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PubMed2003

MTA1 interacts with MAT1, a cyclin-dependent kinase-activating kinase complex ring finger factor, and regulates estrogen receptor transactivation functions.

The transcriptional activity of estrogen receptor-alpha is controlled by coregulators. MTA1 (metastasis-associated protein 1) represses estrogen receptor-alpha-driven transcription by recruiting histone deacetylases (HDACs) to the estrogen response element containing target gene chromatin in breast cancer cells. Using a yeast two-hybrid screen with the MTA1 C-terminal domain as bait, we identified MAT1 (ménage á trois 1) as an MTA1-binding protein. MAT1 is an assembly/targeting factor for cyclin-dependent kinase-activating kinase (CAK), which has been shown to functionally interact with general transcriptional factor TFIIH, a known inducer of ER transactivation. We show that estrogen signaling promotes nuclear translocation of MAT1 and that MTA1 interacts with MAT1 both in vitro and in vivo. MAT1 binds to the C-terminal 389-441 amino acids GATA domain and N-terminal 1-164 amino acids bromo-domain of MTA1, whereas MTA1 binds to the N-terminal ring finger domain of the MAT1. In addition, MAT1 interacts with the activation function 2 domain of ER and colocalizes with ER in activated cells. MTA1 deregulation in breast cancer cells led to its interactions with the CAK complex components, ER, and HDAC2. Accordingly, MTA1 inhibited CAK stimulation of ER transactivation that was partially relieved by HDAC inhibitor trichostatin A, suggesting that MTA1 might inhibit CAK-induced transactivation function of ER by recruiting HDAC. Furthermore, MTA1 overexpression inhibited the ability of CAK complex to phosphorylate ER. Together, these findings identified MAT1 as a target of MTA1 and provided new evidence to suggest that the transactivation functions of ER might be influenced by the regulatory interactions between CAK and MTA1 in breast cancer cells.

Base Sequence↗
PubMed1994

p40MO15 associates with a p36 subunit and requires both nuclear translocation and Thr176 phosphorylation to generate cdk-activating kinase activity in Xenopus oocytes.

p40MO15, a cdc2-related protein, is the catalytic subunit of the kinase (CAK, cdk-activating kinase) responsible for Thr161/Thr160 phosphorylation and activation of cdk1/cdk2. We have found that strong overexpression of p40MO15 only moderately increases CAK activity in Xenopus oocytes, indicating that a regulatory CAK subunit (possibly a cyclin-like protein) limits the ability to generate CAK activity in p40MO15 overexpressing oocytes. This 36 kDa subunit was microsequenced after extensive purification of CAK activity. Production of Xenopus CAK activity was strongly reduced in enucleated oocytes overexpressing p40MO15 and p40MO15 shown to contain a nuclear localization signal required for nuclear translocation and generation of CAK activity. p40MO15 was found to be phosphorylated on Ser170 and Thr176 by proteolytic degradation, radiosequencing of tryptic peptides and mutagenesis. Thr176 phosphorylation is required and Ser170 phosphorylation is dispensable for p40MO15 to generate CAK activity upon association with the 36 kDa regulatory subunit. Finally, Thr176 and Ser170 phosphorylations are not intramolecular autophosphorylation reactions. Taken together, the above results identify protein-protein interactions, nuclear translocation and phosphorylation (by an unidentified kinase) as features of p40MO15 that are required for the generation of active CAK.

Amino Acid Sequence↗
PubMed2000

NAK is an IkappaB kinase-activating kinase.

Phosphorylation of IkappaB by the IkappaB kinase (IKK) complex is a critical step leading to IkappaB degradation and activation of transcription factor NF-kappaB. The IKK complex contains two catalytic subunits, IKKalpha and IKKbeta, the latter being indispensable for NF-kappaB activation by pro-inflammatory cytokines. Although IKK is activated by phosphorylation of the IKKbeta activation loop, the physiological IKK kinases that mediate responses to extracellular stimuli remain obscure. Here we describe an IKK-related kinase, named NAK (NF-kappaB-activating kinase), that can activate IKK through direct phosphorylation. NAK induces IkappaB degradation and NF-kappaB activity through IKKbeta. Endogenous NAK is activated by phorbol ester tumour promoters and growth factors, whereas catalytically inactive NAK specifically inhibits activation of NF-kappaB by protein kinase C-epsilon (PKCepsilon). Thus, NAK is an IKK kinase that may mediate IKK and NF-kappaB activation in response to growth factors that stimulate PKCepsilon activity.

