Dopamine-D2S receptor inhibition of calcium influx,adenylyl cyclase,and mitogen-activated protein kinase in pituitary cells: distinct Galpha and Gbetagamma requirements

The G protein specificity of multiple signaling pathways of the dopamine-D2S (short form) receptor was investigated in GH4ZR7 lactotroph cells. Activation of the dopamine-D2S receptor inhibited forskolin-induced cAMP production, reduced BayK8644- activated calcium influx, and blocked TRH-mediated p42/p44 MAPK phosphorylation. These actions were blocked by pretreatment with pertussis toxin (PTX), indicating mediation by G(i/o) proteins. D2S stimulation also decreased TRH-induced MAPK/ERK kinase phosphorylation. TRH induced c-Raf but not B-Raf activation, and the D2S receptor inhibited both TRH-induced c-Raf and basal B-Raf kinase activity. After PTX treatment, D2S receptor signaling was rescued in cells stably transfected with individual PTX-insensitive Galpha mutants. Inhibition of adenylyl cyclase was partly rescued by Galpha(i)2 or Galpha(i)3, but Galpha(o) alone completely reconstituted D2S-mediated inhibition of BayK8644-induced L-type calcium channel activation. Galpha(o) and Galpha(i)3 were the main components involved in D2S-mediated p42/44 MAPK inhibition. In cells transfected with the carboxyl-terminal domain of G protein receptor kinase to inhibit Gbetagamma signaling, only D2S-mediated inhibition of calcium influx was blocked, but not inhibition of adenylyl cyclase or MAPK. These results indicate that the dopamine-D2S receptor couples to distinct G(i/o) proteins, depending on the pathway addressed, and suggest a novel Galpha(i)3/Galpha(o)-dependent inhibition of MAPK mediated by c-Raf and B-Raf-dependent inhibition of MAPK/ERK kinase.     

Molecular basis of melanocortin-4 receptor for AGRP inverse agonism

We have investigated receptor structural components of the melanocortin-4 receptor (MC4R) responsible for ligand-dependent inverse agonism. We utilized agouti-related protein (AGRP), an inverse agonist which reduces MC4R basal cAMP production, as a tool to determine the molecular mechanism. We tested a series of chimeric receptors and utilized MC4R and MC1R as templates, in which AGRP is an inverse agonist for MC4R but not for MC1R. Our results indicate that replacements of the extracellular loops 1, 2 and 3 of MC4R with the corresponding regions of MC1R did not affect AGRP inverse agonist activity. However, replacement of the N terminus of MC4R with the same region of MC1R decreases AGRP inverse agonism. Replacement of transmembrane domains 3, 4, 5 and 6 of MC4R with the corresponding regions of MC1R did not affect AGRP inverse agonist activity but mutation of D90A in transmembrane 2 (TM2) and D298A in TM7 abolished AGRP inverse activity. Deletion of the distal MC4R C terminus fails to maintain AGRP mediated reduction in basal cAMP production although it maintains NDP-MSH mediated cAMP production. In conclusion, our results indicate that the N terminus and the distal C terminus of MC4R do appear to play important roles in AGRP inverse agonism but not NDP-MSH mediated receptor activation. Our results also indicate that the residues D90 in TM2 and D298 in TM7 of hMC4R are involved in not only NDP-MSH mediated receptor activation but also AGRP mediated inverse agonism.     

Induction of neurite outgrowth in PC12 cells by the bacterial nucleoside N6-methyldeoxyadenosine is mediated through adenosine A2a receptors and via cAMP and MAPK signaling pathways

We have previously shown that N(6)-methyldeoxyadenosine (MDA) is an inducer of differentiation in several tumor cells. Here we show that in addition to its ability to induce neurite-outgrowth in PC12 cells, MDA also significantly enhances the nerve-growth factor-mediated neurite outgrowth of these cells. Thus, MDA acts synergistically with NGF to repress cdc2 and cdk2 synthesis and to enhance tyrosine hydroxylase synthesis. To further elucidate the mechanisms of action of MDA, we investigated the effect of this drug on various signaling pathways. The neuritogenesis observed in PC12 following MDA treatment is mediated through activation of adenylyl cyclase in a PKA independent process and through the recruitment of the p44/p42 MAPK pathway. Furthermore, the adenosine A(2a) receptor antagonist ZM 241385 prevents the MDA-induced neuritogenesis, suggesting that MDA mediates its effect via this adenylyl cyclase-coupled A(2a) receptor. Collectively, these findings suggest that, in PC12 cells, the MDA-induced neuritogenesis requires the recruitment of adenosine A(2a) receptor, the stimulation of adenylate cyclase, and the activation of the p44/42MAP kinase cascade.     

