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First published online 18 March 2008
doi: 10.1242/jcs.018713

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Transforming signals resulting from sustained activation of the PDGFβ receptor in mortal human fibroblasts

Center for Immunology and Microbial Disease, Albany Medical College, MC-151, 47 New Scotland Avenue, Albany, NY 12208, USA

View larger version (24K): [in this window] [in a new window]   Fig. 1. The effect of the C39S and W32S E5 mutants in HDFs. (A) The amino acid sequence of the BPV E5 protein showing the positions of the amino acid substitutions present in the W32S and C39S mutants. Boxed region indicates the predicted transmembrane (TM) domain. (B) Extracts of HDFs stably expressing wild-type (wt) E5, W32S, C39S or no viral oncogene (control) were immunoprecipitated (IP) with an anti-E5 or anti-PDGFβR (PR) antiserum. Immunoprecipitates then were subjected to immunoblotting for PDGFβR (PR), E5 or phosphotyrosine (PY) as indicated. Mature (m) and precursor (p) forms of the PDGFβR (PR) are indicated by the arrows on the left. (C) HDFs expressing wild-type (wt) E5, the W32S or C39S mutant, or no viral oncogene (control) were plated at 1.6x105 cells per 60 mm dish and counted in triplicate at various times thereafter. The mean number of cells with the standard error is plotted. (D,E) A focus-forming assay was performed by infecting HDFs with recombinant retroviruses expressing wild-type (wt) E5, C39S, W32S or no viral oncogene (control) and then maintaining cells at confluence as described in the Materials and Methods. In D, Crystal Violet-stained monolayers are shown for visualization of foci. In E, the number of foci induced by E5, C39S and W32S were counted, averaged, corrected for virus titer and expressed as the mean percent (with standard error) relative to the number of foci induced by wild-type E5.

View larger version (28K): [in this window] [in a new window]   Fig. 2. Recruitment of substrates to the PDGFβR in HDFs expressing the different E5 proteins. (A) HDFs stably expressing the indicated E5 protein, v-Sis or no viral oncogene (control) were lysed after growing to 85-95% confluence, and cell extracts were immunoprecipitated (IP) with a PDGFβR antiserum. PDGFβR (PR) IPs were subjected to immunoblotting for p85-PI3K (PI3-K), SHP2, PLC-, p120RasGAP or Grb2. The uppermost portion of each blot was probed for PDGFβR to determine the amount of total receptor that was immunoprecipitated. For each substrate, a representative blot from several different experiments is shown. (B) The relative band intensities of the indicated substrates that co-immunoprecipitated with the PDGFβR as depicted in A were quantitated using the Image J program and normalized for total receptor amounts. Each normalized value was converted to a percentage relative to the normalized value for the wild-type E5-expressing HDFs, and the mean percentage from several different experiments with standard error is shown. This illustrates the amount of substrate bound to the PDGFβR relative to that in the wild-type E5-expressing HDFs.

View larger version (51K): [in this window] [in a new window]   Fig. 3. Status of downstream signaling intermediates in HDFs expressing the different E5 proteins. (A, upper panel of B) Whole-cell extracts of proliferating HDFs expressing the indicated E5 protein, v-Sis or no viral oncoprotein (control) were subjected to immunoblotting. In A, blots were probed with antibodies specific for phosphorylated (p) forms of JNK, AKT, ERK1/2 or c-Jun, and then stripped and reprobed with pan-specific antibodies detecting the total amount of each protein. JNK isoforms 1 and 2 are indicated on the right. In B (upper panel) the blot was probed with a cyclin D3-specific antibody and then reprobed with actin antibodies. In B (lower panel) E5-expressing HDFs were either untreated (–) or treated with of 20 µM LY294002 (LY) or 310 nM wortmannin (W) for 24 hours. Whole-cell lysates were prepared and immunoblotted for cyclin D3 and β-tubulin.

View larger version (40K): [in this window] [in a new window]   Fig. 4. Gab1 tyrosine phosphorylation and association with SHP2 and Grb2 correlate with the enhanced growth phenotype. SHP2 (A) or Gab1 (B) was immunoprecipitated (IP) from lysates of HDFs expressing no (control) or the indicated viral oncoprotein. Immunoprecipitates were subjected to immunoblotting for Gab1, SHP2, Grb2 or phosphotyrosine (PY) as indicated. For the graphs shown below representative blots, the relative band intensities for the Gab1 and Grb2 blots in A, and the PY and Grb2 blots in B were quantitated using the Image J program and normalized for SHP2 (A) or total Gab1 (B) amounts. These normalized values are expressed as a percentage relative to the amount of co-immunoprecipitation or PY-Gab1 observed in the wild-type E5-expressing HDFs. The mean values from two different experiments with standard error are shown.

