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Results for “Adaptor Protein Complex 1”

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PubMed2006

Epsin 1 is a polyubiquitin-selective clathrin-associated sorting protein.

Epsin 1 engages several core components of the endocytic clathrin coat, yet the precise mode of operation of the protein remains controversial. The occurrence of tandem ubiquitin-interacting motifs (UIMs) suggests that epsin could recognize a ubiquitin internalization tag, but the association of epsin with clathrin-coat components or monoubiquitin is reported to be mutually exclusive. Here, we show that endogenous epsin 1 is clearly an integral component of clathrin coats forming at the cell surface and is essentially absent from caveolin-1-containing structures under normal conditions. The UIM region of epsin 1 associates directly with polyubiquitin chains but has extremely poor affinity for monoubiquitin. Polyubiquitin binding is retained when epsin synchronously associates with phosphoinositides, the AP-2 adaptor complex and clathrin. The enrichment of epsin within clathrin-coated vesicles purified from different tissue sources varies and correlates with sorting of multiubiquitinated cargo, and in cultured cells, polyubiquitin, rather than non-conjugable monoubiquitin, promotes rapid internalization. As epsin interacts with eps15, which also contains a UIM region that binds to polyubiquitin, epsin and eps15 appear to be central components of the vertebrate poly/multiubiquitin-sorting endocytic clathrin machinery.

Adaptor Proteins, Signal Transducing↗

PubMed2004

c-Src regulates clathrin adapter protein 2 interaction with beta-arrestin and the angiotensin II type 1 receptor during clathrin- mediated internalization.

Beta-arrestins are multifunctional adapters involved in the internalization and signaling of G protein-coupled receptors (GPCRs). They target receptors to clathrin-coated pits (CCPs) through binding with clathrin and clathrin adapter 2 (AP-2) complex. They also act as transducers of signaling by recruiting c-Src kinase to certain GPCRs. Here we sought to determine whether c-Src regulates the recruitment of AP-2 to beta-arrestin and the angiotensin II (Ang II) type 1 receptor (AT1R) during internalization. We show that the agonist stimulation of native AT1R in vascular smooth muscle cells (VSMCs) induces the formation of an endogenous complex containing c-Src, beta-arrestins and AP-2. In vitro studies using coimmunoprecipitation experiments and a yeast three-hybrid assay reveal that c-Src stabilizes the agonist-independent association between beta-arrestin2 and the beta-subunit of AP-2 independently of the kinase activity of c-Src. However, although c-Src expression promoted the rapid dissociation of AP-2 from both beta-arrestin and AT1R after receptor stimulation, a kinase-inactive mutant of c-Src failed to induce the dissociation of AP-2 from the agonist-occupied receptor. Thus, the consequence of c-Src in regulating the dissociation of AP-2 from the receptor was also examined on the internalization of AT1R by depleting c-Src in human embryonic kidney (HEK) 293 cells using a small interfering RNA strategy. Experiments in c-Src depleted cells reveal that AT1R remained mostly colocalized with AP-2 at the plasma membrane after Ang II stimulation, consistent with the observed delay in receptor internalization. Moreover, coimmunoprecipitation experiments in c-Src depleted HEK 293 cells and VSMCs showed an increased association of AP-2 to the agonist-occupied AT1R and beta-arrestin, respectively. Together, our results support a role for c-Src in regulating the dissociation of AP-2 from agonist-occupied AT1R and beta-arrestin during the clathrin-mediated internalization of receptors and suggest a novel function for c-Src kinase in the internalization of AT1R.

Adaptor Protein Complex 2↗

PubMed2006

The Bcl10-Malt1 complex segregates Fc epsilon RI-mediated nuclear factor kappa B activation and cytokine production from mast cell degranulation.

