Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.24 (mitogen-activated protein kinase)
 95,810 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

CXCR2 is a seven-transmembrane receptor that transduces intracellular signals in response to the chemokines interleukin-8, melanoma growth-stimulatory activity/growth-regulatory protein, and other ELR motif-containing CXC chemokines by coupling to heterotrimeric GTP-binding proteins. In this study, we explored the mechanism responsible for ligand-induced CXCR2 endocytosis. Here, we demonstrate that dynamin, a component of clathrin-mediated endocytosis, is essential for CXCR2 endocytosis and resensitization. In HEK293 cells, dynamin I K44A, a dominant-negative mutant of dynamin that inhibits the clathrin-mediated endocytosis, blocks the ligand-stimulated CXCR2 sequestration. Furthermore, co-expression of dynamin I K44A significantly delays dephosphorylation of CXCR2 after ligand stimulation, suggesting that clathrin-mediated endocytosis plays an important role in receptor dephosphorylation and resensitization. In addition, ligand-mediated receptor down-regulation is attenuated when receptor internalization is inhibited by dynamin I K44A. Interestingly, inhibition of receptor endocytosis by dynamin I K44A does not affect the CXCR2-mediated stimulation of mitogen-activated protein kinase. Most significantly, our data indicate that the ligand-stimulated receptor endocytosis is required for CXCR2-mediated chemotaxis in HEK293 cells. Taken together, our findings suggest that clathrin-mediated CXCR2 internalization is crucial for receptor endocytosis, resensitization, and chemotaxis.
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PMID:Role of clathrin-mediated endocytosis in CXCR2 sequestration, resensitization, and signal transduction. 1019 23

Agonist-promoted internalization of some G protein-coupled receptors has been shown to mediate receptor desensitization, resensitization, and down-regulation. In this study, we investigated whether opioids induced internalization of the human and rat kappa opioid receptors stably expressed in Chinese hamster ovary cells, the potential mechanisms involved in this process and its possible role in activation of mitogen-activated protein (MAP) kinase. Exposure of the human kappa receptor to the agonists U50,488H, U69,593, ethylketocyclazocine, or tifluadom, but not etorphine, promoted receptor internalization. However, none of these agonists induced significant internalization of the rat kappa opioid receptor. U50, 488H-induced human kappa receptor internalization was time- and concentration-dependent, with 30-40% of the receptors internalized following a 30-min exposure to 1 microM U50,488H. Agonist removal resulted in the receptors gradually returning to the cell surface over a 60-min period. The antagonist naloxone blocked U50, 488H-induced internalization without affecting internalization itself, while pretreatment with pertussis toxin had no effect on U50, 488H-induced internalization. In contrast, incubation with sucrose (0.4-0.8 M) significantly reduced U50,488H-induced internalization of the kappa receptor. While co-expression of the wild type GRK2, beta-arrestin, or dynamin I had no effect on kappa receptor internalization, co-expression of the dominant negative mutants GRK2-K220R, beta-arrestin (319-418), or dynamin I-K44A significantly inhibited receptor internalization. Whether receptor internalization is critical for MAP kinase activation was next investigated. Co-expression of dominant negative mutants of beta-arrestin or dynamin I, which greatly reduced U50,488H-induced internalization, did not affect MAP kinase activation by the agonist. In addition, etorphine, which did not promote human kappa receptor internalization, was able to fully activate MAP kinase. Moreover, U50,488H or etorphine stimulation of the rat kappa receptor, which did not undergo internalization, also effectively activated MAP kinase. Thus, U50,488H-induced internalization of the human kappa opioid receptor in Chinese hamster ovary cells occurs via a GRK-, beta-arrestin-, and dynamin I-dependent process that likely involves clathrin-coated pits. In addition, internalization of the kappa receptor is not required for activation of MAP kinase.
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PMID:U50,488H-induced internalization of the human kappa opioid receptor involves a beta-arrestin- and dynamin-dependent mechanism. Kappa receptor internalization is not required for mitogen-activated protein kinase activation. 1020 34

