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.     

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.     

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.     

Phosphorylation of human microtubule-associated protein tau by protein kinases of the AGC subfamily

Intraneuronal inclusions made of hyperphosphorylated microtubule-associated protein tau are a defining neuropathological characteristic of Alzheimer's disease, and of several other neurodegenerative disorders. Many phosphorylation sites in tau are S/TP sites that flank the microtubule-binding repeats. Others are KXGS motifs in the repeats. One site upstream of the repeats lies in a consensus sequence for AGC kinases. This site (S214) is believed to play an important role in the events leading from normal, soluble to filamentous, insoluble tau. Here, we show that all AGC kinases tested phosphorylated S214. RSK1 and p70 S6 kinase also phosphorylated the neighbouring T212, a TP site that conforms weakly to the AGC kinase consensus sequence. MSK1 phosphorylated S214, as well as S262, a KXGS site in the first repeat, and S305 in the second repeat.     

Biochemical purification uncovers mammalian sterile 3 (MST3) as a new protein kinase for multifunctional protein kinases AMPK and SIK3

The AMP-activated protein kinase (AMPK) and AMPK-related kinase salt-inducible kinase 3 (SIK3) regulate many important biological processes ranging from metabolism to sleep. Liver kinase B1 is known to phosphorylate and activate both AMPK and SIK3, but the existence of other upstream kinases was unclear. In this study, we detected liver kinase B1–independent AMPK-related kinase phosphorylation activities in human embryonic kidney cells as well as in mouse brains. Biochemical purification of this phosphorylation activity uncovered mammalian sterile 20–like kinase 3 (MST3). We demonstrate that MST3 from human embryonic kidney cells could phosphorylate AMPK and SIK3 in vivo. In addition, recombinant MST3 expressed in and purified from Escherichia coli could directly phosphorylate AMPK and SIK3 in vitro. Moreover, four other members of the MST kinase family could also phosphorylate AMPK or SIK3. Our results have revealed new kinases able to phosphorylate and activate AMPK and SIK3.     

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.     

Microtubule affinity–regulating kinase 4 with an Alzheimer's disease-related mutation promotes tau accumulation and exacerbates neurodegeneration

Accumulation of the microtubule-associated protein tau is associated with Alzheimer''s disease (AD). In AD brain, tau is abnormally phosphorylated at many sites, and phosphorylation at Ser-262 and Ser-356 plays critical roles in tau accumulation and toxicity. Microtubule affinity–regulating kinase 4 (MARK4) phosphorylates tau at those sites, and a double de novo mutation in the linker region of MARK4, ΔG316E317D, is associated with an elevated risk of AD. However, it remains unclear how this mutation affects phosphorylation, aggregation, and accumulation of tau and tau-induced neurodegeneration. Here, we report that MARK4^ΔG316E317D increases the abundance of highly phosphorylated, insoluble tau species and exacerbates neurodegeneration via Ser-262/356–dependent and –independent mechanisms. Using transgenic Drosophila expressing human MARK4 (MARK4^wt) or a mutant version of MARK4 (MARK4^ΔG316E317D), we found that coexpression of MARK4^wt and MARK4^ΔG316E317D increased total tau levels and enhanced tau-induced neurodegeneration and that MARK4^ΔG316E317D had more potent effects than MARK4^wt. Interestingly, the in vitro kinase activities of MARK4^wt and MARK4^ΔG316E317D were similar. When tau phosphorylation at Ser-262 and Ser-356 was blocked by alanine substitutions, MARK4^wt did not promote tau accumulation or exacerbate neurodegeneration, whereas coexpression of MARK4^ΔG316E317D did. Both MARK4^wt and MARK4^ΔG316E317D increased the levels of oligomeric forms of tau; however, only MARK4^ΔG316E317D further increased the detergent insolubility of tau in vivo. Together, these findings suggest that MARK4^ΔG316E317D increases tau levels and exacerbates tau toxicity via a novel gain-of-function mechanism and that modification in this region of MARK4 may affect disease pathogenesis.     

