Figure 4

The Common Docking (CD) domain of MAP kinase mediated the binding to the WW domain of ArhGAP9. a. Flag-tagged full-length ArhGAP9 (wild type or the R246,247A mutant, RR) was expressed 293T cells and the lysates were incubated with GST fusion of (i) the CD domain of Erk2 (Erk2-CD-GST, residues 300–358), (ii) a fragment of Erk2 deleted of the CD domain (Erk2-ΔCD-GST, residues 1–300) or (iii) GST alone as a control. Bound ArhGAP9 was detected by western blotting with α-Flag. (iv) Total cell lysates were immunoblotted with α-Flag. b. Acidic residues in the CD domain of MAP kinases are important for interaction with ArhGAP9. (i) Alignment of the Common Docking (CD) domains of Erk2, p38α and Jnk1, the acidic residues mutated to alanine are indicated in superscript. Flag-tagged Erk2 (wild type or D316A, D319A or E320A mutants) or p38α (wild type or D312A, D315A or E316A mutants) was expressed 293T cells. The lysates were incubated with (ii) ArhGAP9-WW-GST or (iii) GST alone. Bound Erk2 or p38α was detected by western blotting with α-Flag. (iv) Total cell lysates were immunoblotted with α-Flag. c. The activation loop of MAP kinase was not involved in binding with ArhGAP9. Flag-tagged Erk2 (wild type or T183, Y185A mutant) and p38α (wild type or T180, Y182A mutant) were expressed in 293T cells. The lysates were incubated with immobilized (i) ArhGAP9-WW-GST or (ii) GST alone. Bound Erk2 or p38α was detected by western blotting with α-Flag. (iii) Total cell lysates were immunoblotted with α-Flag. d. Far-UV CD spectra of WW of ArhGAP9 in complex with MAP kinase CD domain peptides. (i) The CD spectra of the WW domain of ArhGAP9 indicating that the protein folded properly and has mostly β-sheets and random coils. (ii-iv) CD spectra of ArhGAP9 WW domain in complex with Jnk1, p38α and Erk2 peptides, respectively. Conformational changes were observed for the case of ArhGAP9 WW domain in complex with Erk2 and p38α peptides. As for Jnk1, little effect in the spectra profile was observed when compared with WW domain alone, indicating that no significant binding of the Jnk1 peptide had occurred. e. Structural alignment of the ArhGAP9-binding regions in the CD domains of Erk2, p38α and Jnk1. (i) A stereo view of structural alignment of Erk2 (red) with p38α (maroon) (ii) A stereo view of structural alignment of Erk2 with Jnk1. The residues are shown in stick model and the key mutated residues of Erk2 are marked.