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First published online 16 November 2004
doi: 10.1242/jcs.01526

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ARAP3 is transiently tyrosine phosphorylated in cells attaching to fibronectin and inhibits cell spreading in a RhoGAP-dependent manner

¹ Department of Surgery, University of Melbourne, Level 5 Clinical Sciences Building, Royal Melbourne Hospital, VIC 3050, Australia
² Laboratory of Cellular Oncology, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA
³ Cooperative Research Centre for Cellular Growth Factors, Post Office, Royal Melbourne Hospital, VIC 3050, Australia
⁴ Ludwig Institute for Cancer Research, PO Box 2008, Royal Melbourne Hospital, VIC 3050, Australia
⁵ Department of Surgery, Austin Campus, Austin and Repatriation Medical Centre, VIC 3084, Australia

View larger version (26K): [in a new window]   Fig. 1. Mouse ARAP3 domain organisation and generation of ARAP3 and ARAP3SAM. (A) Diagram of mouse ARAP3 showing sterile alpha motif (SAM), pleckstrin homology (PH), ADP-ribosylation factor GTPase activating protein (ArfGAP), RhoGAP and Ras association (RA) domains (boxes) and tyrosine phosphorylation sites, Y1399 and Y1404 (YY). The percent amino acid sequence identity with human ARAP3 is indicated. (B) Top: diagram of the ARAP3 gene 5'-end showing alternative splicing (dotted line) via a cryptic splice acceptor site (CSA). Splicing is predicted to generate ARAP3 transcripts with different translation start sites (Kozak ATG) encoding ARAP3 isoforms with the N-terminal SAM domain (see A) or lacking a SAM domain (ARAP3SAM). Coding and non-coding exons (grey and white boxes) and introns (horizontal line) are indicated. Bottom: plasmids containing cDNAs corresponding to the differentially spliced ARAP3 transcripts were transfected in 293T cells. Whole cell lysate (WCL) from transfected cells and from mouse bone marrow-derived mast cells (BMMC) were analysed by SDS-PAGE and western blotting (WB) with an anti-ARAP3 antibody.

View larger version (27K): [in a new window]   Fig. 2. Mouse ARAP3 exhibits Arf GAP and Rho GAP activity in vitro. (A) FLAG-tagged ARAP3 was transiently expressed in 293T cells then immunoprecipitated with a FLAG monoclonal antibody. Arf GAP activity was assayed using bacterially expressed Arf5 pre-loaded with [-32P]GTP in the presence of 360 µM phosphatidic acid (PA), 45 µM PtdIns(4,5)P2 (PIP2) and/or 1 µM PtdIns(3,4,5)P3 (PIP3). GAP activity is expressed as the rate of GTP hydrolysis (per minute). (B) FLAG-tagged forms of ARAP3, AGAP1 and ACAP1 were expressed in 293T cells and assayed for Arf GAP activity towards bacterially expressed Arf1, Arf5 or Arf6 as described in A. Reaction buffers included 1 µM PtdIns(3,4,5)P3 (ARAP3) or 45 µM PtdIns(4,5)P2 plus 360 µM PA (AGAP1 and ACAP1). (C) A FLAG-tagged truncated ARAP3, (PH3-PH5), which includes the Rho GAP domain and flanking PH domains only, was expressed in 293T cells and immunopurified with a FLAG antibody. His-tagged RhoA, Rac1 or Cdc42 substrates were pre-loaded with [-32P]GTP then incubated for the indicated times in the presence (?) or absence () of ARAP3 PH3-PH5. The results are expressed as the percentage [-32P]GTP that remains bound to the indicated Rho-family GTPases and therefore indicates the rate of GTP hydrolysis. The data are representative of two experiments.

