Abstract 472: Nek3 kinase in breast cancer pathogenesis: The role of paxillin phosphorylation

Abstract

Abstract Breast cancer progression to metastatic disease is responsible for the majority of breast cancer related deaths. The acquisition of a motile and invasive phenotype is a critical step in the metastatic process. The hormone prolactin (PRL) is increasingly recognized as contributing to the development and progression of human breast cancer. In particular, data from our laboratory has shown that PRL serves as a chemoattractant for breast cancer cells and can stimulate the formation of the focal-adhesion complex that is necessary for cell motility. PRL mediates its biological effects through binding to the PRL receptor (PRLr) and initiates several downstream signaling cascades, including activation of the serine/threonine kinase, Nek3. Nek3 regulates breast cancer cell survival, motility and invasion and has been shown to interact with the focal-adhesion adaptor protein, paxillin, in a PRL-dependent manner. Interestingly, we have determined that knockdown of Nek3 by siRNA significantly decreases the phosphorylation of paxillin, which suggests that paxillin may represent a novel substrate of Nek3. Given these findings, we sought to further elucidate the role of Nek3 in the regulation of paxillin phosphorylation and function in breast cancer. Co-immunoprecipitation analysis using mutant truncated proteins demonstrated that Nek3 interacts with the C-terminal region of paxillin, which contains 4 tandem LIM domains. Additionally, to determine the specific site(s) of phosphorylation an in vitro kinase assay was performed and it was determined that Nek3 directly phosphorylates paxillin at T401 located in the LIM2 domain. It is known that LIM2 and LIM3 comprise the focal adhesion targeting motif of paxillin and phosphorylation in this region plays an important role in modulating its activity. Ongoing studies will determine the biological effects of Nek3-mediated phosphorylation at paxillin T401 by examining the effect on breast cancer cell motility and invasion as well as the effect on paxillin localization and function in the focal adhesion complex. These studies will provide novel mechanistic insight into how Nek3-mediated phosphorylation of paxillin contributes to the pathogenesis of breast cancer. We propose that Nek3 is a potential novel therapeutic target for the treatment of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 472. doi:1538-7445.AM2012-472