Role of cAMP inhibition of p44/p42 mitogen-activated protein kinase in potentiation of protein secretion in rat lacrimal gland

We previously found that addition of cAMP and a Ca(2+)/PKC-dependent agonist causes synergism or potentiation of protein secretion from rat lacrimal gland acini. In the present study we determined whether cAMP decreases p44/p42 mitogen-activated protein kinase (MAPK) activity in the lacrimal gland. Since we know that activation of MAPK attenuates protein secretion stimulated by Ca(2+)- and PKC-dependent agonists, we also determined whether this activation causes potentiation of secretion. Freshly prepared rat lacrimal gland acinar cells were incubated with dibutyryl cAMP (DBcAMP), carbachol (a cholinergic agonist), phenylephrine (an alpha(1)-adrenergic agonist), or epidermal growth factor (EGF). The latter three agonists are known to activate p44/p42 MAPK. p44/p42 MAPK activity and protein secretion were measured. As measured by Western blot analysis, DBcAMP inhibited both basal and agonist-stimulated p44/p42 MAPK activity. Cellular cAMP levels were increased by 1) using two different cell-permeant cAMP analogs, 2) activating adenylyl cyclase (L-858051), or 3) activation of G(s)-coupled receptors (VIP). The cell-permeant cAMP analogs, L-858051, and VIP inhibited basal p44/p42 MAPK activity by 50, 40, and 40%, respectively. DBcAMP and VIP inhibited carbachol- and EGF-stimulated MAPK activity. cAMP, but not VIP, inhibited phenylephrine-stimulated MAPK activity. Potentiation of secretion was detected when carbachol, phenylephrine, or EGF was simultaneously added with DBcAMP. We conclude that increasing cellular cAMP levels inhibits p44/p42 MAPK activity and that this could account for potentiation of secretion obtained when cAMP was elevated and Ca(2+) and PKC were increased by agonists.     

PGE2-induced hypertrophy of cardiac myocytes involves EP4 receptor-dependent activation of p42/44 MAPK and EGFR transactivation

Upon induction of cyclooxygenase-2 (COX-2), neonatal ventricular myocytes (VMs) mainly synthesize prostaglandin E2 (PGE2). The biological effects of PGE2 are mediated through four different G protein-coupled receptor (GPCR) subtypes (EP(1-4)). We have previously shown that PGE2 stimulates cAMP production and induces hypertrophy of VMs. Because the EP4 receptor is coupled to adenylate cyclase and increases in cAMP, we hypothesized that PGE2 induces hypertrophic growth of cardiac myocytes through a signaling cascade that involves EP4-cAMP and activation of protein kinase A (PKA). To test this, we used primary cultures of VMs and measured [3H]leucine incorporation into total protein. An EP4 antagonist was able to partially block PGE2 induction of protein synthesis and prevent PGE2-dependent increases in cell surface area and activity of the atrial natriuretic factor promoter, which are two other indicators of hypertrophic growth. Surprisingly, a PKA inhibitor had no effect. In other cell types, G protein-coupled receptor activation has been shown to transactivate the epidermal growth factor receptor (EGFR) and result in p42/44 mitogen-activated protein kinase (MAPK) activation and cell growth. Immunoprecipitation of myocyte lysates demonstrated that the EGFR was rapidly phosphorylated by PGE2 in VMs, and the EP4 antagonist blocked this. In addition, the selective EGFR inhibitor AG-1478 completely blocked PGE2-induced protein synthesis. We also found that PGE2 rapidly phosphorylated p42/44 MAPK, which was inhibited by the EP4 antagonist and by AG-1478. Finally, the p42/44 MAPK inhibitor PD-98053 (25 micromol/l) blocked PGE2-induced protein synthesis. Altogether, we believe these are the first data to suggest that PGE2 induces protein synthesis in cardiac myocytes in part via activation of the EP4 receptor and subsequent activation of p42/44 MAPK. Activation of p42/44 MAPK is independent of the common cAMP-PKA pathway and involves EP4-dependent transactivation of EGFR.     

