Figure 6. Ptsn mediates Rap1... | Rockefeller University Press

Figure 6.

Ptsn mediates Rap1 dephosphorylation. (A) Silencing of Ptsn expression promotes Rap1 phosphorylation. αIIb(SBP)β3(D119A)–expressing U2-OS cells were transduced with a lentivirus encoding an shRNA against Ptsn or sh-CTRL (scrambled) and then transfected with a plasmid encoding Myc-tagged Rap1 in combination with a vector encoding mCherry-tagged Ptsn or empty vector. Rap1 was immunoprecipitated (IP) using an anti-Myc antibody, and phosphorylation of Rap1 was revealed by an antibody recognizing phosphorylated PKA substrates (pRap1). (B) As a control, the phosphorylation of CREB was assayed in Ptsn-depleted cells using an antibody that specifically recognizes the phosphorylated form of CREB. U2-OS cells were treated with 10 μM forskolin for 30 min to induce CREB phosphorylation. (C) The phosphorylation of endogenous Rap1 was assessed by gel mobility shift assay. WT, Ptsn-null, or β-Ptsn–reconstituted Ptsn-null Jurkat T cells described in Fig. 3 were left untreated or treated with H-89. The mobility of Rap1 was measured on an immunoblot of proteins separated on 12% SDS-PAGE. The mobility of the lower band was reduced in Ptsn-null cells compared with WT and β-Ptsn–reconstituted Ptsn-null cells or Ptsn-null cells treated with a PKA inhibitor, H-89. (D) Phosphorylation-resistant Rap1 mutant (S180A) rescues RIAM MIT complex assembly. αIIb(SBP)β3(D119A)–expressing U2-OS cells were transduced with a lentivirus encoding a shRNA against Ptsn and transfected with a bicistronic plasmid encoding Myc-tagged Rap1 in combination with Flag-RIAM. RIAM was immunoprecipitated using an anti-Flag antibody, and the associated β3 integrin was revealed by immunoblotting and quantified by infrared spectroscopy. Bar graphs represent mean ± SEM (n = 3 independent experiments). Significance was tested with one-way ANOVA with Tukey post-test; *, P < 0.05,**, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Source data are available for this figure: SourceData F6.

Ptsn mediates Rap1 dephosphorylation. (A) Silencing of Ptsn expression promotes Rap1 phosphorylation. α_IIb(SBP)β₃(D119A)–expressing U2-OS cells were transduced with a lentivirus encoding an shRNA against Ptsn or sh-CTRL (scrambled) and then transfected with a plasmid encoding Myc-tagged Rap1 in combination with a vector encoding mCherry-tagged Ptsn or empty vector. Rap1 was immunoprecipitated (IP) using an anti-Myc antibody, and phosphorylation of Rap1 was revealed by an antibody recognizing phosphorylated PKA substrates (pRap1). (B) As a control, the phosphorylation of CREB was assayed in Ptsn-depleted cells using an antibody that specifically recognizes the phosphorylated form of CREB. U2-OS cells were treated with 10 μM forskolin for 30 min to induce CREB phosphorylation. (C) The phosphorylation of endogenous Rap1 was assessed by gel mobility shift assay. WT, Ptsn-null, or β-Ptsn–reconstituted Ptsn-null Jurkat T cells described in Fig. 3 were left untreated or treated with H-89. The mobility of Rap1 was measured on an immunoblot of proteins separated on 12% SDS-PAGE. The mobility of the lower band was reduced in Ptsn-null cells compared with WT and β-Ptsn–reconstituted Ptsn-null cells or Ptsn-null cells treated with a PKA inhibitor, H-89. (D) Phosphorylation-resistant Rap1 mutant (S180A) rescues RIAM MIT complex assembly. α_IIb(SBP)β₃(D119A)–expressing U2-OS cells were transduced with a lentivirus encoding a shRNA against Ptsn and transfected with a bicistronic plasmid encoding Myc-tagged Rap1 in combination with Flag-RIAM. RIAM was immunoprecipitated using an anti-Flag antibody, and the associated β₃ integrin was revealed by immunoblotting and quantified by infrared spectroscopy. Bar graphs represent mean ± SEM (n = 3 independent experiments). Significance was tested with one-way ANOVA with Tukey post-test; *, P < 0.05,**, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Source data are available for this figure: SourceData F6.