Figure 4.
Figure 4. pY599 is a recruitment site for the protein tyrosine phosphatase SHP2. (A) Total 32D cell lysates were incubated with immobilized peptide Y599 (lane A) and pY599 (lanes B-D). To prove phosphoselectivity of found interactions, soluble phosphorylated or unphosphorylated peptides (0.5 mg/mL, Y599, lane C; pY599, lane D) were included in the reactions to compete for binding proteins. Bound protein was subjected to a Western blot analysis for SHP2. Bands were densitometrically analyzed and relative intensities are depicted under each lane. (B) Interaction of pY599 (but not pY589) of the entire Flt3 protein and SHP2 was shown in a pulldown experiment. For this purpose, Cos-1 cells were transiently transfected with Flt3-WT, Flt3-Y589F, and Flt3-Y599F constructs. After stimulation of the appropriate samples with FL for 10 minutes, cells were lysed and the lysate was incubated with 2 μg GST-SHP2 (N+C)-SH2 fusion protein. Pulled-down proteins were separated by SDS-PAGE on an 8% gel and blotted, and the membranes were probed for Flt3. To confirm efficient transfection and stimulation of each sample, Flt3 was immunoprecipitated from the lysates and immunoblotted for Flt3 and phosphotyrosine. (C) pY599 of Flt3 binds to SHP2 in living cells. The 32D transfectants of either WT- or Y599F-FLt3 were starved for 4 hours from cytokines before they were stimulated with FL (100 ng/mL) for 7 minutes. Cells were lysed and subjected to immunoprecipitation with an antibody against Flt3. Immunoprecipitated proteins were separated by SDS gel electrophoresis, electrotransferred to Immobilon P, followed by probing with an antibody against SHP2. The filter was consecutively stripped and reprobed with a Flt3 antibody to ensure equal loading and a phosphotyrosine antibody to reveal stimulation of Flt3 phosphorylation. (D) To examine SHP2 phosphorylation, 32D transfectants of either WT-Flt3 or Y599F-Flt3 were starved for 4 hours from cytokines before they were stimulated with FL (100 ng/mL) for the indicated periods of time. SHP2 was immunoprecipitated from cell lysates, run out on an 8% gel, and immunoblotted for phosphotyrosine and SHP2, respectively. (E) Direct interaction of pY599 with SHP2-SH2 was demonstrated by incubation of purified GST fusion protein (1 μg) with immobilized Y599 peptide (lane A) or pY599 (lanes B-D) and 0.5 mg/mL of competing soluble pY599 (lane C) or Y599 (lane D) peptide, respectively. After 2-hour incubation end-over-end, immobilized peptide beads were boiled in Laemmli buffer and subjected to a Western blot probing for GST. One representative experiment is shown of at least 2 performed with consistent results.

pY599 is a recruitment site for the protein tyrosine phosphatase SHP2. (A) Total 32D cell lysates were incubated with immobilized peptide Y599 (lane A) and pY599 (lanes B-D). To prove phosphoselectivity of found interactions, soluble phosphorylated or unphosphorylated peptides (0.5 mg/mL, Y599, lane C; pY599, lane D) were included in the reactions to compete for binding proteins. Bound protein was subjected to a Western blot analysis for SHP2. Bands were densitometrically analyzed and relative intensities are depicted under each lane. (B) Interaction of pY599 (but not pY589) of the entire Flt3 protein and SHP2 was shown in a pulldown experiment. For this purpose, Cos-1 cells were transiently transfected with Flt3-WT, Flt3-Y589F, and Flt3-Y599F constructs. After stimulation of the appropriate samples with FL for 10 minutes, cells were lysed and the lysate was incubated with 2 μg GST-SHP2 (N+C)-SH2 fusion protein. Pulled-down proteins were separated by SDS-PAGE on an 8% gel and blotted, and the membranes were probed for Flt3. To confirm efficient transfection and stimulation of each sample, Flt3 was immunoprecipitated from the lysates and immunoblotted for Flt3 and phosphotyrosine. (C) pY599 of Flt3 binds to SHP2 in living cells. The 32D transfectants of either WT- or Y599F-FLt3 were starved for 4 hours from cytokines before they were stimulated with FL (100 ng/mL) for 7 minutes. Cells were lysed and subjected to immunoprecipitation with an antibody against Flt3. Immunoprecipitated proteins were separated by SDS gel electrophoresis, electrotransferred to Immobilon P, followed by probing with an antibody against SHP2. The filter was consecutively stripped and reprobed with a Flt3 antibody to ensure equal loading and a phosphotyrosine antibody to reveal stimulation of Flt3 phosphorylation. (D) To examine SHP2 phosphorylation, 32D transfectants of either WT-Flt3 or Y599F-Flt3 were starved for 4 hours from cytokines before they were stimulated with FL (100 ng/mL) for the indicated periods of time. SHP2 was immunoprecipitated from cell lysates, run out on an 8% gel, and immunoblotted for phosphotyrosine and SHP2, respectively. (E) Direct interaction of pY599 with SHP2-SH2 was demonstrated by incubation of purified GST fusion protein (1 μg) with immobilized Y599 peptide (lane A) or pY599 (lanes B-D) and 0.5 mg/mL of competing soluble pY599 (lane C) or Y599 (lane D) peptide, respectively. After 2-hour incubation end-over-end, immobilized peptide beads were boiled in Laemmli buffer and subjected to a Western blot probing for GST. One representative experiment is shown of at least 2 performed with consistent results.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal