Abstract 529: Phosphorylation of Thr165 within the activation loop of Nek3 kinase is necessary for its pro-migratory function in breast cancer cells
Authors: Katherine M. Harrington, Charles V. Clevenger
Journal: Cancer Research (2015)

Abstract The polypeptide hormone prolactin (PRL) is increasingly recognized as contributing to the development and progression of human breast cancer. Activation of the prolactin receptor (PRLr) by PRL contributes to the growth, survival and motility of human breast cancer cells. The PRL/PRLr complex activates multiple signaling pathways, including the serine/threonine kinase, Nek3 (NIMA-related kinase 3). PRL acts as a chemoattractant for breast cancer cells, accompanied by reorganization of the cytoskeleton. However, the molecular mechanisms by which PRLr signaling contributes to the invasive breast cancer cellular phenotype is not fully understood. Nek3 [NIMA (never in mitosis gene a)-related kinase 3] is a member of the serine/threonine (S/T) Nek protein kinases. Data from our laboratory suggests that Nek3 may contribute to breast cancer pathogenesis through regulation of cell migration/invasion, apoptosis, and cytoskeletal reorganization. Therefore, targeting Nek3 in breast cancer cells represents a novel therapeutic approach to potentially improve the treatment of breast cancer. We previously demonstrated that PRL/PRLr-signaling activates Nek3, however the molecular mechanisms of Nek3 kinase activation remain to be elucidated. Therefore, to better understand the mechanism of PRL-dependent regulation of Nek3 kinase activity, we focused on determining whether Nek3 activity is regulated by phosphorylation and to identify the potential sites(s) of autophosphorylation. Through site-directed mutagenesis of Nek3 residues in the activation segment, we identified threonine residue 165 (Thr165), as a major site that regulates the catalytic activity of Nek3. In addition to being autophosphorylated in vitro, Nek3 Thr165 was also phosphorylated in response to the PRL-MEK-ERK1/2 signaling pathway in PRL-stimulated breast cancer cells. We show here that phosphorylation at Thr165 Nek3 plays an important regulatory role in cell migration and adhesion, steps that are critical in the invasive process leading to tumor metastasis. In order to test this, we expressed wild-type Nek3 and a nonphosphorylatable Nek3 mutant (T165V) in the breast cancer cell lines T47D and MCF7. We found that expression of T165V Nek3 resulted in a significant decrease in cell adhesion to the basement membrane matrix, Matrigel, while additionally inhibiting cell motility in a wound healing assay. Ongoing studies are further examining the role of Nek3 in regulating focal adhesion dynamics and cytoskeletal remodeling. Citation Format: Katherine M. Harrington, Charles V. Clevenger. Phosphorylation of Thr165 within the activation loop of Nek3 kinase is necessary for its pro-migratory function in breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 529. doi:10.1158/1538-7445.AM2015-529