Microtubule Binding and Trapping at the Tip of Neurites Regulate Tau Motion in Living Neurons

During the development of neurons, the microtubule-associated tau proteins show a graded proximo-distal distribution in axons. In tauopathies such as Alzheimer's disease, tau accumulates in the somatodendritic compartment. To scrutinize the determinants of tau's distribution and motion, we constructed photoactivatable green fluorescent protein (GFP)-tagged tau fusion proteins and recorded their distribution after focal activation in living cells. Simulation showed that the motion of tau was compatible with diffusion/reaction as opposed to active transport/reaction. Effective diffusion constants of 0.7–0.8 μm²/second were calculated in neurites of PC12 cells and primary cortical neurons. Furthermore, tau's amino terminal projection domain mediated binding and enrichment of tau at distal neurites indicating that the tip of a neurite acts as an adsorber trapping tau protein. Treatment with taxol, incorporation of disease-related tau modifications, experimentally induced hyperphosphorylation and addition of preaggregated amyloid β peptides (Aβ) increased the effective diffusion constant compatible with a decreased binding to microtubules. Distal enrichment was present after taxol treatment but was suppressed at disease-relevant conditions. The data suggest that (i) dynamic binding of tau to microtubules and diffusion along microtubules and (ii) trapping at the tip of a neurite both contribute to its distribution during development and disease.     

Interaction of tau with the neural membrane cortex is regulated by phosphorylation at sites that are modified in paired helical filaments

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.     

Tuning microtubule-based transport through filamentous MAPs: the problem of dynein

We recently proposed that regulating the single-to-multiple motor transition was a likely strategy for regulating kinesin-based transport in vivo . In this study, we use an in vitro bead assay coupled with an optical trap to investigate how this proposed regulatory mechanism affects dynein-based transport. We show that tau's regulation of kinesin function can proceed without interfering with dynein-based transport. Surprisingly, at extremely high tau levels – where kinesin cannot bind microtubules (MTs) – dynein can still contact MTs. The difference between tau's effects on kinesin- and dynein-based motility suggests that tau can be used to tune relative amounts of plus-end and minus-end-directed transport. As in the case of kinesin, we find that the 3RS isoform of tau is a more potent inhibitor of dynein binding to MTs. We show that this isoform-specific effect is not because of steric interference of tau's projection domains but rather because of tau's interactions with the motor at the MT surface. Nonetheless, we do observe a modest steric interference effect of tau away from the MT and discuss the potential implications of this for molecular motor structure.     

MARKing tau for tangles and toxicity

In healthy neurons, tau proteins regulate microtubule function in the axon. In the brains of individuals with Alzheimer's disease, tau is hyperphosphorylated and aggregated into intraneuronal deposits called neurofibrillary tangles (NFTs). Hyperphosporylation dislodges tau from the microtubule surface, potentially resulting in compromised axonal integrity and the accumulation of toxic tau peptides. Recent biochemical and animal model studies have re-evaluated tau phosphorylation and other aspects of neurofibrillar pathology. The results indicate that phosphorylation of tau's microtubule-binding domain by the protein kinase MARK primes tau for hyperphosphorylation by the kinases GSK-3 and Cdk5, which in turn triggers the aggregation of tau into filaments and tangles. Toxic consequences for the neuron might be exacerbated by tangle formation but are already evident during the early steps of the process.     

Phosphorylation that detaches tau protein from microtubules (Ser262, Ser214) also protects it against aggregation into Alzheimer paired helical filaments

