spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 20 June 2006
doi: 10.1242/jcs.03015

Journal of Cell Science 119, 2921-2934 (2006)
Published by The Company of Biologists 2006
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Related articles in JCS
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Veracini, L.
Right arrow Articles by Benistant, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Veracini, L.
Right arrow Articles by Benistant, C.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Two distinct pools of Src family tyrosine kinases regulate PDGF-induced DNA synthesis and actin dorsal ruffles

Laurence Veracini*, Mélanie Franco*, Anthony Boureux, Valérie Simon, Serge Roche and Christine Benistant{ddagger}

CNRS FRE2593 CRBM, 1919 route de Mende, 34293 Montpellier CEDEX 05, France


Figure 1
View larger version (25K):

[in a new window]
  		Fig. 1. Membrane cholesterol and Cav-3DGV inhibit PDGF-induced DNA synthesis in fibroblasts. (A) CD and CO inhibit PDGF-induced DNA synthesis. Top panel: discontinuous stimulation for PDGF-induced DNA synthesis. Bottom panel: an example (left) and the statistical analysis (right) of the inhibitory effects of cholesterol depleting agents on PDGF-induced BrdU incorporation. Quiescent NIH 3T3 cells grown on coverslips were treated or not (NS) with CD (10 mM) or CO (0.5 U/ml) as indicated, rinsed and stimulated with PDGF (25 ng/ml) in the presence of BrdU. In some cases, cells have been incubated with CD in the presence of 25 µM soluble cholesterol before stimulation with PDGF (CD+Cholesterol). (B) Caveolin-1-GFP (Cav-1-GFP) and caveolin-3DGV (Cav-3DGV) inhibit mitogenesis induced by continuous PDGF stimulation. Top panel: continuous stimulation for PDGF-induced DNA synthesis. Bottom panel: an example (left) and the statistical analysis (right) of the inhibitory effect of indicated caveolin constructs on PDGF-induced BrdU incorporation. Cells transfected with the indicated constructs were made quiescent and stimulated for 18 hours with PDGF with or without soluble cholesterol (20 µM) as indicated and in the presence of BrdU. GFP was co-transfected with Cav-3DGV to visualise transfected cells. An example of BrdU incorporation obtained from Cav-3DGV transfected cells is indicated by arrows. (C) PDGF-induced DNA synthesis is reduced in cells grown in LPDS. NIH 3T3 cells were grown in 5% fetal calf serum (standard) or 5% LPDS in the presence or absence of soluble cholesterol (25 µM) as indicated for 30 hours and serum-starved for 24 hours before PDGF stimulation. An example (left) and the statistical analysis (right) of the inhibitory effects of chronically depleting cholesterol on PDGF-induced BrdU incorporation. Cells were fixed and BrdU incorporation was analysed by immunofluorescence as described in the Materials and Methods. The means and the s.d. from three to five independent experiments are shown.

 

Figure 2
View larger version (46K):

[in a new window]
  		Fig. 2. Membrane-cholesterol depletion does not affect PDGF-induced receptor, Ras and ERK activation. (A) CD does not affect PDGF receptor activation. NIH 3T3 cells were serum starved, treated with CD, rinsed and stimulated with PDGF for 10 minutes as indicated. PDGFRß was immunoprecipitated from indicated lysate with {alpha}PR4 followed by in vitro kinase assay (left panel). The position of [32P]PDGFRß is shown. Total cell lysates treated as indicated were directly subjected to western blotting with anti-phosphotyrosine antibody (4G10) (right panel). The location of molecular markers (Mr) is also shown. (B) CD does not affect PDGF-induced Ras (left panel) and ERK (right panel) activation. Lysates were either directly subjected to western blotting with anti-Ras (total Ras), anti-ERK (ERK) and anti-phospho ERK (pERKs) antibodies or incubated with Sepharose-bound GST-RalGDS binding domain and western blotting with anti-Ras antibodies (Ras-GTP) as indicated.

 

Figure 3
View larger version (27K):