Mast cells are pivotal effector cells in IgE-mediated allergic inflammatory diseases. Central for mast cell activation are signals from the IgE receptor FcepsilonRI, which induce cell degranulation with the release of preformed mediators and de novo synthesis of proinflammatory leukotrienes and cytokines. How these individual mast cell responses are differentially controlled is still unresolved. We identify B cell lymphoma 10 (Bcl10) and mucosa-associated lymphoid tissue 1 (Malt1) as novel key regulators of mast cell signaling. Mice deficient for either protein display severely impaired IgE-dependent late phase anaphylactic reactions. Mast cells from these animals neither activate nuclear factor kappaB (NF-kappaB) nor produce tumor necrosis factor alpha or interleukin 6 upon FcepsilonRI ligation even though proximal signaling, degranulation, and leukotriene secretion are normal. Thus, Bcl10 and Malt1 are essential positive mediators of FcepsilonRI-dependent mast cell activation that selectively uncouple NF-kappaB-induced proinflammatory cytokine production from degranulation and leukotriene synthesis.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Involvement of Rabphilin3 in endocytosis through interaction with Rabaptin5.

Rabphilin3 and rabaptin5 are downstream target molecules of the Rab3 and -5 subfamily small G proteins that are implicated in exocytosis and endocytosis, respectively. We examined here the physical and functional relationship between the Rab3-rabphilin3 and Rab5-rabaptin5 systems. Rabphilin3 interacted with rabaptin5 at the N-terminal region (amino acids 1-280), which GTP-Rab3A interacted with. The interaction of rabphilin3 with rabaptin5 was inhibited by guanosine 5'-(3-O-thio)triphosphate-Rab3A. Overexpression of the N-terminal fragment of rabphilin3 (amino acids 1-280) inhibited the receptor-mediated endocytosis of transferrin, and this inhibition was overcome by co-transfection with a dominant active mutant of Rab3A or rabaptin5 in PC12 and HeLa cells. These results suggest that rabphilin3, free of GTP-Rab3A, regulates endocytosis through interaction with rabaptin5 after rabphilin3 complexed with GTP-Rab3A regulates exocytosis.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Requirement of a macromolecular signaling complex for beta adrenergic receptor modulation of the KCNQ1-KCNE1 potassium channel.

Sympathetic nervous system (SNS) regulation of cardiac action potential duration (APD) is mediated by beta adrenergic receptor (betaAR) activation, which increases the slow outward potassium ion current (IKS). Mutations in two human I(KS) channel subunits, hKCNQ1 and hKCNE1, prolong APD and cause inherited cardiac arrhythmias known as LQTS (long QT syndrome). We show that betaAR modulation of I(KS) requires targeting of adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (PKA) and protein phosphatase 1 (PP1) to hKCNQ1 through the targeting protein yotiao. Yotiao binds to hKCNQ1 by a leucine zipper motif, which is disrupted by an LQTS mutation (hKCNQ1-G589D). Identification of the hKCNQ1 macromolecular complex provides a mechanism for SNS modulation of cardiac APD through IKS.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1998

Involvement of Src in the vitamin D signaling in human keratinocytes.

1,25-dihydroxyvitamin D3 (VD) is a modulator of growth and differentiation of many cell types, including keratinocytes. We have recently shown in cultured keratinocytes that VD induces tyrosine phosphorylation of proteins involved in signal transduction, such as Shc. In an attempt to identify VD-responsive tyrosine kinases, we studied the effects of VD on the activity of the nonreceptor tyrosine kinase Src. Although VD did not stimulate Src activity in keratinocytes cultured in standard media containing 0.15 mM calcium, preincubation of the cells with 1.8 mM Ca2+ caused a rapid activation of Src in response to VD (10(-8)-10(-7) M). Elevation of calcium concentration alone caused an increase in Src activity as well, but the peak of Src activity was delayed (60 min vs. 15 min) and approximately 2-fold lower in comparison with VD-treated cells. VD treatment also induced tyrosine dephosphorylation of Src and a formation of an Src-Shc-Grb2 complex. Taken together, these findings imply that Src is involved in VD signaling in keratinocytes.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Localization of the MP1-MAPK scaffold complex to endosomes is mediated by p14 and required for signal transduction.