One of the hallmarks of Alzheimer's disease is the abnormal state of the microtubule-associated protein tau in neurons. It is both highly phosphorylated and aggregated into paired helical filaments, and it is commonly assumed that the hyperphosphorylation of tau causes its detachment from microtubules and promotes its assembly into PHFs. We have studied the relationship between the phosphorylation of tau by several kinases (MARK, PKA, MAPK, GSK3) and its assembly into PHFs. The proline-directed kinases MAPK and GSK3 are known to phosphorylate most Ser-Pro or Thr-Pro motifs in the regions flanking the repeat domain of tau: they induce the reaction with several antibodies diagnostic of Alzheimer PHFs, but this type of phosphorylation has only a weak effect on tau-microtubule interactions and on PHF assembly. By contrast, MARK and PKA phosphorylate several sites within the repeats (notably the KXGS motifs including Ser262, Ser324, and Ser356, plus Ser320); in addition PKA phosphorylates some sites in the flanking domains, notably Ser214. This type of phosphorylation strongly reduces tau's affinity for microtubules, and at the same time inhibits tau's assembly into PHFs. Thus, contrary to expectations, the phosphorylation that detaches tau from microtubules does not prime it for PHF assembly, but rather inhibits it. Likewise, although the phosphorylation sites on Ser-Pro or Thr-Pro motifs are the most prominent ones on Alzheimer PHFs (by antibody labeling), they are only weakly inhibitory to PHF assembly. This implies that the hyperphosphorylation of tau in Alzheimer's disease is not directly responsible for the pathological aggregation into PHFs; on the contrary, phosphorylation protects tau against aggregation.     

In vitro assembly of tau protein: mapping the regions involved in filament formation

Unraveling the mechanism of self-assembly of the protein tau into paired helical filaments (PHFs) is a crucial step toward the understanding of Alzheimer's and other neuropathological diseases at the molecular level. In an effort to map the role of different regions of tau in the mechanism of self-assembly, we have studied the polymerization ability of different tau fragments using an in vitro assay. Our results indicate that the N-terminal domain interferes with tau's ability to polymerize in vitro. The effect seems to be size dependent. Particularly, an isoform of tau from the peripheral nervous system, which has a much larger N-terminal domain, was found unable to form filaments in our in vitro assay. This finding can explain why in Alzheimer's patients PHFs only accumulate in the neurons from the central nervous system. We also report that a short segment of tau located in the third microtubule binding repeat (residues 317 to 335, peptide 1/2R) is probably the minimal segment of that region able to grow into filaments in vitro and in the presence of heparin. In contrast with whole peptide 1/2R, peptides corresponding to either the N-terminal or C-terminal halves of this segment were unable to form filaments. Finally, our polymerization studies of peptides from the C-terminal domain reveal a short sequence spanning residues 391 to 407 that grows into filaments in vitro. This tau segment forms filaments regardless of whether is incubated with heparin. Moreover, such filaments differ in diameter and morphology, suggesting a different mechanism of self-assembly.     

Interaction of tau isoforms with Alzheimer's disease abnormally hyperphosphorylated tau and in vitro phosphorylation into the disease-like protein

The microtubule-associated protein tau is a family of six isoforms that becomes abnormally hyperphosphorylated and accumulates in neurons undergoing neurodegeneration in the brains of patients with Alzheimer disease (AD). We investigated the isoform-specific interaction of normal tau with AD hyperphosphorylated tau (AD P-tau). We found that the binding of AD P-tau to normal human recombinant tau was tau4L > tau4S > tau4 and tau3L > tau3S > tau3, and that its binding to tau4L was greater than to tau3L. AD P-tau also inhibited the assembly of microtubules promoted by each tau isoform and caused disassembly when added to preassembled microtubules. This inhibition and depolymerization of microtubules by the AD P-tau corresponded directly to the degree of its interaction with the different tau isoforms. In vitro hyperphosphorylation of recombinant tau (P-tau) conferred AD P-tau-like characteristics. Like AD P-tau, P-tau interacted with and sequestered normal tau and inhibited microtubule assembly. These studies suggest that the AD P-tau interacts preferentially with the tau isoforms that have the amino-terminal inserts and four microtubule binding domain repeats and that hyperphosphorylation of tau appears to be sufficient to acquire AD P-tau characteristics. Thus, lack of amino-terminal inserts and extra microtubule binding domain repeat in fetal human brain might be protective from Alzheimer's neurofibrillary degeneration.     

