The binding and phosphorylation of Thr231 is critical for Tau's hyperphosphorylation and functional regulation by glycogen synthase kinase 3beta

Neuropathological hallmarks of Alzheimer's disease are extracellular senile plaques and intracellular neurofibrillary lesions. The neurofibrillary lesions mainly consist of the hyperphosphorylated microtubule-associated protein Tau predominantly expressed in the axon of CNS neurons. Hyperphosphorylation of Tau negatively affects its binding to tubulin and decreases the capacity to promote microtubule assembly. Among a number of proline-directed kinases capable of phosphorylating paired helical filament-Tau, glycogen synthase kinase 3beta (GSK3beta) was first identified as a Tau protein kinase I and has been demonstrated to phosphorylate Tau both in vivo and in vitro. However, the phosphorylation mechanism of Tau by GSK3beta remained unclear. In this study, we show that the T231 is the primary phosphorylation site for GSK3beta and the Tau227-237 (AVVRTPPKSPS) derived from Tau containing T231P232 motif is identified as the GSK3beta binding site with high affinity of a Kd value 0.82 +/- 0.16 mumol/L. Our results suggest that direct binding and phosphorylation of T231P232 motif by GSK3beta induces conformational change of Tau and consequentially alters the inhibitory activity of its N-terminus that allows the phosphorylation of C-terminus of Tau by GSK3beta. Furthermore, hyperphosphorylation reduces Tau's ability to promote tubulin assembly and to form bundles in N18 cells. T231A mutant completely abolishes Tau phosphorylation by GSK3beta and retains the ability to promote tubulin polymerization and bundle formation. Taken together, these results suggest that phosphorylation of T231 by GSK3beta may play an important role in Tau's hyperphosphorylation and functional regulation.     

Novel phosphorylation sites in tau from Alzheimer brain support a role for casein kinase 1 in disease pathogenesis

Tau in Alzheimer disease brain is highly phosphorylated and aggregated into paired helical filaments comprising characteristic neurofibrillary tangles. Here we have analyzed insoluble Tau (PHF-tau) extracted from Alzheimer brain by mass spectrometry and identified 11 novel phosphorylation sites, 10 of which were assigned unambiguously to specific amino acid residues. This brings the number of directly identified sites in PHF-tau to 39, with an additional six sites indicated by reactivity with phosphospecific antibodies to Tau. We also identified five new phosphorylation sites in soluble Tau from control adult human brain, bringing the total number of reported sites to nine. To assess which kinases might be responsible for Tau phosphorylation, we used mass spectrometry to determine which sites were phosphorylated in vitro by several kinases. Casein kinase 1delta and glycogen synthase kinase-3beta were each found to phosphorylate numerous sites, and each kinase phosphorylated at least 15 sites that are also phosphorylated in PHF-tau from Alzheimer brain. A combination of casein kinase 1delta and glycogen synthase kinase-3beta activities could account for over three-quarters of the serine/threonine phosphorylation sites identified in PHF-tau, indicating that casein kinase 1delta may have a role, together with glycogen synthase kinase-3beta, in the pathogenesis of Alzheimer disease.     

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.     

Protein kinase MARK/PAR-1 is required for neurite outgrowth and establishment of neuronal polarity

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.     

Species specificity of the cytokine function of phosphoglucose isomerase

In Alzheimer's disease, neurofibrillary degeneration results from the aggregation of abnormally phosphorylated Tau proteins into paired helical filaments. These Tau variants displayed specific epitopes that are immunoreactive with anti-phospho-Tau antibodies such as AT100. As shown in in vitro experiments, glycogen synthase kinase 3 beta (GSK3beta) and protein kinase A (PKA) may be key kinases in these phosphorylation events. In the present study, Tau was microinjected into Xenopus oocytes. Surprisingly, in this system, AT100 was generated without any GSK3beta and PKA contribution during the progesterone or insulin-induced maturation process. Our results demonstrate that a non-modified physiological process in a cell model can generate the most specific Alzheimer epitope of Tau pathology.     

