Rac1b, a variant of rac1, interacts with calmodulin
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Date
2014-04-10
Authors
Khanna, Neha
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Abstract
Rac1b, splice isoform of Rac1, is a member of the Rho family of small GTP-binding proteins that has been found to be up-regulated in the cancers of breast, colon and the lung. Rac1b consists of an additional 19 amino acid insertion [VGETYGKDITSRGKDKPIA] close to the switch II domain, a region important for the interaction of Rac1 with various regulators and effectors. This insertion leads to the intracellular predominance of active GTP-bound form of Rac1b and also renders it ineffective to interact with Rho guanine nucleotide dissociation inhibitors (Rho GDI’s).
Previously, a 14 amino acid region [AVKYLECSALTQRG] essential for calmodulin (CaM) binding has been established in Rac1. A similar region also exists in Rac1b. In the present work, we have determined that as for Rac1, Rac1b also interacts with calmodulin in a calcium dependent manner. We have also demonstrated that Rac1b binds to calmodulin directly. However, the putative CaM binding region in the two proteins differ as the commercially synthesized CaM binding peptide for Rac1 failed to compete with Rac1b for binding to calmodulin.
In addition, using the PAK-CRIB domain in pull down assays that interacts with the GTP-bound form of Rac1b, we have established that CaM plays an important role in the activation of Rac1b. Experiments using W7, the inhibitor for CaM, revealed that activation of Rac1b in the presence of W7 is reduced in response to growth factor agonists such as Heregulin β-1 and EGF. However, it was observed that the addition of W7 has no role in in vitro GDP/GTP binding to Rac1b. Molecular modeling and docking studies were also carried out to predict the possible sites in Rac1b that potentially can interact with CaM.
In summary, the results presented here demonstrate that CaM interacts with Rac1b in a calcium dependent manner. Additionally, CaM plays an important role in the activation of Rac1b thus indicating a role for CaM in cancer progression.
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Keywords
Rac1b, Calmodulin