Phosphorylation of tau at Ser214 mediates its interaction with 14-3-3 protein: implications for the mechanism of tau aggregation

The microtubule associated protein tau is a major component of neurofibrillary tangles in Alzheimer disease brain, however the neuropathological processes behind the formation of neurofibrillary tangles are still unclear. Previously, 14-3-3 proteins were reported to bind with tau. 14-3-3 Proteins usually bind their targets through specific serine/threonine –phosphorylated motifs. Therefore, the interaction of tau with 14-3-3 mediated by phosphorylation was investigated. In this study, we show that the phosphorylation of tau by either protein kinase A (PKA) or protein kinase B (PKB) enhances the binding of tau with 14-3-3 in vitro . The affinity between tau and 14-3-3 is increased 12- to 14-fold by phosphorylation as determined by real time surface plasmon resonance studies. Mutational analyses revealed that Ser214 is critical for the phosphorylation-mediated interaction of tau with 14-3-3. Finally, in vitro aggregation assays demonstrated that phosphorylation by PKA/PKB inhibits the formation of aggregates/filaments of tau induced by 14-3-3. As the phosphorylation at Ser214 is up-regulated in fetal brain, tau's interaction with 14-3-3 may have a significant role in the organization of the microtubule cytoskeleton in development. Also as the phosphorylation at Ser214 is up-regulated in Alzheimer's disease brain, tau's interaction with 14-3-3 might be involved in the pathology of this disease.     

The microtubule binding of Tau and high molecular weight Tau in apoptotic PC12 cells is impaired because of altered phosphorylation

Although the importance of the microtubule network throughout cell life is well established, the dynamics of microtubules during apoptosis, a regulated cell death process, is unclear. In a previous study (Davis, P. K., and Johnson, G. V. (1999) Biochem. J. 340, 51-58) we demonstrated that the phosphorylation of the microtubule-associated protein tau was increased during neuronal PC12 cell apoptosis. The purpose of this study was to determine whether the increased tau phosphorylation that occurred during apoptosis impaired the microtubule binding capacity of tau. This study is the first demonstration that microtubule-binding by tau and high molecular weight tau is significantly impaired as a result of altered phosphorylation during a naturally occurring process, apoptosis. Furthermore, co-immunofluorescence studies reveal for the first time that tau populations within an apoptotic neuronal PC12 cell exhibit differential phosphorylation. In control PC12 cells, Tau-1 staining (Tau-1 recognizes an unphosphorylated epitope) is evident throughout the entire cell body. In contrast, Tau-1 immunoreactivity in apoptotic PC12 cells is retained in the nuclear/perinuclear region but is significantly decreased in the cytoplasm up to the plasma membrane. The selective distribution of phosphorylated tau in apoptotic PC12 cells indicates that tau likely plays a significant role in the cytoskeletal changes that occur during apoptosis.     

Relationship between phase resetting and the free-running period in Djungarian hamsters.

A data set of 293 phase shifts was analyzed in order to determine the relationship between phase resetting and the free-running period (tau) in Djungarian hamsters. Phase shifts in response to a 15-min light pulse were assigned to one of two groups (tau short, less than 24 hr; tau long, greater than 24 hr), and two phase response curves (PRCs) were constructed. The two PRCs differed predominantly in the advance region, which extended so far into the subjective day of PRClong that a dead zone was lacking. The functional significance of PRC differences was assessed by computer simulations of entrainment to varying skeleton photoperiods and entrainment to a 12-hr skeleton photoperiod with varying tau's. Results from these simulations confirmed the theoretical predictions by Pittendrigh and Daan: Stability of entrainment under varying photoperiods depended on the ratio of the PRC slopes at the phases illuminated by light (SE/SM). This ratio was always larger than 1 for PRClong. It approached 0 for PRCshort as soon as the evening light illuminated the dead zone; this occurred for entrainment to very short photoperiods. Stability of entrainment to lights-off was in general better for PRClong than for PRCshort, especially if PRClong was used in combination with tau long. This suggests that it can be advantageous for stability of entrainment to lights-off to express a tau greater than 24 hr in combination with a PRC lacking a dead zone. Stability of entrainment under varying tau's was not much different for PRClong or PRCshort. However, stability of entrainment deteriorated for PRClong in combination with short tau's, whereas it deteriorated for PRCshort in combination with long tau's.     

