Phosphatase Cascade Research Articles

Nuclear Envelope Phosphatase 1-Regulatory Subunit 1 (Formerly TMEM188) Is the Metazoan Spo7p Ortholog and Functions in the Lipin Activation Pathway

Lipin-1 catalyzes the formation of diacylglycerol from phosphatidic acid. Lipin-1 mutations cause lipodystrophy in mice and acute myopathy in humans. It is heavily phosphorylated, and the yeast ortholog Pah1p becomes membrane-associated and active upon dephosphorylation by the Nem1p-Spo7p membrane complex. A mammalian ortholog of Nem1p is the C-terminal domain nuclear envelope phosphatase 1 (CTDNEP1, formerly "dullard"), but its Spo7p-like partner is unknown, and the need for its existence is debated. Here, we identify the metazoan ortholog of Spo7p, TMEM188, renamed nuclear envelope phosphatase 1-regulatory subunit 1 (NEP1-R1). CTDNEP1 and NEP1-R1 together complement a nem1Δspo7Δ strain to block endoplasmic reticulum proliferation and restore triacylglycerol levels and lipid droplet number. The two human orthologs are in a complex in cells, and the amount of CTDNEP1 is increased in the presence of NEP1-R1. In the Caenorhabditis elegans embryo, expression of nematode CTDNEP1 and NEP1-R1, as well as lipin-1, is required for normal nuclear membrane breakdown after zygote formation. The expression pattern of NEP1-R1 and CTDNEP1 in human and mouse tissues closely mirrors that of lipin-1. CTDNEP1 can dephosphorylate lipins-1a, -1b, and -2 in human cells only in the presence of NEP1-R1. The nuclear fraction of lipin-1b is increased when CTDNEP1 and NEP1-R1 are co-expressed. Therefore, NEP1-R1 is functionally conserved from yeast to humans and functions in the lipin activation pathway.

Negative modulation of bone morphogenetic protein signaling by Dullard during wing vein formation in Drosophila

Studies in Xenopus have shown that the C-terminal domain phosphatase-like domain (CPD) phosphatase Dullard is essential for proper neural development via inhibition of bone morphogenetic protein (BMP) signaling receptors. In contrast, the orthologous budding yeast Nem1 and human Dullard have been shown to dephosphorylate the phosphatidate phosphatases yeast Smp2/Pah1 and human Lipin, and the relationship between phospholipid metabolism and BMP signaling remain unsolved. Here we report evidence that the Dullard-Lipin phosphatase cascade in Drosophila can regulate BMP signaling, most likely by affecting the function of the nuclear envelope. Manipulating expression levels of either the Drosophila Dullard gene, d-dullard (ddd) or the Lipin gene, DmLpin affected wing vein formation in a manner suggesting a negative effect on BMP signaling. Furthermore, both genes exhibit genetic interaction with BMP signaling pathway components, and can affect the levels of phosphorylated-Mothers against dpp (p-Mad). Although changing ddd expression levels did not have an obvious effect on overall nuclear envelope morphology as has been shown for yeast nem1, the nuclear import machinery components Importin-β and RanGAP were mislocalized and membrane lipid staining was altered in cells overexpressing ddd. Considering the known genetic interaction between Nup84 complex nucleoporins and nem1 in yeast, and the recently reported requirement for components from the orthologous nucleoporin complex in the nuclear translocation of Drosophila Mad (Chen & Xu 2010), it is likely that the role of Drosophila Dullard in regulating membrane lipid homeostasis is conserved and is critical for normal BMP signaling.

