Identification of amino acids involved in Cdc42-calmodulin interaction and regulation of Cdc42 activation

• Main Author: Grewal, Navneet
• Other Authors: Bhullar, Rajinder (Oral Biology)
• Published: 2015
• Subjects: Cdc42
• Online Access: http://hdl.handle.net/1993/30716

Cdc42 is a member of Rho family of Ras GTPase superfamily and has been shown to regulate actin cytoskeleton re-organization and filopodia formation. Calmodulin (CaM) is a calcium modulating protein and regulates calcium dependent signal transduction pathways in the cell. According to CaM target database analysis, amino acid region 151-163 of Cdc42 has a potential CaM binding domain that interacts with CaM. In the present work, we have investigated putative CaM binding region in Cdc42. In addition the role of basic amino acids K153 and K163 within this region in Cdc42 interaction with CaM and effect on Cdc42 activity was elucidated. GST-Cdc42 M (Δ151-163), GST-Cdc42K153A and GST-Cdc42K163A mutants were generated. Binding assay experiments showed that amino acid region 151-163 in Cdc42 is an important regulatory domain for CaM binding. Results also demonstrated that K163A mutant showed significantly reduced binding to CaM, whereas Cdc42 K153A showed reduced but non-significant decrease in its interaction with CaM. A previous study in our laboratory has shown that CaM plays critical role in maintaining basal activity of Cdc42 suggesting that K153A and K163A mutants may play a role in regulating this basal activity. In CHRF 288-11 cells expressing mutant forms of Cdc42 (K153A & K163A), basal activation was significantly decreased as compared to wild type Cdc42. The decrease in basal activity in Cdc42 mutants was not due to an inability to bind GTP. In summary, the results demonstrated that K163 in Cdc42 is a critical amino acid for CaM interaction and in the regulation of basal activity of Cdc42. === October 2015