[Show abstract][Hide abstract] ABSTRACT: Protein kinases of the microtubule affinity-regulating kinase (MARK) family were originally discovered because of their ability to phosphorylate certain sites in tau protein (KXGS motifs in the repeat domain). This type of phosphorylation is enhanced in abnormal tau from Alzheimer brain tissue and causes the detachment of tau from microtubules. MARK-related kinases (PAR-1 and KIN1) occur in various organisms and are involved in establishing and maintaining cell polarity. Herein, we report the ability of MARK2 to affect the differentiation and outgrowth of cell processes from neuroblastoma and other cell models. MARK2 phosphorylates tau protein at the KXGS motifs; this results in the detachment of tau from microtubules and their destabilization. The formation of neurites in N2a cells is blocked if MARK2 is inactivated, either by transfecting a dominant negative mutant, or by MARK2 inhibitors such as hymenialdisine. Alternatively, neurites are blocked if the target KXGS motifs on tau are rendered nonphosphorylatable by point mutations. The results suggest that MARK2 contributes to the plasticity of microtubules needed for neuronal polarity and the growth of neurites.
Molecular Biology of the Cell 12/2002; 13(11):4013-28. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have studied biochemical and structural parameters of several missense and deletion mutants of tau protein (G272V, N279K, DeltaK280, P301L, V337M, R406W) found in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). The mutant proteins were expressed on the basis of both full-length tau (htau40) and constructs derived from the repeat domain. They were analyzed with respect to the capacity to enhance microtubule assembly, binding of tau to microtubules, secondary structure content, and aggregation into Alzheimer-like paired helical or straight filaments. We find that the mutations cause a moderate decrease in microtubule interactions and stabilization, and they show no gross structural changes compared with the natively unfolded conformation of the wild-type protein, but the aggregation into PHFs is strongly enhanced, particularly for the mutants DeltaK280 and P301L. This gain of pathological aggregation would be consistent with the autosomal dominant nature of the disease.
[Show abstract][Hide abstract] ABSTRACT: 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.
Molecular Biology of the Cell 07/1998; 9(6):1495-512. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
European Journal of Biochemistry 03/1998; 252(3):542-52. · 3.58 Impact Factor