Regulatory Cascades of Protein Phosphatases: Implications for Cancer Treatment

Coordinated coupling of biochemical reactions involving protein phosphorylation and dephosphorylation represents the hallmark of the intracellular signal transduction machinery. Distinct classes of enzymes known as kinases and phosphatases respectively drive these reactions. Alterations in activity of such signaling intermediates, either due to mutations in the corresponding genes or epigenetic modulation of their expression levels, is often the cause of many cancers. The role of kinases during signal transduction has been extensively investigated over the past several decades and the consensus view is that subsets of kinases form distinct cascades of signaling pathways. Further, the extensive crosstalk that exists between these cascades leads to a complex network configuration for the signaling machinery. Inhibitors of many of these kinases are now being exploited in cancer therapy. In contrast to this, regulation by cellular phosphatases has generally been considered to occur through isolated interactions between a given phosphatase and its target substrate. Emerging evidence, however, is beginning to suggest that phosphatases also inter-regulate each other, and that such interactions can lead to the formation of discrete phosphatase-specific cascades. A phosphatase cascade may be defined broadly as a series of successive dephosphorylation reactions that occur within a cell and are catalyzed by phosphatases which are activated sequentially. In general, the term phosphatase cascade refers to cascades that include two or more phosphatase members. The crosstalk between such regulatory axes of phosphatases and kinase cascades provides for complex modes of regulation, with non-linear signal input/output relationships. This review discusses the implications of such phosphatase-constituted regulatory elements for both signal processing and transmission. Further, we also explore the potential that insights on the functioning of phosphatase cascades offers, for the development of new and selective strategies for cancer therapy.

Bistable Trypanosome Switch

A glycosome-associated switch regulates transition of trypanosomes from host- to vector-specific forms.

A novel phosphatase cascade regulates differentiation in Trypanosoma brucei via a glycosomal signaling pathway

In the mammalian bloodstream, the sleeping sickness parasite Trypanosoma brucei is held poised for transmission by the activity of a tyrosine phosphatase, TbPTP1. This prevents differentiation of the transmissible "stumpy forms" until entry into the tsetse fly, whereupon TbPTP1 is inactivated and major changes in parasite physiology are initiated to allow colonization of the arthropod vector. Using a substrate-trapping approach, we identified the downstream step in this developmental signaling pathway as a DxDxT phosphatase, TbPIP39, which is activated upon tyrosine phosphorylation, and hence is negatively regulated by TbPTP1. In vitro, TbPIP39 promotes the activity of TbPTP1, thereby reinforcing its own repression, this being alleviated by the trypanosome differentiation triggers citrate and cis-aconitate, generating a potentially bistable regulatory switch. Supporting a role in signal transduction, TbPIP39 becomes rapidly tyrosine-phosphorylated during differentiation, and RNAi-mediated transcript ablation in stumpy forms inhibits parasite development. Interestingly, TbPIP39 localizes in glycosomes, peroxisome-like organelles that compartmentalize the trypanosome glycolytic reactions among other enzymatic activities. Our results invoke a phosphatase signaling cascade in which the developmental signal is trafficked to a unique metabolic organelle in the parasite: the glycosome. This is the first characterized environmental signaling pathway targeted directly to a peroxisome-like organelle in any eukaryotic cell.

P2Y 1 receptors inhibit long-term depression in the prefrontal cortex

Long-term depression (LTD) is a form of synaptic plasticity that may contribute to information storage in the central nervous system. Here we report that LTD can be elicited in layer 5 pyramidal neurons of the rat prefrontal cortex by pairing low frequency stimulation with a modest postsynaptic depolarization. The induction of LTD required the activation of both metabotropic glutamate receptors of the mGlu1 subtype and voltage-sensitive Ca 2+ channels (VSCCs) of the T/R, P/Q and N types, leading to the stimulation of intracellular inositol trisphosphate (IP3) receptors by IP3 and Ca 2+. The subsequent release of Ca 2+ from intracellular stores activated the protein phosphatase cascade involving calcineurin and protein phosphatase 1. The activation of purinergic P2Y 1 receptors blocked LTD. This effect was prevented by P2Y 1 receptor antagonists and was absent in mice lacking P2Y 1 but not P2Y 2 receptors. We also found that activation of P2Y 1 receptors inhibits Ca 2+ transients via VSCCs in the apical dendrites and spines of pyramidal neurons. In addition, we show that the release of ATP under hypoxia is able to inhibit LTD by acting on postsynaptic P2Y 1 receptors. In conclusion, these data suggest that the reduction of Ca 2+ influx via VSCCs caused by the activation of P2Y 1 receptors by ATP is the possible mechanism for the inhibition of LTD in prefrontal cortex.