GSK-3beta directly phosphorylates and activates MARK2/PAR-1

In Alzheimer disease (AD), the microtubule-associated protein tau is found hyperphosphorylated in paired helical filaments. Among many phosphorylated sites in tau, Ser-262 is the major site for abnormal phosphorylation of tau in AD brain. The kinase known to phosphorylate this particular site is MARK2, whose activation mechanism is yet to be studied. Our first finding that treatment of cells with LiCl, a selective inhibitor of another major tau kinase, glycogen synthase kinase-3beta (GSK-3beta), inhibits phosphorylation of Ser-262 of tau led us to investigate the possible involvement of GSK-3beta in MARK2 activation. In vitro kinase reaction revealed that recombinant GSK-3beta indeed phosphorylates MARK2, whereas it failed to phosphorylate Ser-262 of tau. Our further findings led us to conclude that GSK-3beta phosphorylates MARK2 on Ser-212, one of the two reported phosphorylation sites (Thr-208 and Ser-212) found in the activation loop of MARK2. Down-regulation of either GSK-3beta or MARK2 by small interfering RNAs suppressed the level of phosphorylation on Ser-262. These results, respectively, indicated that GSK-3beta is responsible for phosphorylating Ser-262 of tau through phosphorylation and activation of MARK2 and that the phosphorylation of tau at this particular site is predominantly mediated by a GSK-3beta-MARK2 pathway. These findings are of interest in the context of the pathogenesis of AD.     

Spred1 and TESK1--two new interaction partners of the kinase MARKK/TAO1 that link the microtubule and actin cytoskeleton

The signaling from MARKK/TAO1 to the MAP/microtubule affinity-regulating kinase MARK/Par1 to phosphorylated microtubule associated proteins (MAPs) renders microtubules dynamic and plays a role in neurite outgrowth or polarity development. Because hyperphosphorylation of Tau at MARK target sites is a hallmark of Alzheimer neurodegeneration, we searched for upstream regulators by the yeast two-hybrid approach and identified two new interaction partners of MARKK, the regulatory Sprouty-related protein with EVH-1 domain1 (Spred1) and the testis-specific protein kinase (TESK1). Spred1-MARKK binding has no effect on the activity of MARKK; therefore, it does not change microtubule (MT) stability. Spred1-TESK1 binding causes inhibition of TESK1. Because TESK1 can phosphorylate cofilin and thus stabilizes F-actin stress fibers, the inhibition of TESK1 by Spred1 makes F-actin fibers dynamic. A third element in this interaction triangle is that TESK1 binds to and inhibits MARKK. Thus, in Chinese hamster ovary (CHO) cells the elevation of MARKK results in MT disruption (via activation of MARK/Par1 and phosphorylation of MAPs), but this can be blocked by TESK1. Similarly, enhanced TESK1 activity results in increased stress fibers (via phospho-cofilin), but this can be blocked by elevating Spred1. Thus, the three-way interaction between Spred1, MARKK, and TESK1 represents a pathway that links regulation of both the microtubule- and F-actin cytoskeleton.     

Microtubule/MAP-affinity regulating kinase (MARK) is activated by phenylarsine oxide in situ and phosphorylates tau within its microtubule-binding domain

Tau is a microtubule-associated protein (MAP) that is functionally modulated by phosphorylation and that is hyperphosphorylated in several neurodegenerative diseases. Because phosphorylation regulates both normal and pathological tau functioning, it is of interest to identify the signaling pathways and enzymes capable of modulating tau phosphorylation in vivo. Previously, it was demonstrated that in SH-SY5Y human neuroblastoma cells and rat primary cortical cultures tau is phosphorylated at Ser262/356, within its microtubule-binding domain, by a staurosporine-sensitive protein kinase in response to the vicinal thiol-directed agent phenylarsine oxide. The current study demonstrates the presence of a 100-kDa protein kinase activity in SH-SY5Y cells that associates with microtubules, phosphorylates tau at Ser262/356, is activated by phenylarsine oxide, and is inhibited by the protein kinase inhibitor staurosporine. Isolation of individual protein bands from a polyacrylamide gel revealed two closely spaced proteins containing Ser262/356-directed protein kinase activity. Mass spectrometry analysis indicated that these protein bands correspond to the 100-kDa microtubule/MAP-affinity regulating kinase (MARK), which has been shown previously to phosphorylate tau within its microtubule-binding domain. Immunoblot analysis of the protein kinase bands confirmed this finding, providing the first demonstration that activation of endogenous MARK results in increased tau phosphorylation within its microtubule-binding domain in situ.     

