Investigate the interaction between the regulatory subunit B55δ and B56γ3 of protein phosphatase 2A and its selected substrates by a molecular imaging approach

摘要

Protein phosphatase 2A (PP2A) is a major eukaryotic protein serine-threonine phosphatase that comprises three subunits including a scaffolding A subunit a catalytic C subunit (PP2Ac) and a variable regulatory B subunit The highly variable B subunits have been believed to control the subcellular localization and substrate specificity of the PP2A holoenzyme However how the substrate specificity of PP2A is determined by the variable B subunits remains largely unclear Hyperphosphorylation of paraprotein target paratarg-7 (pP-7) is the strongest molecularly defined risk factor for monoclonal gammopathy of undetermined significance (MGUS) multiple myeloma (MM) and Waldenstrom’s macroglobulinemia (WM) It has been demonstrated that accumulation of pP-7 is due to deregulation of the PP2A in targeting to p-P7 in which inefficient dephosphorylation of p-p7 is caused by substitution of the regulatory subunit B55δ with B56γ3 in the PP2A-p-P7 complex in patients compared to that of healthy control individuals Surprisingly no mutation was found in cDNA of B55δ and expression levels of B55δ mRNA and protein are not different from that in healthy individuals In addition the B55δ-containing PP2A complex in the p-P7 carrier was functional in association and reacting with the known substrate securin These findings prompted us to investigate the interaction between different B subunit-containing PP2As and P7 or securin and to unveil the underlying mechanism of dysregulated targeting of PP2A in MGUS MM and WM By employing indirect fluorescence staining out data suggested that B55δ was mostly expressed in the cytoplasm whereas B56γ3 predominantly displayed a ubiquitous distribution Securin was distributed in either entire cells or expressed in cytoplasm of cells In addition P7 showed an exclusively cytoplasmic distribution that co-localized with mitochondria Next we ectopically co-expressed exogenous securin or P7 and B55δ or B56γ3 and performed co-immunoprecipitation analysis on interaction between securin or P7 and B55δ or B56γ3 Surprisingly securin undergoes protein degradation while co-expressing with B55δ or B56γ3 and the level of securin upon co-expression of B55δ or B56γ3 could be reversed by treating with proteasome inhibitor MG132 Results of co-IP showed that securin can associate with either B55δ or B56γ3 In contrast to securin the results of co-IP indicated that P7 physically associates with B55δ but not B56γ3 To gain more insights regarding the spatial distribution of the complex formed between securin or P7 and B55δ or B56γ3 We performed bimolecular fluorescence complementation (BiFC) Results of BiFC analysis showed that marked BiFC signals of various combinations of YN- YC-fused P7 or securin and YN- YC-fused B55δ or B56γ3 were detected only when A? was co-expressed BiFC analysis of various pairs of YN- or YC-fused securin and B55δ or B56γ3 revealed that the highest BiFC signals were generated from the combination of securin-YC and YN-B55δ and YC-securin and YN-B56γ3 complexes On the other hand marked BiFC signals were detected in the combinations of YN-P7 and B56γ3-YC and P7-YC and YN-B55δ complexes Taken together we found association between PP2A B55δ or B56γ3 with securin and association between P7 and B55δ but not B56γ3 by co-IP analysis On the other hand we successfully applied BiFC to visualize the complexes of B55δ or B56γ3 and securin or P7 in cells In agreement with the finding of co-IP BiFC results confirmed that securin associates with either B55δ or B56γ3 In contrast to securin BiFC results indicate that P7 associates with either B55δ or B56γ3 whereas the finding of co-IP demonstrates that P7 associates with B55δ but not B56γ3 Future investigations on the discrepancy of results of P7 association with different B subunits owing to the methods may provide more insights into the regulation of substrate specificity of PP2A and the dysregulation of PP2A regulatory subunits targeting to paratarget proteins in MGUS/MM/WM patients

獎項日期	2017 12月 1
原文	English
監督員	Chi-Wu Chiang (Supervisor)