View larger version (37K): [in a new window]   Fig. 3. Src-kinases phosphorylate ARAP3 at sites including Y1399 and/or Y1404 in cells and can phosphorylate ARAP3 in vitro. (A) ARAP3 (left panels) or FLAG-ARAP3 (right panels) were expressed with Lyn, Src or alone (none) in 293T cells. Cell lysates were immunoprecipitated (IP) with an ARAP3 antibody and analysed by SDS-PAGE and western blotting (WB) with phosphotyrosine (pY), ARAP3, Src or Lyn antibodies. (B) FLAG-ARAP3 (ARAP3 WT) and FLAG-ARAP3 with tyrosines 1399 and 1404 substituted for phenylalanines (Y1399/1404F) were expressed (+) with Lyn (L) or Src (S) in 293T cells. Cell lysates were immunoprecipitated (IP) with a FLAG antibody and analysed as in A with anti-pY and anti-ARAP3 antibodies. (C) FLAG-ARAP3 expressed in 293T cells and purified with a FLAG antibody was incubated with the indicated concentrations of purified Src kinase domain (upper panels) or with increasing amounts of immunopurified baculovirally expressed Lyn (lower panels) in in vitro kinase buffer containing ATP. Samples were analysed by SDS-PAGE and immunoblotting with the indicated antibodies.

View larger version (35K): [in a new window]   Fig. 4. ARAP3 is tyrosine phosphorylated during attachment of HEK293 cells to the ECM substrate fibronectin. (A) Diagram of ARAP3 proteins introduced into tet-inducible Flp-InTM T-RExTM HEK293 cell lines. Crosses designate domains with mutated `catalytic arginine' residues required for GAP activity. (B) Stable Flp-InTM T-RExTM HEK293 cell lines (pools of >20 isogenic clones) harbouring stably integrated cDNAs for the indicated ARAP3 proteins were treated (+) or not treated (-) with 2 µg/ml doxycycline (DOX) for 16 hours. Cell lysates (20 µg protein) were analysed by SDS-PAGE and western blotting (WB) with antibodies against ARAP3, p120RasGAP and p85 (the PI 3-kinase regulatory subunit). (C) HEK293 Flp-InTM cell lines treated for 16 hours with doxycycline to induce expression of the indicated ARAP3 proteins were trypsinised, washed and resuspended in serum-free medium for 1 hour. Suspension cells (0 minutes) or cells plated onto fibronectin (FN)-coated plates for the indicated times were lysed and immunoprecipitated with anti-FLAG then subjected to SDS-PAGE and western analysis with anti-phosphotyrosine (pY) then anti-FLAG antibodies (right panels). (D) The indicated HEK293 Flp-InTM cell lines were treated as in C. Suspension cells were pre-incubated with 15 µM LY294002 (+) or DMSO (-) for 20 minutes and the cells plated onto fibronectin for 30 minutes. FLAG-ARAP3 proteins were analysed as described in C. (E) Serum-starved 293T cells expressing ARAP3 were harvested and held in suspension for 1 hour then incubated for 30 minutes with the indicated concentrations of the Src inhibitors PP2 and SU6656 and the negative control compound PP3. Cells were plated onto fibronectin-coated plastic (10 µg/ml) for 30 minutes and lysed. Samples were analysed by western blotting with phosphotyrosine and ARAP3 antibodies. (F) 293T cells were transfected with plasmids encoding ARAP3 (2 µg) and the indicated amounts of a dominant interfering Src mutant (Src KD). Cells were plated on fibronectin for 30 minutes and samples of the cell lysate analysed as described in E.

View larger version (61K): [in a new window]   Fig. 7. ARAP3 inhibits spreading of HEK293 cells on fibronectin in a Rho GAP-dependent manner. (A) The rate of spreading of serum starved, doxycycline-treated (2 µg/ml for 16 hours) Flp-InTM 293 control cells (filled boxes) versus those expressing ARAP3 (filled triangles), R93 8L (filled diamonds), R527K (open diamonds), R527K/R938L (open boxes) and Y1399/1404F (open triangles) was evaluated by determining the percentage of cells elaborating lamellipodia-like protrusion(s) at the indicated times (minutes). >200 cells were scored at each time point. Data are the means ± s.d. of duplicate determinations from two experiments. (B) The indicated Flp-InTM 293 inducible cell lines were treated with doxycycline (2 µg/ml) for 16 hours, trypsinised, washed and suspensed in serum-free medium for 1 hour. Cells were plated on coverslips pre-treated with 10 µg/ml fibronectin and cells fixed and stained with crystal violet at the indicated times. Cell morphology was visualized by differential interference contrast (DIC) light microscopy. Representative images from two experiments are shown.