Eukaryotic cells use the extracellular signal regulated kinase (ERK) cascade to connect cell-surface receptors to intracellular targets. Although various signals are routed through the ERK pathway, cells respond accordingly to a given stimulus. To regulate proper signal transduction, scaffolds and adaptors are employed to organize specific signaling units. The scaffold protein MP1 (MEK1 partner) assembles a scaffold complex in the ERK cascade. We show that p14 functions as an adaptor protein, which is required and sufficient to localize MP1 to endosomes. Reduction of MP1 or p14 protein levels by siRNAi results in defective signal transduction. Therefore, our results suggest that the endosomal localization of the p14/MP1-MAPK scaffold complex is crucial for signal transduction.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Hypoxia-induced energy stress regulates mRNA translation and cell growth.

Oxygen (O2) deprivation, or hypoxia, has profound effects on cell metabolism and growth. Cells can adapt to low O2 in part through activation of hypoxia-inducible factor (HIF). We report here that hypoxia inhibits mRNA translation by suppressing multiple key regulators, including eIF2alpha, eEF2, and the mammalian target of rapamycin (mTOR) effectors 4EBP1, p70S6K, and rpS6, independent of HIF. Hypoxia results in energy starvation and activation of the AMPK/TSC2/Rheb/mTOR pathway. Hypoxic AMP-activated protein kinase (AMPK) activation also leads to eEF2 inhibition. Moreover, hypoxic effects on cellular bioenergetics and mTOR inhibition increase over time. Mutation of the TSC2 tumor suppressor gene confers a growth advantage to cells by repressing hypoxic mTOR inhibition and hypoxia-induced G1 arrest. Together, eIF2alpha, eEF2, and mTOR inhibition represent important HIF-independent mechanisms of energy conservation that promote survival under low O2 conditions.

AMP-Activated Protein Kinases↗

PubMed2001

UV light-induced degradation of RNA polymerase II is dependent on the Cockayne's syndrome A and B proteins but not p53 or MLH1.

It has been hypothesized that the degradation of the largest subunit of RNA polymerase II (polIILS) is required for transcription-coupled repair (TCR) of UV light-induced transcription-blocking lesions. In this study we further investigated the mechanism of UV-induced degradation of polIILS using cell lines with specific defects in TCR or in the recovery of RNA synthesis. It was found that the hypophosphorylated IIa form of polIILS rapidly decreased following UV-irradiation in all cell lines tested. Inhibition of proteasome activity resulted in an increase of the hyperphosphorylated IIo form of polIILS in UV-irradiated cells, while inhibition of CTD-kinases resulted in the retention of the IIa form. In UV-irradiated Cockayne's syndrome cells, which are defective in TCR, the levels of the IIo form increased in a similar manner as when proteasome inhibitors were added to UV-irradiated normal cells. In contrast, TCR-deficient HCT116 cells, which lack the mismatch repair protein MLH1, showed proficient degradation of polIILS as did cells with deficiencies in the recovery of RNA synthesis following UV-irradiation due to defective p53. Furthermore, we found that proteasome function was important for the recovery of mRNA synthesis even in TCR-deficient HCT116 cells. Our results suggest that proteasome-mediated degradation of polIILS is preceded by phosphorylation of the C-terminal domain of polIILS and requires the CS-A and CS-B but not MLH1 or p53 proteins. Furthermore, our results suggest that following UV-irradiation, the degradation of polIILS is required for the efficient recovery of mRNA synthesis but not for TCR per se.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Signaling from the IL-2 receptor to the nucleus.

Interleukin-2 has pleiotropic actions on the immune system and plays a vital role in the modulation of immune responses. Our current understanding of IL-2 signaling has resulted from in vitro studies that have identified the signaling pathways activated by IL-2, including the Jak-STAT pathways, and from in vivo studies that have analyzed mice in which IL-2, each chain of the receptor, as well a number of signaling molecules have been individually targeted by homologous recombination. Moreover, mutations in IL-2Ralpha, gamma(c) and Jak3 have been found in patients with severe combined immunodeficiency. In addition, with the discovery that two components of the receptor, IL-2Rbeta and gamma(c), are shared by other cytokine receptors, we have an enhanced appreciation of the contributions of these molecules towards cytokine specificity, pleiotropy and redundancy.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Antioxidants enhance mammalian proteasome expression through the Keap1-Nrf2 signaling pathway.