Microtubule affinity-regulating kinase 1 (MARK1) is activated by electroconvulsive shock in the rat hippocampus

Electroconvulsive shock (ECS) induces phosphorylation and dephosphorylation of many signaling molecules in the rat brain. While studying phosphorylated proteins in the rat brain after ECS, we observed a 100-kDa protein that cross-reacted with anti-phospho-p70 S6 kinase antibody, which was subsequently purified and identified as microtubule affinity-regulating kinase 1 (MARK1). Purified MARK1 was phosphorylated at the Ser and Thr residues. MARK1 activation and subsequent Tau phosphorylation in the hippocampus after ECS was confirmed by an in-gel kinase assay using tau protein as a substrate. MARK1 was maximally activated between 2 and 5 min after ECS, and Tau phosphorylation at Ser262 was also increased at 2 min and lasted to 1 h after ECS. Taken together, we concluded that ECS activated MARK1 and subsequently phosphorylated Tau at Ser262. Both MARK1 activity and Tau phosphorylation were increased in the rat hippocampus after chronic ECS where axonal remodeling was apparent. In order to investigate the physiologic stimuli which are involved in the activation of MARK1, SH-SY 5Y cells were treated with brain-derived neurotrophic factor or 60 mm KCl. Both stimuli were capable of inducing MARK activation.     

In vivo and in vitro phosphorylation at Ser-493 in the glutamate (E)-segment of neurofilament-H subunit by glycogen synthase kinase 3beta

Neurofilament (NF), a major neuronal intermediate filament, is composed of three subunits, NF-L, NF-M, and NF-H. All three subunits contain a well conserved glutamate (E)-rich region called "E-segment" in the N terminus of the tail region. Although the E-segments of NF-L and NF-M are phosphorylated by casein kinases, it has not been observed in NF-H. Using mass spectrometric analysis, we identified phosphorylation of the E-segment of NF-H, prepared from rat spinal cords, at Ser-493 and Ser-501 in the Ser-Pro sequences. The E-segment kinase was isolated from rat brain extract using column chromatography and identified as glycogen synthase kinase (GSK) 3beta. GSK3beta was shown to phosphorylate at Ser-493 in vitro by phosphopeptide mapping and site-directed mutagenesis, and in vivo in HEK293 cells using the phospho-Ser-493 antibody, but did not phosphorylate Ser-501. GSK3beta preferred Ser-493 to the KSP-repeated sequences for phosphorylation sites in the NF-H tail domain. Moreover, Ser-493 was a better phosphorylation site for GSK3beta than other proline-directed protein kinases, Cdk5/p35 and ERK. GSK3beta in the spinal cord extract was associated with NF cytoskeletons. Taken together, we concluded that Ser-493 in the E-segment of NF-H is phosphorylated by GSK3beta in rat spinal cords.     

Phosphorylation sites on tau identified by nanoelectrospray mass spectrometry: differences in vitro between the mitogen-activated protein kinases ERK2, c-Jun N-terminal kinase and P38, and glycogen synthase kinase-3beta

The stress-activated kinases c-Jun N-terminal kinase (JNK) and p38 are members of the mitogen-activated protein (MAP) kinase family and take part in signalling cascades initiated by various forms of stress. Their targets include the microtubule-associated protein tau, which becomes hyperphosphorylated in Alzheimer's disease. It is necessary, as a forerunner for in vivo studies, to identify the protein kinases and phosphatases that are responsible for phosphate turnover at individual sites. Using nanoelectrospray mass spectrometry, we have undertaken an extensive comparison of phosphorylation in vitro by several candidate tau kinases, namely, JNK, p38, ERK2, and glycogen synthase kinase 3beta (GSK3beta). Between 10 and 15 sites were identified for each kinase. The three MAP kinases phosphorylated Ser202 and Thr205 but not detectably Ser199, whereas conversely GSK3beta phosphorylated Ser199 but not detectably Ser202 or Thr205. Phosphorylated Ser404 was found with all of these kinases except JNK. The MAP kinases may not be strictly proline specific: p38 phosphorylated the nonproline sites Ser185, Thr245, Ser305, and Ser356, whereas ERK2 was the most strict. All of the sites detected except Thr245 and Ser305 are known or suspected phosphorylation sites in paired helical filament-tau extracted from Alzheimer brains. Thus, the three MAP kinases and GSK3beta are importantly all strong candidates as tau kinases that may be involved in the pathogenic hyperphosphorylation of tau in Alzheimer's disease.     