Amino Acid Sequence↗
PubMed1997

Genes for Tfb2, Tfb3, and Tfb4 subunits of yeast transcription/repair factor IIH. Homology to human cyclin-dependent kinase activating kinase and IIH subunits.

Genes for the Tfb2, Tfb3, and Tfb4 subunits of yeast RNA polymerase transcription factor IIH (TFIIH) are described. All three genes are essential for cell viability, and antibodies against Tfb3 specifically inhibit transcription in vitro. A C-terminal deletion of Tfb2 caused a defect in nucleotide excision repair, as shown by UV sensitivity of the mutant strain and loss of nucleotide excision repair activity in cell extracts (restored by the addition of purified TFIIH). An interaction between Tfb3 and the Kin28 subunit of TFIIH was detected by the two-hybrid approach, consistent with a role for Tfb3 in linking kinase and core domains of the factor. The deduced amino acid sequence of Tfb2 is similar to that of the 52-kDa subunit of human TFIIH, while Tfb3 is identified as a RING finger protein homologous to the 36-kDa subunit of murine CAK (cyclin-dependent kinase activating kinase) and to the 32-kDa subunit of human TFIIH. Tfb4 is homologous to p34 of human TFIIH and is identified as the weakly associated 37-kDa subunit of the yeast factor. These and other findings reveal a one-to-one correspondence and high degree of sequence similarity between the entire set of yeast and human TFIIH polypeptides.

Amino Acid Sequence↗
PubMed1996

Biochemical characterization of the human cyclin-dependent protein kinase activating kinase. Identification of p35 as a novel regulatory subunit.

The activation of cyclin-dependent protein kinases (Cdks) is dependent upon site-specific phosphorylation and dephosphorylation reactions, as well as positive and negative regulatory subunits. The human Cdk-activating protein kinase (Cak1) is itself a Cdc2-related cyclin-dependent protein kinase that associates with cyclin H. The present study utilized specific anti-Cak1 antibodies and immunoaffinity chromatography to identify additional Cak1-associated proteins and potential target substrates. Immunoprecipitation of metabolically labeled human osteosarcoma cells revealed a number of Cak1-associated proteins, including p95, p37 (cyclin H), and a 35-kDa protein that was further characterized herein. Microsequence analysis obtained after limited proteolysis revealed peptide fragments that are similar, but not identical to, human and yeast cyclins, thus identifying p35 as a cyclin-like regulatory subunit. The greatest sequence similarity of human p35 is with Mcs2, a yeast cyclin that is essential for cell cycle progression. Immunoaffinity chromatography performed under nondenaturing conditions afforded the isolation of enzymatically active Cak1 from cell lysates, enabling studies of kinase autophosphorylation and comparative substrate utilization. Immunoaffinity-purified Cak1 phosphorylated monomeric Cdc2 and Cdk2, but not Cdk4; the phosphorylation of both Cdc2 and Cdk2 were increased in the presence of recombinant cyclin A. These studies indicate that the Cak1 catalytic subunit, like Cdc2 and Cdk2, associates with multiple regulatory partners and suggests that subunit composition may be an important determinant of this multifunctional enzyme.

Amino Acid Sequence↗
PubMed2005

A novel pathway for presynaptic mitogen-activated kinase activation via AMPA receptors.

AMPA-type glutamate receptors play a key role in mediating postsynaptic responses of excitatory neurotransmitters. It is now well accepted that AMPA receptors are also present at the presynapse, where they are thought to modulate neurotransmitter release. However, the mechanisms through which they control synaptic vesicle traffic have remained elusive. We used cultured hippocampal neurons and growth cone particles prepared from fetal rat brain to investigate the functional role of presynaptic AMPA receptors. We show here that stimulation of presynaptic AMPA receptors induces activation of mitogen-activated protein kinase (MAPK) through a nonreceptor tyrosine kinase-dependent and Na+/Ca2+-independent mechanism. This pathway is activated predominantly in axonal growth cones compared with the somatodendritic compartment. After stimulation of presynaptic AMPA receptors, synapsin I is phosphorylated at MAPK-specific sites. These events are paralleled by a prominent increase in evoked synaptic vesicle recycling that is blocked by the specific MAPK inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one. Similarly, in synaptosomes isolated from adult brain, AMPA stimulation induces MAPK activation and phosphorylation of synapsin I at MAPK-dependent sites and enhances significantly synaptic vesicle recycling. These results reveal a novel pathway for activation of presynaptic MAPK and suggest a role of this pathway in the regulation of short-term presynaptic plasticity.