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.     

Akt/PKB kinase phosphorylates separately Thr212 and Ser214 of tau protein in vitro

Microtubule-associated protein tau contains a consensus motif for protein kinase B/Akt (Akt), which plays an essential role in anti-apoptotic signaling. The motif encompasses the AT100 double phospho-epitope (Thr212/Ser214), a specific marker for Alzheimer's disease (AD) and other neurodegenerations, raising the possibility that it could be generated by Akt. We studied Akt-dependent phosphorylation of tau protein in vitro. We found that Akt phosphorylated both Thr212 and Ser214 in the longest and shortest tau isoforms as determined using phospho site-specific antibodies against tau. Akt did not phosphorylate other tau epitopes, including Tau-1, AT8, AT180, 12E8 and PHF-1. The Akt-phosphorylated tau retained its initial electrophoretic mobility. Immunoprecipitation studies with phospho-specific Thr212 and Ser214 antibodies revealed that only one of the two sites is phosphorylated per single tau molecule, resulting in tau immunonegative for AT100. Mixed kinase studies showed that prior Ser214 phosphorylation by Akt blocked protein kinase A but not GSK3beta activity. On the other hand, GSK3beta selectively blocked Ser214 phosphorylation, which was prevented by lithium. The results suggest that Akt may be involved in AD-specific phosphorylation of tau at the AT100 epitope in conjunction with other kinases. Our data suggest that phosphorylation of tau by Akt may play specific role(s) in Akt-mediated anti-apoptotic signaling, particularly relevant to AD and other neurodegenerations.     

Control of AMPK-related kinases by USP9X and atypical Lys(29)/Lys(33)-linked polyubiquitin chains

AMPK (AMP-activated protein kinase)-related kinases regulate cell polarity as well as proliferation and are activated by the LKB1-tumour suppressor kinase. In the present study we demonstrate that the AMPK-related kinases, NUAK1 (AMPK-related kinase 5) and MARK4 (microtubule-affinity-regulating kinase 4), are polyubiquitinated in vivo and interact with the deubiquitinating enzyme USP9X (ubiquitin specific protease-9). Knockdown of USP9X increased polyubiquitination of NUAK1 and MARK4, whereas overexpression of USP9X inhibited ubiquitination. USP9X, catalysed the removal of polyubiquitin chains from wild-type NUAK1, but not from a non-USP9X-binding mutant. Topological analysis revealed that ubiquitin monomers attached to NUAK1 and MARK4 are linked by Lys(29) and/or Lys(33) rather than the more common Lys(48)/Lys(63). We find that AMPK and other AMPK-related kinases are also polyubiquitinated in cells. We identified non-USP9X-binding mutants of NUAK1 and MARK4 and find that these are hyper-ubiquitinated and not phosphorylated at their T-loop residue targeted by LKB1 when expressed in cells, suggesting that polyubiquitination may inhibit these enzymes. The results of the present study demonstrate that NUAK1 and MARK4 are substrates of USP9X and provide the first evidence that AMPK family kinases are regulated by unusual Lys(29)/Lys(33)-linked polyubiquitin chains.     

Regulation of the AMPK-related protein kinases by ubiquitination

How can a constitutively active 'master' kinase with numerous downstream targets preferentially phosphorylate one or more of these without influencing all simultaneously? How might such a system be switched off? The characterization of the role of deubiquitination in regulating the phosphorylation and activation of AMPK (AMP-activated protein kinase)-related kinases by LKB1 suggests a novel and interesting mechanism for conferring signal transduction specificity and control at the kinase substrate level. In this issue of the Biochemical Journal, Al-Hakim et al. show that the AMPK-related kinases NUAK1 (AMPK-related kinase 5) and MARK4 (microtubule-affinity-regulating kinase 4) are polyubiquitinated in vivo and that they serve as substrates of the deubiquitinating enzyme USP9X; furthermore, the first evidence is provided for regulation of AMPK-related kinase family members mediated via unusual Lys(29)/Lys(33) polyubiquitin chains, rather than the more common Lys(48)/Lys(63) linkages.     

