Proceedings of The Physiological Society

King's College London (2011) Proc Physiol Soc 22, C10

Oral Communications

Dynamic association of tau with neuronal membranes is regulated by phosphorylation and the tyrosine kinase fyn

A. Pooler1, A. Usardi1, C. J. Evans1, K. L. Philpott2, W. Noble1, D. P. Hanger1

1. Neuroscience, King's College London, London, United Kingdom. 2. Neuroscience Centre of Excellence, GlaxoSmithKline, Harrow, United Kingdom.


Tau is an abundant cytosolic protein which regulates cytoskeletal stability by interacting with microtubules in a phosphorylation-dependent manner. In Alzheimer’s disease, tau becomes hyperphosphorylated and forms paired helical filament (PHF) aggregates and intracellular tangles. Although tau is largely cytosolic, approximately 20% of tau is associated with the plasma membrane, at least in rat pheochromocytoma cells. Membrane-association of tau may be regulated by its phosphorylation state. We therefore determined whether tau is associated with the neuronal membrane, and investigated the mechanisms regulating this association. We performed cellular fractionation by differential centrifugation in cultured rat cortical neurons and analyzed tau in cytosolic and membrane fractions using western blotting. Data were analyzed by two-way ANOVA followed by the post-hoc Dunnett's multiple comparison test. Significance level was set to P < 0.05. Fractionation of cortical neurons revealed that approximately 10% of neuronal tau is membrane-associated. This tau was only weakly recognised by PHF-1, a phospho-dependent tau antibody, but was strongly detected by tau-1, an antibody against tau dephosphorylated at Ser199/202. Isolation of plasma membrane-associated proteins by biotinylation demonstrated the association of tau with the plasma membrane in neurons. Pre-treatment of neurons with tau kinase inhibitors revealed that inhibition of either glycogen synthase kinase-3 (GSK-3) by lithium, or casein kinase 1 (CK1) by IC261, strongly dephosphorylated tau and increased the amount of tau in the membrane relative to the cytosolic fraction. The effect of IC261 on membrane-association of tau was reversible and four hours after treatment, tau localization in treated cells was similar to that in control cells. The effect of IC261 was prevented by pre-treatment with okadaic acid to inhibit phosphatase activity. Mutation of serine/threonine residues in the N-terminal half of tau to glutamate, to mimic a permanent state of phosphorylation, prevented tau localisation to membranes in transfected CHO cells. In contrast, intracellular localisation of tau was unaffected by mutation of serine/threonine residues to glutamate in the C-terminal half of the protein. Inhibiting CK1 in neurons lacking the tyrosine kinase fyn induced tau dephosphorylation but did not increase its membrane association. Furthermore, inhibition of CK1 increased binding of neuronal tau to the fyn-SH3 domain. We conclude that dephosphorylation of tau, especially at CK1 and GSK-3 sites, promotes trafficking of tau from the cytosolic compartment to the neuronal membrane and requires the presence of fyn.

Where applicable, experiments conform with Society ethical requirements