Animals↗
PubMed1984

Characterization of calmodulin-activated protein kinase activity of rat adipocyte endoplasmic reticulum fraction.

Calmodulin-activated protein kinase activity in the endoplasmic reticulum fraction of rat adipocytes was identified and characterized. The major endogenous protein substrate of the calmodulin-activated kinase activity has an apparent molecular weight of 54,000 as determined by sodium dodecyl sulfate gel electrophoresis. The calmodulin-activated component of the activity was saturated at 10 microM ATP. Calcium or calmodulin alone did not increase the activity, but the simultaneous presence of calcium and calmodulin increased activity three to four-fold. Half-maximal activation of this activity occurred at 8 microM Ca2+. The addition of increasing amounts of calmodulin caused a concentration-dependent activation in the presence of calcium, which was saturable at high calmodulin concentrations. Magnesium was required for activity, with half-maximal activity occurring at 230 microM. The antipsychotic drug trifluoperazine inhibited the activation of the protein kinase activity by calmodulin, but had a negligible effect on the basal activity. Half-maximal inhibition occurred at 63 microM. Phosphorylation of the 54,000 mol. wt band was independent of cAMP, cGMP and the combination of cAMP and cAMP-dependent protein kinase. Calmodulin-activated protein kinase phosphorylated both phosphoserine and phosphothreonine residues in the 54,000 mol. wt substrate. These experiments have partially characterized a calmodulin-activated protein kinase activity from adipocytes, which appears to be a unique activity of unknown function.

Adenosine Triphosphate↗
PubMed2000

Src family kinase activity is required for Kit-mediated mitogen-activated protein (MAP) kinase activation, however loss of functional retinoblastoma protein makes MAP kinase activation unnecessary for growth of small cell lung cancer cells.

Small cell lung cancer (SCLC) is characterized by multiple genetic alterations that include inactivation of the retinoblastoma protein (Rb), the establishment of several autocrine loops including that induced by coexpression of stem cell factor (SCF) and Kit, and the ectopic expression and activation of Src family kinases. Previous studies have shown that Lck associates with, and becomes activated by, Kit after SCF stimulation of SCLC cells. In the present study, we have demonstrated that PP1, a pharmacological inhibitor of Src kinases, blocked SCF-mediated activation of mitogen-activated protein (MAP) kinase, but it also inhibited Kit activation. However, MAP kinase activation was more sensitive than Kit activation to the effects of PP1. Overexpression of Lck reduced the sensitivity of MAP kinase activation to PP1 without altering the sensitivity of Kit activation, which suggested a role for Lck in SCF-mediated MAP kinase activation. Inducible expression of a dominant negative Lck inhibited MAP kinase activation in a dose-dependent manner, which confirmed that Src family kinase activity is required for SCF-induced MAP kinase activation. The growth of cells that expressed dominant negative Lck was unaffected, however, despite the inhibition of MAP kinase. Growth was also unaffected by the inhibition of the MAP kinase pathway using PD 98059, but sensitivity to the MAP/extracellular signal-regulated kinase kinase inhibitor could be partially restored by expression of wild-type Rb. Therefore, MAP kinase activation seems to be dispensable for the growth of SCLC only in the absence of Rb expression. These data suggest that the SCF/Kit autocrine loop, through activation of Lck and subsequently MAP kinase, and the mutational inactivation of Rb contribute to the loss of G1-S phase checkpoint regulation during the pathogenesis of SCLC. Furthermore, the data demonstrate that, in established SCLC cell lines, proliferative signal transduction initiated by Kit is mediated by pathways other than the classic MAP kinase pathway.

Blotting, Western↗
PubMed1999

Local renin-angiotensin system and mitogen-activated protein kinase activation in rat aorta.