Deactivation of L-type Ca Current by Inhibition Controls LTP at Excitatory Synapses in the Cerebellar Nuclei

Long-term potentiation (LTP) of mossy fiber EPSCs in the cerebellar nuclei is controlled by synaptic inhibition from Purkinje neurons. EPSCs are potentiated by a sequence of excitation, inhibition, and disinhibition, raising the question of how these stimuli interact to induce plasticity. Here, we find that synaptic excitation, inhibition, and disinhibition couple to different calcium-dependent signaling pathways. In LTP induction protocols, constitutively active calcineurin can replace synaptic excitation, and constitutively active alpha-CaMKII can replace calcium influx associated with resumption of spiking upon disinhibition. Additionally, nimodipine can replace hyperpolarization, indicating that inhibition of firing decreases Ca influx through L-type Ca channels, providing a necessary signal for LTP. Together, these data suggest that potentiation develops after a calcineurin priming signal combines with an alpha-CaMKII triggering signal if and only if L-type Ca current is reduced. Thus, hyperpolarization induced by synaptic inhibition actively controls excitatory synaptic plasticity in the cerebellar nuclei.

Integration of a Phosphatase Cascade with the Mitogen-activated Protein Kinase Pathway Provides for a Novel Signal Processing Function

We mathematically modeled the receptor-dependent mitogen-activated protein kinase (MAPK) signaling by incorporating the regulation through cellular phosphatases. Activation induced the alignment of a phosphatase cascade in parallel with the MAPK pathway. A novel regulatory motif was, thus, generated, providing for the combinatorial control of each MAPK intermediate. This ensured a non-linear mode of signal transmission with the output being shaped by the balance between the strength of input signal and the activity gradient along the phosphatase axis. Shifts in this balance yielded modulations in topology of the motif, thereby expanding the repertoire of output responses. Thus, we identify an added dimension to signal processing wherein the output response to an external stimulus is additionally filtered through indicators that define the phenotypic status of the cell.

A phosphatase cascade by which rewarding stimuli control nucleosomal response.

Dopamine orchestrates motor behaviour and reward-driven learning. Perturbations of dopamine signalling have been implicated in several neurological and psychiatric disorders, and in drug addiction. The actions of dopamine are mediated in part by the regulation of gene expression in the striatum, through mechanisms that are not fully understood. Here we show that drugs of abuse, as well as food reinforcement learning, promote the nuclear accumulation of 32-kDa dopamine-regulated and cyclic-AMP-regulated phosphoprotein (DARPP-32). This accumulation is mediated through a signalling cascade involving dopamine D1 receptors, cAMP-dependent activation of protein phosphatase-2A, dephosphorylation of DARPP-32 at Ser 97 and inhibition of its nuclear export. The nuclear accumulation of DARPP-32, a potent inhibitor of protein phosphatase-1, increases the phosphorylation of histone H3, an important component of nucleosomal response. Mutation of Ser 97 profoundly alters behavioural effects of drugs of abuse and decreases motivation for food, underlining the functional importance of this signalling cascade.

A Conserved Phosphatase Cascade that Regulates Nuclear Membrane Biogenesis

A newly emerging family of phosphatases that are members of the haloacid dehalogenase superfamily contain the catalytic motif DXDX(T/V). A member of this DXDX(T/V) phosphatase family known as Dullard was recently shown to be a potential regulator of neural tube development in Xenopus. (Satow R., et al (2002) Biochem. Biophys. Res. Commun. 295, 85–91). Herein, we demonstrate that human Dullard and the yeast protein Nem1p perform similar functions in mammalian cells and yeast cells, respectively. In addition to similarity in primary sequence, Dullard and Nem1p possess similar domains, they show similar substrate preferences, and both localize to the nuclear envelope. Additionally, we show that human Dullard can rescue the aberrant nuclear envelope morphology of nem1∆ yeast cells, functionally replacing Nem1p. Finally, Nem1p, has been shown to deposphorylate the yeast phosphatidic acid phosphatase Smp2p (Santos‐Rosa et al (2005) EMBO J 24, 1931–41) and we show that Dullard dephosphorylates the mammalian phospatidic acid phosphatase, lipin. Therefore, we propose that Dullard participates in a unique phosphatase cascade regulating nuclear membrane biogenesis and that this cascade is conserved from yeast to mammals

