Functional analysis of the domains involved in regulating nuclear localization of the B56γ3 regulatory subunit of protein phosphatase 2A

  • 高 士綱

Student thesis: Master's Thesis

Abstract

Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase and participates in numerous cellular processes in eukaryotic cells The PP2A holoenzyme comprises three subunits including the scaffolding A subunit the catalytic C subunit and the variable regulatory B subunit The regulatory B subunits are determine the substrate specificity and subcellular localization of PP2A Our study has focused on B56γ3 which belongs to the B’ (B56/PR61) family and has been shown to contribute to the major tumor suppressor activity of PP2A B56γ3 is ubiquitously distributed in the entire cell ranging from the cytoplasm to the nucleus Sequence analysis predicted four putative nuclear localization signals (NLSs) which cluster in the C-terminus of B56γ3 and were denoted as NLS1 NLS2 NLS3 and NLS4 NLS1 is separated from the other NLSs by a 49-amino acid linker domain Previously our laboratory has shown that Ser440 in the linker domain can be phosphorylated by CK1 and AMPK and regulates B56γ3 nuclear localization In addition our laboratory has identified two domains involved in regulating B56γ3 nuclear localization including the 49-amino acid linker domain (aa 413 to aa461) and the domain encompassing aa 306 to aa 405 which contains the 100 amino acids upstream of the putative NLS1 Loss of either of these domains renders B56γ3 predominantly cytoplasmic In this study we identified the putative NLS1 as an additional domain involved in regulating B56γ3 nuclear localization and characterized the function of each domain in regulating B56γ3 nuclear localization We found that the linker domain (aa 413-aa 461) can target a cytoplasmic localized GFP-fused pyruvate kinase (GFP-PK) to the nucleus Neither NLS1 nor the domain encompassing aa306 to aa405 alone can target GFP-PK to the nucleus Nevertheless the segment encompassing both NLS1 and 100 amino acids upstream of NLS1 (aa 306 to aa 405) can drive the nuclear localization of GFP-PK Next we addressed whether these regions mediate B56γ3 nuclear localization by binding to importin-? (Imp-?) or importin-β (Imp-β) By co-immunoprecipitation analysis we found that full-length B56γ3 the segment containing aa 306 to aa 405 and the segment containing aa 306 to aa 412 can associate with both Imp-? and Imp-β However the linker domain and NLS1 associate only with Imp-? Results of in vitro pull down analysis demonstrate that the domain encompassing aa 306 to aa 405 can directly associate with Imp-β The phosphorylation defective Ser440A mutant of B56γ3 showed reduced nuclear localization when compared to that of the wild-type and reduced association with Imp-? without affecting association with Imp-β Furthermore knockdown of Imp-? or Imp-β impairs the activity of the full-length B56γ3 the segment encompassing aa 306 to 412 and the linker domain in driving GFP-PK into the nucleus Together our data suggest that three domains including residues 306-405 residues 406-412 and residues 413-461 may cooperate to associate with Imp-? and/or Imp-β to mediate B56γ3 nuclear localization
Date of Award2014 Sep 1
Original languageEnglish
SupervisorChi-Wu Chiang (Supervisor)

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