We previously reported that endogenous angiotensin II is released to cause mitogen-activated protein (MAP) kinase stimulation in the media portion of the vasculature. In this study, we examined whether a functional renin-angiotensin system is indeed present within the media of the vasculature. In rat aortic strips, endothelium removal produced an increase of MAP kinase activity. The MAP kinase activation was inhibited either by the renin inhibitor pepstatin A or by the angiotensin-converting enzyme inhibitor captopril. The degree of the inhibition of the MAP kinase activation by pepstatin A, captopril and the angiotensin receptor antagonist losartan was almost the same. Pepstatin A inhibited MAP kinase activation induced by renin but not by angiotensin I and angiotensin II. Captopril inhibited the MAP kinase activation induced by angiotensin I but not by angiotensin II. In nephrectomized rat aortic strips, endothelium removal also produced an increase in MAP kinase activity, but the MAP kinase activation was considerably small and minimally inhibited by losartan. Nephrectomy produced a marked decrease in plasma renin activity. These findings suggest that an apparently fully intact and functional renin-angiotensin system is present in the media of the rat vasculature and this system serves to increase MAP kinase activity. It appears that renin plays the determining role in the regulation of angiotensin generation also in the media and the major source of the renin is renin of kidney origin.

Angiotensin I↗
PubMed1999

Vascular mitogen-activated protein kinase activity is enhanced via angiotensin system in spontaneously hypertensive rats.

The vascular structural remodeling function may be altered in genetically hypertensive animals, spontaneously hypertensive rats (SHR). To examine this possibility, we measured the activity of mitogen-activated protein (MAP) kinases, enzymes believed to be involved in the pathway for cell proliferation, in rat aorta strips, and examined whether the endothelium removal-induced MAP kinase activation function is altered in SHR and whether vascular angiotensin and endothelin systems are responsible for the alteration of MAP kinase activation in SHR. Male 4-week-old SHR and age-matched Wistar Kyoto rats (WKY) supplied by Charles River Japan were used. Endothelium-denuded aorta strips were incubated at 37 degrees C in medium. MAP kinase activity after incubation was time-dependently increased in strips from SHR and WKY. MAP kinase activation was greater in SHR than in WKY aorta strips. Similarly, MAP kinase activation was enhanced in aorta strips from 4-week-old SHR and stroke prone SHR supplied by the Diseases Model Cooperative Research Association (Kyoto, Japan). In aorta strips from SHR and WKY, the angiotensin receptor antagonist, losartan, and the endothelin receptor antagonist, cyclo (D-alpha-aspartyl-L-prolyl-D-valyl-L-leucyl-D-tryptophyl)(BQ123), caused concentration-dependent inhibition of MAP kinase activation. The losartan-induced but not BQ123-induced inhibition of MAP kinase activation was greater in SHR than in WKY aorta strips. Angiotensin II caused a concentration-dependent increase in MAP kinase activity and the angiotensin II-induced MAP kinase activation was greater in SHR than in WKY aorta strips. These results indicate that endothelium removal-induced MAP kinase activation is enhanced in aorta strips from young SHR, suggesting that vascular structural remodeling function may be enhanced in SHR. It appears that the enhancement of MAP kinase activation results, at least in part, from enhanced function of vascular angiotensin system in SHR.

Angiotensin II↗
PubMed1996

Role of receptor desensitization, phosphatase induction and intracellular cyclic AMP in the termination of mitogen-activated protein kinase activity in UTP-stimulated EAhy 926 endothelial cells.

We have investigated the mechanisms that bring about the termination of mitogen-activated protein kinase (MAP kinase) activation in response to UTP in EAhy 926 endothelial cells. UTP-stimulated MAP kinase activity was transient, returning to basal values by 60 min. At this time MAP kinase activation was desensitized; re-application of UTP did not further activate MAP kinase, full re-activation of MAP kinase being only apparent after a 1-2 h wash period. However, activation of MAP kinase by UTP could be sustained beyond 60 min by preincubation of the cells with the protein synthesis inhibitor cycloheximide. UTP also stimulated expression of MAP kinase phosphatase-1 and this was abolished after pretreatment with cycloheximide. Pretreatment of cells with forskolin abolished the initial activation of MAP kinase kinase or c-Raf-1 by UTP, but only affected MAP kinase activity during prolonged stimulation. The effect of forskolin on prolonged MAP kinase activation was also prevented by cycloheximide. These results suggest that the termination of MAP kinase activity in response to UTP involves a number of interacting mechanisms including receptor desensitization and the induction of a phosphatase. However, several pieces of evidence do not support a major role for MAP kinase phosphatase-1 in termination of the MAP kinase signal. Raising intracellular cyclic AMP may also be involved but only after an initial protein-synthesis step and by a mechanism that does not involve the inactivation of c-Raf-1 or MAP kinase kinase.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1998

Identification of mitogen-activated protein kinase-activated protein kinase-2 as a vimentin kinase activated by okadaic acid in 9L rat brain tumor cells.

