I PP2A 1 Affects Tau Phosphorylation via Association with the Catalytic Subunit of Protein Phosphatase 2A

Abstract

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 ⌬C2). Double immunofluorescence staining showed that I 1 PP2A and I 1 PP2A ⌬C2 increased Tau phosphorylation and impaired the microtubule network and neurite outgrowth in PC12 cells treated with nerve growth factor.

Cytoplasmic Retention of Protein Phosphatase 2A Inhibitor 2 (I2PP2A) Induces Alzheimer-like Abnormal Hyperphosphorylation of Tau

“…Our data are consistent with our previous observation that I 1 PP2A , another endogenous inhibitor of PP2A, impairs MT network and neurite outgrowth via hyperphosphorylation of Tau (41). Although our data do not imply a direct role of I 2 PP2A in microtubule instability, it is well recognized that once Tau is abnormally hyperphosphorylated, it can disrupt microtubules by sequestering normal microtubule-associated proteins (61).…”

“…PP2A has been reported to interact physically with its endogenous inhibitors I 1 PP2A and I 2 PP2A and its cleavage fragments and thus inhibits its activity (31,41). In the present study, we validated the newly developed cellular model in which cytoplasmic localization of I 2 PP2A leads to increased physical association with PP2Ac and inhibits its activity.…”

“…The functional aftermath of abnormal hyperphosphorylation of Tau is the disruption of microtubule stability, which could reduce the number and the length of neurite outgrowth (41). To investigate this possibility, we differentiated the stable cell lines of I 2 PP2A WT and I 2 PP2A AA-AAA with 100 ng/ml NGF for 5 days and then carried out immunofluorescence using rabbit polyclonal antibody to HA and mouse monoclonal antibody DM1A to tubulin.…”

“…Each experimental condition was calculated using the average number of 5 fields/well for 3-4 wells. The numbers of cells bearing neurites and the length of the neurites were analyzed by 25-30 randomly selected NGFdifferentiated PC12 cells using ImageJ software, as described previously (41).…”

GBA deficiency promotes SNCA/α-synuclein accumulation through autophagic inhibition by inactivated PPP2A

“…43,44 PPP2C phosphorylation at Tyr307 decreases PPP2A activity by 90% in vitro, 19 and is increased by the loss of GBA function. To determine the mechanism of PPP2A inactivation, the methylation status of PPP2A was examined.…”