Limits...
XB130/Tks5 scaffold protein interaction regulates Src-mediated cell proliferation and survival.

Moodley S, Hui Bai X, Kapus A, Yang B, Liu M - Mol. Biol. Cell (2015)

Bottom Line: Yeast two-hybrid screening suggests that XB130 interacts with another scaffold protein, Tks5.Structure-function studies showed that the fifth SH3 domain of Tks5 binds to the N-terminus of XB130, which contains polyproline-rich motifs.Furthermore, down-regulation of XB130 and/or Tks5 inhibited serum- and growth factor-induced Src activation and downstream phosphorylation of PI3K and Akt.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Science, University of Toronto, Toronto, ON M5S 1A8, Canada Latner Thoracic Surgery Research Laboratories, Toronto General Research Institute, University Health Network, Toronto, ON M5G 1L7, Canada.

No MeSH data available.


Related in: MedlinePlus

Down-regulation of XB130 and/or Tks5 leads to inhibition of cell proliferation and accumulation of cells in G1 phase. (A) Cell counting of BEAS-2B cells at 24 , 48 , 72, and 96 h shows that siRNA down-regulation of XB130, Tks5, or both XB130 and Tks5 reduced the number of cells/milliliter after 72 and 96 h. (B) BrdU colorimetric incorporation assay shows that siRNA of XB130 and/or Tks5 significantly decreases DNA synthesis. (C) PI staining and flow cytometry analysis shows that siRNA of XB130 and Tks5 results in accumulation of cells in the G1 phase and a decrease of cells in the S and G2 phases. Data from A–C are summarized from three independent experiments and presented as mean ± SD. *p < 0.05, **p < 0.01 compared with controls (nontransfected BEAS-2B cells and nontargeting siRNA–transfected cells). (D) Western blot analysis shows that the proliferative marker Ki67 is reduced, whereas p21 is up-regulated, in XB130 and/or Tks5 siRNA cell lysates. (E) Expression of XB130 and Tks5 in BEAS-2B cells was reduced by specific siRNA. Note: no transfection (Control), transfection of nontargeting siRNA (Control siRNA), or transfection of XB130 and/or Tks5 siRNA. GAPDH is shown as a loading control for D and E.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4666142&req=5

Figure 4: Down-regulation of XB130 and/or Tks5 leads to inhibition of cell proliferation and accumulation of cells in G1 phase. (A) Cell counting of BEAS-2B cells at 24 , 48 , 72, and 96 h shows that siRNA down-regulation of XB130, Tks5, or both XB130 and Tks5 reduced the number of cells/milliliter after 72 and 96 h. (B) BrdU colorimetric incorporation assay shows that siRNA of XB130 and/or Tks5 significantly decreases DNA synthesis. (C) PI staining and flow cytometry analysis shows that siRNA of XB130 and Tks5 results in accumulation of cells in the G1 phase and a decrease of cells in the S and G2 phases. Data from A–C are summarized from three independent experiments and presented as mean ± SD. *p < 0.05, **p < 0.01 compared with controls (nontransfected BEAS-2B cells and nontargeting siRNA–transfected cells). (D) Western blot analysis shows that the proliferative marker Ki67 is reduced, whereas p21 is up-regulated, in XB130 and/or Tks5 siRNA cell lysates. (E) Expression of XB130 and Tks5 in BEAS-2B cells was reduced by specific siRNA. Note: no transfection (Control), transfection of nontargeting siRNA (Control siRNA), or transfection of XB130 and/or Tks5 siRNA. GAPDH is shown as a loading control for D and E.

Mentions: Unlike XB130, the role of Tks5 in cell growth, proliferation, and survival is unknown. Because we showed that XB130 and Tks5 directly interact, we hypothesized that, depending on the extracellular stimulus, XB130 and Tks5 may act in conjunction to regulate cell growth and proliferation and cell survival. A 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium viability screen showed that down-regulation of XB130 or Tks5 in BEAS-2B cells with specific small interfering RNA (siRNA) resulted in inhibition of cell viability in a dose-dependent manner (unpublished data). A growth curve shows that at 72 and 96 h after siRNA transfection, down-regulation of XB130 and/or Tks5 significantly reduced the numbers of cells compared with the growth of nontransfected control BEAS-2B cells and BEAS-2B cells transfected with a nontargeting siRNA (Figure 4A). To tease out the role of XB130 and Tks5 in DNA synthesis, we performed a bromodeoxyuridine (BrdU) incorporation assay. Down-regulation of XB130 or Tks5 resulted in significantly lower levels of BrdU incorporation as compared with cells receiving no siRNA or cells transfected with nontargeting siRNA (Figure 4B). To analyze cell cycle progression, we performed propidium iodide (PI) staining followed by flow cytometry analysis. Down-regulation of XB130 and/or Tks5 results in the accumulation of cells in the G1 (resting) phase and a decrease of cells in the DNA synthesis and G2 cell growth phase as compared with the control or nontargeting siRNA–transfected cells (Figure 4C). Moreover, expression of the cell-proliferative marker Ki67 was decreased, whereas a cell cycle regulation marker, p21, was increased in cell lysates from BEAS-2B cells transfected with XB130 and/or Tks5 siRNA (Figure 4D). The evidence of down-regulation of XB130 and Tks5 by respective siRNAs is shown in Figure 4E. Of interest, down-regulation of both XB130 and Tks5 resulted in similar inhibition on cell growth, BrdU incorporation, cell cycle progression, and individual siRNA of XB130 or Tks5 with no additive effect (Figure 4).