Organization of intermediate filament, a major component of cytoskeleton, is regulated by protein phosphorylation/dephosphorylation, which is a dynamic process governed by a balance between the activities of involved protein kinases and phosphatases. Blocking dephosphorylation by protein phosphatase inhibitors such as okadaic acid (OA) leads to an apparent activation of protein kinase(s) and to genuine activation of phosphatase-regulated protein kinase(s). Treatment of 9L rat brain tumor cells with OA results in a drastically increased phosphorylation of vimentin, an intermediate filament protein. In-gel renaturing assays and in vitro kinase assays using vimentin as the exogenous substrate indicate that certain protein kinase(s) is activated in OA-treated cells. With specific protein kinase inhibitors, we show the possible involvement of the cdc2 kinase- and p38 mitogen-activated protein kinase (p38MAPK)-mediated pathways in this process. Subsequent in vitro assays demonstrate that vimentin may serve as an excellent substrate for MAPK-activated protein kinase-2 (MAPKAPK-2), the downstream effector of p38MAPK, and that MAPKAPK-2 is activated with OA treatment. Comparative analysis of tryptic phosphopeptide maps also indicates that corresponding phosphopeptides emerged in vimentin from OA-treated cells and were phosphorylated by MAPKAPK-2. Taken together, the results clearly demonstrate that MAPKAPK-2 may function as a vimentin kinase in vitro and in vivo. These findings shed new light on the possible involvement of the p38MAPK signaling cascade, via MAPKAPK-2, in the maintenance of integrity and possible physiological regulation of intermediate filaments.

Animals↗
PubMed2004

Constitutive p21-activated kinase (PAK) activation in breast cancer cells as a result of mislocalization of PAK to focal adhesions.

Cytoskeletal remodeling is critical for cell adhesion, spreading, and motility. p21-activated kinase (PAK), an effector molecule of the Rho GTPases Rac and Cdc42, has been implicated in cytoskeletal remodeling and cell motility. PAK kinase activity and subcellular distribution are tightly regulated by rapid and transient localized Rac and Cdc42 activation, and by interactions mediated by adapter proteins. Here, we show that endogenous PAK is constitutively activated in certain breast cancer cell lines and that this active PAK is mislocalized to atypical focal adhesions in the absence of high levels of activated Rho GTPases. PAK localization to focal adhesions in these cells is independent of PAK kinase activity, NCK binding, or GTPase binding, but requires the association of PAK with PIX. Disruption of the PAK-PIX interaction with competitive peptides displaces PAK from focal adhesions and results in a substantial reduction in PAK hyperactivity. Moreover, disruption of the PAK-PIX interaction is associated with a dramatic decrease of PIX and paxillin in focal adhesions, indicating that PAK localization to these structures via PIX is required for the maintenance of paxillin- and PIX-containing focal adhesions. Abnormal regulation of PAK localization and activity may contribute to the tumorigenic properties of certain breast cancer cells.

Adaptor Proteins, Signal Transducing↗
PubMed1992

Endothelin rapidly stimulates mitogen-activated protein kinase activity in rat mesangial cells.

Mitogen-activated protein (MAP) kinases are regarded as switch kinases in the phosphorylation cascade initiated by various agonists. We have investigated whether endothelins (ET), which are constrictor and mitogenic isopeptides, can increase MAP kinase activity in rat mesangial cells, using bovine myelin basic protein (MBP) as a substrate for an in vitro kinase assay. Treatment of quiescent mesangial cells with ET-1 rapidly stimulated a kinase activity which phosphorylated exogenous MBP. This stimulation was dose-dependent, with threshold responses at 1 nM-ET-1. Epidermal growth factor and thrombin also activated this kinase in mesangial cells. We also examined the ET signal transduction pathways leading to activation of MBP kinase. Pertussis toxin had no effect on ET-stimulated MBP kinase activity. Stimulation of protein kinase C by phorbol ester increased MBP kinase activity, and down-regulation of PKC partially inhibited ET-stimulated MBP kinase as well as phorbol ester-stimulated MBP kinase activity. Interestingly, genestein, an inhibitor of protein tyrosine kinases, partially inhibited MBP kinase stimulated by ET but not by phorbol esters. These results suggest that ET stimulates MBP kinase activity in rat mesangial cells via at least two pathways: one which is protein kinase C-dependent and a second one that involves a protein tyrosine kinase. Finally, by raising rabbit antibodies against the two forms of MAP kinase, p44mapk and p42mapk, we demonstrated that both isoforms are expressed in mesangial cells. Antibody alpha 1 Cp42 specifically immunoprecipitated p42mapk and allowed us to demonstrate that ET stimulates MBP kinase activity in the p42mapk immunocomplex. In conclusion, we have provided evidence that, in rat mesangial cells, MAP kinases are rapidly activated by ET-1, a regulatory process that involves at least protein kinase C activation and also a contribution of a tyrosine kinase not yet characterized.

