Phosphorylation inhibits turnover of the tau protein by the proteasome: influence of RCAN1 and oxidative stress

Abstract

Hyperphosphorylated tau proteins accumulate in the paired helical filaments of neurofibrillary tangles seen in such tauopathies as Alzheimer's disease. In the present paper we show that tau turnover is dependent on degradation by the proteasome (inhibited by MG132) in HT22 neuronal cells. Recombinant human tau was rapidly degraded by the 20 S proteasome in vitro, but tau phosphorylation by GSK3beta (glycogen synthase kinase 3beta) significantly inhibited proteolysis. Tau phosphorylation was increased in HT22 cells by OA [okadaic acid; which inhibits PP (protein phosphatase) 1 and PP2A] or CsA [cyclosporin A; which inhibits PP2B (calcineurin)], and in PC12 cells by induction of a tet-off dependent RCAN1 transgene (which also inhibits PP2B). Inhibition of PP1/PP2A by OA was the most effective of these treatments, and tau hyperphosphorylation induced by OA almost completely blocked tau degradation in HT22 cells (and in cell lysates to which purified proteasome was added) even though proteasome activity actually increased. Many tauopathies involve both tau hyperphosphorylation and the oxidative stress of chronic inflammation. We tested the effects of both cellular oxidative stress, and direct tau oxidative modification in vitro, on tau proteolysis. In HT22 cells, oxidative stress alone caused no increase in tau phosphorylation, but did subtly change the pattern of tau phosphorylation. Tau was actually less susceptible to direct oxidative modification than most cell proteins, and oxidized tau was degraded no better than untreated tau. The combination of oxidative stress plus OA treatment caused extensive tau phosphorylation and significant inhibition of tau degradation. HT22 cells transfected with tau-CFP (cyan fluorescent protein)/tau-GFP (green fluorescent protein) constructs exhibited significant toxicity following tau hyperphosphorylation and oxidative stress, with loss of fibrillar tau structure throughout the cytoplasm. We suggest that the combination of tau phosphorylation and tau oxidation, which also occurs in tauopathies, may be directly responsible for the accumulation of tau aggregates.

Suppression of Autophagy and Activation of Glycogen Synthase Kinase 3beta Facilitate the Aggregate Formation of Tau

“…Several studies have provided evidence that phosphorylation of key sites on tau has a strong impact on the normal function of tau and likely contributes to its pathological role including the tendency of aggregate formation [26,27]. It has been also reported that phosphorylation of tau impedes turnover of tau, resulting in the intracellular accumulation of phosphorylated tau [28]. In this study, the phosphorylation of T4 by GSK3β resulted in significantly increased intracellular level of tau, indicating that phosphorylation of T4 by GSK3β causes the intracellular accumulation, which is supported by a previous report that phosphorylated tau is less efficiently degraded [28].…”

“…It has been also reported that phosphorylation of tau impedes turnover of tau, resulting in the intracellular accumulation of phosphorylated tau [28]. In this study, the phosphorylation of T4 by GSK3β resulted in significantly increased intracellular level of tau, indicating that phosphorylation of T4 by GSK3β causes the intracellular accumulation, which is supported by a previous report that phosphorylated tau is less efficiently degraded [28]. Present data demonstrate that extensive phosphorylation of wild type tau by active GSK3β makes tau less efficient in the autophagic degradation.…”

Protein Oxidation in Aging: Does It Play a Role in Aging Progression?

“…However, in AD, tau undergoes hyperphosphorylation, which reduces the proteasomal susceptibility of the protein (131). Hyperphosphorylation results in conformational changes of tau.…”

Regulation of RCAN1 Protein Activity by Dyrk1A Protein-mediated Phosphorylation

“…The finding that the overexpression of RCAN1 caused the hyperphosphorylation of Tau (35), and our previous observation that Dyrk1A phosphorylated Tau at Thr 212 and Ser 404 residues prompted us to examine the effect of RCAN1 and Caln on Tau phosphorylation. Western blot analysis was performed with lysates of HEK293T cells that had been transiently transfected with a Tau expression plasmid, either in the presence or absence of a plasmid encoding RCAN1 or Caln A. Phosphorylation of Thr 212 and Ser 404 residues was detected with phosphoThr 212 -Tau (p-Tau212)-specific and phospho-Ser 404 -Tau (p-Tau404)-specific antibodies, respectively.…”