Molecular Dissociation of the Role of PSD-95 in Regulating Synaptic Strength and LTD

The postsynaptic density protein PSD-95 influences synaptic AMPA receptor (AMPAR) content and may play a critical role in LTD. Here we demonstrate that the effects of PSD-95 on AMPAR-mediated synaptic responses and LTD can be dissociated. Our findings suggest that N-terminal-domain-mediated dimerization is important for PSD-95's effect on basal synaptic AMPAR function, whereas the C-terminal SH(3)-GK domains are also necessary for localizing PSD-95 to synapses. We identify PSD-95 point mutants (Q15A, E17R) that maintain PSD-95's influence on basal AMPAR synaptic responses yet block LTD. These point mutants increase the proteolysis of PSD-95 within its N-terminal domain, resulting in a C-terminal fragment that functions as a dominant negative likely by scavenging critical signaling proteins required for LTD. Thus, the C-terminal portion of PSD-95 serves a dual function. It is required to localize PSD-95 at synapses and as a scaffold for signaling proteins that are required for LTD.

A conserved phosphatase cascade that regulates nuclear membrane biogenesis

A newly emerging family of phosphatases that are members of the haloacid dehalogenase superfamily contains the catalytic motif DXDX(T/V). A member of this DXDX(T/V) phosphatase family known as Dullard was recently shown to be a potential regulator of neural tube development in Xenopus [Satow R, Chan TC, Asashima M (2002) Biochem Biophys Res Commun 295:85-91]. Herein, we demonstrate that human Dullard and the yeast protein Nem1p perform similar functions in mammalian cells and yeast cells, respectively. In addition to similarity in primary sequence, Dullard and Nem1p possess similar domains and show similar substrate preferences, and both localize to the nuclear envelope. Additionally, we show that human Dullard can rescue the aberrant nuclear envelope morphology of nem1Delta yeast cells, functionally replacing Nem1p. Finally, Nem1p, has been shown to deposphorylate the yeast phosphatidic acid phosphatase Smp2p [Santos-Rosa H, Leung J, Grimsey N, Peak-Chew S, Siniossoglou S (2005) EMBO J 24:1931-1941], and we show that Dullard dephosphorylates the mammalian phospatidic acid phosphatase, lipin. Therefore, we propose that Dullard participates in a unique phosphatase cascade regulating nuclear membrane biogenesis, and that this cascade is conserved from yeast to mammals.

System properties of ErbB receptor signaling for the understanding of cancer progression

An intracellular signal transduction network constitutes an assembled machinery to control the dynamics of kinase-phosphatase cascade and gene expression. Spatio-temporal analyses of the cellular process can explain the biochemical role of the receptor tyrosine kinases in cancer development from a system point of view.

