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First published online 6 May 2003
doi: 10.1242/jcs.00466

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The SH4-Unique-SH3-SH2 domains dictate specificity in signaling that differentiate c-Yes from c-Src

Justin M. Summy^*,,1, Yong Qian^*,2, Bing-Hua Jiang¹, Anne Guappone-Koay¹, Amanda Gatesman¹, Xianglin Shi² and Daniel C. Flynn^1,

¹ Department of Microbiology, Immunology, and Cell Biology, and Mary Babb Randolph Cancer Center, West Virginia University School of Medicine, PO Box 9300, Morgantown, WV 26506, USA
² National Institute for Occupational Safety and Health, Pathology and Physiology Research Branch, Health Effects Laboratory Division, Morgantown, WV 26505, USA

View larger version (24K): [in a new window]   Fig. 1. Src527F/c-Yes chimeric constructs. Src527F/c-Yes chimeras are depicted subdivided into the following functional domains: SH4 domain; Unique domain; SH3 domain; SH2 domain; protein tyrosine kinase (PTK) domain; and regulatory sequence (Reg). Gray boxes represent domains from c-Yes; white boxes represent domains from Src527F. The generation of the Y3527F, Y2527F, Y32527F, Y4U32527F, Y4U527F, Y4527F and YU527F chimeric constructs has been described previously (Summy et al., 2000). All proteins were expressed in CEF via the Rous Sarcoma Virus (RSV) vector.

View larger version (55K): [in a new window]   Fig. 2. Effects of Src527F/c-Yes chimeras on cellular phosphotyrosine. cell morphology, and the actin cytoskeleton. (A) 30 µg of day 12 RIPA lysates from mock-transfected CEF or cells expressing Src527F, LA29, or the chimeric constructs were separated by 8% SDS-PAGE. Lysates were transferred to PVDF membrane, and probed with a rabbit anti-phosphotyrosine antibody Molecular weight markers are shown on the left side of the figure. Protein bands of note are highlighted by their Mr, on the right side of the figure. (B) 50 µg of cell lysate (as used in Fig. 2A) was resolved by 8% SDS-PAGE, followed by western transfer and probed with the anti-pp85 cortactin antibody. (C) By using the same lysates as in Fig. 2A, 30 µg of lysate was resolved by 8% SDS-PAGE and western blot analysis performed with rabbit anti-src antibodies to quantify the steady state levels of Src and the chimeric constructs (top panel), or with anti-phosphoY416 to detect the activation state of the Src or chimeric constructs.

View larger version (102K): [in a new window]   Fig. 3. Expression of Src527F/c-Yes N-terminal chimeras in CEF and their effects on cell morphology and the actin cytoskeleton. (A) The SH4-Unique domains of c-Yes prevent Src527F from affecting cell morphology. Confluent CEF that were uninfected, or expressing Y4U32527F, Y32527F Y4U527F, Y4527F or YU527F were photographed at day 12 post-transfection in 100 mm tissue culture plates (Falcon). Cells were photographed at 40x total magnification using a Plan 2 filter. (B) The SH4-Unique domains of c-Yes prevent Src527F from affecting actin filament integrity. CEF expressing Y4U32527F, Y32527F Y4U527F, Y4527F or YU527F were fixed at 50% confluence on coverslips in 3.7% formaldehyde, permeablized in 0.4% Triton X-100, and stained with rhodamine-phalloidin (2 µg/ml). Cells were visualized via Zeiss LSM 510 confocal microscopy (63x objective). Bars, 10 µm.

View larger version (28K): [in a new window]   Fig. 4. Y4U32527F is associated with the Triton X-100 insoluble cytoskeletal fraction. Mock-transfected CEF or cells expressing c-Src, Src527F or Y4U32527F were grown to confluence in 100 mm dishes. Cells were incubated in 1 ml of CSK buffer for 1, 4 or 10 minutes. The Triton-soluble fractions were collected, and the Triton-insoluble material at each time-point was solublized in RIPA buffer. 50 µg of cell lysates from each fraction were separated by 8% SDS-PAGE, transferred to PVDF membrane, blocked with 5% nonfat milk/TBS-T, and probed with rabbit anti-Src. Results are shown for mock-transfected CEF, c-Src, Src527F and Y4U32527F. C, CSK buffer; R, RIPA buffer. Cytoskeletal-associated proteins are defined in the R fraction.

