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PubMed1998

Effect of nutritional state on the formation of a complex involving insulin receptor IRS-1, the 52 kDa Src homology/collagen protein (Shc) isoform and phosphatidylinositol 3'-kinase activity.

The Src homology and collagen protein (Shc) is tyrosine phosphorylated in response to insulin; however, evidence for its interaction with insulin receptor (IR) in normal tissues is missing. Interactions between IR, Shc and regulatory subunits of the phosphatidylinositol 3'-kinase (PI 3'-kinase) were characterized in the present study in liver and muscles of chickens submitted to various nutritional states. A chicken liver Shc cDNA fragment encoding a 198 amino acid long fragment, including the phosphotyrosine binding domain was sequenced. It shows 89% homology with the corresponding human homologue. The amounts of the three Shc isoforms (66, 52 and 46 kDa) and Shc messenger were not altered by the nutritional state. Shc tyrosine phosphorylation was decreased by fasting in both liver and muscle. Importantly, Shc was immunoprecipitated by IR antibody (mostly the 52 kDa isoform) or by alphaIRS-1(mostly the 46 kDa isoform). IR-Shc association was decreased by fasting and restored by refeeding. In liver, alphaShc immunoprecipitated the three forms of regulatory subunits of PI 3'-kinase and a PI 3'-kinase activity which was decreased by fasting. In muscle, alphaShc immunoprecipitated only the p85 isoform; the associated PI 3'-kinase activity was not altered by the nutritional state. Conversely, in both tissues anti-p85 antibody precipitated only the 52 kDa Shc isoform. In liver, antibodies to insulin receptor substrate-1 (alphaIRS-1), Shc or IR immunoprecipitated the three regulatory subunits of PI 3'-kinase and an equal PI 3'-kinase activity, without any residual activity left in the supernatants, suggesting the presence of a large complex involving IR, IRS-1, Shc (mainly the 52 kDa isoform) and PI 3'-kinase activity. The presence of another complex containing IRS-1 and the 46 kDa Shc isoform, but no PI 3'-kinase activity, is suggested.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Biphasic activation of p21ras by endothelin-1 sequentially activates the ERK cascade and phosphatidylinositol 3-kinase.

Endothelin-1 (ET-1) induces cell proliferation and differentiation through multiple G-protein-linked signaling systems, including p21ras activation. Whereas p21ras activation and desensitization by receptor tyrosine kinases have been extensively investigated, the kinetics of p21ras activation induced by engagement of G-protein-coupled receptors remains to be fully elucidated. In the present study we show that ET-1 induces a biphasic activation of p21ras in rat glomerular mesangial cells. The first peak of activation of p21ras, at 2-5 min, is mediated by immediate association of phosphorylated Shc with the guanosine exchange factor Sos1 via the adaptor protein Grb2. This initial activation of p21ras results in activation of the extracellular signal-regulated kinase (ERK) cascade. We demonstrate that ET-1 signaling elicits a negative feedback mechanism, modulating p21ras activity through ERK-dependent Sos1 phosphorylation, findings which were confirmed using an adenovirus MEK construct. Subsequent to p21ras and ERK deactivation, Sos1 reverts to the non-phosphorylated condition, enabling it to bind again to the Grb2/Shc complex, which is stabilized by persistent Shc phosphorylation. However, the resulting secondary activation of p21ras at 30 min does not lead to ERK activation, correlating with intensive, ET-1-induced expression of MAP kinase phosphatase-1, but does result in increased p21ras-associated phosphatidylinositol 3-kinase activity. Our data provide evidence that ET-1-induced biphasic p21ras activation causes sequential stimulation of divergent downstream signaling pathways.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Overexpression of CIN85 suppresses the growth of herpes simplex virus in HeLa cells.

