ShcA and Grb2 mediate polyoma middle T antigen-induced endothelial transformation and Gab1 tyrosine phosphorylation

Abstract

Middle T antigen (PymT) is the principal transforming component of polyomavirus, and rapidly induces hemangiomas in neonatal mice. PymT, a membrane-associated scaffold, recruits and activates Src family tyrosine kinases, and, once tyrosine phosphorylated, binds proteins with PTB and SH2 domains such as ShcA, phosphatidylinositol 3-kinase (PI3K) and phospholipase Cgamma-1 (PLCgamma-1). To explore the pathways required for endothelial transformation in vivo, we introduced PymT mutant forms into mice. PymT variants unable to bind PI3K and PLCgamma-1 directly induced hemangiomas similarly to wild type, but a mutant unable to bind ShcA was transformation compromised. Requirement for a ShcA PTB domain- binding site was suppressed by replacing this motif in PymT with YXN sequences, which bind the Grb2 SH2 domain upon phosphorylation. Surprisingly, PymT recruitment of ShcA and Grb2 correlated with PI3K activation. PymT mimics activated receptor tyrosine kinases by forming a ShcA-Grb2-Gab1 complex, thus inducing Gab1 tyrosine phosphorylation, which itself is associated with PI3K. Therefore, PymT activation of ShcA-Grb2 signaling is critical for endothelial transformation, and PymT can stimulate Grb2 signaling to both the MAP kinase and PI3K pathways.

Fig. 1. Schematic overview of the recombinant retroviruses transducing wild-type and mutant PymT.

Fig. 2. PymT-associated in vitro kinase activity and in vivo tyrosine phosphorylation of PymT mutant proteins in retrovirally transduced fibroblast and endothelioma cell lines. (A) PymT-associated tyrosine kinase activity. NIH 3T3, F111/cl106 or F111/cl605 fibroblasts or endothelioma cells were starved in serum-free medium for 16–20 h and lysed. PymT proteins were immunoprecipitated and subjected to kinase assays in vitro. Phosphorylated proteins were separated by SDS–PAGE and visualized by autoradiography. Electrophoretic mobilities of PymT proteins (white arrowheads) and the p85 subunit of PI3K (black arrowheads) are indicated. Due to the low phosphorylation levels of the triple mutant Y250,315,322F, five times the reaction products compared with the other samples were loaded. For each PymT mutant, two independently derived endothelioma cell lines are shown. (B) Tyrosine phosphorylation of PymT proteins in cells. PymT proteins were immunoprecipitated from lysates of NIH 3T3 fibroblasts or endothelioma cells expressing wild-type or mutant PymT. The precipitates were electrophoresed and immunoblotted with anti-pTyr or anti-PymT antibodies. The electrophoretic mobility of the mutant PymT proteins is indicated by the arrowheads.

Fig. 2. PymT-associated in vitro kinase activity and in vivo tyrosine phosphorylation of PymT mutant proteins in retrovirally transduced fibroblast and endothelioma cell lines. (A) PymT-associated tyrosine kinase activity. NIH 3T3, F111/cl106 or F111/cl605 fibroblasts or endothelioma cells were starved in serum-free medium for 16–20 h and lysed. PymT proteins were immunoprecipitated and subjected to kinase assays in vitro. Phosphorylated proteins were separated by SDS–PAGE and visualized by autoradiography. Electrophoretic mobilities of PymT proteins (white arrowheads) and the p85 subunit of PI3K (black arrowheads) are indicated. Due to the low phosphorylation levels of the triple mutant Y250,315,322F, five times the reaction products compared with the other samples were loaded. For each PymT mutant, two independently derived endothelioma cell lines are shown. (B) Tyrosine phosphorylation of PymT proteins in cells. PymT proteins were immunoprecipitated from lysates of NIH 3T3 fibroblasts or endothelioma cells expressing wild-type or mutant PymT. The precipitates were electrophoresed and immunoblotted with anti-pTyr or anti-PymT antibodies. The electrophoretic mobility of the mutant PymT proteins is indicated by the arrowheads.

