Tyrosine hydroxylase phosphorylation is under the control of serine 40.

Tyrosine hydroxylase catalyzes the initial and rate-limiting step in the biosynthesis of the neurotransmitter dopamine. The phosphorylation state of Ser40 and Ser31 is believed to exert a direct effect on the enzymatic activity of tyrosine hydroxylase. Interestingly, some studies report that Ser31 phosphorylation affects Ser40 phosphorylation, while Ser40 phosphorylation has no effect on Ser31 phosphorylation, a process named hierarchical phosphorylation. Here, we provide a detailed investigation into the signal transduction mechanisms regulating Ser40 and Ser31 phosphorylation in dopaminergic mouse MN9D and Neuro2A cells. We find that cyclic nucleotide signaling drives Ser40 phosphorylation, and that Ser31 phosphorylation is strongly regulated by ERK signaling. Inhibition of ERK1/2 with UO126 or PD98059 reduced Ser31 phosphorylation, but surprisingly had no effect on Ser40 phosphorylation, contradicting a role for Ser31 in the regulation of Ser40. Moreover, to elucidate a possible hierarchical mechanism controlling tyrosine hydroxylase phosphorylation, we introduced tyrosine hydroxylase variants in Neuro2A mouse neuroblastoma cells that mimic either phosphorylated or unphosphorylated serine residues. When we introduced a Ser40Ala tyrosine hydroxylase variant, Ser31 phosphorylation was completely absent. Additionally, neither the tyrosine hydroxylase variant Ser31Asp, nor the variant Ser31Ala had any significant effect on basal Ser40 phosphorylation levels. These results suggest that tyrosine hydroxylase is not controlled by hierarchical phosphorylation in the sense that first Ser31 has to be phosphorylated and subsequently Ser40, but, conversely, that Ser40 phosphorylation is essential for Ser31 phosphorylation. Overall our study suggests that Ser40 is the crucial residue to target so as to modulate tyrosine hydroxylase activity.

Transforming growth factor ß (TGFß) induces NUAK kinase expression to fine-tune its signaling output.

TGFß signaling via SMAD proteins and protein kinase pathways up- or down-regulates the expression of many genes and thus affects physiological processes, such as differentiation, migration, cell cycle arrest, and apoptosis, during developmental or adult tissue homeostasis. We here report that NUAK family kinase 1 (NUAK1) and NUAK2 are two TGFß target genes. NUAK1/2 belong to the AMP-activated protein kinase (AMPK) family, whose members control central and protein metabolism, polarity, and overall cellular homeostasis. We found that TGFß-mediated transcriptional induction of NUAK1 and NUAK2 requires SMAD family members 2, 3, and 4 (SMAD2/3/4) and mitogen-activated protein kinase (MAPK) activities, which provided immediate and early signals for the transient expression of these two kinases. Genomic mapping identified an enhancer element within the first intron of the NUAK2 gene that can recruit SMAD proteins, which, when cloned, could confer induction by TGFß. Furthermore, NUAK2 formed protein complexes with SMAD3 and the TGFß type I receptor. Functionally, NUAK1 suppressed and NUAK2 induced TGFß signaling. This was evident during TGFß-induced epithelial cytostasis, mesenchymal differentiation, and myofibroblast contractility, in which NUAK1 or NUAK2 silencing enhanced or inhibited these responses, respectively. In conclusion, we have identified a bifurcating loop during TGFß signaling, whereby transcriptional induction of NUAK1 serves as a negative checkpoint and NUAK2 induction positively contributes to signaling and terminal differentiation responses to TGFß activity.

PARP-1 attenuates Smad-mediated transcription.

