Mechanism of inhibition of PP2A activity and abnormal hyperphosphorylation of tau by I2(PP2A)/SET

Abstract

Protein phosphatase-2A (PP2A) activity, which is compromised in Alzheimer disease brain, is regulated by two endogenous inhibitors, one of them being I(2)(PP2A), a 277 amino acid long protein also known as SET. Here we report that both the amino terminal fragment (I(2NTF); aa 1-175) and the carboxy terminal fragment (I(2CTF); aa 176-277) of I(2)(PP2A) inhibit PP2A by binding to its catalytic subunit PP2Ac and cause hyperphosphorylation of tau. The C-terminal acidic region in I(2CTF) and Val 92 in I(2NTF) are essential for their association with PP2Ac and inhibition of the phosphatase activity.

Fig. 1. Inhibition of PP2A activity by I₂^PP2A and its interaction with PP2Ac

(A) NIH3T3 cells were transfected with different amounts of pCMV2B-I₂^PP2A to express FLAG-tagged I₂^PP2A or, as a control, with vector alone. After 48 hours transfection, cells were lysed and expression of PP2Ac, I₂^PP2A, and, as a loading control, β-actin were determined by Western blots. (B) The relative PP2Ac expression level was normalized by the expression of β-actin. PP2Ac expression level did not show any significant difference among different amounts of expression of I₂^PP2A in NIH3T3 cells. One way ANOVA, p = 0.1369; t-test, vector vs. 1 μg, p = 0.2189; vector vs. 2 μg, p = 0.5031; vector vs. 3 μg cDNA, p = 0.1992. (C) PP2A was immunoprecipitated from cell lysates with anti-PP2Ac and the phosphatase activity assayed colorimetrically using pNPP as a substrate. I₂^PP2A inhibited the PP2A activity in a pCMV2B-I₂^PP2A dose-dependent fashion. *p<0.05 compared with control. (D) GST-pull down assay. Rat brain extract (Ext.), used as a source of PP2A holoenzyme, was incubated with Sepharose 4B beads bearing GST, GST-I₂^PP2A, or GST-PP2A-A. After washing, bound PP2Ac, PP2A-B, and PP2A-A were detected by Western blots. The GST, GST-I₂^PP2A and GST-PP2A-A used in pull down assay are shown in the lowest panel. GST-I₂^PP2A pulled down PP2Ac (PP2A catalytic subunit) but not PR55α (PP2A Bα regulatory subunit) or PR65α (PP2A A regulatory subunit). From left to right, Lane 2 (Ext.) is input, and Lane 3 (GST/Ext.) is a negative control. Positions of protein size markers are indicated on the left of the panel. Error bars in panels B and C indicate means ± SE; n=3.

Fig. 2. Intracellular localization of I_2FL, I_2NTF, and I_2CTF with reference to tau

(A) I_2FL-myc, I_2NTF-myc, and I_2CTF-myc cDNA were generated using pEGFP-N3/I₂^PP2A (wt) as a template. The PCR inserts as well as pcDNA3.1 were digested with BamHl and Xhol. The products were subsequently ligated into pcDNA3.1 vector and transformed into E. Coli DH4-α. (B) After amplification, the purified plasmids were digested with BamHl and Xhol, separated by electrophoresis, and sequenced. (C) Left panel: Western blots specific for the C-terminal myc tag of each protein confirmed the expression of I_2FL and I_2NTF. Right panel: Western blots specific for the C-terminal myc tag of each protein confirmed the expression of I_2FL and I_2CTF. (D) Immunocytochemical staining of HEK293FT cells transfected with GFP-Tau and pcDNA3.1 or pcDNA3.1-I_2FL, -I_2NTF, or -I_2CTF. I_2FL tagged with c-myc was localized exclusively in the nucleus of HEK293FT cells. I_2NTF tagged with c-myc was diffused all over the HEK293FT cells. I_2CTF tagged with c-myc, although concentrated in the nucleus, was also found distributed within the cytoplasm. Magnification bar = 10 μm.