Distinct roles for Galpha(i)2 and Gbetagamma in signaling to DNA synthesis and Galpha(i)3 in cellular transformation by dopamine D2S receptor activation in BALB/c 3T3 cells

Control of cell proliferation depends on intracellular mediators that determine the cellular response to external cues. In neuroendocrine cells, the dopamine D2 receptor short form (D2S receptor) inhibits cell proliferation, whereas in mesenchymal cells the same receptor enhances cell proliferation. Nontransformed BALB/c 3T3 fibroblast cells were stably transfected with the D2S receptor cDNA to study the G proteins that direct D2S signaling to stimulate cell proliferation. Pertussis toxin inactivates G(i) and G(o) proteins and blocks signaling of the D2S receptor in these cells. D2S receptor signaling was reconstituted by individually transfecting pertussis toxin-resistant Galpha(i/o) subunit mutants and measuring D2-induced responses in pertussis toxin-treated cells. This approach identified Galpha(i)2 and Galpha(i)3 as mediators of the D2S receptor-mediated inhibition of forskolin-stimulated adenylyl cyclase activity; Galpha(i)2-mediated D2S-induced stimulation of p42 and p44 mitogen-activated kinase (MAPK) and DNA synthesis, whereas Galpha(i)3 was required for formation of transformed foci. Transfection of toxin-resistant Galpha(i)1 cDNA induced abnormal cell growth independent of D2S receptor activation, while Galpha(o) inhibited dopamine-induced transformation. The role of Gbetagamma subunits was assessed by ectopic expression of the carboxyl-terminal domain of G protein receptor kinase to selectively antagonize Gbetagamma activity. Mobilization of Gbetagamma subunits was required for D2S-induced calcium mobilization, MAPK activation, and DNA synthesis. These findings reveal a remarkable and distinct G protein specificity for D2S receptor-mediated signaling to initiate DNA synthesis (Galpha(i)2 and Gbetagamma) and oncogenic transformation (Galpha(i)3), and they indicate that acute activation of MAPK correlates with enhanced DNA synthesis but not with transformation.     

Dictyostelium discoideum lipids modulate cell-cell cohesion and cyclic AMP signaling.

During Dictyostelium discoideum development, cell-cell communication is mediated through cyclic AMP (cAMP)-induced cAMP synthesis and secretion (cAMP signaling) and cell-cell contact. Cell-cell contact elicits cAMP secretion and modulates the magnitude of a subsequent cAMP signaling response (D. R. Fontana and P. L. Price, Differentiation 41:184-192, 1989), demonstrating that cell-cell contact and cAMP signaling are not independent events. To identify components involved in the contact-mediated modulation of cAMP signaling, amoebal membranes were added to aggregation-competent amoebae in suspension. The membranes from aggregation-competent amoebae inhibited cAMP signaling at all concentrations tested, while the membranes from vegetative amoebae exhibited a concentration-dependent enhancement or inhibition of cAMP signaling. Membrane lipids inhibited cAMP signaling at all concentrations tested. The lipids abolished cAMP signaling by blocking cAMP-induced adenylyl cyclase activation. The membrane lipids also inhibited amoeba-amoeba cohesion at concentrations comparable to those which inhibited cAMP signaling. The phospholipids and neutral lipids decreased cohesion and inhibited the cAMP signaling response. The glycolipid/sulfolipid fraction enhanced cohesion and cAMP signaling. Caffeine, a known inhibitor of cAMP-induced adenylyl cyclase activation, inhibited amoeba-amoeba cohesion. These studies demonstrate that endogenous lipids are capable of modulating amoeba-amoeba cohesion and cAMP-induced activation of the adenylyl cyclase. These results suggest that cohesion may modulate cAMP-induced adenylyl cyclase activation. Because the complete elimination of cohesion is accompanied by the complete elimination of cAMP signaling, these results further suggest that cohesion may be necessary for cAMP-induced adenylyl cyclase activation in D. discoideum.     

Mapping structural determinants within third intracellular loop that direct signaling specificity of type 1 corticotropin-releasing hormone receptor