The adaptor protein CIN85 is widely distributed in different tissues and has three Src homology 3 (SH3) domains, a proline-rich region (PRR), and a coiled-coil domain. During studies on the function of CIN85, it was reported to form a complex with herpes simplex virus 1 (HSV-1) infected cell protein 0 (ICP0), which plays a key role in enabling viral replication. Here, we demonstrate that plaque formation by HSV-1 is reduced on HeLa cells expressing CIN85 ectopically. The PRR of CIN85 was found to be essential for the inhibition of virus growth, whereas the three SH3 domains were not required. CIN85 also suppressed HSV-1 growth in Chinese hamster ovary (CHO) cells expressing the receptor for herpes simplex virus entry (herpes virus entry mediator A; HVEM). However, immunoprecipitation experiments showed that CIN85 did not interact with HVEM directly, indicating that CIN85 is not involved in the HSV-1 cell-entry pathway, but rather in another downstream pathway. Collectively, our data indicate that CIN85 might play an important role in HSV-1 infection.

Adaptor Proteins, Signal Transducing↗
PubMed2000

G protein-coupled receptors regulate Na+,K+-ATPase activity and endocytosis by modulating the recruitment of adaptor protein 2 and clathrin.

Inhibition of Na(+),K(+)-ATPase (NKA) activity in renal epithelial cells by activation of G protein-coupled receptors is mediated by phosphorylation of the catalytic alpha-subunit followed by endocytosis of active molecules. We examined whether agonists that counteract this effect do so by dephosphorylation of the alpha-subunit or by preventing its internalization through a direct interaction with the endocytic network. Oxymetazoline counteracted the action of dopamine on NKA activity, and this effect was achieved not by preventing alpha-subunit phosphorylation, but by impaired endocytosis of alpha-subunits into clathrin vesicles and early and late endosomes. Dopamine-induced inhibition of NKA activity and alpha-subunit endocytosis required the interaction of adaptor protein 2 (AP-2) with the catalytic alpha-subunit. Phosphorylation of the alpha-subunit is essential because dopamine failed to promote such interaction in cells lacking the protein kinase C phosphorylation residue (S18A). Confocal microscopy confirmed that oxymetazoline prevents incorporation of NKA molecules into clathrin vesicles by inhibiting the ability of dopamine to recruit clathrin to the plasma membrane. Dopamine decreased the basal levels of inositol hexakisphosphate (InsP(6)), whereas oxymetazoline prevented this effect. Similar increments (above basal) in the concentration of InsP(6) induced by oxymetazoline prevented AP-2 binding to the NKA alpha-subunit in response to dopamine. In conclusion, inhibition of NKA activity can be reversed by preventing its endocytosis without altering the state of alpha-subunit phosphorylation; increased InsP(6) in response to G protein-coupled receptor signals blocks the recruitment of AP-2 and thereby clathrin-dependent endocytosis of NKA.

Adaptor Protein Complex alpha Subunits↗
PubMed2003

The adaptor complex 2 directly interacts with the alpha 1b-adrenergic receptor and plays a role in receptor endocytosis.

Using the yeast two-hybrid system, we identified the mu 2 subunit of the clathrin adaptor complex 2 as a protein interacting with the C-tail of the alpha 1b-adrenergic receptor (AR). Direct association between the alpha 1b-AR and mu 2 was demonstrated using a solid phase overlay assay. The alpha 1b-AR/mu 2 interaction occurred inside the cells, as shown by the finding that the transfected alpha 1b-AR and the endogenous mu 2 could be coimmunoprecipitated from HEK-293 cell extracts. Mutational analysis of the alpha 1b-AR revealed that the binding site for mu 2 does not involve canonical YXX Phi or dileucine motifs but a stretch of eight arginines on the receptor C-tail. The binding domain of mu 2 for the receptor C-tail involves both its N terminus and the subdomain B of its C-terminal portion. The alpha 1b-AR specifically interacted with mu 2, but not with the mu 1, mu 3, or mu 4 subunits belonging to other AP complexes. The deletion of the mu 2 binding site in the C-tail markedly decreased agonist-induced receptor internalization as demonstrated by confocal microscopy as well as by the results of a surface receptor biotinylation assay. The direct association of the adaptor complex 2 with a G protein-coupled receptor has not been reported so far and might represent a common mechanism underlying clathrin-mediated receptor endocytosis.

