Fig. 5. (A) Binding of Mbt to Rho-type GTPases. HA-tagged Mbt or Mbt^H19,22L were transiently expressed in HEK293 cells alone or co-expressed with Myc-tagged Cdc42, Rac1 or Rho1, or the corresponding activated forms. Cell lysates (Lysates) were analysed using western blot (WB) with anti-HA or anti-Myc antibodies to indicate expression of the corresponding proteins. For co-immunoprecipitation, cell lysates were incubated with an anti-HA antibody and GTPases bound to immunopurified Mbt were detected by western blot using the anti-Myc antibody (IP, -HA; WB, -Myc). The presence of Mbt in the immunoprecipitates was verified by western blot (IP, -HA; WB, -HA). The binding of Cdc42 and Cdc42^G12V to Mbt is disrupted by mutation of the PBD (Mbt^H19,22L). (B) Mbt interacts with Cdc42 in a GTP-dependent manner. The protein extract from HEK293 cells transiently transfected with the pcDNA3-HA-Mbt construct was incubated either with unloaded, or with GDP- or GTP-loaded GST-Cdc42 protein. The GST protein and any bound cellular proteins were recovered by precipitation with glutathione-sepharose beads and separated by SDS-PAGE. Mbt protein was detected by western blot using the anti-HA antibody. As a control, total lysates from pcDNA3-HA-Mbt transfected HEK293 cells are shown (input). The presence of equal amounts of the GST-Cdc42 was verified with an anti-GST antibody. (C) Sequence alignment of the p21-binding domain (PBD) and the kinase inhibitory domain (KID) of human PAK1 and D-PAK (group I PAKs) with the corresponding sequences of human PAK4, PAK5, PAK6 and Mbt (group II PAKs). Highly conserved amino acids are indicated in light grey, amino acids only conserved between PAK1/D-PAK or PAK4-6/Mbt are shown in black and dark grey, respectively. The serine 144 autophosphorylation site and lysine 141 (black circles), which are involved in regulating PAK1 kinase activity are not present in PAK4-6/Mbt. The histidine residues that were mutated to create a Cdc42 binding-deficient Mbt protein are indicated by triangles. (D) Regulation of Mbt kinase activity. HEK293 cells were transfected with either an empty pcDNA3 vector (control), with HA-tagged Mbt, Mbt^H19,22L or Mbt^T525A alone or in combination with Myc-tagged Cdc42 or Cdc42^G12V. Expression of Mbt was verified by western blot (WB). Mbt proteins were immunopurified from cell lysates using an anti-HA antibody and incubated in kinase buffer with myelin basic protein (MBP) as a substrate in the presence of [-³²P]ATP. Autophosphorylation of Mbt and MBP phosphorylation were analysed by autoradiography.