The type 1 corticotropin-releasing hormone receptor (CRH-R1) influences biological responses important for adaptation to stressful stimuli, through activation of multiple downstream effectors. The structural motifs within CRH-R1 that mediate G protein activation and signaling selectivity are unknown. The aim of this study was to gain insights about important structural determinants within the third intracellular loop (IC3) of the human CRH-R1α important for cAMP and ERK1/2 pathways activation and selectivity. We investigated the role of the juxtamembrane regions of IC3 by mutating amino acid cassettes or specific residues to alanine. Although simultaneous tandem alanine mutations of both juxtamembrane regions Arg(292)-Met(295) and Lys(311)-Lys(314) reduced ligand binding and impaired signaling, all other mutant receptors retained high affinity binding, indistinguishable from wild-type receptor. Agonist-activated receptors with tandem mutations at the proximal or distal terminal segments enhanced activation of adenylyl cyclase by 50-75% and diminished activation of inositol trisphosphate and ERK1/2 by 60-80%. Single Ala mutations identified Arg(292), Lys(297), Arg(310), Lys(311), and Lys(314) as important residues for the enhanced activation of adenylyl cyclase, partly due to reduced inhibition of adenylyl cyclase activity by pertussis toxin-sensitive G proteins. In contrast, mutation of Arg(299) reduced receptor signaling activity and cAMP response. Basic as well as aliphatic amino acids within both juxtamembrane regions were identified as important for ERK1/2 phosphorylation through activation of pertussis toxin-sensitive G proteins as well as G(q) proteins. These data uncovered unexpected roles for key amino acids within the highly conserved hydrophobic N- and C-terminal microdomains of IC3 in the coordination of CRH-R1 signaling activity.     

Natriuretic peptide receptor-C signaling and regulation

The natriuretic peptides (NP) are a family of three polypeptide hormones termed atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). ANP regulates a variety of physiological parameters by interacting with its receptors present on the plasma membrane. These are of three subtypes NPR-A, NPR-B, and NPR-C. NPR-A and NPR-B are guanylyl cyclase receptors, whereas NPR-C is non-guanylyl cyclase receptor and is coupled to adenylyl cyclase inhibition or phospholipase C activation through inhibitory guanine nucleotide regulatory protein (Gi). ANP, BNP, CNP, as well as C-ANP(4-23), a ring deleted peptide that specifically interacts with NPR-C receptor inhibit adenylyl cyclase activity through Gi protein. Unlike other G-protein-coupled receptors, NPR-C receptors have a single transmembrane domain and a short cytoplasmic domain of 37 amino acids, which has a structural specificity like those of other single transmembrane domain receptors. A 37 amino acid cytoplasmic peptide is sufficient to inhibit adenylyl cyclase activity with an apparent Ki similar to that of ANP(99-126) or C-ANP(4-23). In addition, C-ANP(4-23) also stimulates phosphatidyl inositol (PI) turnover in vascular smooth muscle cells (VSMC) which is attenuated by dbcAMP and cAMP-stimulatory agonists, suggesting that NPR-C receptor-mediated inhibition of adenylyl cyclase and resultant decreased levels of cAMP may be responsible for NPR-C-mediated stimulation of PI turnover. Furthermore, the activation of NPR-C receptor by C-ANP(4-23) and CNP inhibits the mitogen-activated protein kinase activity stimulated by endothelin-3, platelet-derived growth factor, phorbol-12 myristate 13-acetate, suggesting that NPR-C receptor might also be coupled to other signal transduction system or that there may be an interaction of the NPR-C receptor and some other signaling pathways. In this review article, NPR-C receptor coupling to different signaling pathways and their regulation will be discussed.     

Dopamine-dependent cyclic AMP generating system in chick retina and its relation to melatonin biosynthesis

Stimulation of a D₄-like dopamine (DA) receptors inhibits a cAMP-dependent increase in serotonin N-acetyltransferase activity and melatonin biosynthesis in the chick retina. In order to gain more insight into the molecular mechanisms underlying this suppressive action of DA, the effects of selective stimulation of the D2-family of DA receptors (including the D₄-subtype) on cAMP formation were examined in chick retina using two experimental approaches: measurements of adenylyl cyclase activity in retinal homogenates, and cAMP accumulation in eye cup preparations prelabeled with [³H]adenine. The DA-sensitive adenylyl cyclase system is well expressed in chick retina. DA increased both basal and forskolin-stimulated adenylyl cyclase activity. This effect of DA was antagonized by SCH 23390 (a blocker of D1-family of DA receptors) and not affected by sulpiride (a D2-family blocker). Incubation of retinal homogenates with quinpirole (a predominant agonist of D₃/D₄ DA receptor subtypes) did not produce any major changes in adenylyl cyclase activity. On the other hand, activation of D₄-like DA receptor subtype by quinpirole decreased forskolin-stimulated cAMP formation in intact chick retinas maintained in “eye-cup” preparations. It is suggested that D₄-like DA receptors regulating melatonin biosynthesis in chick retina may be indirectly linked to the cAMP generating system.     