Proteasomes degrade damaged proteins formed during oxidative stress, thereby promoting cell survival. Neurodegenerative and other age-related disorders are associated with reduced proteasome activity. We show herein that expression of most subunits of 20S and 19S proteasomes, which collectively assemble the 26S proteasome, was enhanced up to threefold in livers of mice following treatment with dithiolethiones, which act as indirect antioxidants. Subunit protein levels and proteasome activity were coordinately increased. No induction was seen in mice where the transcription factor Nrf2 was disrupted. Promoter activity of the PSMB5 subunit of the 20S proteasome increased with either Nrf2 overexpression or treatment with antioxidants in mouse embryonic fibroblasts. Tandem antioxidant response elements in the proximal promoter of PSMB5 that controlled these responses were identified. We propose that induction of the 26S proteasome through the Nrf2 pathway represents an important indirect action of these antioxidants that can contribute to their protective effects against chronic diseases.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Activation of the tumour suppressor kinase LKB1 by the STE20-like pseudokinase STRAD.

The LKB1 gene encodes a serine/threonine kinase mutated in Peutz-Jeghers cancer syndrome. Despite several proposed models for LKB1 function in development and in tumour suppression, the detailed molecular action of LKB1 remains undefined. Here, we report the identification and characterization of an LKB1-specific adaptor protein and substrate, STRAD (STe20 Related ADaptor). STRAD consists of a STE20- like kinase domain, but lacks several residues that are indispensable for intrinsic catalytic activity. Endogenous LKB1 and STRAD form a complex in which STRAD activates LKB1, resulting in phosphorylation of both partners. STRAD determines the subcellular localization of wild-type, but not mutant LKB1, translocating it from nucleus to cytoplasm. One LKB1 mutation previously identified in a Peutz-Jeghers family that does not compromise its kinase activity is shown here to interfere with LKB1 binding to STRAD, and hence with STRAD-dependent regulation. Removal of endogenous STRAD by siRNA abrogates the LKB1-induced G(1) arrest. Our results imply that STRAD plays a key role in regulating the tumour suppressor activities of LKB1.

AMP-Activated Protein Kinase Kinases↗

PubMed2004

Pdlim2, a novel PDZ-LIM domain protein, interacts with alpha-actinins and filamin A.

PURPOSE: To characterize properties of Pdlim2, a novel PDZ and LIM domain-containing protein. METHODS: cDNA encoding Pdlim2 was identified in a cDNA library of transcripts expressed in the tissues of the rat eye irido-corneal angle. The expression pattern of the Pdlim2 gene was studied by Northern blot analysis and in situ hybridization. Proteins interacting with Pdlim2 were identified by pull-down assay and mass spectrometry. Intracellular localization of Pdlim2 was investigated by confocal microscopy. RESULTS: Rat Pdlim2 protein belongs to the ALP subfamily of proteins containing the PDZ domain in the N-terminal portion and the LIM domain in the C-terminal portion of the protein. The Pdlim2 gene was specifically expressed in the corneal epithelial cells, but not in the corneal stroma and endothelium nor in other ocular tissues. Pdlim2 was also expressed in the lung. In rat corneal and lung extracts, alpha-actinin-1, alpha-actinin-4, filamin A, and myosin heavy polypeptide 9 were co-immunoprecipitated with Pdlim2. Myosin VI was co-immunoprecipitated with Pdlim2 from corneal but not lung extracts. alpha-Actinins were the most abundant among immunoprecipitated proteins. Direct interaction of Pdlim2 with alpha-actinins and filamin was confirmed using pull-down assays and gel overlay assay with purified proteins. Pdlim2 and alpha-actinins were co-localized mainly to stress fibers after transfection into COS-7 cells. In transfected COS-7 cells, complexes of Pdlim2 and alpha-actinin-1 were preferentially located along the basal aspect. CONCLUSIONS: These results suggest that Pdlim2, like other ALP subfamily members, may act as an adapter that directs other proteins to the cytoskeleton.