Aluminum↗
PubMed1997

Tumor-necrosis factor-alpha modulates mitogen-activated protein kinase activity of epidermal-growth-factor-stimulated MCF-7 breast cancer cells.

Tumor-necrosis factor(TNF)-alpha inhibited in a dose-dependent fashion the proliferation of epidermal-growth-factor(EGF)-stimulated MCF-7 breast cancer cells with an IC50 of 0.25 nM. A comparable TNF-alpha-mediated inhibition of p42/44 mitogen-activated protein (MAP) kinase activity was observed in 10 nM EGF-stimulated cells. The MAP kinase activity dropped 50% within 3 min of TNF-alpha (1 nM) addition to EGF-stimulated MCF-7 cells. EGF and TNF-alpha, when added independently, led to a transient stimulation of MAP kinase activity with maximal activations within 6-8 min and 1-2 min, respectively. These observations suggest that MAP kinase activity in EGF-stimulated MCF-7 cells is modulated by the growth-inhibitory receptor pathways of TNF-alpha. Phosphorylation measurements on western blots determined the involvement of several individual MAP kinases, namely p42/44 MAP kinases, p38 MAP kinase and c-Jun N2-terminal kinase 1 (JNK1), in EGF and TNF-alpha-induced signalling. Phosphorylation of p42 and p38 MAP kinases only was observed after treatment with either TNF-alpha or EGF. A combination of both ligands inhibited p42 and p38 MAP kinase phosphorylation in MCF-7 cells. In contrast, no JNK1 phosphorylation was detected in these cells. Simultaneous addition of okadaic acid, a potent inhibitor of phosphatases 1 and 2A, blocked the decay of EGF-stimulated MAP kinase activity over 40 min. TNF-alpha added to EGF-stimulated and okadaic-acid-treated cells increased the MAP kinase activity twofold within 1 min. Similarly, okadaic acid treatment partly reverted the TNF-alpha-inhibited growth of MCF-7 cells. These experiments suggest that phosphatases are involved in the rapid shut-down by TNF-alpha of p42 MAP kinase activity.

Breast Neoplasms↗
PubMed1998

Nociceptin (ORL-1) and mu-opioid receptors mediate mitogen-activated protein kinase activation in CHO cells through a Gi-coupled signaling pathway: evidence for distinct mechanisms of agonist-mediated desensitization.

The recently identified 17-amino acid peptide nociceptin (orphanin FQ) is the endogenous ligand for the opioid receptor-like-1 (ORL-1) receptor. A physiologic role for nociceptin (OFQ) activation of the ORL-1 receptor (OFQR) may be to modulate opioid-induced analgesia. The molecular mechanism by which nociceptin (OFQ) and ORL-1 (OFQR) modify opioid-stimulated effects, however, is unclear. Both ORL-1 (OFQR) and opioid receptors mediate pertussis toxin (PTX)-sensitive signal transduction, indicating these receptors are capable of coupling to Gi/Go proteins. This study determines that nociceptin stimulates an intracellular signaling pathway, leading to activation of mitogen-activated protein (MAP) kinase in CHO cells expressing ORL-1 receptor (OFQR). Nociceptin (OFQ)-stimulated MAP kinase activation was inhibited by PTX or by expression of the carboxyl terminus of beta-adrenergic receptor kinase (betaARKct), which specifically blocks Gbetagamma-mediated signaling. Expression of the proline-rich domain of SOS (SOS-PRO), which inhibits SOS interaction with p21ras, also attenuated nociceptin (OFQ)-stimulated MAP kinase activation. The phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin and LY294002 reduced nociceptin (OFQ)-stimulated MAP kinase activation, whereas inhibition of protein kinase C (PKC) activity by bisindolylmaleimide I or cellular depletion of PKC had no effect. In a similar manner, in cells expressing mu-opioid receptor, [D-Ala2,N-Me-Phe4,Gly-ol]-enkephalin (DAMGO; a mu-opioid receptor-selective agonist) stimulated PTX-sensitive MAP kinase activation that was inhibited by wortmannin, LY294002, betaARKct expression, or SOS-PRO expression but not affected by inhibition of PKC activity. These results indicate that both ORL-1 (OFQR) and mu-opioid receptors mediate MAP kinase activation via a signaling pathway using the betagamma-subunit of Gi, a PI-3K, and SOS, independent of PKC activity. In cells expressing both ORL-1 (OFQR) and mu-opioid receptors, pretreatment with nociceptin decreased subsequent nociceptin (OFQ)- or DAMGO-stimulated MAP kinase activation. In contrast, pretreatment of cells with DAMGO decreased subsequent DAMGO-stimulated MAP kinase but had no effect on subsequent nociceptin (OFQ)-stimulated MAP kinase activation. These results demonstrate that nociceptin (OFQ) activation of ORL-1 (OFQR) can modulate mu-opioid receptor signaling in a cellular system.