Molecular characterization of human melanocortin-3 receptor ligand-receptor interaction

Melanocortin-3 receptor (MC3R), primarily expressed in the hypothalamus, plays an important role in the regulation of energy homeostasis. MC3R-deficient (MC3R(-)(/)(-)) mice demonstrate increased fat mass, higher feeding efficiency, hyperleptinaemia, and mild hyperinsulinism. At least one specific mutation of MC3R has been identified to be associated with human obesity. Functional analysis of this altered MC3R (I183N) has indicated that the mutation completely abolishes agonist-mediated receptor activation. However, the specific molecular determinants of MC3R responsible for ligand binding and receptor signaling are currently unknown. The present study is to determine the structural aspects of MC3R responsible for ligand binding and receptor signaling. On the basis of our theoretical model for MC1R, using mutagenesis, we have examined 19 transmembrane domain amino acids selected for these potential roles in ligand binding and receptor signaling. Our results indicate that (i) substitutions of charged amino acid residues E131 in transmembrane domain 2 (TM2), D154 and D158 in TM3, and H298 in TM6 with alanine dramatically reduced NDP-MSH binding affinity and receptor signaling, (ii) substitutions of aromatic amino acids F295 and F296 in TM6 with alanine also significantly decreased NDP-MSH binding and receptor activity, (iii) substitutions of D121in TM2 and D332 in TM7 with alanine resulted in the complete loss of ligand binding, ligand induced receptor activation, and cell surface protein expression, and (iv) interestingly, substitution of L165 in TM3 with methionine or alanine switched antagonist SHU9119 into a receptor agonist. In conclusion: Our results suggest that TM3 and TM6 are important for NDP-MSH binding, while D121 in TM2 and D332 in TM7 are crucial for receptor activity and signaling. Importantly, L165 in TM3 is critical for agonist or antagonist selectivity. These results provide important information about the molecular determinants of hMC3R responsible for ligand binding and receptor signaling.     

Assessment of the extracellular and intracellular actions of sphingosine 1-phosphate by using the p42/p44 mitogen-activated protein kinase cascade as a model.

We have investigated the extracellular and intracellular actions of sphingosine 1-phosphate (S1P) by using cultured airway smooth muscle cells. We have demonstrated that exogenous S1P elicited an activation of mitogen-activated protein kinase (p42/p44 MAPK) that was abolished by pertussis toxin (0.1 microg/mL, 24 h), which was used to inactivate Gi. The effect of exogenous S1P might therefore be attributed to an action at a putative Gi-coupled receptor. The regulation of the p42/p44 MAPK cascade by S1P was also shown to include a protein kinase C (PKC)-dependent intermediate step. Platelet-derived growth factor (PDGF) stimulates intracellular S1P formation and was therefore used to evaluate the intracellular action of S1P. This has previously been investigated by others using the sphingosine kinase inhibitors D,L-threo-dihydrosphingosine and N,N-dimethylsphingosine. We have demonstrated here that both inhibitors block the PDGF-dependent activation of p42/p44 MAPK. However, both are also PKC inhibitors, which might account for their effect because PDGF utilises PKC as an intermediate in the regulation of the p42/p44 MAPK cascade. Significantly, sphingosine, which is the substrate of sphingosine kinase and a PKC inhibitor, blocked the activation of p42/p44 MAPK by PDGF with an almost identical concentration dependence compared with D,L-threo-dihydrosphingosine and N,N-dimethylsphingosine. Therefore, the use of so-called sphingosine kinase inhibitors might lead to misleading interpretations because of their additional effect on PKC. Other approaches, such as oligodeoxynucleotide anti-sense against sphingosine kinase, are required to address the intracellular role of S1P.     

Nerve growth factor signaling involves interaction between the Trk A receptor and lysophosphatidate receptor 1 systems: nuclear translocation of the lysophosphatidate receptor 1 and Trk A receptors in pheochromocytoma 12 cells