The versatile cytokine transforming growth factor ß (TGF-ß) regulates cellular growth, differentiation, and migration during embryonic development and adult tissue homeostasis. Activation of TGF-ß receptors leads to phosphorylation of Smad2 and Smad3, which oligomerize with Smad4 and accumulate in the nucleus where they recognize gene regulatory regions and orchestrate transcription. Termination of Smad-activated transcription involves Smad dephosphorylation, nuclear export, or ubiquitin-mediated degradation. In an unbiased proteomic screen, we identified poly(ADP-ribose) polymerase-1 (PARP-1) as a Smad-interacting partner. PARP-1 dissociates Smad complexes from DNA by ADP-ribosylating Smad3 and Smad4, which attenuates Smad-specific gene responses and TGF-ß-induced epithelial-mesenchymal transition. Thus, our results identify ADP-ribosylation of Smad proteins by PARP-1 as a key step in controlling the strength and duration of Smad-mediated transcription.

FoxK2 is required for cellular proliferation and survival.

FoxK2 is a forkhead transcription factor expressed ubiquitously in the developing murine central nervous system. Here we investigated the role of FoxK2 in vitro and focused on proliferation and cellular survival. Knockdown of FoxK2 results in a decrease in BrdU incorporation and H3 phosphorylation, suggesting attenuation of proliferation. In the absence of growth factors, FoxK2 knockdown results in a dramatic increase in caspase 3 activity and propidium iodide positive cells, indicative of cell death. Additionally, knockdown of FoxK2 results in an increase in the mRNA of Gadd45α, Gadd45γ, as well as an increase in the phosphorylation of the mTOR dependent kinase p70S6K. Rapamycin treatment completely blocked the increase in p70S6K and synergistically potentiated the decrease in H3 phosphorylation upon FoxK2 knockdown. To gain more insight into the proapoptotic effects upon FoxK2 knockdown we screened for changes in Bcl2 genes. Upon FoxK2 knockdown both Puma and Noxa were significantly upregulated. Both genes were not inhibited by rapamycin treatment, instead rapamycin increased Noxa mRNA. FoxK2 requirement in cellular survival is further emphasized by the fact that resistance to TGFß-induced cell death was greatly diminished after FoxK2 knockdown. Overall our data suggest FoxK2 is required for proliferation and survival, that mTOR is part of a feedback loop partly compensating for FoxK2 loss, possibly by upregulating Gadd45s, whereas cell death upon FoxK2 loss is induced in a Bcl2 dependent manner via Puma and Noxa.

Boosting endogenous dopamine production: a novel therapeutic approach for Parkinson's disease.

Innovative therapeutic strategies are urgently needed for Parkinson's disease due to limited efficacy of current treatments and a weak therapeutic pipeline. In this forum article, we propose targeting tyrosine hydroxylase phosphorylation as a novel mechanism of action to address this critical need.

Lmx1a is an activator of Rgs4 and Grb10 and is responsible for the correct specification of rostral and medial mdDA neurons.

The LIM homeodomain transcription factor Lmx1a is a very potent inducer of stem cells towards dopaminergic neurons. Despite several studies on the function of this gene, the exact in vivo role of Lmx1a in mesodiencephalic dopamine (mdDA) neuronal specification is still not understood. To analyse the genes functioning downstream of Lmx1a, we performed expression microarray analysis of LMX1A-overexpressing MN9D dopaminergic cells. Several interesting regulated genes were identified, based on their regulation in other previously generated expression arrays and on their expression pattern in the developing mdDA neuronal field. Post analysis through in vivo expression analysis in Lmx1a mouse mutant (dr/dr) embryos demonstrated a clear decrease in expression of the genes Grb10 and Rgs4, in and adjacent to the rostral and dorsal mdDA neuronal field and within the Lmx1a expression domain. Interestingly, the DA marker Vmat2 was significantly up-regulated as a consequence of increased LMX1A dose, and subsequent analysis on Lmx1a-mutant E14.5 and adult tissue revealed a significant decrease in Vmat2 expression in mdDA neurons. Taken together, microarray analysis of an LMX1A-overexpression cell system resulted in the identification of novel direct or indirect downstream targets of Lmx1a in mdDA neurons: Grb10, Rgs4 and Vmat2.

TGFbeta activates mitogen- and stress-activated protein kinase-1 (MSK1) to attenuate cell death.

