Electronic Thesis and Dissertation Repository

Degree

Master of Science

Program

Physiology

Supervisor

Dr. John Di Guglielmo

Abstract

The TGFβ pathway, which regulates cell proliferation and differentiation, has also been shown to induce non-small cell lung cancer cell (NSCLC) migration and invasion. The TGFβ pathway is initiated through the binding of TGFβ to cell surface Ser/Thr kinase receptors. Activated receptors then phosphorylate intracellular signaling proteins, termed Smads, which translocate into the nucleus to regulate transcriptional responses. The protein Smad anchor for receptor activation (SARA) facilitates the phosphorylation of Smads and allows for efficient signal transduction. On the other hand, the inhibitory Smad, Smad7, recruits the E3 ubiquitin ligase, Smurf2, to catalyze the degradation of TGFβ receptors. Since the signaling and degradation pathways target active receptor complexes, SARA and Smurf2-Smad7 may interact with common TGFβ receptors. Therefore, the Smurf2-Smad7 complex may affect SARA steady state levels and influence TGFβ signaling. I hypothesized that Smurf2-Smad7 induces SARA degradation through an ubiquitin-dependent pathway. I observed that SARA steady state levels decrease in the presence of Smurf2 and Smad7, and this is dependent on the HECT E3 ubiquitin ligase activity of Smurf2. In addition, I observed that SARA interacts with ubiquitinated proteins and is protected from degradation by the pharmacological inhibition of the proteasome. Finally, I assessed the functional outcome of reducing endogenous SARA levels. I observed that siRNA directed at SARA decreased both TGFβ-dependent Smad2 phosphorylation as well as EMT. These data suggest that the interplay between SARA and Smurf2-Smad7 complexes can influence TGFβ receptor signaling and may provide for a novel approach in targeting this pathway in NSCLC.

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