Adaptor Protein Complex 2↗
PubMed1999

The Bcl-3 oncoprotein acts as a bridging factor between NF-kappaB/Rel and nuclear co-regulators.

The proto-oncoprotein Bcl-3 is a member of the IkappaB family and is present predominantly in the nucleus. To gain insight into specific nuclear functions of Bcl-3 we have isolated proteins that interact with its ankyrin repeat domain. Using the yeast two-hybrid-system we identified four novel binding partners of Bcl-3 in addition to NF-kappaB p50 and p52, previously known to associate with Bcl-3. The novel Bcl-3 interactors Jab1, Pirin, Tip60 and Bard1 are nuclear proteins which also bind to other transcription factors including c-Jun, nuclear factor I (NFI), HIV-1 Tat or the tumor suppressor and PolII holoenzyme component Brca1, respectively. Bcl-3, p50, and either Bard1, Tip60 or Pirin are sequestered into quarternary complexes on NF-kappaB DNA binding sites, whereas Jab1 enhances p50-Bcl-3-DNA complex formation. Furthermore, the histone acetylase Tip60 enhances Bcl-3-p50 activated transcription through an NF-kappaB binding site, indicating that quarternary complexes containing Bcl-3 interactors modulate NF-kappaB driven gene expression. These data implicate Bcl-3 as an adaptor between NF-kappaB p50/p52 and other transcription regulators and suggest that its gene activation function may at least in part be due to recruitment of the Tip60 histone actetylase.

Acetyltransferases↗
PubMed2005

Aberrant beta-catenin signaling in tuberous sclerosis.

The pathology associated with tuberous sclerosis complex (TSC) shows diverse phenotypes that suggest abnormal signaling of multiple pathways. Besides the negative regulatory role of the TSC1/TSC2 proteins on mTOR, we have reported an effect on beta-catenin signaling at the level of the degradation complex in vitro. The TSC1/TSC2 complex associates with GSK3 and Axin and promotes beta-catenin degradation to inhibit Wnt-stimulated TCF/LEF-dependent transcription. Here, we show that beta-catenin and its effectors, cyclin D1 and connexin 43, were up-regulated in TSC-related angiomyolipomas and lymphangioleiomyomatosis. This was supported by the failure of three disease-causing TSC2 missense mutants to inhibit Wnt signaling. Further, the interaction between TSC1/TSC2 and components of the beta-catenin degradation complex was dependent on Wnt stimulation such that binding of tuberin to GSK3 and Axin was reduced in the presence of Wnt whereas the tuberin-Dishevelled interaction was increased. GSK3 activity played a role in regulating the assembly/stability of the degradation complex. Inhibition of GSK3 by lithium chloride reduced its association with TSC1 whereas disruption of GSK3-phosphorylation sites in TSC1 reduced interaction between TSC2 and TSC1. Collectively, our data provide further evidence that beta-catenin signaling plays a role in TSC pathogenesis in vivo and suggest a novel role of GSK3 in modulating the TSC1/TSC2 complex through TSC1 phosphorylation.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Phosphoinositide 3-kinase regulates beta2-adrenergic receptor endocytosis by AP-2 recruitment to the receptor/beta-arrestin complex.

Internalization of beta-adrenergic receptors (betaARs) occurs by the sequential binding of beta-arrestin, the clathrin adaptor AP-2, and clathrin. D-3 phosphoinositides, generated by the action of phosphoinositide 3-kinase (PI3K) may regulate the endocytic process; however, the precise molecular mechanism is unknown. Here we demonstrate that betaARKinase1 directly interacts with the PIK domain of PI3K to form a cytosolic complex. Overexpression of the PIK domain displaces endogenous PI3K from betaARK1 and prevents betaARK1-mediated translocation of PI3K to activated beta2ARs. Furthermore, disruption of the betaARK1/PI3K interaction inhibits agonist-stimulated AP-2 adaptor protein recruitment to the beta2AR and receptor endocytosis without affecting the internalization of other clathrin dependent processes such as internalization of the transferrin receptor. In contrast, AP-2 recruitment is enhanced in the presence of D-3 phospholipids, and receptor internalization is blocked in presence of the specific phosphatidylinositol-3,4,5-trisphosphate lipid phosphatase PTEN. These findings provide a molecular mechanism for the agonist-dependent recruitment of PI3K to betaARs, and support a role for the localized generation of D-3 phosphoinositides in regulating the recruitment of the receptor/cargo to clathrin-coated pits.