Actinin↗

PubMed2005

Mip1, an MEKK2-interacting protein, controls MEKK2 dimerization and activation.

Mitogen-activated protein kinase (MAPK) cascades are central components of the intracellular signaling networks used by eukaryotic cells to respond to a wide spectrum of extracellular stimuli. An MAPK is activated by an MAPK kinase, which in turn is activated by an MAPK kinase kinase (MAP3K). However, little is known about the molecular aspects of the regulation and activation of large numbers of MAP3Ks that are crucial in relaying upstream receptor-mediated signals through the MAPK cascades to induce various physiological responses. In this study, we identified a novel MEKK2-interacting protein, Mip1, that regulates MEKK2 dimerization and activation by forming a complex with inactive and nonphosphorylated MEKK2. In particular, Mip1 prevented MEKK2 activation by blocking MEKK2 dimer formation, which in turn blocked JNKK2, c-Jun N-terminal kinase 1 (JNK1), extracellular signal-regulated kinase 5, and AP-1 reporter gene activation by MEKK2. Furthermore, we found that the endogenous Mip1-MEKK2 complex was dissociated transiently following epidermal growth factor stimulation. In contrast, the knockdown of Mip1 expression by siRNA augmented the MEKK2-mediated JNK and AP-1 reporter activation. Together, our data suggest a novel model for MEKK2 regulation and activation.

Adaptor Proteins, Signal Transducing↗

PubMed2002

The Hermansky-Pudlak syndrome 1 (HPS1) and HPS2 genes independently contribute to the production and function of platelet dense granules, melanosomes, and lysosomes.

Hermansky-Pudlak syndrome (HPS) is an inherited hemorrhagic disease affecting the related subcellular organelles platelet dense granules, lysosomes, and melanosomes. The mouse genes for HPS, pale ear and pearl, orthologous to the human HPS1 and HPS2 (ADTB3A) genes, encode a novel protein of unknown function and the beta(3)A subunit of the AP-3 adaptor complex, respectively. To test for in vivo interactions between these genes in the production and function of intracellular organelles, mice doubly homozygous for the 2 mutant genes were produced by appropriate breeding. Cooperation between the 2 genes in melanosome production was evident in increased hypopigmentation of the coat together with dramatic quantitative and qualitative alterations of melanosomes of the retinal pigment epithelium and choroid of double mutant mice. Lysosomal and platelet dense granule abnormalities, including hyposecretion of lysosomal enzymes from kidneys and depression of serotonin concentrations of platelet dense granules were likewise more severe in double than single mutants. Also, lysosomal enzyme concentrations were significantly increased in lungs of double mutant mice. Interaction between the 2 genes was specific in that effects on organelles were confined to melanosomes, lysosomes, and platelet dense granules. Together, the evidence indicates these 2 HPS genes function largely independently at the whole organism level to affect the production and function of all 3 organelles. Further, the increased lysosomal enzyme levels in lung of double mutant mice suggest a cause of a major clinical problem of HPS, lung fibrosis. Finally, doubly mutant HPS mice are a useful laboratory model for analysis of severe HPS phenotypes.

Adaptor Protein Complex 3↗

PubMed2005

TAB2, TRAF6 and TAK1 are involved in NF-kappaB activation induced by the TNF-receptor, Edar and its adaptator Edaradd.

Activation of the NF-kappaB pathway by the TNF-receptor Edar (Ectodysplasin receptor) and its downstream adaptator Edaradd (Edar-associated death domain) is essential for the development of hair follicles, teeth, exocrine glands and other ectodermal derivatives. Dysfunction of Edar signalling causes hypohidrotic/anhidrotic ectodermal dysplasia (ED), a disorder characterized by sparse hair, lack of sweat glands and malformation of teeth. The Edar signalling pathway stimulates NF-kappaB transcription factors via an activation of the IkappaB kinase (IKK) complex. To gain further insight into the mechanism of IKK activation by Edar and Edaradd, we performed a yeast two-hybrid screen and isolated TAB2 (TAK1-binding protein 2) as a binding partner of Edaradd. TAB2 is an adaptator protein that brigdes TRAF6 (TNF-receptor-associated factor 6) to TAK1 (TGFbeta-activated kinase 1), allowing TAK1 activation and subsequent IKK activation. Here, we show that endogenous and overexpressed TAB2, TRAF6 and TAK1 co-immunoprecipitated with Edaradd in 293 cells. Moreover, we show that dominant negative forms of TAB2, TRAF6 and TAK1 blocked the NF-kappaB activation induced by Edaradd. These results support the involvement of the TAB2/TRAF6/TAK1 signalling complex in the Edar signal transduction pathway and have important implications for our understanding of NF-kappaB activation and EDs in human.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Inactivation of the tuberous sclerosis complex-1 and -2 gene products occurs by phosphoinositide 3-kinase/Akt-dependent and -independent phosphorylation of tuberin.