Calcineurin

Also known as... Protein phosphatase 2B (PP2B) and occasionally Ca2+–calmodulin-activated protein phosphatase. Only the Human Genome Organization nomenclature committee calls it PP3. What is it and what does it do? Calcineurin is a phosphoprotein serine/threonine phosphatase, activated physiologically by Ca2+–calmodulin. In plain language, calcineurin couples intracellular Ca2+ to dephosphorylation of selected substrates. The biological consequences are diverse and depend on the cellular context. Thus, in mammals, calcineurin contributes to regulation of enzymes and ion channels, chemotaxis and neuronal growth cone pathfinding, membrane vesicle trafficking, development of cardiac valves and osteoclasts, and genetic programs of activation in lymphocytes and differentiation in skeletal muscle. What does it look like? Calcineurin is a dimer of an A catalytic subunit and a B subunit (Figure 1). Calmodulin becomes tightly associated with calcineurin only in the presence of elevated, but physiological, levels of Ca2+. In mammals three isoforms of calcineurin A (Aα, Aβ, Aγ) and two isoforms of calcineurin B (B1, B2) are expressed from separate genes. Where can it be found? Calcineurin is present in all eukaryotes investigated except higher plants. It is found in all tissues in mammals, with notably high levels in brain. Calcineurin can have a pivotal role in intracellular signalling even at relatively low levels, however, as exemplified in T lymphocytes and skeletal muscle. In budding yeast, calcineurin has a role in coordinating adaptation to environmental stress both through the calcineurin–Crz1p transcriptional pathway and through post-translational mechanisms. What are its substrates? Substrates that contribute to transcriptional signalling, in particular nuclear factor of activated T cells (NFAT), have sometimes overshadowed calcineurin’s substrates in non-transcriptional pathways, which constitute a vast majority of the identified substrates. Substrate selection is determined in part by a docking site in calcineurin that recognizes the consensus PxIxIT motif in NFAT and some other substrates. Since not all substrates depend on binding at this site, there are probably additional substrate recognition sites. Another contribution to substrate selection is made by scaffold proteins, e.g. AKAP79, which target the phosphatase to neuronal synapses or other cellular sites. Calcineurin also delegates work to protein phosphatase 1 (PP1) through a phosphatase cascade, in which dephosphorylation of the PP1 antagonists DARPP-32 or inhibitor-1 by calcineurin relieves their inhibitory effect on PP1, and allows PP1 to act on its own preferred substrates. What are its inhibitors? Cyclosporin A (CsA) and FK506 bind tightly to the abundant intracellular proteins cyclophilin A and FKBP12, respectively, and the resulting ligand–protein complex binds to calcineurin and impedes access of protein substrates to the active site. Blockade of a biological process by CsA and independently by FK506 is diagnostic for calcineurin involvement. Other inhibitors that find frequent experimental use are the autoinhibitory peptide from calcineurin and fragments of the regulatory proteins DSCR1/MCIP/calcipressin/Rcn1p, Cabin1/cain, and AKAP79. Does it have any medical relevance? Calcineurin signalling is prominent in transplant rejection and autoimmune disease, where the inhibitors CsA and FK506 are used clinically, and is being studied for its contribution to myocardial hypertrophy and to virulence in fungal pathogens.

A DARPP Side to ERK

Drug addiction is associated with changes in neuronal function that can be considered a form of learning. Many drugs of abuse enhance dopaminergic neurotransmission in the nucleus accumbens and are thereby thought to influence the plasticity of glutamatergic corticostriatal neurons (suggesting effects on neurons that receive both dopaminergic and glutamatergic input). Valjent et al . uncovered converging effects of dopaminergic and glutamatergic signals in response to drugs of abuse that led to activation of extracellular signal-regulated kinase (ERK, a target of dopaminergic and glutamatergic activity that has been implicated in synaptic plasticity) in striatal neurons. D -amphetamine injection stimulated ERK phosphorylation in a subset of mouse dopamine D1 receptor (D1R)-bearing neurons in dorsal striatum and nucleus accumbens. D -amphetamine also stimulated phosphorylation of the D1R targets dopamine and cAMP-regulated phosphoprotein of M r 32,000 (DARPP-32) and the glutamate receptor 1 subunit (GluR1). Phosphorylation of ERK and GluR1 in response to d -amphetamine was inhibited by D1R blockade or knockout, whereas pharmacological blockade of the glutamatergic N methyl-D-aspartate receptor inhibited ERK phosphorylation but not that of GluR1 or DARPP-32. DARPP-32 knockout attenuated striatal ERK phosphorylation in response to d -amphetamine or other drugs of abuse. Further, mutation of a DARPP-32 threonine residue implicated in its inhibition of protein phosphatase-1 (Thr-34) abolished cocaine or d -amphetamine-mediated phosphorylation of striatal ERK and of the ERK substrate Elk-1 but did not affect ERK activation in the prefrontal cortex. DARPP-32 knockout, Thr-34 mutation, or pharmacological blockade of the ERK signaling pathway also inhibited behavioral sensitization to cocaine. DARPP-32 both promoted ERK phosphorylation and inhibited ERK dephosphorylation. Gould and Manji provide context and discuss implications of this research in a Commentary. E. Valjent, V. Pascoli, P. Svenningsson, S. Paul, H. Enslen, J.-C. Corvol, A. Stipanovich, J. Caboche, P. J. Lombroso, A. C. Nairn, P. Greengard, D. Hervé, J.-A. Girault, Regulation of a protein phosphatase cascade allows convergent dopamine and glutamate signals to activate ERK in the striatum. Proc. Natl. Acad. Sci. U.S.A. 102 , 491-496 (2005). [Abstract] [Full Text] T. D. Gould, H. K. Manji, DARPP-32: A molecular switch at the nexus of reward pathway plasticity. Proc. Natl. Acad. Sci. U.S.A. 102 , 253-254 (2005). [Full Text]