Dynamin plays a critical role in the membrane fission mechanism that mediates regulated endocytosis of many G protein-coupled receptors. In addition, dynamin is required for ligand-induced activation of mitogen-activated protein kinase by certain receptors, raising a general question about the role of dynamin in mitogenic signal transduction. Here we report that endocytosis of mu and delta opioid receptors is not required for efficient ligand-induced activation of mitogen-activated protein kinase. Nevertheless, mitogenic signaling mediated by these receptors is specifically dynamin-dependent. Thus a functional role of dynamin in mitogenic signaling can be dissociated from its role in receptor-mediated endocytosis, suggesting a previously unidentified and distinct role of dynamin in signal transduction by certain G protein-coupled receptors.
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PMID:Dissociation of functional roles of dynamin in receptor-mediated endocytosis and mitogenic signal transduction. 1045 21

Agonist-elicited receptor sequestration is strikingly different for the alpha(2A)- versus alpha(2B)-adrenergic receptor (alpha(2)-AR) subtypes; the alpha(2B)-AR undergoes rapid and extensive disappearance from the HEK 293 cell surface, whereas the alpha(2A)-AR does not (Daunt, D. A., Hurt, C., Hein, L., Kallio, J., Feng, F., and Kobilka, B. K. (1997) Mol. Pharmacol. 51, 711-720; Eason, M. G., and Liggett, S. B. (1992) J. Biol. Chem. 267, 25473-25479). Since recent reports suggest that endocytosis is required for some G protein-coupled receptors to stimulate the mitogen-activated protein (MAP) kinase cascade (Daaka, Y., Luttrell, L. M., Ahn, S., Della Rocca, G. J., Ferguson, S. S., Caron, M. G., and Lefkowitz, R. J. (1998) J. Biol. Chem. 273, 685-688; Luttrell, L. M., Daaka, Y., Della Rocca, G. J., and Lefkowitz, R. J. (1997) J. Biol. Chem. 272, 31648-31656; Ignatova, E. G., Belcheva, M. M., Bohn, L. M., Neuman, M. C., and Coscia, C. J. (1999) J. Neurosci. 19, 56-63), we evaluated the differential ability of these two subtypes to activate MAP kinase. We observed no correlation between subtype-dependent agonist-elicited receptor redistribution and receptor activation of the MAP kinase cascade. Furthermore, incubation of cells with K(+)-depleted medium eliminated alpha(2B)-AR internalization but did not eliminate MAP kinase activation, suggesting that receptor internalization is not a general prerequisite for activation of the MAP kinase cascade via G(i)-coupled receptors. We also noted that neither dominant negative dynamin (K44A) nor concanavalin A treatment dramatically altered MAP kinase activation or receptor redistribution, indicating that these experimental tools do not universally block G protein-coupled receptor internalization.
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PMID:Stimulation of mitogen-activated protein kinase by G protein-coupled alpha(2)-adrenergic receptors does not require agonist-elicited endocytosis. 1045 69

Internalization of activated receptors from the plasma membrane has been implicated in the activation of mitogen-activated protein (MAP) kinase. However, the mechanism whereby membrane trafficking may regulate mitogenic signaling remains unclear. Here we report that dominant-negative dynamin (K44A), an inhibitor of endocytic vesicle formation, abrogates MAP kinase activation in response to epidermal growth factor, lysophosphatidic acid, and protein kinase C-activating phorbol ester. In contrast, dynamin-K44A does not affect the activation of Ras, Raf, and MAP kinase kinase (MEK) by either agonist. Through immunofluorescence and subcellular fractionation studies, we find that activated MEK is present both at the plasma membrane and in intracellular vesicles but not in the cytosol. Our findings suggest that dynamin-regulated endocytosis of activated MEK, rather than activated receptors, is a critical event in the MAP kinase activation cascade.
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PMID:Dynamin is required for the activation of mitogen-activated protein (MAP) kinase by MAP kinase kinase. 1058 93

