Abstract 3323: PRAF2 regulates Rab5-dependent endocytic trafficking of EGFR in neuroblastoma

Abstract

Abstract Receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR), mediate extracellular signaling that activates downstream cell proliferation, differentiation, and migration. Through ligand stimulation, clathrin-mediated endocytosis regulates the EGFR signal transduction pathway. Prenylated Rab acceptor 1 domain family, membrane 2 (PRAF2) is a novel 19-kDa protein with a prenylated Rab acceptor 1 (PRA1) motif and four transmembrane-spanning domains. Similar to PRAF1 and PRAF3, PRAF2 belongs to the PRAF family of vesicle transport-associated proteins. Our lab identified PRAF2 as a candidate prognostic marker of neuroblastic tumors. Using RNA interference, we showed that PRAF2 plays an essential role in neuroblastoma tumorigenesis and metastasis. In the present study, we examined the function of PRAF2 in EGFR endocytic trafficking. We generated stable PRAF2-knockdown (SK-shPRAF2) and PRAF2-overexpressing (SK-PRAF2) neuroblastoma SK-N-SH cell lines and stimulated both cell lines with EGF (10 ng/ml) for various time periods in order to determine the role of PRAF2 in EGFR endocytic trafficking. As judged by immunofluorescence microscopy, we detected larger Rab5-positive punctuate structures in PRAF2-overexpressing SK-PRAF2 cells compared to PRAF2-knockdown SK-shPRAF2 cells or control cells, after 15 min EGF stimulation. After EGF treatment, PRAF2 clearly colocalized with Rab5-positive punctae in SK-PRAF2 cells, suggesting that PRAF2 interacts with Rab5, a critical regulator for receptor endocytosis and early endosome fusion. Moreover, using a receptor internalization assay, we observed a delay in the internalization of EGFR in EGF-treated SK-shPRAF2 cells, in contrast to SK-PRAF2 and control cells. This is in support of our immunofluorescence microscopy results in which Rab5-postive punctae distribution is observed mostly along the membrane of the EGF-treated SK-shPRAF2 cells. Notably, the level of phosphorylation of Akt (Ser473) transiently elevated in EGF-treated SK-PRAF2 cells after 15 min and diminished thereafter, suggesting that overexpression of PRAF2 accelerates the EGFR internalization and degradation. Based on our findings, we propose that PRAF2 affects EGFR endocytosis by regulating early endosome trafficking and Akt activation in neuroblastoma. Citation Format: Lisette P. Yco, Andre S. Bachmann. PRAF2 regulates Rab5-dependent endocytic trafficking of EGFR in neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3323. doi:10.1158/1538-7445.AM2014-3323