The Adaptor Proteins p66Shc and Grb2 Regulate the Activation of the GTPases ARF1 and ARF6 in Invasive Breast Cancer Cells

• Published in: The Journal of biological chemistry Vol. 289; no. 9; pp. 5687 - 5703
• Main Authors: Haines, Eric, Saucier, Caroline, Claing, Audrey
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 28.02.2014; American Society for Biochemistry and Molecular Biology
• Subjects: Adaptor Proteins; ADP-Ribosylation Factor 1 - genetics; ADP-Ribosylation Factor 1 - metabolism; ADP-Ribosylation Factors - genetics; ADP-Ribosylation Factors - metabolism; ARF; Breast Cancer; Breast Neoplasms - genetics; Breast Neoplasms - metabolism; Breast Neoplasms - pathology; Cell Line, Tumor; Enzyme Activation - genetics; Epidermal Growth Factor - genetics; Epidermal Growth Factor - metabolism; Epidermal Growth Factor Receptor (EGFR); ErbB Receptors - genetics; ErbB Receptors - metabolism; Female; GRB2 Adaptor Protein - genetics; GRB2 Adaptor Protein - metabolism; GTPase; Humans; Neoplasm Invasiveness - genetics; Neoplasm Invasiveness - pathology; Shc Signaling Adaptor Proteins - genetics; Shc Signaling Adaptor Proteins - metabolism; Signal Transduction; Src Homology 2 Domain-Containing, Transforming Protein 1; ARF; Epidermal Growth Factor Receptor (EGFR); Adaptor Proteins; Breast Cancer; GTPase
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.516047

Signals downstream of growth factor receptors play an important role in mammary carcinogenesis. Recently, we demonstrated that the small GTPases ARF1 and ARF6 were shown to be activated downstream of the epidermal growth factor receptor (EGFR) and act as a key regulator of growth, migration, and invasion of breast cancer cells. However, the mechanism via which the EGFR recruits and activates ARF1 and ARF6 to transmit signals has yet to be fully elucidated. Here, we identify adaptor proteins Grb2 and p66Shc as important regulators mediating ARF activation. We demonstrate that ARF1 can be found in complex with Grb2 and p66Shc upon EGF stimulation of the basal-like breast cancer MDA-MB-231 cell line. However, we report that these two adaptors regulate ARF1 activation differently, with Grb2 promoting ARF1 activation and p66Shc blocking this response. Furthermore, we show that Grb2 is essential for the recruitment of ARF1 to the EGFR, whereas p66Shc hindered ARF1 receptor recruitment. We demonstrate that the negative regulatory role of p66Shc stemmed from its ability to block the recruitment of Grb2/ARF1 to the EGFR. Conversely, p66Shc potentiates ARF6 activation as well as the recruitment of this ARF isoform to the EGFR. Interestingly, we demonstrate that Grb2 is also required for the activation and receptor recruitment of ARF6. Additionally, we show an important role for p66Shc in modulating ARF activation, cell growth, and migration in HER2-positive breast cancer cells. Together, our results highlight a central role for adaptor proteins p66Shc and Grb2 in the regulation of ARF1 and ARF6 activation in invasive breast cancer cells. ADP-ribosylation factors (ARF) 1 and 6 play an important role in breast cancer cell proliferation, migration, and invasion. In invasive breast cancer cells, EGF-mediated ARF1 and ARF6 activation is regulated by p66Shc and Grb2. Adaptor proteins are required to promote ARF activation. Understanding the signaling mechanisms leading to ARF activation may identify novel therapeutic targets for the treatment of breast cancer.