Fig 1.

The first and the second cysteine residues of the CCaX motif of bCdc42 are prenylated and palmitoylated, respectively. (A) The C-terminal amino acid sequences of human Cdc42 and bCdc42 are shown. Amino acids distinguishing the two isoforms are shown in boldface type. The CaaX motif is underlined. (B) HEK293 cells transfected with FLAG-Cdc42 or FLAG-bCdc42 were incubated in medium containing 100 μM palmitic acid (PA) or 100 μM 17-ODYA (OD) for 6 h. Lysates were immunoprecipitated (IP) with anti-FLAG antibody, and 17-ODYA incorporated into bCdc42 was detected with Alexa Fluor 488-azide using click chemistry. bCdc42 labeled with Alexa Fluor 488 was separated by SDS-PAGE and analyzed by in-gel fluorescence. Cdc42 and bCdc42 protein expression in the immunoprecipitates and cell lysates was analyzed by immunoblotting (IB). (C) HEK293 cells expressing FLAG-bCdc42 were labeled with 100 μM 17-ODYA for 6 h. Geranylgeranyltransferase inhibitor (5 μM; GGTI) or farnesyltransferase inhibitor (5 μM; FTI) was added to the medium 18 h prior to labeling with 17-ODYA. 2-Bromopalmitate (100 μM; 2-BP) or dimethyl sulfoxide (DMSO) was added to the medium 1 h prior to labeling with 17-ODYA. 17-ODYA incorporated into bCdc42 was detected with Alexa Fluor 488-azide. Palmitoylated bCdc42 was quantified (bar graph). Data represent the means ± SDs (n = 3). **, P < 0.01 (Student's t test). (D) HEK293 cells expressing FLAG-bCdc42 were labeled with 30 μM geranylgeranyl-azide (GG-azide) or farnesyl-azide (Far-azide) for 24 h in the presence or absence of 5 μM GGTI or FTI. Geranylgeranyl-azide or farnesyl-azide incorporated into bCdc42 was detected with Alexa Fluor 647-alkyne. (E and F) HEK293 cells transiently transfected with FLAG-Cdc42, wild-type FLAG-bCdc42 (WT), or CCaX motif mutants of FLAG-bCdc42 (SC, CS, and SS) were labeled with 30 μM geranylgeranyl-azide (D) or 100 μM 17-ODYA (E) for 24 h or 6 h, respectively. Geranylgeranylation and palmitoylation were detected with Alexa Fluor 647-alkyne and Alexa Fluor 488-azide, respectively.