Tyrosine phosphorylation of tau regulates its interactions with Fyn SH2 domains, but not SH3 domains, altering the cellular localization of tau

Recent reports have demonstrated that interactions between the microtubule-associated protein tau and the nonreceptor tyrosine kinase Fyn play a critical role in mediating synaptic toxicity and neuronal loss in response to β-amyloid (Aβ) in models of Alzheimer's disease. Disruption of interactions between Fyn and tau may thus have the potential to protect neurons from Aβ-induced neurotoxicity. Here, we investigated tau and Fyn interactions and the potential implications for positioning of these proteins in membrane microdomains. Tau is known to bind to Fyn via its Src-homology (SH)3 domain, an association regulated by phosphorylation of PXXP motifs in tau. Here, we show that Pro216 within the PXXP(213-216) motif in tau plays an important role in mediating the interaction of tau with Fyn-SH3. We also show that tau interacts with the SH2 domain of Fyn, and that this association, unlike that of Fyn-SH3, is influenced by Fyn-mediated tyrosine phosphorylation of tau. In particular, phosphorylation of tau at Tyr18, a reported target of Fyn, is important for mediating Fyn-SH2-tau interactions. Finally, we show that tyrosine phosphorylation influences the localization of tau to detergent-resistant membrane microdomains in primary cortical neurons, and that this trafficking is Fyn-dependent. These findings may have implications for the development of novel therapeutic strategies aimed at disrupting the tau/Fyn-mediated synaptic dysfunction that occurs in response to elevated Aβ levels in neurodegenerative disease.     

Modulation of Tau Phosphorylation Within Its Microtubule-Binding Domain by Cellular Thiols

Tau is a microtubule-stabilizing protein that is functionally modulated by alterations in its phosphorylation state. Because phosphorylation regulates both normal and pathological tau functioning, it is of importance to identify the signaling pathways that regulate tau phosphorylation in vivo. The present study examined changes in tau phosphorylation and function in response to modulation of cellular thiol content. Treatment of cells with phenylarsine oxide, which reacts with vicinal thiols, selectively increased tau phosphorylation within its microtubule-binding domain, at the non-Ser/Thr-Pro sites Ser262/356, while decreasing tau phosphorylation at Ser/ Thr-Pro sites outside this region. This increase in tau phosphorylation correlated with a decrease in the amount of tau associated with the cytoskeleton and decreased microtubule stability. Phenylarsine oxide-induced tau phosphorylation was inhibited by oxidants and by the protein kinase inhibitor staurosporine. Although staurosporine completely eliminated the increase in tau phosphorylation at Ser262/356, as detected by immunostaining with 12E8, it had a comparatively minor effect on the changes in tau localization induced by phenylarsine oxide. The results suggest that regulation of cellular thiols is important for modulating tau phosphorylation and function in situ. Additionally, although phosphorylation of Ser262/356 decreases tau's interaction with the cytoskeleton, phosphorylation of these residues alone is not sufficient for the phenylarsine oxide-induced changes in tau localization.     

The microtubule-associated protein tau forms a triple-stranded left-hand helical polymer

High resolution transmission electron microscopy (TEM) has shown that bovine tau are 2.1 +/- 0.2-nm diameter filaments which are triple-stranded left-hand helical structures composed of three 1.0 +/- 0.2-nm strands. The reported amino acid sequence of human and bovine tau have been computer processed to predict secondary structure. Within the constraints imposed by the images, the secondary structure models and other structural information have been used to calculate tau's maximum and minimum length. The length calculations and secondary structure form the basis for image interpretation. This work indicates that each approximately 1.0-nm strand is a tau polypeptide chain and that the approximately 2.1-nm filament is composed of three separate tau chains (tau3). Bovine tau length measurements indicate that tau trimer filaments are generally longer than a fully extended tau monomer. These measurements indicate that each trimer, tau3, is joined with other trimers to form long tau polymers, (tau3)n. An inverse temperature transition has been found in the circular dichroism spectrum of tau indicating that its structure is less ordered below 20 degrees C and more ordered at 37 degrees C. The implications of this phenomenon with respect to tau's temperature-dependent ability to reconstitute microtubules is discussed and a mechanism for the possible abnormal aggregation of tau into neurofibrillary tangles in Alzheimer's disease is proposed.     