L1-mediated axon growth involves intracellular signaling, but the precise mechanisms involved are not yet clear. We report a role for the mitogen-activated protein kinase (MAPK) cascade in L1 signaling. L1 physically associates with the MAPK cascade components Raf-1, ERK2, and the previously identified p90(rsk) in brain. In vitro, ERK2 can phosphorylate L1 at Ser(1204) and Ser(1248) of the L1 cytoplasmic domain. These two serines are conserved in the L1 family of cell adhesion molecules, also being found in neurofascin and NrCAM. The ability of ERK2 to phosphorylate L1 suggests that L1 signaling could directly regulate L1 function by phosphorylation of the L1 cytoplasmic domain. In L1-expressing 3T3 cells, L1 cross-linking can activate ERK2. Remarkably, the activated ERK localizes with endocytosed vesicular L1 rather than cell surface L1, indicating that L1 internalization and signaling are coupled. Inhibition of L1 internalization with dominant-negative dynamin prevents activation of ERK. These results show that L1-generated signals activate the MAPK cascade in a manner most likely to be important in regulating L1 intracellular trafficking.
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PMID:Activation of the MAPK signal cascade by the neural cell adhesion molecule L1 requires L1 internalization. 1060 64

In previous studies we found that mu-opioids, acting via mu-opioid receptors, inhibit endothelin-stimulated C6 glioma cell growth. In the preceding article we show that the kappa-selective opioid agonist U69,593 acts as a mitogen with a potency similar to that of endothelin in the same astrocytic model system. Here we report that C6 cell treatment with mu-opioid agonists for 1 h results in the inhibition of kappa-opioid mitogenic signaling. The mu-selective agonist endomorphin-1 attenuates kappa-opioid-stimulated DNA synthesis, phosphoinositide turnover, and extracellular signal-regulated kinase phosphorylation. To investigate the role of receptor endocytosis in signaling, we have examined the effects of dynamin-1 and its GTPase-defective, dominant suppressor mutant (K44A) on opioid modulation of extracellular signal-regulated kinase phosphorylation in C6 cells. Overexpression of dynamin K44A in C6 cells does not affect kappa-opioid phosphorylation of extracellular signal-regulated kinase. However, it does block the inhibitory action on kappa-opioid signaling mediated by the kappa-opioid receptor. Our results are consistent with a growing body of evidence of the opposing actions of mu- and kappa-opioids and provide new insight into the role of opioid receptor trafficking in signaling.
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PMID:Mu-opioid agonist inhibition of kappa-opioid receptor-stimulated extracellular signal-regulated kinase phosphorylation is dynamin-dependent in C6 glioma cells. 1064 8

Acting through a number of distinct pathways, many G protein-coupled receptors (GPCRs) activate the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cascade. Recently, it has been shown that in some cases, clathrin-mediated endocytosis is required for GPCR activation of the ERK/MAPK cascade, whereas in others it is not. Accordingly, we compared ERK activation mediated by a GPCR that does not undergo agonist-stimulated endocytosis, the alpha(2A) adrenergic receptor (alpha(2A) AR), with ERK activation mediated by the beta(2) adrenergic receptor (beta(2) AR), which is endocytosed. Surprisingly, we found that in COS-7 cells, ERK activation by the alpha(2A) AR, like that mediated by both the beta(2) AR and the epidermal growth factor receptor (EGFR), is sensitive to mechanistically distinct inhibitors of clathrin-mediated endocytosis, including monodansylcadaverine, a mutant dynamin I, and a mutant beta-arrestin 1. Moreover, we determined that, as has been shown for many other GPCRs, both alpha(2A) and beta(2) AR-mediated ERK activation involves transactivation of the EGFR. Using confocal immunofluorescence microscopy, we found that stimulation of the beta(2) AR, the alpha(2A) AR, or the EGFR each results in internalization of a green fluorescent protein-tagged EGFR. Although beta(2) AR stimulation leads to redistribution of both the beta(2) AR and EGFR, activation of the alpha(2A) AR leads to redistribution of the EGFR but the alpha(2A) AR remains on the plasma membrane. These findings separate GPCR endocytosis from the requirement for clathrin-mediated endocytosis in EGFR transactivation-mediated ERK activation and suggest that it is the receptor tyrosine kinase or another downstream effector that must engage the endocytic machinery.
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PMID:Role of endocytosis in the activation of the extracellular signal-regulated kinase cascade by sequestering and nonsequestering G protein-coupled receptors. 1067 89