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.     

The development of cell processes induced by tau protein requires phosphorylation of serine 262 and 356 in the repeat domain and is inhibited by phosphorylation in the proline-rich domains

The differentiation of neurons and the outgrowth of neurites depends on microtubule-associated proteins such as tau protein. To study this process, we have used the model of Sf9 cells, which allows efficient transfection with microtubule-associated proteins (via baculovirus vectors) and observation of the resulting neurite-like extensions. We compared the phosphorylation of tau23 (the embryonic form of human tau) with mutants in which critical phosphorylation sites were deleted by mutating Ser or Thr residues into Ala. One can broadly distinguish two types of sites, the KXGS motifs in the repeats (which regulate the affinity of tau to microtubules) and the SP or TP motifs in the domains flanking the repeats (which contain epitopes for antibodies diagnostic of Alzheimer's disease). Here we report that both types of sites can be phosphorylated by endogenous kinases of Sf9 cells, and that the phosphorylation pattern of the transfected tau is very similar to that of neurons, showing that Sf9 cells can be regarded as an approximate model for the neuronal balance between kinases and phosphatases. We show that mutations in the repeat domain and in the flanking domains have opposite effects. Mutations of KXGS motifs in the repeats (Ser262, 324, and 356) strongly inhibit the outgrowth of cell extensions induced by tau, even though this type of phosphorylation accounts for only a minor fraction of the total phosphate. This argues that the temporary detachment of tau from microtubules (by phosphorylation at KXGS motifs) is a necessary condition for establishing cell polarity at a critical point in space or time. Conversely, the phosphorylation at SP or TP motifs represents the majority of phosphate (>80%); mutations in these motifs cause an increase in cell extensions, indicating that this type of phosphorylation retards the differentiation of the cells.     

PAR-1 kinase plays an initiator role in a temporally ordered phosphorylation process that confers tau toxicity in Drosophila

Multisite hyperphosphorylation of tau has been implicated in the pathogenesis of neurodegenerative diseases including Alzheimer's disease (AD). However, the phosphorylation events critical for tau toxicity and mechanisms regulating these events are largely unknown. Here we show that Drosophila PAR-1 kinase initiates tau toxicity by triggering a temporally ordered phosphorylation process. PAR-1 directly phosphorylates tau at S262 and S356. This phosphorylation event is a prerequisite for the action of downstream kinases, including glycogen synthase kinase 3 (GSK-3) and cyclin-dependent kinase-5 (Cdk5), to phosphorylate several other sites and generate disease-associated phospho-epitopes. The initiator role of PAR-1 is further underscored by the fact that mutating PAR-1 phosphorylation sites causes a much greater reduction of overall tau phosphorylation and toxicity than mutating S202, one of the downstream sites whose phosphorylation depends on prior PAR-1 action. These findings begin to differentiate the effects of various phosphorylation events on tau toxicity and provide potential therapeutic targets.     

Prostate-derived Sterile 20-like Kinases (PSKs/TAOKs) Phosphorylate Tau Protein and Are Activated in Tangle-bearing Neurons in Alzheimer Disease