Transforming growth factor-ß (TGFß) binding to its receptor leads to intracellular phosphorylation of Smad2 and Smad3, which oligomerize with Smad4. These complexes accumulate in the nucleus and induce gene transcription. Here we describe mitogen- and stress-activated kinase 1 (MSK1) as an antagonist of TGFß-induced cell death. Induction of MSK1 activity by TGFß depends on Smad4 and p38 MAPK activation. Knockdown of GADD45, a Smad4-induced upstream regulator of p38 MAPK prevents TGFß-induced p38 and MSK1 activity. MSK1 functionally regulates pro-apoptotic BH3-only BCL2 proteins, as MSK1 knockdown reduces Bad phosphorylation and enhances Noxa and Bim expression, leading to enhanced TGFß-induced caspase-3 activity and cell death. This finding suggests that MSK1 represents a pro-survival pathway bifurcating downstream of p38 and antagonizes the established pro-apoptotic p38 MAPK function. Furthermore, EGF could reverse all the effects observed after MSK1 knockdown. Monitoring the status of MSK1 activity in cancer promises new therapeutic targets as inactivating both MSK1 and EGF signaling may (re)-sensitize cells to TGFß-induced cell death.

NUAK1 and NUAK2 Fine-Tune TGF-ß Signaling.

Transforming growth factor-ß (TGF-ß) signaling plays a key role in governing various cellular processes, extending from cell proliferation and apoptosis to differentiation and migration. Due to this extensive involvement in the regulation of cellular function, aberrant TGF-ß signaling is frequently implicated in the formation and progression of tumors. Therefore, a full understanding of the mechanisms of TGF-ß signaling and its key components will provide valuable insights into how this intricate signaling cascade can shift towards a detrimental course. In this review, we discuss the interplay between TGF-ß signaling and the AMP-activated protein kinase (AMPK)-related NUAK kinase family. We highlight the function and regulation of these kinases with focus on the pivotal role NUAK1 and NUAK2 play in regulating TGF-ß signaling. Specifically, TGF-ß induces the expression of NUAK1 and NUAK2 that regulates TGF-ß signaling output in an opposite manner. Besides the focus on the TGF-ß pathway, we also present a broader perspective on the expression and signaling interactions of the NUAK kinases to outline the broader functions of these protein kinases.

Regulation of FoxO activity by CBP/p300-mediated acetylation.

Forkhead box, class O (FoxO) transcription factors are inhibited by insulin-induced FoxO phosphorylation. Recently, acetylation of FoxO factors by calcium response element-binding (CREB)-binding protein (CBP) and/or p300 has been identified as a novel regulatory pathway, although the exact consequences of acetylation remain unclear. We propose that binding of CBP/p300 to FoxO factors is essential for FoxO-mediated transcription. CBP and p300 act as FoxO cofactors by weakening histone-DNA interactions. Acetylation of FoxO factors, however, attenuates FoxO-mediated transcriptional activity by disrupting the interaction between FoxO factors and target DNA. Therefore, acetylation shifts the function of FoxO from cell-cycle arrest and protection against oxidative stress towards cell death.

MCL1 as a Therapeutic Target in Parkinson's Disease?

Dopamine neurons in the substantia nigra (SN) pars compacta are selectively lost during the progression of Parkinson's disease (PD). Recent work performed on the role of the Bcl2 family (highly specialized proteins which control cellular survival and death) in midbrain dopamine neurons has led to the identification of the Bcl2 factor Mcl1 as a weak link in the survival of these neurons. We hypothesize that the regulation of BCL2 proteins may explain this selective vulnerability, and may even provide a novel therapeutic opportunity - strengthening weak links such as MCL1 could result in a delay or complete abrogation of cell death during PD.

Insulin signaling in the central nervous system: learning to survive.