Regulation of a protein phosphatase cascade allows convergent dopamine and glutamate signals to activate ERK in the striatum

Many drugs of abuse exert their addictive effects by increasing extracellular dopamine in the nucleus accumbens, where they likely alter the plasticity of corticostriatal glutamatergic transmission. This mechanism implies key molecular alterations in neurons in which both dopamine and glutamate inputs are activated. Extracellular signal-regulated kinase (ERK), an enzyme important for long-term synaptic plasticity, is a good candidate for playing such a role. Here, we show in mouse that d-amphetamine activates ERK in a subset of medium-size spiny neurons of the dorsal striatum and nucleus accumbens, through the combined action of glutamate NMDA and D1-dopamine receptors. Activation of ERK by d-amphetamine or by widely abused drugs, including cocaine, nicotine, morphine, and Delta(9)-tetrahydrocannabinol was absent in mice lacking dopamine- and cAMP-regulated phosphoprotein of M(r) 32,000 (DARPP-32). The effects of d-amphetamine or cocaine on ERK activation in the striatum, but not in the prefrontal cortex, were prevented by point mutation of Thr-34, a DARPP-32 residue specifically involved in protein phosphatase-1 inhibition. Regulation by DARPP-32 occurred both upstream of ERK and at the level of striatal-enriched tyrosine phosphatase (STEP). Blockade of the ERK pathway or mutation of DARPP-32 altered locomotor sensitization induced by a single injection of psychostimulants, demonstrating the functional relevance of this regulation. Thus, activation of ERK, by a multilevel protein phosphatase-controlled mechanism, functions as a detector of coincidence of dopamine and glutamate signals converging on medium-size striatal neurons and is critical for long-lasting effects of drugs of abuse.

Cyclosporin A, FK506 and rapamycin produce multiple, temporally distinct, effects on memory following single-trial, passive avoidance training in the chick

Few studies have used a pharmaco–behavioural methodology to directly investigate roles for the calcium-dependent protein phosphatase calcineurin (CaN) in memory formation, due partly to the absence of specific inhibitory agents. A number of drugs with different inhibitory profiles were used to examine this issue in groups of chicks trained on a single-trial, passive-avoidance task. Bilateral intracranial administration of the immunosuppressants FK506 and cyclosporin A (CyA) led to two temporally distinct effects, distinguished by the concentration of drug required and the effective time of administration relative to training. In addition to inhibiting CaN, CyA and FK506 inhibit distinct classes of peptidyl prolyl- cis/trans-isomerases (PPIases). Other agents known to inhibit these enzymes, including the Map kinase inhibitor Rapamycin, also induced memory deficits in a complex, dose- and time-of-administration-dependent, manner. The data fail to conclusively implicate CaN in memory formation, but are consistent with proposals that a phosphatase cascade may participate in an early stage of information storage. PPIases may be required at a later stage to catalyse the folding of new or translocated proteins, the synthesis of which is required for formation of long-term memory, although other possible explanations for the data remain to be investigated.

Expression of atrial natriuretic peptide receptor-A antagonizes the mitogen-activated protein kinases (Erk2 and P38MAPK) in cultured human vascular smooth muscle cells.