The tuberous sclerosis complex (TSC) is a genetic disorder that is caused through mutations in either one of the two tumor suppressor genes, TSC1 and TSC2, that encode hamartin and tuberin, respectively. Interaction of hamartin with tuberin forms a heterodimer that inhibits signaling by the mammalian target of rapamycin to its downstream targets: eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). During mitogenic sufficiency, the phosphoinositide 3-kinase (PI3K)/Akt pathway phosphorylates tuberin on Ser-939 and Thr-1462 that inhibits the tumor suppressor function of the TSC complex. Here we show that tuberin-hamartin heterodimers block protein kinase C (PKC)/MAPK- and phosphatidic acid-mediated signaling toward mammalian target of rapamycin-dependent targets. We also show that two TSC2 mutants derived from TSC patients are defective in repressing phorbol 12-myristate 13-acetate-induced 4E-BP1 phosphorylation. PKC/MAPK signaling leads to phosphorylation of tuberin at sites that overlap with and are distinct from Akt phosphorylation sites. Phosphorylation of tuberin by phorbol 12-myristate 13-acetate was reduced by treatment of cells with either bisindolylmaleimide I or UO126, inhibitors of PKC and MAPK/MEK (MAPK/ERK kinase), respectively, but not by wortmannin (an inhibitor of PI3K). This work reveals that both PI3K-independent and -dependent mechanisms modulate tuberin phosphorylation in vivo.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Ecabet sodium inhibits Helicobacter pylori lipopolysaccharide-induced activation of NADPH oxidase 1 or apoptosis of guinea pig gastric mucosal cells.

Helicobacter pylori LPS activates a homolog of gp91(phox), NADPH oxidase 1 (Nox1), in guinea pig gastric mucosal cells cultured in 10% FBS-containing medium. RT-PCR and Northern hybridization demonstrated that H. pylori LPS stimulated expression of Nox1 and a novel p47(phox) homolog (Noxo1) mRNAs with a peak at 4 h, followed by upregulation of superoxide anion (O2-) generation. Pretreatment with 10 mg/ml of a nonabsorbable antigastric ulcer drug, ecabet sodium (ecabet), completely blocked these two mRNA expressions and the upregulation of O2- production. Under low (0.1%)-FBS conditions, H. pylori LPS predominantly caused apoptosis of the cells. Ecabet completely blocked the LPS-triggered phosphorylation of transforming growth factor-beta-activated kinase 1 (TAK1) and TAK1-binding protein 1, activation of caspase 8, loss of mitochondrial membrane potential, release of cytochrome c, activation of caspase 3, and appearance of apoptotic cells. In contrast, ecabet had no effect on ethanol- or etoposide-initiated apoptosis. The ecabet-pretreated cells exhibited the responsiveness to H. pylori LPS, similarly as untreated control cells did, when ecabet was removed by washing before the addition of H. pylori LPS. Incubation of H. pylori LPS with ecabet eliminated the toxic effects of the LPS, and nondenatured polyacrylamide gel electrophoresis indicated the formation of higher molecular mass complexes between H. pylori LPS and ecabet, suggesting that ecabet may interact with H. pylori LPS and block the activation of Toll-like receptor 4 (TLR4). Our results suggest that ecabet may suppress TLR4-mediated inflammation or accelerated apoptosis caused H. pylori infection.

Abietanes↗