I1PP2A affects Tau phosphorylation via association with the catalytic subunit of protein phosphatase 2A

In Alzheimer disease (AD) brain, the level of I-1(PP2A), a 249-amino acid long endogenous inhibitor of protein phosphatase 2A(PP2A), is increased, the activity of the phosphatase is decreased, and the microtubule-associated protein Tau is abnormally hyperphosphorylated. However, little is known about the detailed regulatory mechanism by which PP2A activity is inhibited by I-1(PP2A) and the consequent events in mammalian cells. In this study, we found that both I-1(PP2A) and its N-terminal half I-1(PP2A(1-120)), but neither I-1(PP2A(1-163)) nor I-1(PP2A(164-249)), inhibited PP2A activity in vitro, suggesting an autoinhibition by amino acid residues 121-163 and its neutralization by the C-terminal region. Furthermore, transfection of NIH3T3 cells produced a dose-dependent inhibition of PP2A activity by I-1(PP2A). I-1(PP2A) and PP2A were found to colocalize in PC12 cells. I-1(PP2A) could only interact with the catalytic subunit of PP2A (PP2Ac) and had no interaction with the regulatory subunits of PP2A (PP2A-A or PP2A-B) using a glutathione S-transferase-pulldown assay. The interaction was further confirmed by coimmunoprecipitation of I-1(PP2A) and PP2Ac from lysates of transiently transfected NIH3T3 cells. The N-terminal isotype specific region of I-1(PP2A) was required for its association with PP2Ac as well as PP2A inhibition. In addition, the phosphorylation of Tau was significantly increased in PC12/Tau441 cells transiently transfected with full-length I-1(PP2A) and with PP2Ac-interacting I-1(PP2A) deletion mutant 1-120 (I-1(PP2A)Delta C2). Double immunofluorescence staining showed that I-1(PP2A) and I-1(PP2A)Delta C2 increased Tau phosphorylation and impaired the microtubule network and neurite outgrowth in PC12 cells treated with nerve growth factor.