Figure 5

ArhGAP competes with MAP kinase kinases (MAPKKs) to suppress MAP kinase activation. a. Coexpression of MKK6 dimished the binding of p38α to the WW domain of ArhGAP9. p38α-Flag was transfected alone or together with HA-tagged activated mutant of MKK6 in 293T cells and the lysates were incubated with (i) ArhGAP9-WW-GST or (ii) GST alone as a control. Bound p38α was detected by western blotting with α-Flag. (iii, iv) Total cell lysates were immunoblotted with α-Flag or α-HA, respectively. b. Overexpression of MKK6 diminished the binding between ArhGAP9 and p38α. Flag-tagged p38α, HA-tagged MKK6 and ArhGAP9 were expressed in 293T cells as indicated in the figure. The immunocomplex of p38α was analyzed by western blotting with (i) α-ArhGAP9 and (ii) α-Flag. (iii-v) Total lysates were immunoblotted with α-ArhAGAP9, α-Flag and α-HA, respectively. c. Reduction of ArhGAP9 binding to Erk2 by MEKoverexpression in vivo. Flag-tagged Erk2, HA-tagged MEK2 and ArhGAP9 were transiently transfected in 293T cells as indicated in the figure. Immunoprecipitation was carried out with α-ArhGAP9, followed by western blotting with (i) α-ArhGAP9 α-Flag and (ii) α-HA. Western blotting of the total lysates with (iii) α-phospho-Erk showed that Erk2 was activated when coexpressed with MEK2. Western blotting of the total lysates with (iv) α-ArhAGP9, (v) α-Flag and (vi) α-HA showed the expression of ArhGAP9, Erk2 and MEK2 in the total cell lysates. d. MAP kinase binding has no significant effect on ArhGAP9 RhoGAP activity. ArhGAP9 (wild type or the GAP-inactive mutant, R578K) was transfected with myc-tagged Cdc42, as well as Flag-tagged Erk2 or p38α in 293T cells, as indicated in the figure. (i) The lysates were incubated with immobilized PBD-GST followed by detection of the relative amounts of active cdc42 precipitated by western blotting with α-myc. (ii) GST alone was used as a control. (iii-v) Total cell lysates were immunoblotted with α-ArhGAP, α-Flag or α-myc, rspectively. e. ArhGAP9 binding suppresses MAP kinase activation by EGFR. ArhGAP9 [wild type or the triple mutant W242K, R246A, R247A (WRR)] was expressed in 293T cells alone or with EGFR and Flag-tagged p38α, as indicated in the figure. (i-v) Total cell lysates were immunoblotted with antibodies to EGFR, phosphotyrosine, ArhGAP9, Flag epitope or phospho-p38α. f. RhoGAP activity of ArhGAP9 is not required for its suppression of MAP kinase activation. Wild type ArhGAP9 or the mutants R578K (GAP-inactive), WW-1 (W219K) and WW-2 (W242K) were cotransfected with EGFR and p38α in 293T cells. (i-iv) Total cell lysates were immunoblotted with antibodies to phosphotyrosine, ArhGAP9, Flag epitope or phospho-p38α.