Cooperative phosphorylation of the tumor suppressor phosphatase and tensin homologue (PTEN) by casein kinases and glycogen synthase kinase 3beta

The phosphatase and tensin homologue (PTEN) tumor suppressor is a phosphatidylinositol D3-phosphatase that counteracts the effects of phosphatidylinositol 3-kinase and negatively regulates cell growth and survival. PTEN is itself regulated by phosphorylation on multiple serine and threonine residues in its C terminus. Previous work has implicated casein kinase 2 (CK2) as the kinase responsible for this phosphorylation. Here we showed that CK2 does not phosphorylate all sites in PTEN and that glycogen synthase kinase 3beta (GSK3beta) also participates in PTEN phosphorylation. Although CK2 mainly phosphorylated PTEN at Ser-370 and Ser-385, GSK3beta phosphorylated Ser-362 and Thr-366. More importantly, prior phosphorylation of PTEN at Ser-370 by CK2 strongly increased its phosphorylation at Thr-366 by GSK3beta, suggesting that the two may synergize. Using RNA interference, we showed that GSK3 phosphorylates PTEN in intact cells. Finally, PTEN phosphorylation was affected by insulin-like growth factor in intact cells. We concluded that multiple kinases, including CK2 and GSK3beta, participate in PTEN phosphorylation and that GSK3beta may provide feedback regulation of PTEN.     

Spectroscopic Studies of GSK3β Phosphorylation of the Neuronal Tau Protein and Its Interaction with the N-terminal Domain of Apolipoprotein E

Alzheimer disease neurons are characterized by extraneuronal plaques formed by aggregated amyloid-β peptide and by intraneuronal tangles composed of fibrillar aggregates of the microtubule-associated Tau protein. Tau is mostly found in a hyperphosphorylated form in these tangles. Glycogen synthase kinase 3β (GSK3β) is a proline-directed kinase generally considered as one of the major players that (hyper)phosphorylates Tau. The kinase phosphorylates mainly (Ser/Thr)-Pro motifs and is believed to require a priming activity by another kinase. Here, we use an in vitro phosphorylation assay and NMR spectroscopy to characterize in a qualitative and quantitative manner the phosphorylation of Tau by GSK3β. We find that three residues can be phosphorylated (Ser-396, Ser-400, and Ser-404) by GSK3β alone, without priming. Ser-404 is essential in this process, as its mutation to Ala prevents all activity of GSK3β. However, priming enhances the catalytic efficacy of the kinase, as initial phosphorylation of Ser-214 by the cAMP-dependent protein kinase (PKA) leads to the rapid modification by GSK3β of four regularly spaced additional sites. Because the regular incorporation of negative charges by GSK3β leads to a potential parallel between phospho-Tau and heparin, we investigated its interaction with the heparin/low density lipoprotein receptor binding domain of human apolipoprotein E. We indeed observed an interaction between the GSK3β-promoted regular phospho-pattern on Tau and the apolipoprotein E fragment but none in the absence of phosphorylation or the presence of an irregular phosphorylation pattern by the prolonged activity of PKA. Apolipoprotein E is therefore able to discriminate and interact with specific phosphorylation patterns of Tau.     

Glycogen synthase kinase 3beta is tyrosine phosphorylated by PYK2

Glycogen synthase kinase 3beta (GSK3beta) is a Ser/Thr kinase that is involved in numerous cellular activities. GSK3beta is activated by tyrosine phosphorylation. However, very little is known about the tyrosine kinases that are responsible for phosphorylating GSK3beta. In this report, we investigated the ability of the calcium-dependent tyrosine kinase, proline-rich tyrosine kinase 2 (PYK2) to tyrosine phosphorylate GSK3beta. In transfected CHO cells, it was demonstrated that PYK2 tyrosine phosphorylates GSK3beta in situ. The two kinases also coimmunoprecipitated. Furthermore, GSK3beta was tyrosine phosphorylated in vitro by an active, wild type PYK2, but not by the inactive, kinase dead form of PYK2. Therefore, this study is the first to demonstrate that GSK3beta is a substrate of PYK2 both in vitro and in situ.     

The direct activation of MIK,a germinal center kinase (GCK)-like kinase,by MARK,a maize atypical receptor kinase,suggests a new mechanism for signaling through kinase-dead receptors

Signaling by receptor protein kinases (RPKs) involves their dimerization and transphosphorylation. However, atypical RPKs with kinase-defective domains have been described recently. Some of them are essential for proper signaling in animal systems, although the precise mechanism involved is unknown in most cases. Here we describe the cloning and characterization of an atypical plant receptor kinase from maize, MARK, which does not phosphorylate in vitro. A yeast two-hybrid approach has allowed us to identify a new germinal center kinase (GCK)-related protein, MIK, that interacts with MARK. Interestingly, the interaction of the intracellular domain of MARK with the regulator domain of MIK strongly induces MIK kinase activity. As some GCK-related proteins connect cell-surface receptors to the intracellular MAPK cascades, the activation of MIK by direct interaction with MARK could illustrate a new mechanism for signaling through atypical RPKs.     

