1. Cdc42.GTP directly activates PAK4.
Cdc42.GTP directly activates PAK4. (A) A schematic of the pull‐down protocol from COS7 cell lysates is shown. The PAK4 proteins were recovered as indicated, and assayed for activity towards glutathione S‐transferase (GST)–Raf13 (20 μg) in the presence of 10 μM [γ32‐P] ATP. The pS474 was probed using CS#3241. (B) Schematic of the active Cdc42–Gly6–PAK4 chimaera (Flag‐C–PAK4). (C) The relative in vitro kinase activity of purified PAK4 tested in three independent experiments was calculated after subtracting PAK4 KD (that is, background value) and setting the Flag–PAK4 value to 1. The bars represent the standard error for each measurement. (D) The efficiency of green fluorescent protein–GEFH1 binding to various Flag–PAK4 constructs as indicated was tested by coimmunoprecipitation. CA, constitutively active; Cat, PAK4(286–591); CRIB, Cdc42–Rac interaction/binding; GFP, green fluorescent protein; GST, glutathione S‐transferase; HA, haemagglutinin; IP, immunoprecipitated; KD, kinase dead; PAK, p21‐activated kinase; WB, western blot; WT, wild type; Δ50=PAK4a(51–591).
Yohendran Baskaran et al. EMBO Rep. 2012;13:
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