[in a new window]
  		Fig. 3. Src mitogenic signalling is affected by membrane cholesterol depletion and Cav-3DGV expression. (A) CD inhibits PDGF-induced SFK activation. SFKs were immunoprecipitated from RIPA cell lysates, which solubilises caveolae, and subjected to an in vitro kinase assay using enolase as a substrate. In addition to conditions used in Fig. 2, kinase activity was also performed on cells treated with CD and left 2 hours for membrane cholesterol replenishment before PDGF stimulation (Recovery) or not. Top panel: an example of SFK activity (32P-Enolase); middle panel: quantification of SFK activity under conditions specified (mean ± s.d. from three independent experiments) and expressed as the ratio of non-stimulated and non-treated cells (control); bottom panel: levels of caveolin-1 and tubulin from RIPA and LB cell lysates. Protein levels were assessed by western blotting of total cell lysates using specific antibodies. (B) CD affects Src-specific tyrosine phosphorylation of Stat3. The level of immunoprecipitated Stat3 and pY705Stat3 from indicated cell lysates is shown. (C) Membrane cholesterol depletion inhibits PDGF-induced Myc induction. Quiescent cells treated or not with cholesterol-depleting agents in the presence or absence of soluble cholesterol were stimulated for 1 hour with PDGF and total RNA was isolated. Northern blot analysis was performed from indicated RNA using a probe specific for Myc or 26S genes as a control of total RNA level (left panel). Myc mRNA level was quantified by real-time quantitative PCR (right panel). The ratio between the mRNA level and that obtained from quiescent non-treated cells was calculated (Myc response). The mean and the s.d. are shown from three to five independent experiments. (D) SFKs are required for early mitogenic signalling. Quiescent cells were incubated with SU6656 (1 µM) for 1 hour before PDGF stimulation, and subjected to a discontinuous stimulation protocol depicted in top panels. Bottom panels: BrdU incorporation (left) was analysed as described in Fig. 1 and SFK activity (right) was analysed by western blotting of the immunoprecipitated SFK with pY416Src antibody specific to the active Src kinases. The ratio between active SFK and SFK levels is shown and is representative of two independent experiments. (E) Expression of Myc overcomes mitogenic inhibition induced by CD. Cells were transfected with a trace amount of GFP construct in the presence of empty vector (mock), and the indicated constructs. Cells were serum starved, treated and stimulated in the presence of BrdU as described in top panel. (F) Expression of Myc overcomes mitogenic inhibition induced by dominant-negative Cav-3DGV. Cells were transfected with trace amounts of GFP construct in the presence of the indicated constructs. Cells were serum starved, and stimulated in the presence of BrdU as described in top panel. BrdU incorporation was analysed and calculated as described in Fig. 1. The mean and the s.d. of three to five independent experiments are shown.

 

Figure 4
View larger version (38K):

[in a new window]
  		Fig. 4. PDGF stimulates two pools of SFK activities with distinct sensitivity to membrane cholesterol. (A) Purification of CEF. 1% Triton X-100 cell lysates of fibroblasts treated as indicated were subjected to a Dounce homogenisation followed by a sucrose gradient fractionation. Fractions were directly subjected to western blotting with an anti-phosphotyrosine (4G10) and caveolin-specific antibody as indicated. The presence of respective protein as well as the fraction number is shown. (B) PDGF-induced SFK activities in caveolae-enriched (CEF) and non-caveolae (NCF) fractions. CEF (2-4) and NCF (7-9) were pooled and treated as in the Materials and Methods. SFK activities and their association with caveolin were measured by western blotting of immunoprecipitated kinases with pY416Src and caveolin antibody respectively. (C) CO regulates SFK-PDGFR association in CEF. In vitro kinase assay was performed with the immunoprecipitated SFK from CEF. The presence of PDGFR was revealed by re-immunoprecipitation of the labelled proteins using specific antibody ({alpha}PR4) as indicated (2nd ip). Antibodies used for immunoprecipitation (ip), cell treatments, SFK, pY416 SFK, heavy chains immunoglobulin (Hc), [32P]SFK and [32 P]PDGFR are indicated. (D) Cholesterol content in CEF. CEF were isolated as described in Materials and Methods from NIH 3T3 cells treated or grown as indicated and from HEK 293 cells expressing Cav3DGV as indicated. Is shown the cholesterol content in CEF relative to the non treated cells (% control). (E) Levels of SFK in CEF and NCF fractions. SFK were immunoprecipitated from two-thirds of CEF and one-quarter of NCF and subjected to western blotting with cst1 antibody. Caveolin-1 and ß 1 integrin levels were detected as specific markers of CEF and NFC respectively and assessed by western blotting of total protein fractions. A quantification of SFK levels is indicated.