Fig. 3. Co-immunoprecipitation of PP2Ac with I_2FL, I_2NTF, or I_2CTF

HEK293FT cells transfected to overexpress c-myc-tagged I_2FL, I_2NTF or I_2CTF were lysed and c-myc tag antibody was used for immunoprecipitation. The pulled down proteins were separated on SDS-polyacrylamide gels, then transferred onto two stacked PVDF membranes of decreasing pore size and analyzed by Western blots. (A) Upper row: Western blots on the 0.45 μm PVDF membrane developed with c-myc tag antibody. Lower row: Western blots on the 0.22 μm PVDF membrane developed with c-myc tag antibody. I_2FL, I_2NTF, and I_2CTF were all immunoprecipitated with the c-myc antibody (see framed bands). (B) Western blots developed with anti-PP2Ac. NC=negative control, i.e. non-treated with the vector. Positions of protein molecular markers are shown on the left of the blots.

Fig. 4. Inhibition of PP2A activity by I_2FL and I_2NTF

(A) Relative PP2A activity in transiently transfected HEK293FT cells with N-terminal myc tagged I_2FL and I_2NTF. I_2FL and I_2NTF reduced PP2A activity to ~70% and ~50%, respectively, in HEK293FT cells. *p<0.05 compared with the vector transfection control. (B) Tau₄₄₁ stably transfected PC12 cells were transiently transfected to express myc tagged I_2FL or I_2NTF and tau phosphorylation at M4 (pT231/pS235), pS404, PHF-1 (pS396/404) and 12E8 (pS262/356), total tau (R134d), as well as transfected I₂^PP2A were detected by Western blots from same amounts of lysates. The myc-tagged proteins were detectedwith anti-myc monoclonal 9E10 (1:100 in 5% defatted milk and 0.1% Tween-20) from Millipore/Chemicon. (C) The level of phosphorylated tau was quantified by densitometry and normalized to the amount of total tau. *p<0.05 compared with the vector transfection control. Positions of protein size markers are indicated on the left of the blots. Positions of I_2FL and I_2NTF bands are labeled with arrows. NSB = non-specific band.

Fig. 5. Inhibition of PP2Ac activity by I_2FL and I_2CTF

(A) Relative PP2A activity in transiently transfected cells with C-terminal myc tagged constructs was detected as described in Fig. 1C. I_2FL reduced PP2A activity to ~69% whereas I_2CTF inhibited PP2A activity to ~47%. *p<0.05 compared with the vector transfection control. (B) Tau phosphorylation at M4 (pT231/pS235), pS404, pS262, 12E8 (pS262/356), total tau (R1334D) and I₂^PP2A were detected by Western blots from same amounts of lysates from I_2FL and I_2CTF transfected cells. The myc-tagged proteins were detected with anti-myc monoclonal 9B11 (1:1,000 in 5% defatted milk and 0.1% Tween-20). (C) The level of phosphorylated tau was quantified by densitometry and normalized using the amount of total tau. *p<0.05 compared with the vector transfection control. The rest of the details are the same as in Figure 4.

Fig. 6. Identification of minimal and critical regions of I₂^PP2A required for interaction with PP2Ac

(A) Schematic diagram of the constructs of functional domains of I₂^PP2A employed for the interaction studies. (B, C) GST-pull down assays with rat brain extract as a source of PP2A holoenzyme. The pull down assays were carried out using 0.5 μg of GST-fusion protein per mg brain extract, except in cases of GST-I_2CTF (0.1 μg was employed), and GST alone (double the amount, i.e. 1 μg was employed). I_2CTF, F4, and I_2NTF but not I_2NTFV92A interacted with PP2Ac. (D) Relative PP2A activity in transiently transfected NIH3T3 cells with C-terminal myc tagged constructs I_2NTF-myc can reduce PP2A activity and mutation of V92 to A in I_2NTF results in a complete loss of this inhibitory activity. *p<0.05.