XB130/Tks5 scaffold protein interaction regulates Src-mediated cell proliferation and survival.

Moodley S, Hui Bai X, Kapus A, Yang B, Liu M - Mol. Biol. Cell (2015)

Down-regulation of XB130 and/or Tks5 leads to inhibition of cell proliferation and accumulation of cells in G1 phase. (A) Cell counting of BEAS-2B cells at 24 , 48 , 72, and 96 h shows that siRNA down-regulation of XB130, Tks5, or both XB130 and Tks5 reduced the number of cells/milliliter after 72 and 96 h. (B) BrdU colorimetric incorporation assay shows that siRNA of XB130 and/or Tks5 significantly decreases DNA synthesis. (C) PI staining and flow cytometry analysis shows that siRNA of XB130 and Tks5 results in accumulation of cells in the G1 phase and a decrease of cells in the S and G2 phases. Data from A–C are summarized from three independent experiments and presented as mean ± SD. *p < 0.05, **p < 0.01 compared with controls (nontransfected BEAS-2B cells and nontargeting siRNA–transfected cells). (D) Western blot analysis shows that the proliferative marker Ki67 is reduced, whereas p21 is up-regulated, in XB130 and/or Tks5 siRNA cell lysates. (E) Expression of XB130 and Tks5 in BEAS-2B cells was reduced by specific siRNA. Note: no transfection (Control), transfection of nontargeting siRNA (Control siRNA), or transfection of XB130 and/or Tks5 siRNA. GAPDH is shown as a loading control for D and E.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4666142&req=5

Figure 4: Down-regulation of XB130 and/or Tks5 leads to inhibition of cell proliferation and accumulation of cells in G1 phase. (A) Cell counting of BEAS-2B cells at 24 , 48 , 72, and 96 h shows that siRNA down-regulation of XB130, Tks5, or both XB130 and Tks5 reduced the number of cells/milliliter after 72 and 96 h. (B) BrdU colorimetric incorporation assay shows that siRNA of XB130 and/or Tks5 significantly decreases DNA synthesis. (C) PI staining and flow cytometry analysis shows that siRNA of XB130 and Tks5 results in accumulation of cells in the G1 phase and a decrease of cells in the S and G2 phases. Data from A–C are summarized from three independent experiments and presented as mean ± SD. *p < 0.05, **p < 0.01 compared with controls (nontransfected BEAS-2B cells and nontargeting siRNA–transfected cells). (D) Western blot analysis shows that the proliferative marker Ki67 is reduced, whereas p21 is up-regulated, in XB130 and/or Tks5 siRNA cell lysates. (E) Expression of XB130 and Tks5 in BEAS-2B cells was reduced by specific siRNA. Note: no transfection (Control), transfection of nontargeting siRNA (Control siRNA), or transfection of XB130 and/or Tks5 siRNA. GAPDH is shown as a loading control for D and E.
Mentions: Unlike XB130, the role of Tks5 in cell growth, proliferation, and survival is unknown. Because we showed that XB130 and Tks5 directly interact, we hypothesized that, depending on the extracellular stimulus, XB130 and Tks5 may act in conjunction to regulate cell growth and proliferation and cell survival. A 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium viability screen showed that down-regulation of XB130 or Tks5 in BEAS-2B cells with specific small interfering RNA (siRNA) resulted in inhibition of cell viability in a dose-dependent manner (unpublished data). A growth curve shows that at 72 and 96 h after siRNA transfection, down-regulation of XB130 and/or Tks5 significantly reduced the numbers of cells compared with the growth of nontransfected control BEAS-2B cells and BEAS-2B cells transfected with a nontargeting siRNA (Figure 4A). To tease out the role of XB130 and Tks5 in DNA synthesis, we performed a bromodeoxyuridine (BrdU) incorporation assay. Down-regulation of XB130 or Tks5 resulted in significantly lower levels of BrdU incorporation as compared with cells receiving no siRNA or cells transfected with nontargeting siRNA (Figure 4B). To analyze cell cycle progression, we performed propidium iodide (PI) staining followed by flow cytometry analysis. Down-regulation of XB130 and/or Tks5 results in the accumulation of cells in the G1 (resting) phase and a decrease of cells in the DNA synthesis and G2 cell growth phase as compared with the control or nontargeting siRNA–transfected cells (Figure 4C). Moreover, expression of the cell-proliferative marker Ki67 was decreased, whereas a cell cycle regulation marker, p21, was increased in cell lysates from BEAS-2B cells transfected with XB130 and/or Tks5 siRNA (Figure 4D). The evidence of down-regulation of XB130 and Tks5 by respective siRNAs is shown in Figure 4E. Of interest, down-regulation of both XB130 and Tks5 resulted in similar inhibition on cell growth, BrdU incorporation, cell cycle progression, and individual siRNA of XB130 or Tks5 with no additive effect (Figure 4).

Bottom Line: Yeast two-hybrid screening suggests that XB130 interacts with another scaffold protein, Tks5.Structure-function studies showed that the fifth SH3 domain of Tks5 binds to the N-terminus of XB130, which contains polyproline-rich motifs.Furthermore, down-regulation of XB130 and/or Tks5 inhibited serum- and growth factor-induced Src activation and downstream phosphorylation of PI3K and Akt.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Science, University of Toronto, Toronto, ON M5S 1A8, Canada Latner Thoracic Surgery Research Laboratories, Toronto General Research Institute, University Health Network, Toronto, ON M5G 1L7, Canada.

No MeSH data available.


Related in: MedlinePlus