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Gastric hyperplasia in mice lacking the putative Cdc42 effector IQGAP1.

ABSTRACT: Human IQGAP1 is a widely expressed 190-kDa Cdc42-, Rac1-, and calmodulin-binding protein that interacts with F-actin in vivo and that can cross-link F-actin microfilaments in vitro. Recent results have implicated IQGAP1 as a component of pathways via which Cdc42 or Rac1 modulates cadherin-based cell adhesion (S. Kuroda et al., Science 281:832-835, 1998), whereas yeast IQGAP-related proteins have been found to play essential roles during cytokinesis. To identify critical in vivo functions of IQGAP1, we generated deficient mice by gene targeting. We demonstrate that IQGAP1 null mutants arise at normal frequency and show no obvious defects during development or for most of their adult life. Loss of IQGAP1 also does not affect tumor development or tumor progression, but mutant mice exhibit a significant (P < 0.0001) increase in late-onset gastric hyperplasia relative to wild-type animals of the same genetic background. While we cannot exclude that functional redundancy with IQGAP2 contributes to the lack of developmental phenotypes, the restricted expression pattern of IQGAP2 is not obviously altered in adult IQGAP1 mutant mice. Thus, IQGAP1 does not serve any essential nonredundant functions during murine development but may serve to maintain the integrity of the gastric mucosa in older animals.

SUBMITTER: Li S

PROVIDER: S-EPMC85173 | biostudies-literature | 2000 Jan

REPOSITORIES: biostudies-literature

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Gastric hyperplasia in mice lacking the putative Cdc42 effector IQGAP1.

Li S S Wang Q Q Chakladar A A Bronson R T RT Bernards A A

Molecular and cellular biology 20000101 2

Human IQGAP1 is a widely expressed 190-kDa Cdc42-, Rac1-, and calmodulin-binding protein that interacts with F-actin in vivo and that can cross-link F-actin microfilaments in vitro. Recent results have implicated IQGAP1 as a component of pathways via which Cdc42 or Rac1 modulates cadherin-based cell adhesion (S. Kuroda et al., Science 281:832-835, 1998), whereas yeast IQGAP-related proteins have been found to play essential roles during cytokinesis. To identify critical in vivo functions of IQGA ...[more]

PMID: 10611248

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Project description:AimTo understand the interaction of human IQGAP1 and CDC42, especially the effects of phosphorylation and a cancer-associated mutation.MethodsRecombinant 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 to make predictions about changes to the structure and flexibility of the protein which occur in the cancer-associated variant.ResultsThe novel, C-terminal region of human IQGAP1 (residues 877-1558) is soluble following expression and purification. It is also capable of binding to CDC42, as judged by crosslinking experiments. Interaction appears to be strongest in the presence of added GTP. The three phosphomimicking mutants had different affinities for CDC42. S1441E had an approximately 200-fold reduction in affinity compared to wild type. This was caused largely by a dramatic reduction in the association rate constant. In contrast, both S1443D and the double variant S1441E/S1443D had similar affinities to the wild type. The cancer-associated variant, M1231I, also had a similar affinity to wild type. However, in the case of this variant, both the association and dissociation rate constants were reduced approximately 10-fold. Molecular modelling of the M1231I variant, based on the published crystal structure of part of the C-terminal region, revealed no gross structural changes compared to wild type (root mean square deviation of 0.564 Å over 5556 equivalent atoms). However, predictions of the flexibility of the polypeptide backbone suggested that some regions of the variant protein had greatly increased rigidity compared to wild type. One such region is a loop linking the proposed CDC42 binding site with the helix containing the altered residue. It is suggested that this increase in rigidity is responsible for the observed changes in association and dissociation rate constants.ConclusionThe consequences of introducing negative charge at Ser-1441 or Ser-1443 in IQGAP1 are different. The cancer-associated variant M1231I exerts its effects partly by rigidifying the protein.

Dataset Information

Gastric hyperplasia in mice lacking the putative Cdc42 effector IQGAP1.

Publications

Gastric hyperplasia in mice lacking the putative Cdc42 effector IQGAP1.

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