FIG. 3.
Identification of the TCoB domains that interact with Pak1. (A) Pak1 cDNA encoding aa 1 to 545 was translated in vitro. 35S-labeled Pak1 was incubated with GST-TCoB or GST fusions of various lengths of TCoB in the GST pull-down assays, and the interaction was then analyzed by SDS-PAGE followed by autoradiography. WT, wild type; CLIP, MHC class II-invariant peptide. (B) Identification of the Pak1 domain that interacts with TCoB. Wild-type GST-Pak1 (aa 1 to 545) and GST-Pak1 fusion proteins containing the kinase domain (aa 270 to 545), N-terminal domain (aa 1 to 132), PIX binding domain (aa 132 to 270), NCK binding domain (aa 1 to 75), CRIB domain (aa 52 to 132), and autoinhibitory domain (aa 75 to 149) were incubated with in vitro-translated TCoB (amino acids 1 to 244). Binding was analyzed with GST pull-down assay followed by SDS-PAGE and autoradiography. PBD, protein binding domain. (C) TCoB stimulates Pak1 activity in vitro. Pak1 enzyme was incubated with either GTP-γS-loaded Cdc42 (6.7 mM) or purified TCoB (6.7, 13.3, or 20 mM) and incubated in kinase buffer containing [γ-32P]ATP, and Pak1 autophosphorylation (auto phos.) was measured by autoradiography. (D) Pak1 enzyme was incubated with 16.7, 33.3, or 50 mM GST protein or GST-TCoB for 30 min, and the ability of Pak1 to phosphorylate MBP was analyzed by in vitro kinase reaction. Ponceau stains for GST loading and Pak1 immunoblot are shown below. (E) Ability of various TCoB domains to stimulate Pak1 activity as determined by in vitro kinase assay. (F) MCF-7 cells were cotransfected with Myc-tagged Pak1, with or without increasing amounts of wild-type TCoB or TCoB (aa 140 to 244). Cells were serum starved for 24 h, and Pak1 activity was measured by immunoprecipitation with Myc-tagged antibody followed by in vitro kinase assay using MBP as a substrate.