Date of Award

2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Biology

Abstract

The chemokine receptor CXCR4 is a member of the G protein-coupled receptor (GPCR) family. The cognate ligand for CXCR4 is the C-X-C chemokine known as CXCL12. The CXCL12/CXCR4 signaling axis is essential for a number of developmental processes including organogenesis, vascularization of the GI tract and hematopoiesis. Dysregulated CXCR4 signaling is also implicated in a variety of pathological conditions such as WHIM (Warts, Hypogammaglobunemia, Infections and myelokathexis) syndrome, cardiovascular disease and cancer. Despite its role in several pathologies, the molecular mechanisms mediating CXCR4 signaling are not completely understood. Upon CXCL12 binding to CXCR4, several signaling pathways are activated including the Akt/mTOR pathway, which mediates several cellular responses including survival, proliferation, invasion, and migration. In the current dissertation project, we aim to elucidate the molecular mechanisms regulating CXCR4 promoted Akt signaling.

Akt is a serine/threonine kinase that is fully activated by a complex multistep process involving phosphorylation of two key amino acid residues. Akt is activated by phosphorylation at threonine residue 308 and serine residue 473 by phosphoinositide-dependent kinase (PDK1) and mechanistic/mammalian target of rapamycin complex 2 (mTORC2), respectively. The mechanisms by which GPCRs promote PDK1 mediated phosphorylation of Akt at T308 are well understood; however the mechanism by which mTORC2 phosphorylates Akt on S473 remain essentially unknown. mTORC2 is a multi-subunit kinase complex comprised of key units including rictor, Sin1 and mTOR, the catalytic subunit. An important subunit referred to as DEPTOR binds to mTOR and inhibits its catalytic activity. Aberrant expression of DEPTOR has been linked to altered mTORC2 kinase activity and Akt signaling in several cancers. However, the mechanisms that regulate DEPTOR expression remain poorly understood.

We set out to understand the mechanisms that regulates DEPTOR levels in cells and how this impacts the mTORC2/Akt axis in response to CXCR4 activation. We show for the first time, that CXCL12 stimulation leads to rapid degradation of DEPTOR through lysosomes and that the ESCRT (Endosomal Sorting Complex Required for Transport) pathway that sorts ubiquitinated membrane receptors, mediates lysosomal degradation of DEPTOR. Pharmacological inhibition of heterotrimeric G protein G, PI3K signaling and siRNA targeting ESCRTs blocks CXCR4 promoted degradation of DEPTOR. We also show that by promoting DEPTOR degradation, the ESCRT pathway mediates Akt signaling promoted by CXCR4. Depletion of ESCRTs by siRNA leads to increased levels of DEPTOR and attenuates CXCR4 promoted, G protein and PI3K dependent Akt activation and signaling, consistent with decreased mTORC2 activity. In addition, ESCRTs likely have a broader role in Akt signaling because ESCRT depletion also attenuates receptor tyrosine kinase promoted Akt activation and signaling. Collectively, our data reveal a novel role for the ESCRT pathway in promoting intracellular signaling, which may begin to identify the signal transduction pathways that are important in the physiological roles of ESCRTs and Akt.

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Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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