Regulation of neuronal morphology by Toca-1, an F-BAR/EFC protein that induces plasma membrane invagination

Actin reorganization is important for regulation of neuronal morphology. Neural Wiskott-Aldrich syndrome protein (N-WASP) is an important regulator of actin polymerization and also known to be strongly expressed in brain. Recently, Toca-1 (transducer of Cdc42-dependent actin assembly) has been shown to be required for Cdc42 to activate N-WASP from biochemical experiments. Toca-1 has three functional domains: an F-BAR/EFC domain at the N terminus, an HR1 at the center, and an SH3 domain at the C terminus. The F-BAR/EFC domain induces tubular invagination of plasma membrane, while Toca-1 binds both N-WASP and Cdc42 through the SH3 domain and the HR1, respectively. However, the physiological role of Toca-1 is completely unknown. Here we have investigated the neural function of Toca-1. Toca-1 is strongly expressed in neurons including hippocampal neurons in developing brain at early times. Knockdown of Toca-1 in PC12 cells significantly enhances neurite elongation. Consistently, overexpression of Toca-1 suppresses neurite elongation through the F-BAR/EFC domain with a membrane invaginating property, suggesting an implication of membrane trafficking in the neural function of Toca-1. In addition, knockdown of N-WASP, to our surprise, also enhances neurite elongation in PC12 cells, which is in clear contrast to the previous report that dominant negative mutants of N-WASP suppress neurite extension in PC12 cells. On the other hand, knockdown of Toca-1 in cultured rat hippocampal neurons enhances axon branching a little but not axon elongation, while knockdown of N-WASP enhances both axon elongation and branching. These results suggest that a vesicle trafficking regulator Toca-1 regulates different aspects of neuronal morphology from N-WASP.     

Inability of tau to properly regulate neuronal microtubule dynamics: a loss-of-function mechanism by which tau might mediate neuronal cell death

Interest in the microtubule-associated protein tau stems from its critical roles in neural development and maintenance, as well as its role in Alzheimer's, FTDP-17 and related neurodegenerative diseases. Under normal circumstances, tau performs its functions by binding to microtubules and powerfully regulating their stability and growing and shortening dynamics. On the other hand, genetic analyses have established a clear cause-and-effect relationship between tau dysfunction/mis-regulation and neuronal cell death and dementia in FTDP-17, but the molecular basis of tau's destructive action(s) remains poorly understood. One attractive model suggests that the intracellular accumulation of abnormal tau aggregates causes cell death, i.e., a gain-of-toxic function model. Here, we describe the evidence and arguments for an alternative loss-of-function model in which tau-mediated neuronal cell death is caused by the inability of affected cells to properly regulate their microtubule dynamic due to mis-regulation by tau. In support of this model, our recent data demonstrate that missense FTDP-17 mutations that alter amino acid residues near tau's microtubule binding region strikingly modify the ability of tau to modulate microtubule dynamics. Additional recent data from our labs support the notion that the same dysfunction occurs in the FTDP-17 regulatory mutations that alter tau RNA splicing patterns. Our model posits that the dynamics of microtubules in neuronal cells must be tightly regulated to enable them to carry out their diverse functions, and that microtubules that are either over-stabilized or under-stabilized, that is, outside an acceptable window of dynamic activity, lead to neurodegeneration. An especially attractive aspect of this model is that it readily accommodates both the structural and regulatory classes of FTDP-17 mutations.     

