Investigate the role of Serine 440 of the regulatory subunit B56γ3 in the assembly of B56γ3 with the protein phosphatase 2A core enzyme and B56γ3 regulation of p27KIP1

  • 洪 正昇

Student thesis: Master's Thesis

Abstract

Protein phosphatase 2A (PP2A) is a trimeric complex that consists of a scaffolding A subunit a catalytic C subunit and a variable regulatory B subunit The B subunits determine the substrate specificity and subcellular localization of the PP2A holoenzyme Accumulated evidence indicates that the B56γ-containing PP2A holoenzyme (PP2A-B56γ) plays a tumor suppressor role PP2A-B56γ3 reduces proliferation of an array of cancer cell lines through regulating levels nuclear localization and activity of p27KIP1 Our lab previously found that both AMP-activated protein kinase (AMPK) and casein kinase I (CKI) regulate phosphorylation of B56γ3 at Ser440 which is critically involved in regulating the nuclear localization of B56γ3 To investigate the role of Ser440 in regulating function of B56γ3 we have obtained anti-phospho-Ser440 (p-S440) custom-made antibody (Ab) to pursue our study However our data showed that the anti-pS440 Ab equally recognized both wild-type B56γ3(B56γ3WT) and the phosphorylation defective mutant B56γ3 S440A Interestingly another phosphorylation defective mutant B56γ3 S440I which was found in human lung carcinoma was poorly recognized by the antibody We found that B56γ3 S440I but not B56γ3 S440A showed significantly reduced association with the A subunit suggesting a defect in recruitment into the PP2A holoenzyme We performed bimolecular fluorescence complementation analysis (BiFC) to investigate the effect of Ser440 mutations on PP2A holoenzyme assembly and showed that BiFC signals were reduced when B56γ3S440A but not S440I bound with PP2A A subunit compared with B56γ3WT We found that both S440I and S440A mutants showed impaired inhibitory function on cell proliferation Besides results of co-immunoprecipitation (co-IP) showed that both B56γ3 S440I and B56γ3 S440A had reduced interaction with p27 KIP1 In consistent with the co-IP data we found that both B56γ3 S440I and B56γ3 S440A showed impaired activity in regulating levels of phosphorylation at Thr157 of p27KIP1 compared with B56γ3WT In summary we demonstrate that Ser440 plays a role in regulating assembly of B56γ3 with the AC core enzyme and in regulating interaction with p27KIP1 Therefore it is possible that mutation at Ser440 may result in loss of tumor suppression activity of PP2A-B56γ3
Date of Award2017 Dec 20
Original languageEnglish
SupervisorChi-Wu Chiang (Supervisor)

Cite this

Investigate the role of Serine 440 of the regulatory subunit B56γ3 in the assembly of B56γ3 with the protein phosphatase 2A core enzyme and B56γ3 regulation of p27KIP1
正昇, 洪. (Author). 2017 Dec 20

Student thesis: Master's Thesis