Abstract
The axonal microtubule-associated phosphoprotein tau interacts with neural plasma membrane (PM) components during neuronal development (Brandt, R., Léger, J., and Lee, G. (1995) J. Cell Biol. 131, 1327-1340). To analyze the mechanism and potential regulation of tau's PM association, a method was developed to isolate PM-associated tau using microsphere separation of surface-biotinylated cells. We show that tau's PM association requires an intact membrane cortex and that PM-associated tau and cytosolic tau are differentially phosphorylated at sites detected by several Alzheimer's disease (AD) diagnostic antibodies (Ser(199)/Ser(202), Thr(231), and Ser(396)/Ser(404)). In polar neurons, the association of endogenous tau phosphoisoforms with the membrane cortex correlates with an enrichment in the axonal compartment. To test for a direct effect of AD-specific tau modifications in determining tau's interactions, a phosphomutant that simulates an AD-like hyperphosphorylation of tau was produced by site-directed mutagenesis of Ser/Thr residues to negatively charged amino acids (Glu). These mutations completely abolish tau's association with the membrane cortex; however, the construct retains its capability to bind to microtubules. The data suggest that a loss of tau's association with the membrane cortex as a result of phosphorylation at sites that are modified during disease contributes to somatodendritic tau accumulation, axonal microtubule disintegration, and neuronal death characteristic for AD.
Highlights
The axonal tau proteins represent a family of closely related low molecular weight phosphoproteins that copolymerize with microtubules and that modulate the dynamic instability of tubulin assembly in cell-free reactions [1,2,3,4]
Isolation of Plasma Membrane-associated Tau by Microsphere Separation of Surface-biotinylated Cells—In PC12 cells stably transfected with adult rat tau cDNA, tau was clearly enriched at the cellular periphery as judged by laser scanning microscopy of cells that had been processed for immunocytochemistry (Fig. 1A)
The method is based on microsphere separation of surface-biotinylated cells and yielded three fractions: a PM fraction that was highly enriched in plasma membrane components, a crude organelle fraction that contained all types of membranous organelles together with residual plasma membrane that did not bind to the beads, and a cytosolic fraction representing the supernatant of a high speed centrifugation of the unbound material (Fig. 1B)
Summary
The axonal tau proteins represent a family of closely related low molecular weight phosphoproteins that copolymerize with microtubules and that modulate the dynamic instability of tubulin assembly in cell-free reactions [1,2,3,4]. Microtubule binding requires the carboxyl-terminal half of the protein containing tau’s microtubule-binding repeat domain, whereas tau’s PM association is mediated by its amino-terminal domain, which protrudes from the microtubule surface when tau is bound to microtubules These interactions are mediated by distinct domains, raising the possibility that tau functions as a linker protein between the microtubule system and axonal PM components. Several AD diagnostic antibodies such as AT-8, AT-180, and PHF-1 detect epitopes containing phosphorylated Ser-Pro or Thr-Pro motifs, which could point to an involvement of prolinedirected kinases, e.g. mitogen-activated protein kinase, glycogen synthase kinase-3, and Cdk, in the disease process These kinases preferentially phosphorylate sites that flank the microtubule-binding domain of tau protein and have only a weak effect on the binding of tau to microtubules [21, 22]. Phosphomutants that simulate a PHFlike hyperphosphorylation of tau in regions that flank the microtubule-binding domain are defective in their association with the membrane cortex, but can still bind to microtubules
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