Interaction of tau protein with model lipid membranes induces tau structural compaction and membrane disruption

The misfolding and aggregation of the intrinsically disordered, microtubule-associated tau protein into neurofibrillary tangles is implicated in the pathogenesis of Alzheimer's disease. However, the mechanisms of tau aggregation and toxicity remain unknown. Recent work has shown that anionic lipid membranes can induce tau aggregation and that membrane permeabilization may serve as a pathway by which protein aggregates exert toxicity, suggesting that the plasma membrane may play dual roles in tau pathology. This prompted our investigation to assess tau's propensity to interact with membranes and to elucidate the mutually disruptive structural perturbations the interactions induce in both tau and the membrane. We show that although highly charged and soluble, the full-length tau (hTau40) is also highly surface active, selectively inserts into anionic DMPG lipid monolayers and induces membrane morphological changes. To resolve molecular-scale structural details of hTau40 associated with lipid membranes, X-ray and neutron scattering techniques are utilized. X-ray reflectivity indicates hTau40s presence underneath a DMPG monolayer and penetration into the lipid headgroups and tailgroups, whereas grazing incidence X-ray diffraction shows that hTau40 insertion disrupts lipid packing. Moreover, both air/water and DMPG lipid membrane interfaces induce the disordered hTau40 to partially adopt a more compact conformation with density similar to that of a folded protein. Neutron reflectivity shows that tau completely disrupts supported DMPG bilayers while leaving the neutral DPPC bilayer intact. Our results show that hTau40s strong interaction with anionic lipids induces tau structural compaction and membrane disruption, suggesting possible membrane-based mechanisms of tau aggregation and toxicity in neurodegenerative diseases.     

Suprachiasmatic nucleus and photic entrainment of circannual rhythms in ground squirrels.

The efficacy of photoperiod as a zeitgeber for entrainment of circannual body weight and estrous rhythms was tested in female golden-mantled ground squirrels maintained for 3 or more years in either a simulated natural photoperiod (SNP) or a fixed LD 14:10 photoperiod (FP). The role of the retinohypothalamic tract--suprachiasmatic nucleus (RHT-SCN) projection in photic entrainment was assessed in animals that sustained destruction of the SCN (SCNX). Circannual rhythms were lengthened by the SNP as compared to the FP. Mean periods (tau's) for neurologically intact animals in the third year of testing were 49.6 +/- 0.3 weeks and 43.1 +/- 1.2 weeks (p less than 0.001) for the SNP and FP groups, respectively; furthermore, 56% and 7% of animals in these groups had tau's not significantly different from 365 days (p less than 0.005), and within-group variability was lower for SNP than for FP squirrels (p less than 0.01). SCNX squirrels differed from animals with the SCN intact (SCNC), as evidenced by higher within-group variability (p less than 0.001); only 29% of SCNX squirrels had tau's not different from 365 days (p less than 0.03 compared to the SCNC group). The coupling between estrous and body weight rhythms that was evident in SCN-intact SNP and FP squirrels was disrupted in SCNX animals. The RHT-SCN pathway is implicated in entrainment and in maintenance of normal phase relations among the several circannual rhythms. In a second experiment, female squirrels were maintained for 2.5 years in an accelerated SNP that compressed two normal annual photocycles into each calendar year. Of 12 squirrels, 3 had tau's that did not differ significantly from 6 months; 6 had tau's equivalent to 12 months; and 3 had tau's significantly different from both 6 months and 12 months. The data suggest that photoperiod is a major zeitgeber for entrainment of golden-mantled ground squirrels circannual rhythms.     

Increased expression of the Ras suppressor Rsu-1 enhances Erk-2 activation and inhibits Jun kinase activation.

