Functional characterization of cancer-associated Gab1 mutations.
Grb2-associated binder (Gab)1 is a docking protein that transduces signals from a variety of tyrosine kinases, including Met and the EGF receptor. While the related protein Gab2 is strongly implicated in human cancer, a role for Gab1 has been less clear. However, a screen for gene mutations in breast cancer identified two somatic mutations in Gab1, Y83C and T387N. In this manuscript we describe functional characterization of these Gab1 mutants, undertaken in the immortalized mammary epithelial cell line MCF-10A. Cells overexpressing Gab1 Y83C and T387N exhibited a more elongated, fibroblastic phenotype compared to wildtype Gab1 controls. Expression of Gab1 or the mutants promoted EGF-independent proliferation in monolayer culture to a similar degree. However, in Matrigel culture, both mutants enhanced the formation of acini exhibiting an aberrant, branched morphology. In addition, expression of the mutants modestly increased Erk activation. To gain further insights into the mechanism of action of these mutations, we mapped Gab1 phosphorylation sites by mass spectrometry. This detected phosphorylation of T387 but not Y83. Cellular stimulation with EGF or HGF led to a transient, or sustained, induction of T387 phosphorylation, respectively. Since T387 corresponds in position to Gab2 T391, which suppresses Gab2 signalling in a phosphorylation-dependent manner, these data support a model in which the T387N mutation abrogates negative feedback regulation of Gab1. These data indicate that aberrant Gab1 signalling can directly contribute to breast cancer progression, and that negative feedback sites in docking proteins can be targeted by oncogenic mutations.
|Authors||Ortiz-Padilla, C.; Gallego-Ortega, D.; Browne, B. C.; Hochgrafe, F.; Caldon, C. E.; Lyons, R. J.; Croucher, D. R.; Rickwood, D.; Ormandy, C. J.; Brummer, T.; Daly, R. J.|
|DOI||10.1038/onc.2012.271 onc2012271 [pii]|
|URL link to publisher's version||http://www.ncbi.nlm.nih.gov/pubmed/22751113|
|OpenAccess link to author's accepted manuscript version||https://publications.gimr.garvan.org.au/open-access/11322|