View larger version (92K): [in a new window]   Fig. 5. Inducible expression of ARAP3 in HEK293 cells affects cell morphology in a Rho GAP-dependent manner. The stable Flp-InTM T-RExTM cell lines indicated were plated onto fibronectin-coated (10 µg/ml) plasticware in the presence (+ Dox) or absence of 2 µg/ml doxycycline (-Dox) for 16 hours. Digital phase contrast microscopic images representative of more than five experiments are shown.

View larger version (24K): [in a new window]   Fig. 6. ARAP3-mediated membrane process formation is Rho GAP dependent, blocked by activated RhoA expression, and enhanced by mutation of the tyrosine phosphorylation sites Y1399 and Y1404. (A) The indicated Flp-InTM T-RexTM 293 cell lines were grown overnight on laminin- or fibronectin-coated dishes in the presence (+) or absence (-) of 2 µg/ml doxycycline (Dox). Multiple random fields of cells were imaged as in Fig. 5 and the proportion of cells with process(es) >1 cell diameter was scored. Between 200 and 350 cells were scored per sample. Data are means ± s.e.m. from duplicate determinations in two independent experiments. (B) Cell lines treated with doxycycline overnight to induce ARAP3 proteins were transfected with plasmids encoding green fluorescent protein (GFP) plus a 10-fold excess of RhoA V14 or empty vector (Vector). Live transfected cells expressing GFP were visualised 24 hours post-transfection by confocal fluorescence microscopy using a water immersion 60x objective.

View larger version (72K): [in a new window]   Fig. 9. Effect of ARAP3 overexpression on endogenous RhoA, Rac1 and Cdc42. Levels of active GTP-bound RhoA, Rac1 and Cdc42 in the indicated Flp-InTM 293 inducible cell lines, treated (+) or not treated (-) with 2 µg/ml doxycycline (DX) were determined by incubating cell lysate with GST fusion proteins containing the Rhotekin RBD (to detect RhoA GTP) or the PAK1 RBD (to detect Rac1 GTP and Cdc42 GTP). Affinity precipitates and samples of whole cell lysate (2% of that used in A.P.) were analysed by SDS-PAGE and western blotting (WB) with the indicated antibodies. The results shown are representative of two experiments.

View larger version (19K): [in a new window]   Fig. 8. ARAP3 does not affect cell adhesion but impairs cell migration. The indicated Flp-InTM T-RexTM HEK293 cell lines were treated with 2 µg/ml doxycycline for 16 hours to induce ARAP3 proteins, harvested and maintained in suspension in serum-free medium for 1 hour, then plated in quadruplicate in microwells (5000 cells/well) that were pre-coated with 2.5, 5, 10 or 20 µg/ml fibronectin. Cells were allowed to adhere for 35 minutes, wells were washed four times, and the relative numbers of adhering cells determined using an MTT assay (Materials and Methods). Data are the mean percentage ± s.d. of maximal levels of adhesion (20 µg/ml fibronectin) in two independent experiments. (B) Equivalent numbers of ARAP3 and R938L Flp-InTM 293 cell lines that had been treated (+) or not treated (-) with doxycycline (Dox) and serum-starved for 16 hours were seeded (5x104 cells in triplicate) on Transwell 8 µ pore filters (coated with 10 µg/ml fibronectin). Filters were suspended in serum-free medium and incubated at 37°C for 8 hours. Cells were removed from the upper side of filters and the relative numbers of cells migrating to the underside was determined by fixing and staining the cells with 1% crystal violet, extracting the dye and determining its absorbance at 590 nm. Data are means ± s.d. and are representative of three independent experiments. Statistical significance was assessed using an unpaired t-test; *P<0.05.

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