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. 2008 Mar 4;105(9):3622-7.
doi: 10.1073/pnas.0709180105. Epub 2008 Feb 21.

Akt and CHIP coregulate tau degradation through coordinated interactions

Chad A Dickey  1 , John Koren, Yong-Jie Zhang, Ya-Fei Xu, Umesh K Jinwal, Morris J Birnbaum, Bobby Monks, Mei Sun, Jin Q Cheng, Cam Patterson, Rachel M Bailey, Judith Dunmore, Sareh Soresh, Carlos Leon, Dave Morgan, Leonard Petrucelli
Affiliations

Akt and CHIP coregulate tau degradation through coordinated interactions

Chad A Dickey et al. Proc Natl Acad Sci U S A. .

Abstract

A hallmark of the pathology of Alzheimer's disease is the accumulation of the microtubule-associated protein tau into fibrillar aggregates. Recent studies suggest that they accumulate because cytosolic chaperones fail to clear abnormally phosphorylated tau, preserving a pool of toxic tau intermediates within the neuron. We describe a mechanism for tau clearance involving a major cellular kinase, Akt. During stress, Akt is ubiquitinated and degraded by the tau ubiquitin ligase CHIP, and this largely depends on the Hsp90 complex. Akt also prevents CHIP-induced tau ubiquitination and its subsequent degradation, either by regulating the Hsp90/CHIP complex directly or by competing as a client protein with tau for binding. Akt levels tightly regulate the expression of CHIP, such that, as Akt levels are suppressed, CHIP levels also decrease, suggesting a potential stress response feedback mechanism between ligase and kinase activity. We also show that Akt and the microtubule affinity-regulating kinase 2 (PAR1/MARK2), a known tau kinase, interact directly. Akt enhances the activity of PAR1 to promote tau hyperphosphorylation at S262/S356, a tau species that is not recognized by the CHIP/Hsp90 complex. Moreover, Akt1 knockout mice have reduced levels of tau phosphorylated at PAR1/MARK2 consensus sites. Hence, Akt serves as a major regulator of tau biology by manipulating both tau kinases and protein quality control, providing a link to several common pathways that have demonstrated dysfunction in Alzheimer's disease.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The CHIP/Hsp90 complex is required for Hsp90 inhibitor-mediated Akt degradation. (A) HeLa cells transfected with siRNA pools for Hsp90α, CHIP, or a nonsilencing control were then transfected with WT Akt and treated with 0.5 μM 17-AAG for an additional 24 h. SiRNAs for both CHIP and Hsp90α prevented Akt degradation. (B) Brain homogenates from eight mice (four CHIP−/− and four CHIP+/+) were analyzed by Western blot using the indicated antibodies. Total Akt (P = 0.02) and pS9 GSK3β (P = 0.007) protein levels were significantly decreased (27% ± 7% and 39% ± 8%) in CHIP−/− mice compared with CHIP+/+ littermates after normalization to GAPDH and total GSK3β, respectively (SI Fig. 7B).
Fig. 2.
Fig. 2.
CHIP binds Akt in a predominantly Hsp70/90-dependent manner, promoting its ubiquitination and degradation regardless of phosphorylation state. (A) CHIP coimmunoprecipitation from brain lysates of Akt1−/− and Akt1+/+ mice showed CHIP binding to endogenous Akt1 only in WT (+/+) mice. (B) HEK293 cells were transfected with HA-tagged WT Akt and myc-tagged WT CHIP, myc-CHIP harboring the K30A mutation, or empty vector. Akt coimmunoprecipitation showed that the K30A mutation in CHIP abrogated Akt/CHIP complex formation. (C) Cells were transfected with WT Akt and ubiquitin and either myc-CHIP or empty vector. After 24 h, 1 μM epoxomicin was added to media for 6 h. Akt coimmunoprecipitation showed enhanced CHIP/Akt complexes and ubiquitination of Akt in the presence of epoxomicin. (D) Cells transfected with CHIP or control siRNA for 72 h were then treated with 0.5 μM 17-AAG and 50 μM cycloheximide, and cells were harvested at indicated time points. CHIP siRNA prevented endogenous Akt degradation.
Fig. 3.
Fig. 3.