Fig. 3. Tumorigenicity of mutant PymT proteins in vivo. (A) The indicated numbers of newborn CD1 mice were inoculated intra peritoneally with 5 × 10⁴ c.f.u. of recombinant retroviruses within 48 h after birth. The number of surviving animals is plotted versus the time after infection. The cause of death was established for all dead animals by necropsy. (B) In the experimental group infected with PymT(Y315,322F) (indicated by open circles), one animal died from injection-related complications within 12 h after inoculation. (C) In the group infected with PymT(245Δ10-YVNQ/YYND) (indicated by filled circles), one animal died from injection-related complications within 12 h after inoculation. n indicates the number of inoculated mice.

Fig. 3. Tumorigenicity of mutant PymT proteins in vivo. (A) The indicated numbers of newborn CD1 mice were inoculated intra peritoneally with 5 × 10⁴ c.f.u. of recombinant retroviruses within 48 h after birth. The number of surviving animals is plotted versus the time after infection. The cause of death was established for all dead animals by necropsy. (B) In the experimental group infected with PymT(Y315,322F) (indicated by open circles), one animal died from injection-related complications within 12 h after inoculation. (C) In the group infected with PymT(245Δ10-YVNQ/YYND) (indicated by filled circles), one animal died from injection-related complications within 12 h after inoculation. n indicates the number of inoculated mice.

Fig. 3. Tumorigenicity of mutant PymT proteins in vivo. (A) The indicated numbers of newborn CD1 mice were inoculated intra peritoneally with 5 × 10⁴ c.f.u. of recombinant retroviruses within 48 h after birth. The number of surviving animals is plotted versus the time after infection. The cause of death was established for all dead animals by necropsy. (B) In the experimental group infected with PymT(Y315,322F) (indicated by open circles), one animal died from injection-related complications within 12 h after inoculation. (C) In the group infected with PymT(245Δ10-YVNQ/YYND) (indicated by filled circles), one animal died from injection-related complications within 12 h after inoculation. n indicates the number of inoculated mice.

Fig. 4. PI3K activity associated with mutant forms of PymT in fibroblasts and endothelioma cells. PymT proteins were immuno precipitated and associated PI3K activity was determined by phosphorylation of mixed phosphatidylinositol/phosphatidylserine substrates in vitro in the presence of [γ-³²P]ATP. Radioactively labeled phospholipids were extracted and separated by thin-layer chromato graphy on silica gel 60 and visualized by autoradiography. The origin of sample application and the chromatographic mobility of PI3-P are indicated.

Fig. 5. (A) ShcA binding to PymT induces the association of ShcA with tyrosine-phosphorylated Gab1. Lysates from NIH 3T3 fibroblasts expressing wild-type or the Y250F mutant of PymT were immuno precipitated with anti-ShcA antibodies. The immunocomplexes were resolved by SDS–PAGE and immunoblotted with (i) anti-pTyr, (ii) anti-Gab1, (iii) anti-Grb2 or (iv) anti-ShcA antibodies. (v) The same lysates were immunoprecipitated with anti-Gab1 antibodies, and then resolved by SDS–PAGE and immunoblotted with the same antibodies. An aliquot of 50 µg of total cell lysates was resolved by SDS–PAGE and immunoblotted with (vi) anti-PymT or (vii) anti-Grb2. (B) A PymT mutant with two YXN motifs replacing the ShcA PTB domain-binding site associates strongly with Grb2. Lysates from NIH 3T3 fibroblasts expressing wild-type or the indicated mutant forms of PymT were immunoprecipitated with anti-Grb2 antibodies. Immunocomplexes were resolved by SDS–PAGE and immunoblotted with (i) anti-PymT, (ii) anti-Grb2 or (iii) anti-Gab1 antibodies.