Adaptor Protein Complex delta Subunits↗
PubMed2001

CD38 is associated with lipid rafts and upon receptor stimulation leads to Akt/protein kinase B and Erk activation in the absence of the CD3-zeta immune receptor tyrosine-based activation motifs.

T lymphocytes can be activated via the T cell receptor (TCR) or by triggering through a number of other cell surface structures, including the CD38 co-receptor molecule. Here, we show that in TCR+ T cells that express a CD3-zeta lacking the cytoplasmic domain, cross-linking with CD38- or CD3-specific monoclonal antibodies induces tyrosine phosphorylation of CD3-epsilon, zeta-associated protein-70, linker for activation of T cells, and Shc. Moreover, in these cells, anti-CD38 or anti-CD3 stimulation leads to protein kinase B/Akt and Erk activation, suggesting that the CD3-zeta-immunoreceptor tyrosine-based activation motifs are not required for CD38 signaling in T cells. Interestingly, in unstimulated T cells, lipid rafts are highly enriched in CD38, including the T cells lacking the cytoplasmic tail of CD3-zeta. Moreover, CD38 clustering by extensive cross-linking with an anti-CD38 monoclonal antibody and a secondary antibody leads to an increased resistance of CD38 to detergent solubilization, suggesting that CD38 is constitutively associated with membrane rafts. Consistent with this, cholesterol depletion with methyl-beta-cyclodextrin substantially reduces CD38-mediated Akt activation while enhancing CD38-mediated Erk activation. CD38/raft association may improve the signaling capabilities of CD38 via formation of protein/lipid domains to which signaling-competent molecules, such as immunoreceptor tyrosine-based activation motif-bearing CD3 molecules and protein-tyrosine kinases, are recruited.

ADP-ribosyl Cyclase↗
PubMed1999

A thrombopoietin receptor mutant deficient in Jak-STAT activation mediates proliferation but not differentiation in UT-7 cells.

Thrombopoietin (TPO) stimulates proliferation and differentiation of cells of the megakaryocytic lineage. It exerts its function by binding and activating c-mpl, a member of the hematopoietic receptor superfamily. Upon binding of TPO to its receptor, numerous signaling events are triggered. These include activation of the Jak-STAT (signal transducers and activators of transcription) pathway, mitogen-activated protein kinase (MAPK), Tec, and phospatidylinositol (PI) 3-kinase and phosphorylation of Shc and Vav. The contribution of different signaling pathways to the induction of specific cellular processes such as proliferation and differentiation is incompletely understood. We have previously described a mutant of c-mpl that fails to activate the Jak-STAT pathway but nevertheless retains its ability to mediate proliferation and activation of most signaling events in the murine hematopoietic precursor cell lines BAF/3 and 32D. We confirm here the ability of this mutant to mediate proliferation in the absence of Jak-STAT activation in the human cell line UT-7 and further show that this mutant fails to mediate TPO-induced megakaryocytic differentiation. Comparison of the signaling capacity of this mutant in UT-7 and BAF/3 cells shows considerable cell-type-specific differences. Whereas in BAF/3 cells the mutant still mediates activation of Shc, MAPK, Vav, and PI 3-kinase at levels comparable to the wild-type receptor, these events are strongly diminished in UT-7 cells expressing the mutant. Furthermore, we show that the C-terminal 25 amino acid residues of the receptor mutant are crucial for the mitogenic response in UT-7 cells.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Platelet-derived growth factor receptor-mediated signal transduction from endosomes.

