CXCR4 is a G-protein coupled receptor (GPCR) in the chemokine receptor subfamily, which mediates directional cell migration towards gradients of SDF ligand. CXCR4 is critical for migration of select neuronal and hematopoietic cell types, as well as for the migration and invasion of breast cancer cells whereby CXCR4 promotes a metastatic phenotype upon aberrant expression. Despite these observations, the biochemical signaling pathways downstream of motility inducing SDF gradients are unknown. To date for CXCR4, and all chemokine GPCRs, signaling profiles are constructed following receptor activation with strictly single and uniform concentrations of ligand. Previously, we identified unique pathways and sustained signaling kinetic signatures only revealed upon stimulation with gradients of SDF, including a novel Arf6-ERK motility pathway required for migration of metastatic breast cancer cells and a sustained Shp2-ERK signaling pathway atypical for GPCRs but reminiscent of growth factor receptor (GFR) signaling during malignancy. These findings have intensified efforts towards examining this unexplored area of gradient dependent signaling. Here, we identify an upstream regulator of sustained signaling kinetics upon SDF gradient sensing (PI3K), as well as identify a novel growth arrest pathway activated upon SDF gradient sensing (p53). Specifically, in AIM I, we further investigate the novel signaling kinetics of the ERK pathway, as well as identify a sustained Akt activation upon SDF gradient sensing. Our results demonstrate that SDF gradient sensing by endogenous CXCR4 promotes sustained ERK and Akt phosphorylation by immunoblot analysis and identify PI3K as an upstream regulator of both pathways, as supported by impaired signaling with PI3K inhibitors wortmannin and LY294002. In Aim II, we demonstrate that SDF gradient sensing by endogenous CXCR4 promotes a robust accumulation of p53, a known tumor suppressor and cell cycle inhibitor, with an associated increase in Ser15 and Ser46 phosphorylation. The SDF gradient-promoted p53 accumulation and Ser15 phosphorylation is not inhibited by the p38 MAPK inhibitor SB 202190, suggesting that despite p38 being a known kinase for p53 at this site, that p53 regulation downstream of CXCR4 signaling is p38-independent. Notably for Aim I and II, PI3K pathways are known to regulate processes critical to the malignant phenotype including cell motility and invadopodia formation, while p53 pathways are known to regulate cell cycle arrest. These aims address a working model that SDF gradient sensing by CXCR4 promotes a migratory phenotype by inducing sustained activation of pathways that promote cell motility, as well as inducing an inhibitory effect on the cell cycle through activation of growth inhibitory pathways. Our data provide a previously unknown link between PI3K motility pathways and p53 cell cycle arrest pathways, both of which are activated upon SDF gradient sensing by CXCR4.
|School:||University of the Sciences in Philadelphia|
|School Location:||United States -- Pennsylvania|
|Source:||MAI 55/06M(E), Masters Abstracts International|
|Subjects:||Cellular biology, Pharmacology, Oncology|
|Keywords:||Cell motility, Cxcr4, Gpcr signaling, P53, Pi3k, Sdf|
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