View larger version (29K): [in a new window]   Fig. 5. Activation of the MAP kinase pathway by Src527F and Src527F/c-Yes chimeras. (A) 500 µg of RIPA lysates from mock-transfected CEF or cells expressing Src527F, Y4U32527F or Y4U527F were immunoprecipitated with an anti-Shc antibody and separated by 10% SDS PAGE. Immunoprecipitated proteins were transferred to PVDF membrane, blocked in 5% nonfat milk/TBS-T and probed with an anti-Grb2 antibody (top panel). The blots were stripped and re-probed with the anti-Shc antibody, immunoreactive against the 46 and 52 kDa isoforms of Shc (bottom panel). (B) 50 µg of RIPA lysates from mock-transfected CEF or cells expressing Src527F, Y4U32527F, or Y4U527F were resolved by 8% SDS-PAGE, transferred to PVDF membrane, blocked in 5% nonfat milk/TBS-T and probed with an anti-phospho-Raf antibody (top panel). Western blot analysis was also performed with an anti-c-Raf antibody to determine relative protein levels (bottom panel). (C) 50 µg of RIPA lysates from mock-transfected CEF or cells expressing Src527F, Y4U32527F or Y4U527F were processed for western blot analysis as described above. Membranes were probed with an anti-phospho-Erk antibody (top panel) or an anti-p42/44 MAPK antibody to determine relative protein levels (bottom panel). (A-C) Lane 1, CEF; lane 2, Src527F; lane 3, Y4U32527F; lane 4, Y4U527F.

View larger version (35K): [in a new window]   Fig. 6. Differential signaling to the PI3K/Akt and RhoA pathway. (A) Akt phosphorylation. 50 µg of RIPA lysates from mock-transfected CEF or cells expressing Src527F or the chimeric constructs were resolved by 8% SDS-PAGE, transferred to PVDF membrane, blocked with 5% nonfat milk/TBS-T, and probed with an anti-phospho-Akt antibody (top panel) or an anti-Akt antibody to determine relative protein levels (bottom panel). (B) PI3K assay. 400 µg of lysates from mock-transfected CEF or cells expressing Src527F or Y4U32527F were immunoprecipitated with an anti-PI3K antibody and subjected to PI3K assay as described in Materials and Methods. Kinase assay products were resolved by thin layer chromatography and visualized by Phosphorimager analysis (top panel). Anti-PI3K immunoprecipitates were also resolved by 8% SDS-PAGE, transferred to PVDF membrane, blocked in 5% nonfat milk/TBS-T, and probed with an anti-PI3K p85 antibody to verify that there were equal amounts of PI3K in the lysates (bottom panel). (C) RhoA-GTP assay. CEF cells expressing the chimeric constructs or Src527F were lysed and 500 µg of cell lysate processed for affinity absorption with GST-Crib, expressing the Crib domain of rhotekin.

View larger version (70K): [in a new window]   Fig. 7. Y4U32527F is unable to induce motility or invasion of CEF cells. (A) Equal numbers of mock transfected CEF cells, or CEF cells expressing Src527F, Y32527F, Y4U527F, Y4U32527F subjected to (A) a transwell migration assay where cells migrated across the transwell and were visualized by staining. (B) The transwell migration assay was repeated and the total number of cells that migrated was isolated and quantified spectrophotometrically, to determine the relative numbers of cells that migrated. The experiment was done in triplicate and error bars indicate standard deviation. (C) A Matrigel invasion assay was also performed under the same conditions with the same cells, where the number of cells capable of invading through the Matrigel were counted. The experiment was carried out twice.

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