Date of Degree

2-2014

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor(s)

Cathy Savage-Dunn

Subject Categories

Biochemistry | Biology | Developmental Biology

Keywords

C. elegans, protein phopshatases, reversible phosphorylation, Sma/Mab pathway, TGF-beta

Abstract

TGF-beta signaling is a conserved signaling pathway among eukaryotes, which controls various normal cellular responses from cell proliferation to cell death. The mutations in its components are found in developmental disorders and cancer. Therefore, this signaling pathway is extensively investigated so that new therapeutic targets could be discovered and novel drugs could be developed. Previous studies suggested the involvement of phosphatases in regulation of TGF-beta signaling, but these studies were performed in cell culture rather than intact organisms. C. elegans is a tractable organism in which to study signaling in vivo. In C. elegans, growth is controled by a conserved TGFβ pathway, the Sma/Mab pathway.

We used a C. elegans RNA interference library of phosphatases to identify genes that cause a body size phenotype. Library-wide screening was carried out in an RNAi-hypersensitive mutant background, rrf-3. To further narrow the candidate pool, we analyzed the body size phenotypes of these candidates using different genetic backgrounds. These analyses allow us to narrow the candidate pool down to 80-candidates.

A Sma/Mab pathway reporter, RAD-SMAD reporter (a kind gift from Dr. Jun Liu, Cornell University), was used to assess whether the candidates regulate body size phenotype in a Sma/Mab pathway-dependent manner or not. The reporter assay revealed 22 likely candidates regulate Sma/Mab signaling directly or indirectly. Among them, 11 candidates were verified as protein phosphatases by sequencing. These include homologues of mammalian PPM1A/B/G and PP1.

Our study revealed that ppm-1 (metal-ion dependent protein phosphatase-1), a human PPM1A/B/G homolog, might act as a potential SMA-3-specific linker phosphatase to regulate the Sma/Mab pathway. This is the first time to show that a homolog of human PPM1A/B/G might act as a potential linker phosphatase of R-Smads to promote TGFβ signaling. Our studies also showed that a homolog of the inhibitory regulatory subunit of human PP1 might synergize with the catalytic subunit in the regulation of TGFβ signaling for the first time. We also discovered the regulatory role of several protein tyrosine phosphatases in this signaling cascade. In summary, this study sheds a light on elucidating the regulatory mechanism of TGFβ signaling pathway, therefore providing insight in various TGFβ signaling-involved human developmental disorders and cancer, and contributing to the development of potential diagnostic markers and therapeutic targets in human diseases.

 
 

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