Within the phosphatase field, identification of regulatory subunits and associated proteins has proven successful in determining the cellular role and potential substrates of phosphatases. This has been especially valuable for the PPP family of phosphatases due to its complex association with myriad of regulatory subunits, which dictate the activity, localization and substrate specificity of the phosphatases. To identify interactors of protein phosphatase 4 (PP4), I established a sensitive affinity purification coupled with mass spectrometry (AP-MS) approach to generate a high-density PP4 interaction network from the soluble fraction of human cell extracts. Our proteomic approach uncovered previously identified as well as new interactors; some may function as auxiliary regulators and others may serve as potential substrates. One of the interactor identified is a novel cytosolic PP4 regulatory subunit, which we termed PP4R4. PP4R4 displays weak homology to the PP2A A subunit of PP2Ac, but interacts specifically with PP4c and does not function as a scaffolding subunit to bridge other known regulatory subunits of PP4c. Remarkably, the PP4 interaction network revealed significant enrichment for proteins involved in transcription elongation and RNA processing. These interactors associate exclusively with PP4R3-PP4R2-PP4c holoenzyme. Consistent with this finding, PP4R3A possesses characteristics resembling that of splicing and transcription factors. We provided evidence suggesting that depletion of PP4c significantly reduced the transcription-elongation regulated genes, JUN and FOS, and altered the exon inclusion of selective genes. Our results define a high-density interaction network for the mammalian PP4 and uncover a potential role PP4 play in regulating the process of transcription elongation and alternative splicing.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/33869 |
| Date | 06 December 2012 |
| Creators | Chen, Ginny |
| Contributors | Gingras, Anne-Claude |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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