View larger version (42K): [in this window] [in a new window]   Fig. 5. p66Shc association with SHP2 and Ser36 phosphorylation correlate with the focus-formation phenotype. (A,B) SHP2 (A) or Shc (B) were immunoprecipitated (IP) from lysates of HDFs expressing no (control) or the indicated viral oncoprotein. IPs were subjected to immunoblotting for Shc (A,B), SHP2 (A) or Ser36 phosphorylated (PS36)-p66Shc (B), as indicated. For the graphs shown below representative blots, the relative band intensities for the p66Shc blot in A and the phospho-Ser36 p66Shc blot in B were quantitated using the Image J program and normalized for total SHP2 and p66Shc amounts, respectively. These normalized values are expressed as a percentage relative to the level of p66Shc-SHP2 co-immunoprecipitation (A) or p66Shc Ser36 phosphorylation (B) observed in the wild-type E5-expressing HDFs. The mean values from two (A) or three (B) different experiments with the standard error are shown.

View larger version (34K): [in this window] [in a new window]   Fig. 6. The effect of siRNA-mediated knockdown of SHP2 on phosphorylation of p66Shc and JNK. (A,B) Whole-cell lysates of E5-expressing HDFs transfected with a control siRNA (250 pmoles) or a SHP2-specific siRNA (250 or 500 pmoles) were subjected to immunoblotting for SHP2, Ser36 phosphorylated p66Shc (PS36-p66Shc) or phosphorylated JNK (pJNK) as indicated. In A, the PS36-p66Shc blot was reprobed for total p66Shc and the SHP2 blot was reprobed for β-tubulin. In B, the pJNK blot was reprobed for total JNK and SHP2. JNK isoforms 1 and 2 are indicated.

View larger version (39K): [in this window] [in a new window]   Fig. 7. The effect of overexpressing wild-type or S36A mutant p66Shc on focus formation and enhanced growth of HDFs. (A) HDFs stably co-expressing wild-type (WT) p66Shc, S36A or the corresponding empty retroviral expression vector (LXSN) with E5, v-Sis or no viral oncogene (Puro) were seeded at an equal density in a 24-well dish. At various times thereafter, cells were trypsinized and counted in triplicate. The mean cell number with standard error is plotted against the time after plating. (B) Whole-cell extracts of cells co-expressing the indicated transgenes were subjected to immunoblot analysis for Ser36 phosphorylated p66Shc (PS36-p66Shc), or phosphorylated AKT or JNK (pAKT or pJNK, respectively). Blots were reprobed for total p66Shc, AKT or JNK. JNK isoforms 1 and 2 are indicated. (C) A focus-forming assay was performed by infecting HDFs expressing wild-type p66Shc, S36A or no transgene (LXSN) with E5- or v-Sis-containing retrovirus. Foci were counted and averaged from several experiments. Focus formation is expressed as the mean percent (with standard error) relative to the number of foci formed by the LXSN-HDFs.

View larger version (58K): [in this window] [in a new window]   Fig. 8. p66Shc forms a complex with p190BRhoGAP in E5- and v-Sis-expressing HDFs. Total Shc (A) or p66Shc (B) was immunoprecipitated (IP) from lysates of HDFs expressing no transgene (control or LXSN-Puro) or the indicated viral oncoprotein in the absence (A) or presence (B) of exogenous wild-type (WT) p66Shc, S36A or empty LXSN. Immunoprecipitates were subjected to immunoblotting for p190B, SHP2 or Shc as indicated.

View larger version (13K): [in this window] [in a new window]   Fig. 9. The effect of specific kinase inhibitors on growth and focus formation of HDFs. (A) Focus formation of HDFs by E5 or v-Sis was assessed in the absence (–) or presence (+) of LY294002 (LY), wortmannin (W), SP600125 (SP) or Y27632. Foci were counted and averaged from two experiments. Focus formation is expressed as the mean percent (with standard error) relative to the number of foci formed by the untreated cells. (B-D) E5-expressing HDFs were seeded at an equal density either into 60 mM dishes (B) or a 24-well dish (C,D). Two days after plating, cells were either left untreated (–) or treated with the above inhibitors. At various times thereafter, cells were trypsinized and counted in triplicate. The mean cell number with standard error is plotted against the time after plating.

View larger version (17K): [in this window] [in a new window]   Fig. 10. Model of the PDGFβR signaling pathways involved in enhanced growth and focus formation of HDFs. See text for explanation.

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