Insulin is best known for its role in peripheral glucose homeostasis. Less studied, but not less important, is its role in the central nervous system. Insulin and its receptor are located in the central nervous system and are both implicated in neuronal survival and synaptic plasticity. Interestingly, over the past few years it has become evident that the effects of insulin, on neuronal survival and synaptic plasticity, are mediated by a common signal transduction cascade, which has been identified as "the PI3K route". This route has turned out to be a major integrator of insulin signaling in the brain. A pronounced feature of this insulin-activated route is that it promotes survival by directly inactivating the pro-apoptotic machinery. Interestingly, it is this same route that is required for the induction of long-term potentiation and depression, basic processes underlying learning and memory. This leads to the hypothesis that the PI3K route forms a direct link between learning and memory and neuronal survival. The implications of this hypothesis are far reaching, since it provides an explanation why insulin has beneficial effects on learning and memory and how synaptic activity can prevent cellular degeneration. Applying this knowledge may provide novel therapeutic approaches in the treatment of neurodegenerative diseases such as Alzheimer's disease.

Survival of midbrain dopamine neurons depends on the Bcl2 factor Mcl1.

Mitochondria-dependent apoptosis plays an important role in the embryonic development of the midbrain dopaminergic system as well as in Parkinson's disease. Central to mitochondria-dependent apoptosis is the Bcl2 family of apoptosis-regulating proteins. However, it was unclear which Bcl2 proteins are important for the survival of dopaminergic neurons. Here, we identify Mcl1 as a critical Bcl2 pro-survival factor in midbrain dopaminergic neurons. Using a chemical biology approach to inhibit various components of the apoptotic machinery in the dopaminergic MN9D cell line or the control neuroblastoma N2A cell line, we find that functional inhibition of Mcl1 with the high affinity small molecule inhibitor UMI-77 results in a rapid and dose-dependent loss of viability, selectively in dopaminergic cells. In-depth analysis of the apoptotic signaling pathway reveals that chemical inhibition of Mcl1 results in the activation of Bax, activation of cleaved caspase-3 and finally cell death. The dependence of mouse dopaminergic midbrain neurons on Mcl1 was confirmed using ex vivo slice cultures from Pitx3GFP/+ and wildtype mice. In mouse dopaminergic midbrain neurons positive for the midbrain dopaminergic marker Pitx3, or tyrosine hydroxylase, UMI-77 treatment caused a dramatic increase in cleaved caspase 3, indicating that Mcl1 activity is required for basal neuronal survival. Overall, our results suggest that Mcl1 is of critical importance to dopaminergic neurons and is a weak link in the chain controlling cellular survival. Boosting the pro-survival function of Mcl1 should be pursued as a therapeutic approach to augment the resilience of midbrain dopaminergic neurons to apoptotic stress in Parkinson's disease.

FoxO6 transcriptional activity is regulated by Thr26 and Ser184, independent of nucleo-cytoplasmic shuttling.

Forkhead members of the 'O' class (FoxO) are transcription factors crucial for the regulation of metabolism, cell cycle, cell death and cell survival. FoxO factors are regulated by insulin-mediated activation of PI3K (phosphoinositide 3-kinase)-PKB (protein kinase B) signalling. Activation of PI3K-PKB signalling results in the phosphorylation of FoxO factors on three conserved phosphorylation motifs, which are essential for the translocation of FoxO factors from the nucleus to the cytosol. FoxO6, however, remains mostly nuclear due to the fact that its shuttling ability is dramatically impaired. FoxO1, FoxO3 and FoxO4 all contain an N- and C-terminal PKB motif and a motif located in the forkhead domain. FoxO6 lacks the conserved C-terminal PKB motif, which is the cause of the shuttling impairment. Since FoxO6 can be considered constitutively nuclear, we investigated whether it is also a constitutively active transcription factor. Our results show that FoxO6 transcriptional activity is inhibited by growth factors, independent of shuttling, indicating that it is not constitutively active. The PKB site in the forkhead domain (Ser184) regulated the DNA binding characteristics and the N-terminal PKB site acted as a growth factor sensor. In summary, FoxO6 is not a constitutively active transcription factor and can be regulated by growth factors in a Thr26- and Ser184-dependent manner, independent of shuttling to the cytosol.