In Alzheimer disease (AD), the microtubule-associated protein tau is highly phosphorylated and aggregates into characteristic neurofibrillary tangles. Prostate-derived sterile 20-like kinases (PSKs/TAOKs) 1 and 2, members of the sterile 20 family of kinases, have been shown to regulate microtubule stability and organization. Here we show that tau is a good substrate for PSK1 and PSK2 phosphorylation with mass spectrometric analysis of phosphorylated tau revealing more than 40 tau residues as targets of these kinases. Notably, phosphorylated residues include motifs located within the microtubule-binding repeat domain on tau (Ser-262, Ser-324, and Ser-356), sites that are known to regulate tau-microtubule interactions. PSK catalytic activity is enhanced in the entorhinal cortex and hippocampus, areas of the brain that are most susceptible to Alzheimer pathology, in comparison with the cerebellum, which is relatively spared. Activated PSK is associated with neurofibrillary tangles, dystrophic neurites surrounding neuritic plaques, neuropil threads, and granulovacuolar degeneration bodies in AD brain. By contrast, activated PSKs and phosphorylated tau are rarely detectible in immunostained control human brain. Our results demonstrate that tau is a substrate for PSK and suggest that this family of kinases could contribute to the development of AD pathology and dementia.     

Comparison of the phosphorylation of microtubule-associated protein tau by non-proline dependent protein kinases

Microtubule-associated protein tau from Alzheimer brain has been shown to be phosphorylated at several ser/thr-pro and ser/thr-X sites (Hasegawa, M. et al., J. Biol. Chem, 267, 17047–17054, 1992). Several proline-dependent protein kinases (PDPKs) (MAP kinase, cdc2 kinase, glycogen synthase kinase-3, tubulin-activated protein kinase, and 40 kDa neurofilament kinase) are implicated in the phosphorylation of the ser-thr-pro sites. The identity of the kinase(s) that phosphorylate that ser/thr-X sites are unknown. To identify the latter kinase(s) we have compared the phosphorylation of bovine tau by several brain protein kinases. Stoichiometric phosphorylation of tau was achieved by casein kinase-1, calmodulin-dependent protein kinase II, Gr kinase, protein kinase C and cyclic AMP-dependent protein kinase, but not with casein kinase-2 or phosphorylase kinase. Casein kinase-1 and calmodulin-dependent protein kinase II were the best tau kinases, with greater than 4 mol and 3 mol³²P incorporated, respectively, into each mol of tau. With the sequential addition of these two kinases,³²P incorporation approached 6 mol. Peptide mapping revealed that the different kinases largely phosphorylate different sites on tau. After phosphorylation by casein kinase-1, calmodulin-dependent protein kinase II, Gr kinase, cyclic AMP-dependent protein kinase and casein kinase-2, the mobility of tau isoforms as detected by SDS-PAGE was decreased. Protein kinase C phosphorylation did not produce such a mobility shift. Our results suggest that one or more of the kinases studied here may participate in the hyperphosphorylation of tau in Alzheimer disease. Such phosphorylation may serve to modulate the activaties of other tau kinases such as the PDPKs.Abbreviations PHF paired helical filaments - A-kinase cyclic AMP-dependent protein kinase - CaM kinase II calcium/calmodulin-dependent protein kinase II - C-kinase calcium-phospholipid-dependent protein kinase - CK-1 casein kinase-1 - CK-2 casein kinase-2 - Gr kinase calcium/calmodulin-dependent protein kinase from rat cerebellum - GSK-3 glycogen synthase kinase-3 - MAP kinase mitogen-activated protein kinase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Phosphorylation of microtubule-associated protein tau by isoforms of c-Jun N-terminal kinase (JNK)

Microtubule-associated protein tau in a hyperphosphorylated state is the major component of the filamentous lesions that define a number of neurodegenerative diseases commonly referred to as tauopathies. Hyperphosphorylation of tau at most sites appears to precede filament assembly. Many of the hyperphosphorylated sites are serine/threonine-proline sequences. Here we show that c-Jun N-terminal kinases JNK1, JNK2 and JNK3 phosphorylate tau at many serine/threonine-prolines, as assessed by the generation of the epitopes of phosphorylation-dependent anti-tau antibodies. Of the three protein kinases, JNK2 phosphorylated the most sites in tau, followed by JNK3 and JNK1. Phosphorylation by JNK isoforms resulted in a greatly reduced ability of tau to promote microtubule assembly. These findings extend the number of candidate protein kinases for the hyperphosphorylation of tau in Alzheimer's disease and other neurodegenerative disorders.     

