![Figure 6. ErbB2 directly binds and activates Dock7 by phosphorylating Tyr-1118. (A) 250 ng of immobilized full-length FLAG-Dock7 protein was incubated in 30 μl of reaction buffer containing 20 μM of cold ATP in the presence or absence of 100 ng ErbB2 kinase for 30 min, washed, and immunoblotted with an anti-pTyr or ErbB2 antibody. Immobilized FLAG-Dock7 was also stained with Coomassie brilliant blue. (B) The schematic structures of Dock7 and the domains are illustrated. Red rectangle, Tyr in the middle region 2. (C) 293T cells were transfected with the plasmid encoding DHR-1, middle region 1, middle region 2, or DHR-2 of Dock7. The lysates of transfected cells were immunoprecipitated with an anti-FLAG antibody, incubated with ErbB2 kinase and ATP, and immunoblotted with an anti-pTyr or ErbB2 antibody. A shift in the mobility of the bands for the tyrosine-phosphorylated protein was observed. The cell lysates were also immunoblotted with an anti-FLAG antibody. (D) The amino acid sequences containing six tyrosine residues in the middle region 2 are shown. (E) Cells were transfected with the plasmid encoding each middle region 2 containing one Tyr-to-Phe mutation. The samples, immunoprecipitated with the anti-FLAG antibody, were incubated with ErbB2 kinase and ATP. A shift in the mobility was observed in bands of the tyrosine-phosphorylated protein. The tyrosine phosphorylation of the constructs and their expression are also shown. (F) Cells were transfected with each full-length Dock7 harboring one Tyr-to-Phe mutation in the middle region 2, immunoprecipitated with anti-FLAG antibody, and incubated with ErbB2 kinase and ATP. The tyrosine phosphorylation of the constructs and their expression are also shown. (G) 250 ng of immobilized full-length FLAG-Dock7 or FLAG-Dock7Y1118F was incubated with ErbB2 kinase and ATP. (H) A comparison of the amino acid sequences surrounding the ErbB2 phosphorylation sites (red squares) of mammalian Dock7 with other homologous proteins is shown. Black, conserved amino acids; grey, nonconserved amino acids. (I and J) Immobilized FLAG-Dock7 or the Y1118F mutant was incubated in 30 μl of reaction buffer containing 20 μM of cold ATP in the presence or absence of ErbB2 kinase and washed. The release of [3H]GDP from GST-Rac1–[3H]GDP or Cdc42–[3H]GDP by immobilized proteins was measured (n = 3). Error bars show ±SD. Data were evaluated by using one-way ANOVA (*, P < 0.01).](https://cdn.rupress.org/rup/content_public/journal/jcb/181/2/10.1083_jcb.200709033/8/jcb_200709033_rgb_fig6.jpeg?Expires=1771765363&Signature=25xtul08xoMGoHovSniQw--PYSASkbgMHRA5KyDFRqgZjmo8k-SfUReQaxJuXYjQ2NmuRQ7-jDdVjBORjn7nSu3UhYKYyad0j59kQp-7K--VdSG1o~D0Q8ruXv1~lZwzVB8ULiam-lkp6hIrNpchkeJSKWbWmjvei6snbU7eEqgeCEgQXi70hRpffnSGFlu8wN6paX7zn9jPjtNgIaBzvFBhCcUNRxkl8YTxzgFgea~rlwjMdZd7X-If6DKYEEYuPEd3RIO-if9GxXhHBdcwzZtN1lHJJGQ-yKUpiE1nf-G-IrhGsVREdkqZzT8Y2OVaRFWxTvHIDlLvoEHBMyiAZQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
ErbB2 directly binds and activates Dock7 by phosphorylating Tyr-1118. (A) 250 ng of immobilized full-length FLAG-Dock7 protein was incubated in 30 μl of reaction buffer containing 20 μM of cold ATP in the presence or absence of 100 ng ErbB2 kinase for 30 min, washed, and immunoblotted with an anti-pTyr or ErbB2 antibody. Immobilized FLAG-Dock7 was also stained with Coomassie brilliant blue. (B) The schematic structures of Dock7 and the domains are illustrated. Red rectangle, Tyr in the middle region 2. (C) 293T cells were transfected with the plasmid encoding DHR-1, middle region 1, middle region 2, or DHR-2 of Dock7. The lysates of transfected cells were immunoprecipitated with an anti-FLAG antibody, incubated with ErbB2 kinase and ATP, and immunoblotted with an anti-pTyr or ErbB2 antibody. A shift in the mobility of the bands for the tyrosine-phosphorylated protein was observed. The cell lysates were also immunoblotted with an anti-FLAG antibody. (D) The amino acid sequences containing six tyrosine residues in the middle region 2 are shown. (E) Cells were transfected with the plasmid encoding each middle region 2 containing one Tyr-to-Phe mutation. The samples, immunoprecipitated with the anti-FLAG antibody, were incubated with ErbB2 kinase and ATP. A shift in the mobility was observed in bands of the tyrosine-phosphorylated protein. The tyrosine phosphorylation of the constructs and their expression are also shown. (F) Cells were transfected with each full-length Dock7 harboring one Tyr-to-Phe mutation in the middle region 2, immunoprecipitated with anti-FLAG antibody, and incubated with ErbB2 kinase and ATP. The tyrosine phosphorylation of the constructs and their expression are also shown. (G) 250 ng of immobilized full-length FLAG-Dock7 or FLAG-Dock7Y1118F was incubated with ErbB2 kinase and ATP. (H) A comparison of the amino acid sequences surrounding the ErbB2 phosphorylation sites (red squares) of mammalian Dock7 with other homologous proteins is shown. Black, conserved amino acids; grey, nonconserved amino acids. (I and J) Immobilized FLAG-Dock7 or the Y1118F mutant was incubated in 30 μl of reaction buffer containing 20 μM of cold ATP in the presence or absence of ErbB2 kinase and washed. The release of [3H]GDP from GST-Rac1–[3H]GDP or Cdc42–[3H]GDP by immobilized proteins was measured (n = 3). Error bars show ±SD. Data were evaluated by using one-way ANOVA (*, P < 0.01).