The ins and outs of FoxO shuttling: mechanisms of FoxO translocation and transcriptional regulation.

FoxO (forkhead box O; forkhead members of the O class) are transcription factors that function under the control of insulin/insulin-like signalling. FoxO factors have been associated with a multitude of biological processes, including cell-cycle, cell death, DNA repair, metabolism and protection from oxidative stress. Central to the regulation of FoxO factors is a shuttling system, which confines FoxO factors to either the nucleus or the cytosol. Shuttling of FoxO requires protein phosphorylation within several domains, and association with 14-3-3 proteins and the nuclear transport machinery. Description of the FoxO-shuttling mechanism contributes to the understanding of FoxO function in relation to signalling and gene regulation.

Ageing and diabetes: implications for brain function.

Diabetes mellitus is associated with moderate cognitive deficits and neurophysiological and structural changes in the brain, a condition that may be referred to as diabetic encephalopathy. Diabetes increases the risk of dementia, particularly in the elderly. The emerging view is that the diabetic brain features many symptoms that are best described as "accelerated brain ageing." The clinical characteristics of diabetic encephalopathy are discussed, as well as behavioural (e.g. spatial learning) and neurophysiological (e.g. hippocampal synaptic plasticity) findings in animal models. Animal models can make a substantial contribution to our understanding of the pathogenesis, which shares many features with the mechanisms underlying brain ageing. By unravelling the pathogenesis, targets for pharmacotherapy can be identified. This may allow treatment or prevention of this diabetic complication in the future. We discuss changes in glutamate receptor subtypes, in second-messenger systems and in protein kinases that may account for the alterations in synaptic plasticity. In addition, the possible role of cerebrovascular changes, oxidative stress, nonenzymatic protein glycation, insulin and alterations in neuronal calcium homeostasis are addressed.

Fine-tuning of Smad protein function by poly(ADP-ribose) polymerases and poly(ADP-ribose) glycohydrolase during transforming growth factor ß signaling.

BACKGROUND: Initiation, amplitude, duration and termination of transforming growth factor ß (TGFß) signaling via Smad proteins is regulated by post-translational modifications, including phosphorylation, ubiquitination and acetylation. We previously reported that ADP-ribosylation of Smads by poly(ADP-ribose) polymerase 1 (PARP-1) negatively influences Smad-mediated transcription. PARP-1 is known to functionally interact with PARP-2 in the nucleus and the enzyme poly(ADP-ribose) glycohydrolase (PARG) can remove poly(ADP-ribose) chains from target proteins. Here we aimed at analyzing possible cooperation between PARP-1, PARP-2 and PARG in regulation of TGFß signaling. METHODS: A robust cell model of TGFß signaling, i.e. human HaCaT keratinocytes, was used. Endogenous Smad3 ADP-ribosylation and protein complexes between Smads and PARPs were studied using proximity ligation assays and co-immunoprecipitation assays, which were complemented by in vitro ADP-ribosylation assays using recombinant proteins. Real-time RT-PCR analysis of mRNA levels and promoter-reporter assays provided quantitative analysis of gene expression in response to TGFß stimulation and after genetic perturbations of PARP-1/-2 and PARG based on RNA interference. RESULTS: TGFß signaling rapidly induces nuclear ADP-ribosylation of Smad3 that coincides with a relative enhancement of nuclear complexes of Smads with PARP-1 and PARP-2. Inversely, PARG interacts with Smads and can de-ADP-ribosylate Smad3 in vitro. PARP-1 and PARP-2 also form complexes with each other, and Smads interact and activate auto-ADP-ribosylation of both PARP-1 and PARP-2. PARP-2, similar to PARP-1, negatively regulates specific TGFß target genes (fibronectin, Smad7) and Smad transcriptional responses, and PARG positively regulates these genes. Accordingly, inhibition of TGFß-mediated transcription caused by silencing endogenous PARG expression could be relieved after simultaneous depletion of PARP-1. CONCLUSION: Nuclear Smad function is negatively regulated by PARP-1 that is assisted by PARP-2 and positively regulated by PARG during the course of TGFß signaling.