Animals↗
PubMed2000

Angiotensin II mediates pressure loading-induced mitogen-activated protein kinase activation in isolated rat aorta.

Vascular hypertrophy occurs during chronic hypertension and contributes to the elevation of peripheral vascular resistance in hypertension. In this study, we examined whether acute pressure overloading of the vascular wall produces activation of mitogen-activated protein (MAP) kinases, enzymes believed to be involved in the pathway for cell proliferation, in isolated perfused rat aortae, and examined whether the mechanical overloading-induced MAP kinase activation is mediated via the vascular angiotensin system. Aortae were perfused with Tyrode solution. Increases in perfusion pressure caused a pressure-dependent increase in MAP kinase activity in endothelium-intact aortae and in endothelium-denuded aortae. The increase in MAP kinase activity induced by pressure loading was inhibited by the angiotensin receptor antagonist, losartan, the renin inhibitor, pepstatin A, and the angiotensin-converting enzyme inhibitor, captopril. Ca(2+) depletion and the Ca(2+) channel antagonist, nifedipine, did not affect the pressure loading-induced MAP kinase activation. The results of the present study suggest that pressure loading of the vascular wall per se can activate MAP kinases in the vasculature and that the MAP kinase activation is mediated at least partly via the vascular angiotensin system. It seems unlikely that the pressure loading-induced increase in MAP kinase activity is mainly mediated via increases in Ca(2+) influx in vascular cells.

Angiotensin II↗
PubMed2001

Mitogen-activated protein kinase activity regulation role of angiotensin and endothelin systems in vascular smooth muscle cells.

To examine whether angiotensin II and endothelins produced in vascular smooth muscle cells can play roles in the regulation of mitogen-activated protein (MAP) kinase activity in vascular smooth muscle cells, we measured the activity of MAP kinases in cultured vascular smooth muscle cells, and determined effects of renin-angiotensin and endothelin systems activators and inhibitors. Angiotensin II and endothelin-1 produced an activation of MAP kinase activity in vascular smooth muscle cells, whereas the angiotensin receptor antagonist, losartan and the endothelin receptor antagonist, cyclo (D-alpha-aspartyl-L-prolyl-D-valyl-L-leucyl-D-tryptophyl, BQ123) inhibited the enzyme activity. MAP kinase activity in vascular smooth muscle cells was also inhibited either by the renin inhibitor pepstatin A or by the angiotensin-converting enzyme inhibitor captopril. The degree of the inhibition of MAP kinase activity by pepstatin A, captopril and losartan was almost the same. Renin produced a considerable increase in MAP kinase activity and the renin-induced MAP kinase activation was inhibited by pepstatin A. The endothelin precursor big endothelin-1 produced an increase of MAP kinase activity in vascular smooth muscle cells, whereas the endothelin-converting enzyme inhibitor phosphoramidon inhibited the enzyme activity. These findings suggest that functional renin-angiotensin system and endothelin system are present in vascular smooth muscle cells and these systems tonically serve to increase MAP kinase activity. It appears that renin or renin-like substances play the determining role in the regulation of renin-angiotensin system in vascular smooth muscle cells.

Angiotensin I↗