Studies were undertaken to determine the effect of the Ras suppressor Rsu-1 on Ras signal transduction pathways in two different cell backgrounds. An expression vector containing the mouse rsu-1 cDNA under the control of a mouse mammary tumor virus promoter was introduced into NIH 3T3 cells and the pheochromocytoma cell line PC12. Cell lines developed in the NIH 3T3 background expressed p33rsu-1 at approximately twice the normal endogenous level. However, PC12 cell clones which expressed p33rsu-1 at an increased level in a regulatable fashion in response to dexamethasone were isolated. Analysis of proteins involved in regulation of Ras and responsive to Ras signal transduction revealed similar changes in the two cell backgrounds in the presence of elevated p33rsu-1. There was an increase in the level of SOS, the guanine nucleotide exchange factor, and an increase in the percentage of GTP-bound Ras. In addition, there was an increase in the amount of p120 Ras-specific GTPase-activating protein (GAP) and GAP-associated p190. However, a decrease in Ras GTPase-activating activity was detected in lysates of the Rsu-1 transfectants, and immunoprecipitated p120 GAP from the Rsu-1 transfectants showed less Ras GTPase-activating activity than GAP from control cells. Activation of Erk-2 kinase by growth factor and tetradecanyol phorbol acetate was greater in the Rsu-1 transfectants than in control cells. However, c-Jun amino-terminal kinase activity (Jun kinase) was not activatable by epidermal growth factor in Rsu-1 PC12 cell transfectants, in contrast to the PC12 vector control cell line. Transient expression of p33rsu-1 in Cos1 cells following cotransfection with either hemagglutinin-tagged Jun kinase or hemagglutinin-tagged Erk-2 revealed that Rsu-1 expression inhibited constitutive Jun kinase activity while enhancing Erk-2 activity. Detection of in vitro binding of Rsu-1 to Raf-1 suggested that in Rsu-1 transfectants, increased activation of the Raf-1 pathway occurred at the expense of activation of signal transduction leading to Jun kinase. These results indicate that inhibition of Jun kinase activation was sufficient to inhibit Ras transformation even in the presence of activated Erk-2.     

Glutathione S-transferase Pi is a dopamine-inducible suppressor of dopamine-induced apoptosis in PC12 cells

The finding that the neurotransmitter dopamine induces apoptosis in neurons implies the existence of a cellular mechanism by which dopaminergic neurons protect themselves from dopamine-induced apoptosis. By profiling the expression of a number of genes in differentiating PC12 cells which exhibit elevated levels of dopamine biosynthesis, we found that expression of glutathione S-transferase class Pi (GSTp) mRNA was selectively up-regulated. Interestingly, dopamine added to the culture medium of PC12 cells also augmented their expression of GSTp mRNA. Suppression of GSTp expression by transfection of its antisense expression vector augmented dopamine-induced apoptosis of PC12 cells. Conversely, overexpression of GSTp made the resultant PC12 transfectants highly resistant to dopamine-induced apoptosis. Transfection of the antisense or sense GSTp expression vectors also resulted in corresponding augmentation or suppression of dopamine-induced activation of cell-associated Jun-N-terminal kinase (JNK), which has been suggested to mediate dopamine-induced apoptosis in neuronal cells. These results indicate that GSTp is a dopamine-inducible suppressor of dopamine-induced apoptosis in PC12 cells, and suggest that this activity is exerted through inhibition of JNK activity.     

The frontotemporal dementia mutation R406W blocks tau's interaction with the membrane in an annexin A2-dependent manner

Changes of the microtubule-associated protein tau are central in Alzheimer's disease (AD) and frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). However, the functional consequence of the FTDP-17 tau mutation R406W, which causes a tauopathy clinically resembling AD, is not well understood. We find that the R406W mutation does not affect microtubule interaction but abolishes tau's membrane binding. Loss of binding is associated with decreased trapping at the tip of neurites and increased length fluctuations during process growth. Tandem affinity purification tag purification and mass spectrometry identify the calcium-regulated plasma membrane-binding protein annexin A2 (AnxA2) as a potential interaction partner of tau. Consistently, wild-type tau but not R406W tau interacts with AnxA2 in a heterologous yeast expression system. Sequestration of Ca(2+) or knockdown of AnxA2 abolishes the differential trapping of wild-type and R406W tau. We suggest that the pathological effect of the R406W mutation is caused by impaired membrane binding, which involves a functional interaction with AnxA2 as a membrane-cytoskeleton linker.