Figure 6.
Figure 6. Biochemical characterizations of DPak3. (A) DPak3 is phosphorylated in S2R+ cells. FLAG-DPak3 was expressed in S2R+ cells and immunoprecipitated with α-FLAG, followed by CIP treatment. Note the disappearance of the up-shifted band of DPak3 (red arrowhead; lane 1) after CIP treatment (black arrowhead; lane 2), indicating that the up-shifted band was caused by phosphorylation. (B) Constitutively active Rac further activates DPak3 phosphorylation in S2R+ cells. DPak3 phosphorylation was further increased by coexpressing with activated Rac1 (Rac1G12V; red arrowhead; lane 1). The Rac-binding defective mutant DPak3H29,31L remained partially phosphorylated (lane 2), whereas the kinase-inactive mutant was unphosphorylated (black arrowhead; lane 3). (C) In vitro kinase assays for wild-type and mutant DPak3. The kinase activities of DPak3 and its mutant forms were assessed by their ability to phosphorylate myelin basic protein (MBP) in the presence of γ-[32P]ATP. Expression of MBP and different DPak3 proteins was detected by Coomassie brilliant blue staining. The kinase activities were normalized against protein expression levels and compared with that of V5-DPak3. Results from three independent experiments were quantified. Note the great increase in the DPak3 kinase activity when it was coexpressed with activated Rac1 (lane 3), the complete loss of kinase activity resulting from the K322A mutation (lane 4), and the constitutive kinase activity of DPak3H29,31L (lane 5). Error bars: standard deviations. (D) DPak3, but not DPak3H29,31L, interacts with activated Rac1 (Rac1G12V) expressed in S2R+ cells (compare lanes 2 and 3). (E) DPak3 is phosphorylated in stage 14 Drosophila embryos. Extracts prepared from stage 14 embryos expressing V5-DPak3 in the mesoderm with twi-GAL4 were subjected to SDS-PAGE (lane 2), together with an extract of S2R+ cells expressing V5-DPak3 as a control (lane 1). Note the similarly up-shifted bands in both lanes (red arrowhead), indicating phosphorylation of V5-DPak3 in vivo. Percentage of the polyacrylamide gels: 6% in A, B, and E; 15% in C; and 12% in D.

Biochemical characterizations of DPak3. (A) DPak3 is phosphorylated in S2R+ cells. FLAG-DPak3 was expressed in S2R+ cells and immunoprecipitated with α-FLAG, followed by CIP treatment. Note the disappearance of the up-shifted band of DPak3 (red arrowhead; lane 1) after CIP treatment (black arrowhead; lane 2), indicating that the up-shifted band was caused by phosphorylation. (B) Constitutively active Rac further activates DPak3 phosphorylation in S2R+ cells. DPak3 phosphorylation was further increased by coexpressing with activated Rac1 (Rac1G12V; red arrowhead; lane 1). The Rac-binding defective mutant DPak3H29,31L remained partially phosphorylated (lane 2), whereas the kinase-inactive mutant was unphosphorylated (black arrowhead; lane 3). (C) In vitro kinase assays for wild-type and mutant DPak3. The kinase activities of DPak3 and its mutant forms were assessed by their ability to phosphorylate myelin basic protein (MBP) in the presence of γ-[32P]ATP. Expression of MBP and different DPak3 proteins was detected by Coomassie brilliant blue staining. The kinase activities were normalized against protein expression levels and compared with that of V5-DPak3. Results from three independent experiments were quantified. Note the great increase in the DPak3 kinase activity when it was coexpressed with activated Rac1 (lane 3), the complete loss of kinase activity resulting from the K322A mutation (lane 4), and the constitutive kinase activity of DPak3H29,31L (lane 5). Error bars: standard deviations. (D) DPak3, but not DPak3H29,31L, interacts with activated Rac1 (Rac1G12V) expressed in S2R+ cells (compare lanes 2 and 3). (E) DPak3 is phosphorylated in stage 14 Drosophila embryos. Extracts prepared from stage 14 embryos expressing V5-DPak3 in the mesoderm with twi-GAL4 were subjected to SDS-PAGE (lane 2), together with an extract of S2R+ cells expressing V5-DPak3 as a control (lane 1). Note the similarly up-shifted bands in both lanes (red arrowhead), indicating phosphorylation of V5-DPak3 in vivo. Percentage of the polyacrylamide gels: 6% in A, B, and E; 15% in C; and 12% in D.

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