Fig. 5. (A) ShcA binding to PymT induces the association of ShcA with tyrosine-phosphorylated Gab1. Lysates from NIH 3T3 fibroblasts expressing wild-type or the Y250F mutant of PymT were immuno precipitated with anti-ShcA antibodies. The immunocomplexes were resolved by SDS–PAGE and immunoblotted with (i) anti-pTyr, (ii) anti-Gab1, (iii) anti-Grb2 or (iv) anti-ShcA antibodies. (v) The same lysates were immunoprecipitated with anti-Gab1 antibodies, and then resolved by SDS–PAGE and immunoblotted with the same antibodies. An aliquot of 50 µg of total cell lysates was resolved by SDS–PAGE and immunoblotted with (vi) anti-PymT or (vii) anti-Grb2. (B) A PymT mutant with two YXN motifs replacing the ShcA PTB domain-binding site associates strongly with Grb2. Lysates from NIH 3T3 fibroblasts expressing wild-type or the indicated mutant forms of PymT were immunoprecipitated with anti-Grb2 antibodies. Immunocomplexes were resolved by SDS–PAGE and immunoblotted with (i) anti-PymT, (ii) anti-Grb2 or (iii) anti-Gab1 antibodies.

Fig. 6. Coordinate association of Grb2 and Gab1 with the PymT(245Δ10-YYND/YVNQ) mutant. 293T cells were transfected with plasmids of vector or v-src alone or PymT and v-src (lanes 1–10 as indicated in the figure). (i) The expression of PymT and its mutants was verified by resolving equivalent amounts of the total cell lysates and immunoblotting with anti-PymT. Equivalent amounts of the lysates were incubated with (iii) Grb2(SH2)–GST or (ii–iv) Gab1(MBD)–GST bound on beads. Specifically bound proteins were eluted and resolved by SDS–PAGE, followed by immunoblotting with anti-PymT, anti-Grb2 or anti-Sos1 antibodies.

Fig. 7. PymT mimics growth factor-induced phosphorylation of Gab1. (A) Parental NIH 3T3 fibroblasts or stable transfectants expressing wild-type (wt) or the Y250,315,322F mutant of PymT were quiesced by growing in serum-free medium for 20 h, then treated with phosphate-buffered saline alone (–), or with 50 ng/ml PDGF-BB for 10 min at 37°C. The lysates were immunoprecipitated with anti-Gab1 antibodies, followed by SDS–PAGE and immunoblotting with anti-pTyr or anti-Gab1 antibodies. (B) Endothelioma cells expressing wild-type or the mutant forms of PymT indicated in the figure were cultured in complete medium and then starved in serum-free medium for 16 h before lysis. A 300 µg aliquot of each lysate was used for immunoprecipitation with anti-Gab2 antibodies, followed by SDS–PAGE and immunoblotting with anti-pTyr antibodies. A 30 µg aliquot of the same lysates was resolved by SDS–PAGE and blotted with anti-Gab2 antibodies.

Fig. 7. PymT mimics growth factor-induced phosphorylation of Gab1. (A) Parental NIH 3T3 fibroblasts or stable transfectants expressing wild-type (wt) or the Y250,315,322F mutant of PymT were quiesced by growing in serum-free medium for 20 h, then treated with phosphate-buffered saline alone (–), or with 50 ng/ml PDGF-BB for 10 min at 37°C. The lysates were immunoprecipitated with anti-Gab1 antibodies, followed by SDS–PAGE and immunoblotting with anti-pTyr or anti-Gab1 antibodies. (B) Endothelioma cells expressing wild-type or the mutant forms of PymT indicated in the figure were cultured in complete medium and then starved in serum-free medium for 16 h before lysis. A 300 µg aliquot of each lysate was used for immunoprecipitation with anti-Gab2 antibodies, followed by SDS–PAGE and immunoblotting with anti-pTyr antibodies. A 30 µg aliquot of the same lysates was resolved by SDS–PAGE and blotted with anti-Gab2 antibodies.