Akt1−/− mice have reduced CHIP levels, and decreased Akt levels leads to lower CHIP expression. (A) Brain homogenates from six mice (three Akt1−/− and three Akt+/+) were analyzed by Western blot for total Akt levels, along with CHIP and pGSK3β S9 levels. For each of these proteins, expression was lower in Akt1−/− mice than in Akt1+/+ mice. Quantitation of CHIP reductions by densitometry showed a significant 17% reduction by Student's t test (P = 0.015) (SI Fig. 11A). (B) Hs578T cells transfected with Akt, CHIP, or control siRNAs for 72 h showed reductions in CHIP levels with either CHIP or Akt siRNA. (C) Cells were transfected in triplicate with the same siRNAs described in A. Real-time PCR for CHIP mRNA showed significantly reduced levels (>50%) in cells transfected with Akt siRNA. Primer specificity was confirmed with CHIP siRNA.
Fig. 4.
Fig. 4.
Reducing total Akt levels enhances phospho-tau degradation by Hsp90 inhibition and ubiquitination; altering PI3K activation reduces HSF1 activity but does not affect tau degradation. (A) Cells were transfected with control or Akt siRNA pools for 72 h and then transfected with V5-tau. After 24-h treatment with 0.5 μM 17-AAG or 100 μM LY294002 plus 17-AAG at the indicated concentrations, lysates were analyzed by Western blot. 17-AAG-mediated reductions in phospho-tau were enhanced by Akt siRNA but not LY294002. (B) Cells transfected with control or Akt siRNA for 48 h were then transfected with V5-tau, HA-ubiquitin, and myc-CHIP or empty vector. V5 coimmunoprecipitation revealed that Akt siRNA enhanced CHIP binding and ubiquitination of tau. (C) Cells were transfected with myc-CHIP and V5-tau or empty vector. CHIP/tau complexes were coimmunoprecipitated with an anti-myc antibody and incubated with 100 ng of recombinant Akt (rAkt) for 16 h. After washing, Western blot showed less tau bound to CHIP in those lysates incubated with rAkt. Inputs confirm the presence of rAkt in the unbound fraction.
Fig. 5.
Fig. 5.
PAR1 and Akt interact, enhancing the acceleration of phospho-tau accumulation; Akt1−/− mice have selectively reduced levels of tau phosphorylated at the PAR1 consensus sites on tau at S262/S356. (A) Cells were transfected with V5-tau and GSK3β alone or with either WT Akt or CA Akt harboring a myristoylation signal for 48 h and analyzed by Western blot. Tau phosphorylated at S396/S404 was elevated in cells cotransfected with each Akt plasmid and GSK3β. Total tau levels were also elevated (V5). Tau phosphorylated at S262/S356 was undetectable (data not shown). GAPDH was used for loading normalization. (B) Cells were transfected with V5-tau and PAR1 alone, or PAR1 together with either WT Akt or CA Akt, for 48 h and analyzed by Western blot. Tau phosphorylated at S396/S404 was elevated in cells cotransfected with each Akt plasmid and PAR1. Total tau levels were also elevated (V5). Tau phosphorylated at S262/S356 was detectable only in cells transfected with PAR1. GAPDH was used for loading normalization. (C Upper) Cells were transfected with WT Akt, myc-PAR1, or both for 48 h. Akt coimmunoprecipitation and myc immunoblotting showed that PAR1 bound to Akt. (C Lower) Reverse coimmunoprecipitation with myc demonstrated Akt binding with overexpressed Akt. Inputs refer to cell lysates before coimmunoprecipitation. (D) Brain homogenates from six mice (three Akt1−/− and three Akt+/+) were analyzed by Western blot using the indicated antibodies. Deletion of Akt1 was confirmed, and tau phosphorylated at S262/S356 was significantly lower in Akt1−/− mice. Total tau and GAPDH levels were unchanged.
Fig. 6.
Fig. 6.
Proposed mechanism by which unconventional Akt activity affects tau degradation and CHIP regulation. (Left) Under normal conditions, stress or Hsp90 inhibition (17-AAG) stimulates the CHIP/Hsp90 complex to recognize both Akt and phospho-tau, targeting them for proteasome-mediated degradation by ubiquitination. A consequence of reduced Akt levels would lead to decreased CHIP levels, either through direct transcriptional regulation or via a feedback mechanism. Akt may also regulate the CHIP/Hsp90 complex directly. (Right) As Akt levels increase with age or disease, the CHIP/Hsp90 complex either targets Akt over phospho-tau or alters CHIP activity. As tau accumulates, Akt and PAR1 synergize to enhance tau phosphorylation. Increased levels of Akt maintain CHIP expression.
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