Relative resistance of Cdk5-phosphorylated CRMP2 to dephosphorylation

Collapsin response mediator protein 2 (CRMP2) binds to microtubules and regulates axon outgrowth in neurons. This action is regulated by sequential phosphorylation by the kinases cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3 (GSK3) at sites that are hyperphosphorylated in Alzheimer disease. The increased phosphorylation in Alzheimer disease could be due to increases in Cdk5 and/or GSK3 activity or, alternatively, through decreased activity of a CRMP phosphatase. Here we establish that dephosphorylation of CRMP2 at the residues targeted by GSK3 (Ser-518/Thr-514/Thr-509) is carried out by a protein phosphatase 1 family member in vitro, in neuroblastoma cells, and primary cortical neurons. Inhibition of GSK3 activity using insulin-like growth factor-1 or the highly selective inhibitor CT99021 causes rapid dephosphorylation of CRMP2 at these sites. In contrast, pharmacological inhibition of Cdk5 using purvalanol results in only a gradual and incomplete dephosphorylation of CRMP2 at the site targeted by Cdk5 (Ser-522), suggesting a distinct phosphatase targets this residue. A direct comparison of dephosphorylation at the Cdk5 versus GSK3 sites in vitro shows that the Cdk5 site is comparatively resistant to phosphatase treatment. The presence of the peptidyl-prolyl isomerase enzyme, Pin1, does not affect dephosphorylation of Ser-522 in vitro, in cells, or in Pin1 transgenic mice. Instead, the relatively high resistance of this site to phosphatase treatment is at least in part due to the presence of basic residues located nearby. Similar sequences in Tau are also highly resistant to phosphatase treatment. We propose that relative resistance to phosphatases might be a common feature of Cdk5 substrates and could contribute to the hyperphosphorylation of CRMP2 and Tau observed in Alzheimer disease.     

LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1

We recently demonstrated that the LKB1 tumour suppressor kinase, in complex with the pseudokinase STRAD and the scaffolding protein MO25, phosphorylates and activates AMP-activated protein kinase (AMPK). A total of 12 human kinases (NUAK1, NUAK2, BRSK1, BRSK2, QIK, QSK, SIK, MARK1, MARK2, MARK3, MARK4 and MELK) are related to AMPK. Here we demonstrate that LKB1 can phosphorylate the T-loop of all the members of this subfamily, apart from MELK, increasing their activity >50-fold. LKB1 catalytic activity and the presence of MO25 and STRAD are required for activation. Mutation of the T-loop Thr phosphorylated by LKB1 to Ala prevented activation, while mutation to glutamate produced active forms of many of the AMPK-related kinases. Activities of endogenous NUAK2, QIK, QSK, SIK, MARK1, MARK2/3 and MARK4 were markedly reduced in LKB1-deficient cells. Neither LKB1 activity nor that of AMPK-related kinases was stimulated by phenformin or AICAR, which activate AMPK. Our results show that LKB1 functions as a master upstream protein kinase, regulating AMPK-related kinases as well as AMPK. Between them, these kinases may mediate the physiological effects of LKB1, including its tumour suppressor function.     

Suppression of tubulin polymerization by the LKB1-microtubule-associated protein/microtubule affinity-regulating kinase signaling

LKB1, a tumor suppressor gene mutated in the Peutz-Jeghers syndrome, encodes a serine/threonine protein kinase. Recent biochemical studies have shown that LKB1 activates 14 AMP-activated protein kinase-related kinases including MARKs (microtubule-associated protein/microtubule affinity-regulating kinases) that regulate microtubule dynamics. Here we show in vitro that LKB1 phosphorylates and activates MARK2, which in turn phosphorylates microtubule-associated protein Tau at the KXGS motif and suppresses tubulin polymerization. In cells, forced expression of LKB1 suppresses microtubule regrowth, whereas LKB1 knockdown accelerates it. We further show that the phosphorylation of Tau by the LKB1-MARK signaling triggers proteasome-mediated degradation of Tau. These results indicate that LKB1 is involved in the regulation of microtubule dynamics through the activation of MARKs.