p104 Binds to Rac1 and Reduces Its Activity during Myotube Differentiation of C2C12 Cell
Figure 2
The carboxy-terminal region of p104 was essential for the interaction with Rac1. (a) Schematic representation of the constructs used in this experiment. p104 was divided into three regions and inserted into the pCMV Taq2B vector. Full-length (FL) 104 contains the entire open reading frame (ORF), and constructs I, II, and III contain 7–352, 353–609, and 611–898 amino acid residues, respectively. (b) p104 interacts with Rac1 through the carboxy-terminal 611–898 amino acid region. NIH3T3 cells were transfected with a Flag-tagged p104 construct. After 24 h, 2 mg of clarified cell lysates was immunoprecipitated with an anti-Flag antibody followed by Western blot analysis using an anti-Rac1 antibody (upper panel). The immunoprecipitated Flag-tagged protein was confirmed with an anti-FLAG antibody (lower panel). (c) The carboxy-terminal region of p104 is required for the association with Rac1 in mammalian cells. Full-length p104 (FL), carboxy-terminal deleted p104 (C), and carboxy-terminus of p104 (C) were inserted into a GFP expression vector (G), and the constructs were transfected into NIH3T3 cells. Twenty-four hours after transfection, the interaction between various truncated forms of p104 and Rac1 was assayed by immunoprecipitation with the anti-Rac1 antibody followed by Western blot analysis using an anti-GFP mouse monoclonal antibody. Ext: cell extract, IgG H: immunoglobulin heavy chain, and IgG L: immunoglobulin light chain. (d) The 814–848 residue of p104 is required for the association with Rac1. Two micrograms of GST-fused p104 I, II, and III was used for the GST pull-down assay. Prepared GST-fused p104 I, II, and III were incubated with 1 mg of mouse brain extract, and bound proteins were then subjected to Western blot analysis to detect Rac1. p104 III (amino acid residues 611–898) was subdivided into three parts, consisting of residues 611–726 (III-1), 698–814 (III-2), and 783–898 (III-3). Each DNA fragment was inserted into a pGEX 4T1 vector and a GST-pull-down assay was carried out. Again, p104III-3 was divided into III-3A (783–848) and III-3B (870–898) and then inserted into the pGEX 4T1 vector. Each construct was used in the in vitro binding assay as described above. A coomassie brilliant blue stained SDS-PAGE gel showed that equal amounts of fusion proteins were used.
