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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

XB130 binds to the fifth SH3 domain of Tks5. (A) Schematic diagram of Tks5, in particular showing the location of the SH3 #5 single–amino acid substitution mutant, tryptophan 1108 to alanine (W1108A). (B) GST-fusion protein pull-down assay and immunoblot of XB130 shows that XB130 binds to only the fifth SH3 domain of Tks5 in COS-7 (transfected with His-XB130), WRO, and TPC-1 cells. (C) Immunoprecipitation of XB130 shows that single amino acid substitution of tryptophan (W) 1108 to alanine (A) in the fifth SH3 domain of Tks5 inhibited coIP between Tks5 and XB130 in COS-7 cells. (D) Immunoprecipitation of XB130 shows that deletion of PX domain of Tks5 (Myc-Tks5 ∆PX) also inhibited Tks5 binding to XB130 in COS-7 cells.
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Figure 2: XB130 binds to the fifth SH3 domain of Tks5. (A) Schematic diagram of Tks5, in particular showing the location of the SH3 #5 single–amino acid substitution mutant, tryptophan 1108 to alanine (W1108A). (B) GST-fusion protein pull-down assay and immunoblot of XB130 shows that XB130 binds to only the fifth SH3 domain of Tks5 in COS-7 (transfected with His-XB130), WRO, and TPC-1 cells. (C) Immunoprecipitation of XB130 shows that single amino acid substitution of tryptophan (W) 1108 to alanine (A) in the fifth SH3 domain of Tks5 inhibited coIP between Tks5 and XB130 in COS-7 cells. (D) Immunoprecipitation of XB130 shows that deletion of PX domain of Tks5 (Myc-Tks5 ∆PX) also inhibited Tks5 binding to XB130 in COS-7 cells.

Mentions: Protein–protein interaction requires specific physical contact and orientation between proteins as a result of electrostatic forces and biochemical processes (Jones and Thornton, 1996). Protein sequences often contain well-known or canonical structural domains and motifs, which are required for specific binding to other proteins (Pawson and Nash, 2000). We probed the protein-binding regions and structural domains of XB130 and Tks5 to determine the specific physical binding sites between these partners. SH3 domains, although similar in overall structures, do not share identical amino acid sequences and thus may have very specific binding partners (Weng et al., 1995). To determine whether XB130 binds specifically to one or more of the SH3 domains of Tks5, we performed a glutathione S-transferase (GST)–fusion protein pull-down assay in which the DNA sequence of each of the five SH3 domains of Tks5 was fused with the GST DNA sequence to express GST-tagged Tks5 SH3-domain recombinant proteins (Figure 2A). The individual GST-tagged Tks5 SH3-domain recombinant proteins were incubated with cell lysates from COS-7 cells, which were transfected with His-XB130–tagged vectors. XB130 was found to bind specifically to the fifth SH3 domain of Tks5 (Figure 2B). Similarly, endogenous XB130 in WRO and TPC-1 cells also bound specifically to the fifth SH3 domain of Tks5 (Figure 2B). The Tks5 protein sequence in the fifth SH3 domain contains an SH3 domain–binding pocket with a protein-binding tripeptide of two sequential tryptophans (W) and a tyrosine (Y) at positions 1108–1110. It has been shown that single–amino acid mutation of W1108 to alanine (W1108A, previously annotated as W1056A; Diaz et al., 2009; see Discussion) abolishes the association of the fifth SH3 domain of Tks5 with ADAM19 (Abram et al., 2003). GST-fusion protein pull-down of GST-tagged Tks5 SH3 #5 containing the single amino acid mutation of W1108A prevented XB130 binding to Tks5 (Figure 2B). Overexpression of His-XB130 and mutant Myc-Tks5 W1108A in COS-7 cells showed that the coIP between XB130 and Tks5 was abolished when the W1108A mutant was used (Figure 2C). It has been demonstrated that the PX domain of Tks5 is critical for its intermolecular interaction with phosphatidylinositol lipids, as well as the translocation of Tks5 and Tks5-bound actin-associated proteins to the cell periphery (Abram et al., 2003). In COS-7 cells, anti-His antibody, used to detect XB130, did not pull down PX domain deleted Myc-Tks5 mutant (Figure 2D).