We report here that the nerve growth factor (NGF) and lysophosphatidate (LPA) receptor signaling systems interact to regulate the p42/p44 MAPK pathway in PC12 cells. This is based upon several lines of evidence. First, the treatment of PC12 cells, which express LPA(1) receptors, with a sub-maximal concentration of LPA and NGF induced synergistic activation of p42/p44 MAPK. Second, the transfection of PC12 cells with LPA(1) receptor anti-sense construct, which reduced the expression of LPA(1), abrogated both LPA- and NGF-stimulated activation of p42/p44 MAPK. Third, the over-expression of recombinant LPA(1) receptor potentiated LPA- and NGF-dependent activation of p42/p44 MAPK. Fourth, the over-expression of C-terminal GRK2 peptide (which sequesters G-protein betagamma subunits) or beta-arrestin I clathrin binding domain (amino acids: 319-418) or pre-treatment of cells with pertussis toxin reduced the LPA- and NGF-dependent stimulation of p42/p44 MAPK. These findings support a model in which the Trk A receptor uses a G-protein-mediated mechanism to regulate the p42/p44 MAPK pathway. Such G-protein-mediated signaling is activated by the LPA(1) receptor as a means of cross-talk regulation with the Trk A receptor. Fifth, the treatment of cells with LPA induced the transactivation of the Trk A receptor. Sixth, LPA and/or NGF stimulated the translocation of tyrosine phosphorylated Trk A receptor and LPA(1) receptor to the nucleus. Taken together, these findings suggest that NGF and LPA exert cross-talk regulation both at the level of p42/p44 MAPK signaling and in the nuclear translocation of LPA(1) and Trk A receptors.     

Activation of MAP kinase by MC4-R through PI3 kinase

The melanocortin 4 receptor (MC4-R) is a G_s-coupled receptor known to increase cAMP production following agonist stimulation. We demonstrate that the mitogen-activated protein kinases p42 (ERK2) and p44 (ERK1) are also activated by MC4-R following treatment with the MC4-R agonist NDP--MSH in stably transfected CHO-K1 cells. This time- and dose-dependent response is abolished by the MC4-R antagonist SHU-9119. p42/p44 MAPK activation is blocked by the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 but not by the protein kinase A (PKA) inhibitor Rp-cAMPS, indicating that that signal activating the p42/p44 MAPK pathway is conveyed through inositol triphosphate.     

Mutation of Asn-391 within the conserved NPXXY motif of the cholecystokinin B receptor abolishes Gq protein activation without affecting its association with the receptor

Among the most conserved regions in the G-protein-coupled receptors is the (N/D)PX(2-3)Y motif of the seventh transmembrane domain (X represents any amino acid). The mutation of the Asn/Asp residue of this motif in different G-protein-coupled receptors was shown to affect the activation of either adenylyl cyclase or phospholipase C. We have mutated the Asn residue (Asn-391) of the NPXXY motif in the CCKBR to Ala and determined the effects of the mutation on binding, signaling, and G-proteins coupling after expression of the mutated receptor in COS cells. The mutated receptor displayed similar expression levels and high affinity CCK binding compared with the wild type CCKBR. However, unlike the wild type CCKBR, the mutated receptor was completely unable to mediate activation of either phospholipase C and protein kinase C-dependent and -independent mitogen-activated protein kinase pathways, indicating an essential role of Asn-391 in CCKBR signaling. Coimmunoprecipitation experiments allowed us to show that the inactive mutant retains an intact capacity to form stable complexes with G(q)alpha subunits in response to CCK. These results indicate that the formation of high affinity CCK-receptor-G(q) protein complexes is not sufficient to activate G(q) and suggest that Asn-391 is specifically involved in G(q) proteins activation.     

Evidence that inhibition of p44/42 mitogen-activated protein kinase signaling is a factor in proteasome inhibitor-mediated apoptosis

The proteasome is emerging as a target for cancer therapy because small molecule inhibitors of its catalytic activity induce apoptosis in both in vitro and in vivo models of human malignancies and are proving to have efficacy in early clinical trials. To further elucidate the mechanism of action of these inhibitors, their impact on signaling through the p44/42 mitogen-activated protein kinase (MAPK) pathway was studied. Proteasome inhibition with either carbobenzoxy-leucyl-leucyl-phenylalaninal or lactacystin led to a loss of dually phosphorylated, activated p44/42 MAPK in A1N4-myc human mammary and MDA-MB-231 breast carcinoma cells in a dose- and time-dependent fashion. This correlated with an induction of the dual specificity MAPK phosphatases (MKP)-1 and -2, and blockade of MKP induction using either actinomycin D or Ro-31-8220 significantly decreased loss of activated p44/42 MAPK. Inhibition of p44/42 MAPK signaling by use of the MAPK kinase inhibitors PD 98059 or U0126, or by use of a dominant negative MAPK construct, enhanced proteasome inhibitor-mediated apoptosis. Conversely, activation of MAPK by epidermal growth factor, or use of a mutant MAPK resistant to MKP-mediated dephosphorylation, inhibited apoptosis. These studies support a role for inactivation of signaling through the p44/42 MAPK pathway in proteasome inhibitor-mediated apoptosis.