Okadaic acid mediates tau phosphorylation via sustained activation of the L-voltage-sensitive calcium channel

doi:10.1016/S0169-328X(03)00294-8

Molecular Brain Research

Volume 117, Issue 2, 7 October 2003, Pages 145-151

https://doi.org/10.1016/S0169-328X(03)00294-8 Get rights and content

Abstract

Accumulation of phosphorylated isoforms of the microtubule-associated protein tau is one hallmark of affected neurons in Alzheimer’s disease (AD). This increase has been attributed to increased kinase or decreased phosphatase activity. Prior studies indicate that one of the kinases that phosphorylates tau (mitogen-activated protein kinase, or MAP kinase) does so at least in part indirectly within intact neuronal cells by phosphorylating and activating the L-voltage-sensitive calcium channel. Resultant calcium influx then fosters tau phosphorylation via one or more calcium-activated kinases. We demonstrate herein that treatment of differentiated SH-SY-5Y human neuroblastoma with the phosphatase inhibitor okadaic acid (OA) similarly may increase tau phosphorylation via sustained activation of the L-voltage-sensitive calcium channel. OA increased phospho-tau as indicated by increased immunoreactivity towards an antibody (PHF-1) directed against paired helical filaments from AD brain. This increase was blocked by co-treatment with the channel antagonist nimodipine. OA treatment increased channel phosphorylation. The increases in calcium influx, PHF-1 immunoreactivity and channel phosphorylation were all attenuated by co-treatment with PD98059, which inhibits MAP kinase activity, suggesting that OA mediates these effects at least in part via sustained activation of MAP kinase. These findings underscore that divergent and convergent kinase and phosphatase activities regulate tau phosphorylation.

Introduction

One hallmark of affected neurons in Alzheimer’s disease (AD) is the accumulation of increased levels of phosphorylated forms of the microtubule-associated protein tau [13], [17]. Several kinases have been implicated in regulation of tau phosphorylation [3], [7], [9], [10], [12], [22], [24], [26], [28], [32], [37], [38] (Hanger et al., 1992). In addition, however, a number of studies indicate that phosphatase activities contribute to increased tau phosphorylation [8], [19], [25], [27], [5], [31]. Okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A, increases levels of phospho-tau in culture and in situ [10], [2], [15], [16], [40], [47], [20], [21], [46], [42], [41]. It has been considered that alterations in phosphatase activity may exert a more profound impact than that of kinase activity [44]. At the very least, it should be kept in mind that the combined impact of kinase and phosphatase activities are likely to be responsible for the net level of tau phosphorylation.

Prior studies from our laboratory point towards a further complexity, in that one of the kinases reported to phosphorylate tau, mitogen-activated protein (MAP) kinase, may increase phospho-tau levels within cells at least in part by an indirect manner as follows. Following treatment of cultured neurons with Abeta, MAP kinase phosphorylates the L-voltage-sensitive calcium channel, inducing calcium influx [11]. Co-treatment with an antagonist of this channel or chelation of cytosolic calcium following channel activation each prevented Abeta-induced tau phosphorylation [11]. Thus, while MAP kinase can phosphorylate tau directly, these data suggested that the major influence of MAP kinase on tau phosphorylation was instead derived by MAP kinase-mediated activation of the L-voltage-sensitive calcium channel. Phosphorylation of tau itself may have then been carried out by activation of one or more calcium-dependent kinases following resultant calcium influx [11].

Inhibition of phosphatase activity, including that achieved by treatment with OA, potentiates MAP kinase activity by maintaining its phosphorylation [1], [23]. We therefore investigated herein whether or not phosphatase inhibition might contribute to increased phospho-tau levels in an indirect manner by maintaining MAP kinase activity and/or activation of the L-voltage-sensitive calcium channel.

Section snippets

Cell culture and treatment

SH-SY-5Y human neuroblastoma cells were cultured in DMEM (Cellgro) containing 10% fetal bovine serum in 5% CO₂. Cultures were differentiated for 7 days with 10 μM retinoic acid, during which time they elaborate extensive neurites that exhibit characteristics of axons [43]. Cells were deprived of serum and treated for 2 h with one or more of the following: the phosphatase inhibitor OA (1 μM), the MAP kinase inhibitor PD98059 (10 μM; RBI, Natick, MA, USA [35]) or nimodipine (1 μM [45]), Prior

The phosphatase inhibitor OA enhances phosphorylation of the L-voltage-sensitive calcium channel

Metabolic radiolabeling indicated that OA induced an approximate 100% increase in phosphorylation of the L-voltage-sensitive calcium channel (Fig. 1; Table 1). While this effect could be due to prevention of dephosphorylation of the channel, OA also sustains activation of MAP kinase, a kinase known to phosphorylate this channel [11], by inhibition of dephosphorylation of the kinase itself. To examine whether or not sustained activation of MAP kinase could be responsible for the increase in

Discussion

The findings of the present study indicate that phosphatase inhibition sustains phosphorylation and activity of the L-voltage-sensitive calcium channel. These findings are consistent with prior studies indicating that multiple phosphatases regulate activity of this channel [14], [18], [33], [34], [49]. The twofold increase in ³²P labeling of channel subunits following OA addition suggests that this channel normally undergoes rapid cycles of phosphorylation and dephosphorylation. It is therefore

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¹: Present address: Department of Pharmacology, College of Medicine, University of Tennessee Center for Health Sciences, Memphis, TN 38163, USA.

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Research reportOkadaic acid mediates tau phosphorylation via sustained activation of the L-voltage-sensitive calcium channel

Abstract

Introduction

Section snippets

Cell culture and treatment

The phosphatase inhibitor OA enhances phosphorylation of the L-voltage-sensitive calcium channel

Discussion

J. Biol. Chem.

Exp. Neurol.

FEBS Lett.

J. Biol. Chem.

J. Chem. Anat.

J. Biol. Chem.

J. Biol. Chem.

FEBS Lett.

FEBS Lett.

Exp. Neurol.

Curr. Biol.

J. Biol. Chem.

Neuron

Neuroscience

J. Biol. Chem.

Trends Neurosci.

Biochem. Biophys. Res. Commun.

FEBS Lett.

J. Biol. Chem.

Contrasting effects of two tumour promoters, phorbol myristate acetate and okadaic acid, on T-cell responses and activation of p42 MAP-kinase/ERK-2

Immunology

The protein phosphatase inhibitor okadaic acid induces heat shock protein expression and neurodegeneration in rat hippocampus in vivo

Exp. Neurol.

Growth cones contain a highly-dynamic population of neurofilament subunits

Cell Motil. Cytoskel.

Hyperphosphorylation of tau and filopodial retraction following microinjection of protein kinase C catalytic subunits

J. Neurosci. Res.

Mitogen activated protein (MAP) kinase transforms tau protein into an Alzheimer-like state

EMBO J.

Research report
Okadaic acid mediates tau phosphorylation via sustained activation of the L-voltage-sensitive calcium channel