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)

XB130 binds to the fifth SH3 domain of Tks5. (A) Schematic diagram of Tks5, in particular showing the location of the SH3 #5 single–amino acid substitution mutant, tryptophan 1108 to alanine (W1108A). (B) GST-fusion protein pull-down assay and immunoblot of XB130 shows that XB130 binds to only the fifth SH3 domain of Tks5 in COS-7 (transfected with His-XB130), WRO, and TPC-1 cells. (C) Immunoprecipitation of XB130 shows that single amino acid substitution of tryptophan (W) 1108 to alanine (A) in the fifth SH3 domain of Tks5 inhibited coIP between Tks5 and XB130 in COS-7 cells. (D) Immunoprecipitation of XB130 shows that deletion of PX domain of Tks5 (Myc-Tks5 ∆PX) also inhibited Tks5 binding to XB130 in COS-7 cells.
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Related In: Results  -  Collection

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Figure 2: XB130 binds to the fifth SH3 domain of Tks5. (A) Schematic diagram of Tks5, in particular showing the location of the SH3 #5 single–amino acid substitution mutant, tryptophan 1108 to alanine (W1108A). (B) GST-fusion protein pull-down assay and immunoblot of XB130 shows that XB130 binds to only the fifth SH3 domain of Tks5 in COS-7 (transfected with His-XB130), WRO, and TPC-1 cells. (C) Immunoprecipitation of XB130 shows that single amino acid substitution of tryptophan (W) 1108 to alanine (A) in the fifth SH3 domain of Tks5 inhibited coIP between Tks5 and XB130 in COS-7 cells. (D) Immunoprecipitation of XB130 shows that deletion of PX domain of Tks5 (Myc-Tks5 ∆PX) also inhibited Tks5 binding to XB130 in COS-7 cells.
Mentions: Protein–protein interaction requires specific physical contact and orientation between proteins as a result of electrostatic forces and biochemical processes (Jones and Thornton, 1996). Protein sequences often contain well-known or canonical structural domains and motifs, which are required for specific binding to other proteins (Pawson and Nash, 2000). We probed the protein-binding regions and structural domains of XB130 and Tks5 to determine the specific physical binding sites between these partners. SH3 domains, although similar in overall structures, do not share identical amino acid sequences and thus may have very specific binding partners (Weng et al., 1995). To determine whether XB130 binds specifically to one or more of the SH3 domains of Tks5, we performed a glutathione S-transferase (GST)–fusion protein pull-down assay in which the DNA sequence of each of the five SH3 domains of Tks5 was fused with the GST DNA sequence to express GST-tagged Tks5 SH3-domain recombinant proteins (Figure 2A). The individual GST-tagged Tks5 SH3-domain recombinant proteins were incubated with cell lysates from COS-7 cells, which were transfected with His-XB130–tagged vectors. XB130 was found to bind specifically to the fifth SH3 domain of Tks5 (Figure 2B). Similarly, endogenous XB130 in WRO and TPC-1 cells also bound specifically to the fifth SH3 domain of Tks5 (Figure 2B). The Tks5 protein sequence in the fifth SH3 domain contains an SH3 domain–binding pocket with a protein-binding tripeptide of two sequential tryptophans (W) and a tyrosine (Y) at positions 1108–1110. It has been shown that single–amino acid mutation of W1108 to alanine (W1108A, previously annotated as W1056A; Diaz et al., 2009; see Discussion) abolishes the association of the fifth SH3 domain of Tks5 with ADAM19 (Abram et al., 2003). GST-fusion protein pull-down of GST-tagged Tks5 SH3 #5 containing the single amino acid mutation of W1108A prevented XB130 binding to Tks5 (Figure 2B). Overexpression of His-XB130 and mutant Myc-Tks5 W1108A in COS-7 cells showed that the coIP between XB130 and Tks5 was abolished when the W1108A mutant was used (Figure 2C). It has been demonstrated that the PX domain of Tks5 is critical for its intermolecular interaction with phosphatidylinositol lipids, as well as the translocation of Tks5 and Tks5-bound actin-associated proteins to the cell periphery (Abram et al., 2003). In COS-7 cells, anti-His antibody, used to detect XB130, did not pull down PX domain deleted Myc-Tks5 mutant (Figure 2D).

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