The neuronal microtubule-associated protein tau plays an important role in establishing cell polarity by stabilizing axonal microtubules that serve as tracks for motor-protein–driven transport processes. To investigate the role of tau in intracellular transport, we studied the effects of tau expression in stably transfected CHO cells and differentiated neuroblastoma N2a cells. Tau causes a change in cell shape, retards cell growth, and dramatically alters the distribution of various organelles, known to be transported via microtubule-dependent motor proteins. Mitochondria fail to be transported to peripheral cell compartments and cluster in the vicinity of the microtubule-organizing center. The endoplasmic reticulum becomes less dense and no longer extends to the cell periphery. In differentiated N2a cells, the overexpression of tau leads to the disappearance of mitochondria from the neurites. These effects are caused by tau's binding to microtubules and slowing down intracellular transport by preferential impairment of plus-end–directed transport mediated by kinesin-like motor proteins. Since in Alzheimer's disease tau protein is elevated and mislocalized, these observations point to a possible cause for the gradual degeneration of neurons.

AT100 is a monoclonal antibody highly specific for phosphorylated Tau in Alzheimer paired helical filaments. Here we show that the epitope is generated by a complex sequence of sequential phosphorylation, first of Ser199, Ser202 and Thr205 (around the epitope of antibody AT8), next of Thr212 by glycogen synthase kinase (GSK)-3beta (a proline-directed kinase), then of Ser214 by protein kinase A (PKA). Conversely, if Ser214 is phosphorylated first it protects Thr212 and the Ser-Pro motifs around the AT8 site against phosphorylation, and the AT100 epitope is not formed. The generation of the AT100 epitope requires a conformation of tau induced by polyanions such as heparin, RNA or poly(Glu), conditions which also favor the formation of paired helical filaments. The Alzheimer-like phosphorylation can be induced by brain extracts. In the extract, the kinases responsible for generating the AT100 epitope are GSK-3beta and PKA, which can be inhibited by their specific inhibitors LiCl and RII, respectively. A cellular model displaying the reaction with AT100 is presented by Sf9 insect cells transfected with Tau. Knowledge of the events and kinases generating the AT100 epitope in cells might allow us to study the degeneration of the cytoskeleton in Alzheimer's disease.

In Alzheimer's disease the neuronal microtubule-associated protein tau becomes highly phosphorylated, loses its binding properties, and aggregates into paired helical filaments. There is increasing evidence that the events leading to this hyperphosphorylation are related to mitotic mechanisms. Hence, we have analyzed the physiological phosphorylation of endogenous tau protein in metabolically labeled human neuroblastoma cells and in Chinese hamster ovary cells stably transfected with tau. In nonsynchronized cultures the phosphorylation pattern was remarkably similar in both cell lines, suggesting a similar balance of kinases and phosphatases with respect to tau. Using phosphopeptide mapping and sequencing we identified 17 phosphorylation sites comprising 80-90% of the total phosphate incorporated. Most of these are in SP or TP motifs, except S214 and S262. Since phosphorylation of microtubule-associated proteins increases during mitosis, concomitant with increased microtubule dynamics, we analyzed cells mitotically arrested with nocodazole. This revealed that S214 is a prominent phosphorylation site in metaphase, but not in interphase. Phosphorylation of this residue strongly decreases the tau-microtubule interaction in vitro, suppresses microtubule assembly, and may be a key factor in the observed detachment of tau from microtubules during mitosis. Since S214 is also phosphorylated in Alzheimer's disease tau, our results support the view that reactivation of the cell cycle machinery is involved in tau hyperphosphorylation.

Glycogen synthase kinase-3L L (GSK-3L L) has been described as a proline-directed kinase which phosphorylates tau protein at several sites that are elevated in Alzheimer paired helical filaments. However, it has been claimed that GSK-3L L can also phosphorylate the non-proline-directed KXGS motifs in the presence of heparin, including Ser262 in the repeat domain of tau, which could induce the detachment of tau from microtubules. We have analyzed the activity of recombinant GSK-3L L and of GSK-3L L preparations purified from tissue, using two-dimensional phosphopeptide mapping, immunoblotting with phosphorylationsensitive antibodies, and phosphopeptide sequencing. The most prominent phosphorylation sites on tau are Ser396 and Ser404 (PHF-1 epitope), Ser46 and Thr50 in the first insert, followed by a less efficient phosphorylation of other Alzheimer phosphoepitopes (antibodies AT-8, etc). We also show that the nonproline-directed activity at KXGS motifs is not due to GSK-3L L itself, but to kinase contaminations in common GSK-3L L preparations from tissues which are activated upon addition of heparin.z 1999 Federation of European Biochemical Societies.

NF-κB-inducing kinase (NIK) mediates non-canonical NF-κB signaling downstream of multiple TNF family members, including BAFF, TWEAK, CD40, and OX40, which are implicated in the pathogenesis of systemic lupus erythematosus (SLE). Here, we show that experimental lupus in NZB/W F1 mice can be treated with a highly selective and potent NIK small molecule inhibitor. Both in vitro as well as in vivo, NIK inhibition recapitulates the pharmacological effects of BAFF blockade, which is clinically efficacious in SLE. Furthermore, NIK inhibition also affects T cell parameters in the spleen and proinflammatory gene expression in the kidney, which may be attributable to inhibition of OX40 and TWEAK signaling, respectively. As a consequence, NIK inhibition results in improved survival, reduced renal pathology, and lower proteinuria scores. Collectively, our data suggest that NIK inhibition is a potential therapeutic approach for SLE.