REGULATION OF AKT SIGNALING BY PHOSPHOINOSITIDE DYNAMICS WITHIN CLATHRIN COATED PITS

Candidate: Rebecca Cabral-Dias
Supervisor: Dr. Costin Antonescu

ABSTRACT

The Epidermal Growth Factor (EGF) Receptor (EGFR) controls many aspects of cell physiology including proliferation, survival, migration, and metabolism. Upon ligand binding, EGFR activates several signaling intermediates, leading to the downstream activation of phosphatidylinositol-3-kinase (PI3K) and Akt, and is simultaneously recruited to clathrin-coated pits. We previously uncovered that perturbation of clathrin, but not of receptor endocytosis, impairs EGF-stimulated activation of Akt signaling, and that some EGFR signaling intermediates such as phosphorylated Gab1 are enriched within a subset of clathrin-coated pits. We proposed that clathrin-coated pits have a direct role to control EGFR signaling at the plasma membrane prior to receptor internalization. How clathrin-coated pits and clathrin accessory proteins may control EGFR signaling was poorly understood, which I have examined here. I uncovered that the clathrin-binding protein target of myb-like 1 (TOM1L1) and the TOM1L1-binding Src-family kinase Fyn are recruited to a subset of clathrin-coated pits with unique properties including distinct lifetimes and protein composition. Knockdown-rescue experiments with novel stable cell lines expressing specific TOM1L1 mutants, including a Clathrin-binding and Fyn-binding mutant, revealed that clathrin-binding by TOM1L1 was essential for EGF-stimulated Akt phosphorylation and Fyn recruitment to clathrin-coated pits.

Perturbation of TOM1L1 or Fyn impaired EGF-stimulated Akt phosphorylation, specifically that of Akt2 but not Akt1. Previous reports indicated that the 5-phosphatase SHIP2 produces phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2) at the plasma membrane and that PI(3,4)P2 selectively activates Akt2. Using total internal reflection fluorescence microscopy and automated image analysis, I uncovered that EGF stimulation also triggered the TOM1L1-and Fyndependent recruitment of the phosphoinositide 5-phosphatase SHIP2 to clathrin-coated pits in ARPE-19 and MCF10A cells. These results suggest that clathrin-coated pit-localized TOM1L1/Fyn signals control localized phosphoinositide dynamics, specifically growth-factor stimulated PI(3,4)P2 production by SHIP2. I propose that a distinct subset of clathrin-coated pits become specialized as signaling-capable structures by enrichment with TOM1L1 and Fyn and are required for EGFR signaling leading to Akt2 activation.

I also defined that the recruitment of SHIP2 to clathrin-coated pits not only required the signaling proteins TOM1L1 and Fyn, but also the clathrin binding adaptors intersectin 1 (ITSN1) and Dab2 in ARPE-19 cells. As well, I uncovered that triple-negative breast cancer MDA-MB- 231 cells minimally recruit SHIP2 to clathrin-coated pits, despite expressing both TOM1L1 and Fyn. MDA-MB-231 cells preferentially express Akt1 and activate significantly less Akt2 following EGF stimulation compared to levels in ARPE-19 cells. Notably, the overexpression of ITSN1 in MDA-MB-231 cells rescues the recruitment of SHIP2 into clathrin-coated pits. These results suggest that there are distinct complements of proteins in clathrin-coated pits in various cells, and that this cell-context specific cohort of clathrin-coated pit proteins establishes different abilities to recruit SHIP2 and activate different Akt isoforms in a cell-specific manner. In other words, this establishes for the first time a mechanism by which clathrin endocytic proteins define the isoform-selective activation of Akt. Due to the significant role EGFR plays in driving growth and survival in triple-negative breast cancer cells, it is important to gain a better understanding of the impact that these distinct complements of proteins in clathrin-coated pits have on EGFR signaling within the plasma membrane. This molecular insight may lead to the development of novel ani-cancer treatments.