 

Figure 5
View larger version (62K):

[in a new window]
  		Fig. 5. Membrane cholesterol and caveolins do not regulate SFK signalling linked to PDGF-induced dorsal ruffle formation. (A) SFKs are required for PDGF-induced dorsal ruffle formation. Top panel: typical effect obtained with SFK inhibition on PDGF-induced dorsal ruffle formation. Bottom panel: statistical analysis of PDGF response (% of cells with dorsal ruffle) in cells expressing indicated construct or treated with the SFK (SU6656 5 µM) or the MEK (U0126 10 µM) inhibitor as shown. (B) Dorsal ruffle formation is not affected by cholesterol depletion. Top panel: typical effect obtained with membrane cholesterol depletion on PDGF-induced dorsal ruffle formation. Bottom panel: statistical analysis of PDGF response (% of cells with dorsal ruffle) of cells expressing indicated caveolin constructs, treated with indicated cholesterol-depleting agents, or grown in LPDS as shown. NIH 3T3 grown on coverslips were transfected or not with indicated construct, serum-starved, treated with SU6656 (5 µM) for 30 minutes and stimulated with PDGF (15 ng/ml) for 10 minutes. Cells were fixed and processed for actin staining using Rhodamine-phalloidin and ectopic protein expression. Cav-3DGV was co-transfected with trace amounts of GFP construct to visualise transfected cells. Cells expressing SrcK– (A) or Cav-3DGV (B) constructs are indicated by arrows. The mean percentage of cells that formed dorsal ruffles and the s.d. from three to five independent experiments are shown.

 

Figure 6
View larger version (52K):

[in a new window]
  		Fig. 6. PDGF-induced dorsal ruffle formation and SFK activation outside caveolae require S1P signalling. (A) S1P signalling regulates PDGF-induced dorsal ruffle formation. Left panel: an illustration of a typical effect obtained with S1P signalling inhibition on PDGF-induced dorsal ruffle formation. Right panel: PDGF response (% of cells with dorsal ruffles) of cells treated with indicated drugs or expressing S1K– as shown. (B) S1P signalling does not affect PDGF-induced BrdU incorporation. Quiescent cells expressing S1K– or treated with indicated drugs were stimulated with PDGF in the presence of BrdU. BrdU incorporation was analysed and calculated as described in Fig. 1. The mean and the s.d. of three to five independent experiments are shown. (C) S1P signalling regulates PDGF-induced SFK activation in NCF. SFKs were immunoprecipitated from NCF as depicted in Fig. 4 with cells treated or not (control) with indicated agents and stimulated or not with PDGF. Levels of SFKs were determined and the in vitro kinase assay performed with denatured enolase as an exogeneous substrate (32P-Enolase). (D) S1P signalling does not regulate SFK-PDGFR association in CEF. SFKs were immunoprecipitated from CEF obtained from cells used in panel C and subjected to an in vitro kinase assay. Autophosphorylation of Src kinases (32P-SFK) as well as the presence of the associated PDGFR (32P-PDGFR) are shown. Cell treatment was as follows: DMS (10 µM) for 30 minutes, U73343 and U73122 (1 µM) for 30 minutes and PTx (500 ng/ml) for 4 hours.

 

Figure 7
View larger version (59K):

[in a new window]
  		Fig. 7. Spatial regulation of Src activity by membrane cholesterol and S1P signalling in PDGF-stimulated cells. NIH 3T3 were transfected with an avian Src construct, synchronised in G0 by serum starvation, treated or not with indicated agent and stimulated or not (NS) for 10 minutes with PDGF as shown. Src was detected with EC10 antibody (red), active Src with anti-pY416 antibody (green) and actin with fluorescent Phalloidin (blue). Left panels: merged imaged of Src and active Src fluorescence. Middle panels: actin. Right panels: merged images of Src, active Src and actin. Pools of active Src at the plasma membrane and associated actinic dorsal ruffles are indicated by arrows.

 

Figure 8
View larger version (21K):

[in a new window]
  		Fig. 8. Abl is an effector of the S1P signalling for PDGF-induced dorsal ruffle formation. (A) Abl, but not other Src mitogenic substrates, is required for PDGF-induced dorsal ruffle formation. PDGF response (percentage of cells with dorsal ruffles) of cells expressing indicated constructs. (B) PDGF-induced Abl but not Stat3 activation requires an S1P signalling pathway. Stat3 was immunoprecipitated from cells treated with the indicated drugs and stimulated or not with PDGF as shown. Abl was detected in the same way as Stat3 and then assayed for in vitro kinase activity. The levels of Stat3 and pY705Stat3 were assessed by western blotting. [32P]Gst-Crk was also detected.

 

Figure 9
View larger version (16K):

[in a new window]
  		Fig. 9. A model illustrating how PDGF uses two distinct pools of SFKs for DNA synthesis and dorsal ruffle formation. PDGF activates SFKs in caveolae (grey) allowing phosphorylation of mitogenic substrates for Myc induction and DNA synthesis. PDGF recruits sphingosine kinase (SK1) to the membrane through a PLC{gamma}-dependent pathway for S1P formation. S1P further activates SFK outside caveolae through an EDG receptor coupled to a heterotrimeric Gi protein allowing Abl activation for dorsal ruffle formation. Additional Src substrates (others) may be expected for induction of this morphological change. S, sphingosine.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2006