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First published online 10 July 2007
doi: 10.1242/jcs.003657

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The membrane targeting and spatial activation of Src, Yes and Fyn is influenced by palmitoylation and distinct RhoB/RhoD endosome requirements

The Beatson Institute for Cancer Research, Cancer Research UK Beatson Laboratories, Garscube Estate, Switchback Road, Bearsden, Glasgow, G61 1BD, UK

View larger version (46K): [in this window] [in a new window]   Fig. 1. Src, Fyn and Yes are distinctly spatially regulated. (A) SYF–/– cells expressing Src-WT (wild-type Src), Yes-WT or Fyn-WT were maintained in serum-free medium (upper panels) or stimulated with PDGF (lower panels). Localization of active SFK was detected using an anti-P-Y416-SFK antibody (Texas Red secondary) and total protein using an anti-Src, anti-Yes or anti-Fyn antibody (FITC secondary). Solid arrows indicate SFK at the cell periphery whereas broken arrows indicate SFKs at the perinuclear region of the cell. Bars, 25 µm. (B) Western blot analysis of SYF–/– cells expressing Src-WT (left panels), Yes-WT (middle panels) or Fyn-WT (right panels) in serum-free medium (–) or stimulated with PDGF. Activation was detected using an anti-P-Y416-SFK antibody (lower panels). Quantification shown in Table S1 in supplementary material.

View larger version (41K): [in this window] [in a new window]   Fig. 2. Activation of all three SFKs requires an intact actin cytoskeleton. (A) SYF–/– cells expressing Src-WT, Yes-WT or Fyn-WT were treated with cytochalasin D (0.3 µg/ml) for 1 hour prior to PDGF stimulation. Localization of active SFK was detected with an anti-P-Y416-SFK antibody (Cy5 secondary), total protein with an anti-Src, anti-Yes or anti-Fyn antibody (FITC secondary) and actin with TRITC-phalloidin. Bars, 25 µm. (B) Lysates from SYF–/– cells expressing Src-WT, Yes-WT or Fyn-WT maintained in suspension (Sus) or plated on fibronectin (FN) for 1 hour (10 µg/ml) were immunoblotted using anti-Src, anti-Yes, anti-Fyn (upper panels) and anti-P-Y416-SFK antibodies (lower panels). Quantification shown in Table S1 in supplementary material.

View larger version (66K): [in this window] [in a new window]   Fig. 3. RhoB is involved in the intracellular targeting of Src but not of Yes and Fyn. (A) The colocalization of Myc-RhoB (upper panels) and Myc-RhoD (lower panels) with Src-WT, Yes-WT or Fyn-WT was studied in SYF–/– cells stimulated with PDGF. Solid arrows indicate colocalization. (B) SYF–/– cells expressing Fyn-WT-GFP and Myc-RhoB or Myc-RhoD were stimulated with PDGF and stained for activated SFK. (C) RhoB+/+ (upper panels) or RhoB–/– (lower panels) cells expressing Src-WT, Yes-WT or Fyn-WT were plated on fibronectin for 1 hour and stained for activated SFK. Solid arrows indicate localization of active protein at the cell periphery whereas broken arrows indicate inactive SFK retained in the perinuclear region. Localization of active SFK was detected using an anti-P-Y416-SFK antibody (Texas Red or Cy5 secondary), total protein using an anti-Src, anti-Yes or anti-Fyn antibody (FITC secondary) and Myc-Rho with an anti-Myc antibody (Texas Red secondary). Bars, 25 µm. Quantification shown in the Tables S1 and S2 in supplementary material.

View larger version (64K): [in this window] [in a new window]   Fig. 4. Palmitoylation contributes to RhoB-dependence of intracellular targeting. (A) SYF–/– cells expressing Src-GFP S3C/S6C, Yes C3S or Fyn-GFP C3S/C6S were maintained in serum-free medium (upper panels) or stimulated with PDGF (lower panels). Solid arrows indicate localization of active SFK at cell periphery whereas broken arrows indicate inactive protein at the perinuclear region. (B) SYF–/– cells expressing Src-GFP S3C/S6C or Fyn-GFP C3S/C6S with Myc-RhoB or Myc-RhoD were stimulated with PDGF. Solid arrows in higher magnification images indicate colocalization between active SFK and Myc-Rho. (C) RhoB+/+ (upper panels) or RhoB–/– cells (lower panels) expressing Src-GFP S3C/S6C, Yes C3S or Fyn-GFP C3S/C6S were plated on fibronectin for 1 hour. Solid arrows indicate active SFK at the cell periphery and broken arrows inactive SFK at the perinuclear region. Localization of active SFK was detected using an anti-P-Y416-SFK antibody (Texas Red or Cy5 secondary), total protein using an anti-Yes antibody (FITC secondary) and Myc-Rho was visualized using an anti-Myc antibody (Texas Red secondary). Bars, 25 µm. Quantification shown in the Tables S1 and S2 in supplementary material.

View larger version (31K): [in this window] [in a new window]   Fig. 5. Knockdown of RhoD impairs translocation of Fyn-GFP and Src-GFP palmitoylation mutant. cDNA from SYF–/– cells expressing (A) Fyn-WT-GFP and an siRNA control or Fyn-WT-GFP and RhoD siRNA or (B) Src-GFP S3C/S6C and an siRNA control or Src-GFP S3C/S6C and RhoD siRNA or (C) Src-WT-GFP and an siRNA control or Src-WT-GFP and RhoD siRNA was reverse transcribed (left panels). Cells from the same experiment were plated for 6 hours in serum-containing medium then stained for anti-P-Y416-SFK (Texas Red secondary). Solid arrows indicate SFK at the cell periphery whereas broken arrows show SFK retained in the perinuclear region. Bars, 25 µm.

View larger version (52K): [in this window] [in a new window]   Fig. 6. FTI inhibitor disrupts RhoB-, but not RhoD-, dependent trafficking of SFKs. SYF–/– cells expressing Src-WT-GFP, Fyn-WT-GFP, Src-GFP S3C/S6C or Fyn-GFP C3S/C6S were treated with 10 µM L-744832 for 2 hours prior to stimulation with PDGF. Active SFK was detected using anti-P-Y416-SFK (Texas Red secondary). Solid arrows indicate SFK at cell periphery whereas broken arrows indicate SFK retained in perinuclear region. Bars, 25 µm. Quantification shown in Table S1 in supplementary material.