Similar requirement for clathrin in EGF- and HGF- stimulated Akt phosphorylation

Stefanie Lucarelli; Rohan Pandey; Gurjeet Judge; Costin N Antonescu

doi:10.1080/19420889.2016.1175696

Similar requirement for clathrin in EGF- and HGF- stimulated Akt phosphorylation

Commun Integr Biol. 2016 Apr 21;9(3):e1175696. doi: 10.1080/19420889.2016.1175696. eCollection 2016 May-Jun.

Authors

Stefanie Lucarelli¹, Rohan Pandey², Gurjeet Judge¹, Costin N Antonescu³

Affiliations

¹ Department of Chemistry and Biology, Ryerson University, Toronto, Ontario, Canada; Graduate Program in Molecular Science, Ryerson University, Toronto, Ontario, Canada.
² Department of Chemistry and Biology, Ryerson University , Toronto, Ontario, Canada.
³ Department of Chemistry and Biology, Ryerson University, Toronto, Ontario, Canada; Graduate Program in Molecular Science, Ryerson University, Toronto, Ontario, Canada; Keenan Research Centre for Biomedical Science of St. Michael's Hospital, Toronto, Ontario, Canada.

Abstract

Receptor tyrosine kinases, such as the epidermal growth factor (EGF) receptor (EGFR) and Met lead to activation of intracellular signals including Akt, a critical regulator of cell survival, metabolism and proliferation. Upon binding their respective ligands, each of these receptors is recruited into clathrin coated pits (CCPs) eventually leading to endocytosis. We have recently shown that phosphorylation of Gab1 and Akt following EGFR activation requires clathrin, but does not require receptor endocytosis. We examined whether clathrin regulates Akt signaling downstream of Met, as it does for EGFR signaling. Stimulation with the Met ligand Hepatocyte Growth Factor (HGF) leads to enrichment of phosphorylated Gab1 (pGab1) within CCPs in ARPE-19 cells. Perturbation of clathrin using the inhibitor pitstop2 decreases HGF-stimulated Akt phosphorylation. These results indicate that clathrin may regulate Met signaling leading to Akt phosphorylation similarly as it does for EGFR signaling.

Keywords: TIRF microscopy; clathrin; endocytosis; membrane traffic; phosphorylation; receptor tyrosine kinase; signaling adaptor; signaling microdomain; signaling scaffold.