The BCL2 code to dopaminergic development and Parkinson's disease.

Continuous, nonrandom cell death during development of the dopaminergic system is carefully orchestrated by locally secreted growth factors and the expression of transcription factors to ensure every neuron is carefully placed in its appropriate position and no 'miswiring' occurs. We hypothesize that the machinery directly responsible for executing cell death is composed of a precisely calibrated array of BCL2 proteins. Paradoxically, these BCL2 proteins, required for proper development of the dopaminergic system, may also cause vulnerability of this system in the adult, as these BCL2 proteins have recently been linked to Parkinson's disease. In addition to the intriguing possibility of finding useful biomarkers for predicting later neurodegeneration, further investigation of these factors could open new paths for treating Parkinson's disease.

Lmx1a encodes a rostral set of mesodiencephalic dopaminergic neurons marked by the Wnt/B-catenin signaling activator R-spondin 2.

Recent developments in molecular programming of mesodiencephalic dopaminergic (mdDA) neurons have led to the identification of many transcription factors playing a role in mdDA specification. LIM homeodomain transcription factor Lmx1a is essential for chick mdDA development, and for the efficient differentiation of ES-cells towards a dopaminergic phenotype. In this study, we aimed towards a more detailed understanding of the subtle phenotype in Lmx1a-deficient (dreher) mice, by means of gene expression profiling. Transcriptome analysis was performed, to elucidate the exact molecular programming underlying the neuronal deficits after loss of Lmx1a. Subsequent expression analysis on brain sections, confirmed that Nurr1 is regulated by Lmx1a, and additional downstream targets were identified, like Pou4f1, Pbx1, Pitx2, C130021l20Rik, Calb2 and Rspo2. In line with a specific, rostral-lateral (prosomer 2/3) loss of expression of most of these genes during development, Nurr1 and C130021l20Rik were affected in the SNc of the mature mdDA system. Interestingly, this deficit was marked by the complete loss of the Wnt/b-catenin signaling activator Rspo2 in this domain. Subsequent analysis of Rspo2-/- embryos revealed affected mdDA neurons, partially phenocopying the Lmx1a mutant. To conclude, our study revealed that Lmx1a is essential for a rostral-lateral subset of the mdDA neuronal field, where it might serve a critical function in modulating proliferation and differentiation of mdDA progenitors through the regulation of the Wnt activator Rspo2.

Insulin modulates hippocampal activity-dependent synaptic plasticity in a N-methyl-d-aspartate receptor and phosphatidyl-inositol-3-kinase-dependent manner.

Insulin and its receptor are both present in the central nervous system and are implicated in neuronal survival and hippocampal synaptic plasticity. Here we show that insulin activates phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB), and results in an induction of long-term depression (LTD) in hippocampal CA1 neurones. Evaluation of the frequency-response curve of synaptic plasticity revealed that insulin induced LTD at 0.033 Hz and LTP at 10 Hz, whereas in the absence of insulin, 1 Hz induced LTD and 100 Hz induced LTP. LTD induction in the presence of insulin required low frequency synaptic stimulation (0.033 Hz) and blockade of GABAergic transmission. The LTD or LTP induced in the presence of insulin was N-methyl-d-aspartate (NMDA) receptor specific as it could be inhibited by alpha-amino-5-phosphonopentanoic acid (APV), a specific NMDA receptor antagonist. LTD induction was also facilitated by lowering the extracellular Mg(2+) concentration, indicating an involvement of NMDA receptors. Inhibition of PI3K signalling or discontinuing synaptic stimulation also prevented this LTD. These results show that insulin modulates activity-dependent synaptic plasticity, which requires activation of NMDA receptors and the PI3K pathway. The results obtained provide a mechanistic link between insulin and synaptic plasticity, and explain how insulin functions as a neuromodulator.