Phosphorylation of microtubule-associated protein tau by Ca2+/calmodulin-dependent protein kinase II in its tubulin binding sites

The paired helical filaments (PHF) found in Alzheimer's disease (AD) brain are composed mainly of the hyperphosphorylated form of microtubule-associated protein tau (PHF-tau). It is well known that tau is a good in vitro substrate for Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II). To establish the phosphorylation sites, the longest human tau (hTau40) was bacterially expressed and phosphorylated by CaM kinase II, followed by digestion with lysyl endoprotease. The digests were subjected to liquid chromatography/mass spectrometry. We found that 5 of 22 identified peptides were phosphorylated. From the tandem mass spectrometry, two phosphorylation sites (serines 262 and 356) were identified in the tubulin binding sites. When tau was phosphorylated by CaM kinase II, the binding of tau to taxol-stabilized microtubules was remarkably impaired. As both serines 262 and 356 are reportedly phosphorylated in PHF-tau, CaM kinase II may be involved in hyperphosphorylation of tau in AD brain.     

Activity of LKB1 and AMPK-related kinases in skeletal muscle: effects of contraction, phenformin, and AICAR

Activation of AMP-activated protein kinase (AMPK) by exercise and metformin is beneficial for the treatment of type 2 diabetes. We recently found that, in cultured cells, the LKB1 tumor suppressor protein kinase activates AMPK in response to the metformin analog phenformin and the AMP mimetic drug 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). We have also reported that LKB1 activates 11 other AMPK-related kinases. The activity of LKB1 or the AMPK-related kinases has not previously been studied in a tissue with physiological relevance to diabetes. In this study, we have investigated whether contraction, phenformin, and AICAR influence LKB1 and AMPK-related kinase activity in rat skeletal muscle. Contraction in situ, induced via sciatic nerve stimulation, significantly increased AMPKalpha2 activity and phosphorylation in multiple muscle fiber types without affecting LKB1 activity. Treatment of isolated skeletal muscle with phenformin or AICAR stimulated the phosphorylation and activation of AMPKalpha1 and AMPKalpha2 without altering LKB1 activity. Contraction, phenformin, or AICAR did not significantly increase activities or expression of the AMPK-related kinases QSK, QIK, MARK2/3, and MARK4 in skeletal muscle. The results of this study suggest that muscle contraction, phenformin, or AICAR activates AMPK by a mechanism that does not involve direct activation of LKB1. They also suggest that the effects of excercise, phenformin, and AICAR on metabolic processes in muscle may be mediated through activation of AMPK rather than activation of LKB1 or the AMPK-related kinases.     

β-carboline compounds, including harmine, inhibit DYRK1A and tau phosphorylation at multiple Alzheimer's disease-related sites

Harmine, a β-carboline alkaloid, is a high affinity inhibitor of the dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) protein. The DYRK1A gene is located within the Down Syndrome Critical Region (DSCR) on chromosome 21. We and others have implicated DYRK1A in the phosphorylation of tau protein on multiple sites associated with tau pathology in Down Syndrome and in Alzheimer's disease (AD). Pharmacological inhibition of this kinase may provide an opportunity to intervene therapeutically to alter the onset or progression of tau pathology in AD. Here we test the ability of harmine, and numerous additional β-carboline compounds, to inhibit the DYRK1A dependent phosphorylation of tau protein on serine 396, serine 262/serine 356 (12E8 epitope), and threonine 231 in cell culture assays and in vitro phosphorylation assays. Results demonstrate that the β-carboline compounds (1) potently reduce the expression of all three phosphorylated forms of tau protein, and (2) inhibit the DYRK1A catalyzed direct phosphorylation of tau protein on serine 396. By assaying several β-carboline compounds, we define certain chemical groups that modulate the affinity of this class of compounds for inhibition of tau phosphorylation.