Oncology and Cell Biology, Institute for Medical Research at North Shore-LIJ, Manhasset, NY, USA


Figure 1
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  		Fig. 1. EGF-stimulated PtdIns(3,4,5)P3 production at the leading edge of lamellipod is rapid and sustained. Quiescent MTLn3 cells were stimulated with 5 nM EGF and fixed at various times. (A) Selected images of EGF-stimulated cells stained with anti-PtdIns(3,4,5)P3 antibody. Bar, 10 µm for all panels. (B) Quantification of leading-edge PtdIns(3,4,5)P3 production following EGF stimulation. The average edge fluorescence (0-0.66 µm from the perimeter) was measured as described in the Materials and Methods, normalized for the edge fluorescence of unstimulated cells and plotted as a function of time. The data are the mean ± s.e.m. from four experiments. (C) Anti-phosphotyrosine immunoprecipitates from EGF-stimulated cells were assayed for PI 3-kinase activity. The data are the mean ± s.e.m. from four experiments.

 

Figure 2
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  		Fig. 2. The kinetics of EGF-stimulated Ras and Rac activation and translocation. (A,B) GST-CRIB (from hPAK) or GST-RBD (from Raf 1) were immobilized on glutathione-Sepharose beads and incubated with EGF-stimulated cell lysates as described. Glutathione-Sepharose pull-downs were separated by 14% SDS-PAGE gel and blotted with anti-Rac or anti-Ras antibody. (A, upper panel) A representative anti-Rac immunoblot of a GST-CRIB pull-down from EGF-stimulated cells. (Lower panel) A representative anti-Ras immunoblot of a GST-RBD pull-down from EGF-stimulated cells. Where indicated, cells were treated with 100 nM wortmannin prior to EGF stimulation. Total Ras and Rac levels did not change during 5 minutes of EGF stimulation (data not shown). (B) Ras (closed diamonds) and Rac (open circles) pull-down assays were quantified by densitometry. Data were expressed as percentage of maximum activity, and show the mean ± s.e.m. from nine experiments. (C) Representative fluorescent images of MTLn3 cells stimulated with EGF for various times and stained with anti-Rac antibodies. Bar, 10 µm. (D) Representative images of EGF-stimulated MTLn3 cells stained with GST-RBD followed by anti-GST antibody, to visualize endogenous activated Ras. Bar, 10 µm.

 

Figure 3
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  		Fig. 3. Inhibition of Ras but not Rac reduces PtdIns(3,4,5)P3 production at the leading edge of lamellipodia. (A,B) MTLn3 cells were transfected with control or Rac1 siRNA. (A) Representative images of control siRNA or Rac1 siRNA-treated cells stimulated with EGF for 3 minutes and stained with anti-PtdIns(3,4,5)P3 antibody are shown. (B) Quantification of leading-edge PtdIns(3,4,5)P3 in siRNA-treated cells. The data are mean ± s.e.m. from four experiments. Bar, 10 µm in all panels. (C,D) MTLn3 cells were transfected with control siRNA or pools of four siRNA duplexes specific for H-Ras, K-Ras or N-Ras. (C) Representative images of control siRNA or K-Ras siRNA-treated cells stimulated with EGF for 0, 1 or 3 minutes and stained with anti-PtdIns(3,4,5)P3 antibody are shown. (D) Quantification of leading-edge PtdIns(3,4,5)P3 in siRNA-treated cells. The data are mean ± s.e.m. from 40 cells per condition, and are representative of two independent experiments.

 

Figure 4
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  		Fig. 4. Specific inhibition of Ras, but not Rac, inhibits EGF-stimulated protrusion. (A) MTLn3 cells were transfected with control or Rac1 siRNA. Time-lapse images of EGF-stimulated cells were recorded (20 seconds per frame) with a 20x 0.4 NA objective, and surface areas were measured using NIH Image. The data show the mean ± s.e.m. from 41 cells per time point. (B) Data from A were normalized to the initial area of each cell. (C) MTLn3 cells were transfected with control siRNA or isoform-specific siRNA targeting H-Ras, K-Ras, N-Ras or both N-Ras and K-Ras. The surface area of control siRNA or Ras siRNA-treated cells was measured after various times of EGF stimulation. Cell areas were normalized to the initial area of each cell. Data show the mean ± s.e.m. from 9-14 cells per time point. (D) Pooled data from two experiments showing maximal EGF-stimulated protrusion in cells treated with control or anti-Ras siRNA. Data are the mean ± s.d.

 

Figure 5
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  		Fig. 5. Specific inhibition of Ras, but not Rac, inhibits EGF-stimulated Arp2/3 recruitment. MTLn3 cells were transfected with control (left panels), Rac1 (middle panels) or K-Ras (right panels) siRNA. Cells were stimulated with EGF for 0, 1 or 3 minutes, fixed and stained with anti-Arp2/3 antibodies.

 

Figure 6
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  		Fig. 6. Rac1 knockdown inhibits formation of focal adhesions. MTLn3 cells were transfected with control (luciferase, upper panels) or Rac1 (lower panels) siRNA. Cells were stimulated with EGF for 0, 1 or 3 minutes, fixed and stained with anti-paxillin antibodies.

 

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© The Company of Biologists Ltd 2007