Figure 3
The ArhGAP9 is a novel MAP kinase docking protein. a. Alignment of the WW domains of ArhGAP9 and ArhGAP12. WW domains of human ArhGAP9 and ArhGAP12 were aligned using ClustalW, highlighted in yellow. The di-Arginine motif at the extreme C terminus of ArhGAP9 WW domain is highlighted in cyan. b. R246 and R247 in the WW domain of human ArhGAP9 are required for binding to MAP kinases. Flag-tagged Erk2, p38α or Jnk1 was expressed in 293T cells and the lysates were incubated with immobilized ArhGAP9-WW-GST, ArhGAP9-WW-(R246, 247A)-GST mutant, or GST alone as a control. Specifically MAP kinases were detected by western blotting with α-Flag. (ii) The expression levels of Erk2, p38α and Jnk1 were confirmed to be equivalent by immunoblotting of the total cell lysates with α-Flag. c. R246 and R247 are required for ArhGAP9 WW domain binding to CD domain of MAP kinase in vitro. Flag-tagged ArhGAP9 (wild type or mutants R246A, R247A or R246, 247A) was expressed in 293T cells and the lysates were incubated with the (i) Erk2-CD-GST or (ii) GST alone. Bound ArhGAP9 was detected by immunoblotting with α-Flag. (iii) Total cell lysates were immunoblotted with α-Flag. d. R246 and R247 are required for ArhGAP9 WW domain binding to MAP kinase in vivo. Flag-tagged Erk2 or p38α was expressed individually or together with full-length ArhGAP9 [wildtype or the R246, 247A mutant (RR)] in 293T cells, as indicated in the figure. The lysates were immunoprecipitated with α-Flag followed by western blotting of the immunocomplexes with (i) α-ArhGAP9 or (ii) α-Flag. Total cell lysates were immunoblotted with (iii) α-ArhGAP9 or (iv) α-Flag. e. K243, P244 and P245 within the WW domain of ArhGAP9 are also important for MAP kinase binding. Flag-tagged Erk2 were transiently expressed either individually or together with full-length ArhGAP9 [wildtype or K243A, P244A and P245A mutants] in 293T cells. The lysates were then subjected to immunoprecipitation with α-Flag. The immunocomplexes were resolved by SDS-PAGE followed by western blotting with (i) α-ArhGAP9 or (ii) α-Flag. Total cell lysates were separated by SDS-PAGE and immunoblotted with (iii) α-ArhGAP9 or (iv) α-Flag.