Although accumulated evidence supports the concept of endosomal signaling of receptor tyrosine kinases, most results are generated from studies of epidermal growth factor receptor (EGFR). It is not clear whether the concept of endosomal signaling could be generally applied to the other receptor tyrosine kinases. For example, platelet-derived growth factor receptor (PDGFR) is very similar to EGFR in terms of both signaling and trafficking; however, little is known about the endosomal signaling of PDGFR. In this research, we applied the same approaches from our recent studies regarding EGFR endosomal signaling to investigate the endosomal signaling of PDGFR. We showed in this communication that we are able to establish a system that allows the specific activation of endosome-associated PDGFR without the activation of the plasma membrane-associated PDGFR and without disrupting the overall endocytosis pathway. By using this system, we showed that endosomal activation of PDGFR recruits various signaling proteins including Grb2, SHC, phospholipase C-gamma1, and the p85alpha subunit of phosphatidylinositol 3-kinase into endosomes and forms signaling complexes with PDGFR. We also showed that endosomal PDGFR signaling is sufficient to activate the major signaling pathways implicated in cell proliferation and survival. Moreover, we demonstrate that endosomal PDGFR signaling is sufficient to generate physiological output including cell proliferation and cell survival.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Phosphotyrosine signaling networks in epidermal growth factor receptor overexpressing squamous carcinoma cells.

Overexpression and enhanced activation of the epidermal growth factor (EGF) receptor are frequent events in human cancers that correlate with poor prognosis. Anti-phosphotyrosine and anti-EGFr affinity chromatography, isotope-coded muLC-MS/MS, and immunoblot methods were combined to describe and measure signaling networks associated with EGF receptor activation and pharmacological inhibition. The squamous carcinoma cell line HN5, which overexpresses EGF receptor and displays sustained receptor kinase activation, was used as a model system, where pharmacological inhibition of EGF receptor kinase by erlotinib markedly reduced auto and substrate phosphorylation, Src family phosphorylation at EGFR Y845, while increasing total EGF receptor protein. Diverse sets of known and poorly described functional protein classes were unequivocally identified by affinity selection, comprising either proteins tyrosine phosphorylated or complexed therewith, predominantly through EGF receptor and Src family kinases, principally 1) immediate EGF receptor signaling complexes (18%); 2) complexes involved in adhesion and cell-cell contacts (34%); and 3) receptor internalization and degradation signals. Novel and known phosphorylation sites could be located despite the complexity of the peptide mixtures. In addition to interactions with multiple signaling adaptors Grb2, SHC, SCK, and NSP2, EGF receptors in HN5 cells were shown to form direct or indirect physical interactions with additional kinases including ACK1, focal adhesion kinase (FAK), Pyk2, Yes, EphA2, and EphB4. Pharmacological inhibition of EGF receptor kinase activity by erlotinib resulted in reduced phosphorylation of downstream signaling, for example through Cbl/Cbl-B, phospholipase Cgamma (PLCgamma), Erk1/2, PI-3 kinase, and STAT3/5. Focal adhesion proteins, FAK, Pyk2, paxillin, ARF/GIT1, and plakophillin were down-regulated by transient EGF stimulation suggesting a complex balance between growth factor induced kinase and phosphatase activities in the control of cell adhesion complexes. The functional interactions between IGF-1 receptor, lysophosphatidic acid (LPA) signaling, and EGF receptor were observed, both direct and/or indirectly on phospho-Akt, phospho-Erk1/2, and phospho-ribosomal S6.

Carcinoma, Squamous Cell↗
PubMed2004

Identification of a novel 14-3-3zeta binding site within the cytoplasmic tail of platelet glycoprotein Ibalpha.

The glycoprotein Ib-V-IX (GPIb-V-IX) complex interacts with subendothelial von Willebrand factor (VWF) to ensure recruitment of platelets at sites of vascular injury, a process that culminates in integrin alpha(IIb)beta(3)-dependent stable adhesion and spreading. Interaction of the 14-3-3zeta adaptor protein with the C-terminal 606-610 phosphoserine motif of the GPIbalpha subunit has been implicated in the control of alpha(IIb)beta(3) activation and cell spreading. In this study, we have examined potentially novel 14-3-3zeta binding sites by expressing mutant forms of GPIbalpha in Chinese-hamster-ovary (CHO) cells. Analysis of a series of neighboring 11-12 residue deletions identified a critical role for the 580-LVAGRRPSALS-590 sequence in promoting GPIbalpha-14-3-3zeta interaction. Development of a phosphospecific antibody demonstrated high levels of phosphorylation of the Ser587 and Ser590 residues in resting platelets (which became dephosphorylated during platelet spreading on VWF), and peptides containing these phosphorylated residues effectively displaced 14-3-3zeta from GPIbalpha. Analysis of single and double alanine substitutions of Ser587 and Ser590 demonstrated a major role for these residues in promoting GPIbalpha-14-3-3zeta binding. Moreover, these cell lines exhibited a defect in cell spreading on immobilized VWF. These studies demonstrate the existence of a second major 14-3-3zeta binding site within the cytoplasmic tail of GPIbalpha that has an important functional role in regulating integrin-dependent cell spreading.

14-3-3 Proteins↗
PubMed1999

SHPS-1 is a scaffold for assembling distinct adhesion-regulated multi-protein complexes in macrophages.

Inhibitory immunoreceptors downregulate signaling by recruiting Src homology 2 (SH2) domain-containing tyrosine and/or lipid phosphatases to activating receptor complexes [1]. There are indications that some inhibitory receptors might also perform other functions [2] [3]. In adherent macrophages, two inhibitory receptors, SHPS-1 and PIR-B, are the major proteins binding to the tyrosine phosphatase SHP-1. SHPS-1 also associates with two tyrosine-phosphorylated proteins (pp55 and pp130) and a protein tyrosine kinase [4]. Here, we have identified pp55 and pp130 as the adaptor molecules SKAP55hom/R (Src-kinase-associated protein of 55 kDa homologue) and FYB/SLAP-130 (Fyn-binding protein/SLP-76-associated protein of 130 kDa), respectively, and the tyrosine kinase activity as PYK2. Two distinct SHPS-1 complexes were formed, one containing SKAP55hom/R and FYB/SLAP-130, and the other containing PYK2. Recruitment of FYB/SLAP-130 to SHPS-1 required SKAP55hom/R, whereas PYK2 associated with SHPS-1 independently. Formation of both complexes was independent of SHP-1 and tyrosine phosphorylation of SHPS-1. Finally, tyrosine phosphorylation of members of the SHPS-1 complexes was regulated by integrin-mediated adhesion. Thus, SHPS-1 provides a scaffold for the assembly of multi-protein complexes that might both transmit adhesion-regulated signals and help terminate such signals through SHP-1-directed dephosphorylation. Other inhibitory immunoreceptors might have similar scaffold-like functions.

Animals↗
PubMed1994

Signal transduction by the PDGF receptors.

The three isoforms of PDGF bind with different affinities to two related tyrosine kinase receptors, denoted the PDGF alpha- and beta-receptors. Ligand binding induces receptor dimerization, creating receptor homo- or heterodimers. Dimerization is accompanied by, and might be a prerequisite for, receptor autophosphorylation and kinase activation. Receptor autophosphorylation serves to regulate the kinase activity and to create binding sites on the receptor molecule for downstream signalling components. The activities of the signalling components are ultimately manifested as specific biological responses. All the currently described PDGF receptor-binding components, e.g. phospholipase C-gamma, members of the src family of cytoplasmic tyrosine kinases, the rasGT-Pase activating protein and p85, the regulatory subunit of phosphatidylinositol 3' kinase, contain a conserved src homology 2-domain, through which the association with the receptor takes place. The receptor-binding components appear to either possess an intrinsic enzymatic activity, or they function as adaptors, which may complex with catalytically active components. For most receptor-binding components, there is insufficient understanding of how binding to the receptor affects the catalytic function. Certain of these components become tyrosine-phosphorylated, i.e. they are substrates for the receptor tyrosine kinase. Moreover, the change in subcellular localization, which most of the receptor binding components undergo in conjunction with receptor binding, could play a critical role. The current efforts of many laboratories are aimed at delineating different PDGF receptor signal transduction pathways and what roles the different receptor-binding components play in the establishment of these pathways.

Amino Acid Sequence↗
PubMed2000

Human Upf proteins target an mRNA for nonsense-mediated decay when bound downstream of a termination codon.

Nonsense-mediated decay (NMD) rids eukaryotic cells of aberrant mRNAs containing premature termination codons. These are discriminated from true termination codons by downstream cis-elements, such as exon-exon junctions. We describe three novel human proteins involved in NMD, hUpf2, hUpf3a, and hUpf3b. While in HeLa cell extracts these proteins are complexed with hUpf1, in intact cells hUpf3a and hUpf3b are nucleocytoplasmic shuttling proteins, hUpf2 is perinuclear, and hUpf1 cytoplasmic. hUpf3a and hUpf3b associate selectively with spliced beta-globin mRNA in vivo, and tethering of any hUpf protein to the 3'UTR of beta-globin mRNA elicits NMD. These data suggest that assembly of a dynamic hUpf complex initiates in the nucleus at mRNA exon-exon junctions and triggers NMD in the cytoplasm when recognized downstream of a translation termination site.

3' Untranslated Regions↗
PubMed2005

Role of charged and hydrophobic residues in the oligomerization of the PYRIN domain of ASC.

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an adaptor protein composed of two homophilic protein-protein interaction domains, a PYRIN domain (PYD) and a caspase recruitment domain. PYD-dependent oligomerization of ASC is thought to play a crucial role in formation of a molecular platform, the inflammasome, which activates caspase-1. When expressed in cells, the PYD of ASC was shown to form cytoplasmic filaments through self-association. Over 70 single point mutants were analyzed for filament formation in cells expressing the mutant proteins. The set of mutations comprised every single amino acid residue with a charged side chain (Arg, Lys, Asp, and Glu) and a large hydrophobic side chain (Ile, Leu, Met, Phe, Pro, and Val). Filament formation of the ASC PYD was prevented by mutation of Lys21, Leu25, Lys26, Pro40, Arg41, Asp48, and Asp51 of helices 2, 3, and 4. These data identify a coherent interaction surface, establishing a molecular model of PYD-PYD complexes with an important role for charge-charge interactions.

Amino Acid Sequence↗
PubMed2001

The sorting nexin, DSH3PX1, connects the axonal guidance receptor, Dscam, to the actin cytoskeleton.

Dock, an adaptor protein that functions in Drosophila axonal guidance, consists of three tandem Src homology 3 (SH3) domains preceding an SH2 domain. To develop a better understanding of axonal guidance at the molecular level, we used the SH2 domain of Dock to purify a protein complex from fly S2 cells. Five proteins were obtained in pure form from this protein complex. The largest protein in the complex was identified as Dscam (Down syndrome cell adhesion molecule), which was subsequently shown to play a key role in directing neurons of the fly embryo to correct positions within the nervous system (Schmucker, D., Clemens, J. C., Shu, H., Worby, C. A., Xiao, J., Muda, M., Dixon, J. E., and Zipursky, S. L. (2000) Cell 101, 671-684). The smallest protein in this complex (p63) has now been identified. We have named p63 DSH3PX1 because it appears to be the Drosophila orthologue of the human protein known as SH3PX1. DSH3PX1 is comprised of an NH(2)-terminal SH3 domain, an internal PHOX homology (PX) domain, and a carboxyl-terminal coiled-coil region. Because of its PX domain, DSH3PX1 is considered to be a member of a growing family of proteins known collectively as sorting nexins, some of which have been shown to be involved in vesicular trafficking. We demonstrate that DSH3PX1 immunoprecipitates with Dock and Dscam from S2 cell extracts. The domains responsible for the in vitro interaction between DSH3PX1 and Dock were also identified. We further show that DSH3PX1 interacts with the Drosophila orthologue of Wasp, a protein component of actin polymerization machinery, and that DSH3PX1 co-immunoprecipitates with AP-50, the clathrin-coat adapter protein. This evidence places DSH3PX1 in a complex linking cell surface receptors like Dscam to proteins involved in cytoskeletal rearrangements and/or receptor trafficking.

Actins↗