To understand the signaling mechanisms of atrial natriuretic peptide (ANP) receptor-A (NPRA), we studied the effect of the ANP/NPRA system on mitogen-activated protein kinases (MAPKs), with particular emphasis on the extracellular-regulated kinase (Erk2) and stress-activated protein kinase (p38MAPK) in cultured human vascular smooth muscle cells (HVSMC). Angiotensin II (ANG II) and platelet-derived growth factor (PDGF) stimulated the immunoreactive Erk2 and p38MAPK activities and their protein levels by 2-4 fold. The pretreatment of cells with ANP significantly inhibited the agonist-stimulated Erk2 and p38MAPK activities and protein expression by 65-75% in HVSMC transiently transfected with NPRA, as compared with only 18-22% inhibition in vector-transfected cells. The pretreatment of cells with KT5823, an inhibitor of cGMP-dependent protein kinase (PKG), reversed the inhibitory effects of ANP on MAPK activities and protein expression by 90-95%. PD98059, which inhibits Erk2 by directly inhibiting the MAPK-kinase (MEK), and SB202192, a selective antagonist of p38MAPK, blocked the Erk2 and p38MAPK activities, respectively. Interestingly, ANP stimulated the MAPK-phosphatase-3 (MKP-3) protein levels by more than 3-fold in HVSMC over-expressing NPRA, suggesting that ANP-dependent inhibition of MAPKs may also proceed by stimulating the phosphatase cascade. These present findings provide the evidence that ANP exerts inhibitory effects on agonist-stimulated MAPKs (Erk2 and p38MAPK) activities and protein levels in a 2-fold manner: by antagonizing the up-stream signaling pathways and by activation of MKP-3 to counter-regulate MAPKs in a cGMP and PKG-dependent manner. Our results identify a signal transduction pathway in HVSMC that could contribute to vascular remodeling and structural changes in human hypertension.

Tyrosine phosphorylation of the mu-opioid receptor regulates agonist intrinsic efficacy.

The mu-opioid receptor (MOR) contains four highly conserved cytoplasmic tyrosine residues that may serve to regulate receptor activity. For Xenopus laevis oocytes coexpressing the rat MOR and the heteromultimeric potassium channel, K(IR)3.1/3.2, pretreatment with insulin produced both a 40% suppression in the basal channel conductance and potentiation of response to the mu-opioid agonist [D-Ala(2),methyl-Phe(4),Gly(5)-ol]enkephalin (DAMGO) to 155% of matched, untreated control cells. Insulin-induced potentiation of the DAMGO response was concentration-dependent and reversed after 1 h. Insulin pretreatment increased the maximal effect of DAMGO, but did not change its EC(50) value. Potentiation of the DAMGO response did not result from a recruitment of MOR to the cell surface, as measured by specific binding of the opioid peptide antagonist [(3)H]d-Phe((3)H)-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH(2) (cyclic) to whole-oocytes, but instead the potentiation was probably caused by an increase in intrinsic efficacy of G protein coupling. The involvement of tyrosine residues on the putative intracellular loops of the MOR was demonstrated with four point-mutated receptors, replacing tyrosine with phenylalanine to create MOR(Y96F), MOR(Y106F), MOR(Y166F), and MOR(Y336F). None of these mutations significantly altered the EC(50) value for DAMGO compared with wild-type MOR, and insulin pretreatment still potentiated the effect of 1 microM DAMGO in oocytes containing either MOR(Y96F) or MOR(Y336F) to 137 +/- 10 and 124 +/- 8%, respectively. However, insulin did not significantly potentiate the DAMGO response with oocytes containing either MOR(Y106F) or MOR(Y166F), suggesting that these two sites were responsible for the insulin-induced opioid potentiation. The tyrosine-kinase inhibitors genistein (100 microM) or K-252a (20 microM) did not block the insulin-induced potentiation of the DAMGO response, but coincubation of insulin with either the MAP kinase inhibitor PD98,059 (20 microM) or phosphatase inhibitor orthovanadate (30 microM) completely blocked the potentiation. The results suggest the hypothesis that the potentiation was caused by dephosphorylation of the two tyrosines in MOR. To test this hypothesis, we measured the recovery rates after insulin treatment. As predicted, tyrosine kinase inhibition by K-252a significantly slowed the reversal and phosphatase inhibition by orthovanadate significantly accelerated the recovery. These findings support a rapid modulatory role for insulin on opioid signal transduction, possibly through the dephosphorylation of the MOR at tyrosines 106 and 166 by an insulin-activated MAP kinase/protein tyrosine phosphatase cascade. We conclude that tyrosine phosphorylation of the mu-opioid receptor regulates receptor-G protein coupling efficacy.

Three Ca2+ levels affect plasticity differently: the LTP zone, the LTD zone and no man's land

Three Ca²⁺ levels affect plasticity differently: the LTP zone, the LTD zone and no man's land