Biochemical analysis of the interactions of IQGAP1 C-terminal domain with CDC42.

ABSTRACT:

Aim

To understand the interaction of human IQGAP1 and CDC42, especially the effects of phosphorylation and a cancer-associated mutation.

Methods

Recombinant CDC42 and a novel C-terminal fragment of IQGAP1 were expressed in, and purified from, Escherichia coli. Site directed mutagenesis was used to create coding sequences for three phosphomimicking variants (S1441E, S1443D and S1441E/S1443D) and to recapitulate a cancer-associated mutation (M1231I). These variant proteins were also expressed and purified. Protein-protein crosslinking using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide was used to investigate interactions between the C-terminal fragment and CDC42. These interactions were quantified using surface plasmon resonance measurements. Molecular modelling was employed

SUBMITTER: Elliott SF 

PROVIDER: S-EPMC3312201 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature