Date of Award

2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pharmacology and Experimental Therapeutics

Abstract

The hypoxia inducible factors (HIF1α, HIF2α, HIF1β) promote transcription of genes that regulate glycolysis and cell survival and growth. Sprouty2 (Spry2) is a modulator of receptor tyrosine kinase signaling and inhibits cell proliferation via different mechanisms. Because of the seemingly opposite actions of the HIF and Spry2 on cellular processes, we hypothesized that Spry2 decreases the protein levels of HIF1α, HIF2α and HIF1β by enhancing the proximity of the HIF subunit to an ubiquitin ligase capable of degrading the subunit. Focusing on HIF1α as a prototypical alpha subunit, in a variety of tumor derived cell lines, Spry2 decreases the protein levels of HIF1α. We showed that this decrease is cause by Spry2 increasing the ubiquitylation and proteosomal degradation of HIF1α by enhancing the amount of pVHL bound to HIF1α. Spry2 also decreases in the mRNA levels of the HIF1α-regulated genes; specifically the genes that regulate glycolysis. Along the same lines, we demonstrated Spry2 decreases the HIF1α-sensitive glucose uptake.

In a cell type dependent manner, Spry2 reduced the protein levels of the other half of the transcription factor, HIF1β or Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT), by enhancing proteosomal degradation of ARNT. Spry2 also associates with ARNT. We showed that Nedd4-1, a ubiquitin ligase, regulates ARNT and participates in the ability of Spry2 to regulate the protein levels of ARNT. As a functional impact, we demonstrated that Spry2 reduced the mRNA levels of the ARNT/Aryl hydrocarbon receptor (AhR) regulated gene cytochrome P450 1A1 (CYP1A1), which regulates metabolism of environmental toxins. Together, these data suggest that Spry2 via Nedd4-1 enhances the proteosomal degradation of ARNT decreasing the mRNA levels of ARNT/AhR regulated genes.

Overall, my dissertation work has unveiled a novel mode of action for Spry2 by which Spry2 enhances the degradation of proteins that regulate gene transcription by associating with both the target protein and E3 ligase that ubiquitylates the target protein. Also, we have shown a new role for Spry2 in modulating biological processes regulated by HIF subunits, such as glucose uptake. This paradigm could contribute towards the tumor suppressive actions of Spry2 and has major implications in toxicology.

Creative Commons License

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

Available for download on Wednesday, June 23, 2021

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