beta-Arrestins can act as adapter molecules, coupling G-protein-coupled receptors to proteins involved in mitogenic as well as endocytic pathways. We have previously identified c-SRC as a molecule that is rapidly recruited to the beta2-adrenergic receptor in a beta-arrestin1-dependent manner. Recruitment of c-SRC to the receptor appears to be involved in pathways leading to receptor internalization and mitogen-activated protein kinase activation. This recruitment of c-SRC to the receptor involves an interaction between the amino-terminal proline-rich region of beta-arrestin1 and the Src homology 3 (SH3) domain of c-SRC, but deletion of the proline-rich domain does not totally ablate the interaction. We have found that a major interaction also exists between beta-arrestin1 and the catalytic or kinase domain (SH1) of c-SRC. We therefore hypothesized that a catalytically inactive mutant of the isolated catalytic subunit, SH1(kinase dead) (SH1(KD)), would specifically block those cellular actions of c-SRC that are mediated by beta-arrestin1 recruitment to the G-protein-coupled receptor. In contrast, the majority of cellular phosphorylations catalyzed by c-SRC, which do not involve interaction with the SH1 domain, would be predicted to be unaffected. The SH1(KD) mutant did indeed block beta2-adrenergic receptor internalization and receptor-stimulated tyrosine phosphorylation of dynamin, actions previously shown to be c-SRC-dependent. In contrast, SAM-68 and whole cell tyrosine phosphorylation by c-SRC was unaffected, indicating that the SH1(KD) mutant did not inhibit c-SRC tyrosine kinase activity in general. These results not only clarify the nature of the beta-arrestin1/c-SRC interaction but also implicate beta-arrestin1 as an important mediator of receptor internalization by recruiting tyrosine kinase activity to the cell surface to phosphorylate key endocytic intermediates, such as dynamin.
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PMID:beta-arrestin1 interacts with the catalytic domain of the tyrosine kinase c-SRC. Role of beta-arrestin1-dependent targeting of c-SRC in receptor endocytosis. 1075 43

The neural cell adhesion molecule L1 mediates the axon outgrowth, adhesion, and fasciculation necessary for proper development of synaptic connections. Mutations of human L1 cause an X-linked mental retardation syndrome termed CRASH (corpus callosum hypoplasia, retardation, aphasia, spastic paraplegia, and hydrocephalus), and L1 knock-out mice display defects in neuronal process extension resembling the CRASH phenotype. Little is known about the biochemical or cellular mechanism by which L1 performs neuronal functions. Here it is demonstrated that clustering of L1 with antibodies or L1 protein in rodent B35 neuroblastoma and cerebellar neuron cultures induced the phosphorylation/activation of the mitogen-activated protein kinases (MAPKs) and extracellular signal-regulated kinases 1 and 2. MAPK activation was essential for L1-dependent neurite outgrowth, because chemical inhibitors [2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one and 1,4-diamino-2, 3-dicyano-1,4-bis(2-aminophenylthio)butadiene] of the MAPK kinase MEK strongly suppressed neurite outgrowth by cerebellar neurons on L1. The nonreceptor tyrosine kinase pp60(c-src) was required for L1-triggered MAPK phosphorylation, as shown in src-minus cerebellar neurons and by expression of the kinase-inactive mutant Src(K295M) in B35 neuroblastoma cells. Phosphatidylinositol 3-kinase (PI3-kinase) and the small GTPase p21(rac) were identified as signaling intermediates to MAPK by phosphoinositide and Rac-GTP assays and expression of inhibitory mutants. Antibody-induced endocytosis of L1, visualized by immunofluorescence staining and confocal microscopy of B35 cells, was blocked by expression of kinase-inactive Src(K295M) and dominant-negative dynamin(K44A) but not by inhibitors of MEK or PI3-kinase. Dynamin(K44A) also inhibited L1 antibody-triggered MAPK phosphorylation. This study supports a model in which pp60(c-src) regulates dynamin-mediated endocytosis of L1 as an essential step in MAPK-dependent neurite outgrowth on an L1 substrate.
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PMID:A MAP kinase-signaling pathway mediates neurite outgrowth on L1 and requires Src-dependent endocytosis. 1081 53


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