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PubMed2002

Identification of multiple binding partners for the amino-terminal domain of synapse-associated protein 97.

Multiprotein complexes mediate static and dynamic functions to establish and maintain cell polarity in both epithelial cells and neurons. Membrane-associated guanylate kinase (MAGUK) proteins are thought to be scaffolding molecules in these processes and bind multiple proteins via their obligate postsynaptic density (PSD)-95/Disc Large/Zona Occludens-1, Src homology 3, and guanylate kinase-like domains. Subsets of MAGUK proteins have additional protein-protein interaction domains. An additional domain we identified in SAP97 called the MAGUK recruitment (MRE) domain binds the LIN-2,7 amino-terminal (L27N) domain of mLIN-2/CASK, a MAGUK known to bind mLIN-7. Here we show that SAP97 binds two other mLIN-7 binding MAGUK proteins. One of these MAGUK proteins, DLG3, coimmunoprecipitates with SAP97 in lysates from rat brain and transfected Madin-Darby canine kidney cells. This interaction requires the MRE domain of SAP97 and surprisingly, both the L27N and L27 carboxyl-terminal (L27C) domains of DLG3. We also demonstrate that SAP97 can interact with the MAGUK protein, DLG2, but not the highly related protein, PALS2. The ability of SAP97 to interact with multiple MAGUK proteins is likely to be important for the targeting of specific protein complexes in polarized cells.

Adaptor Proteins, Signal Transducing↗
PubMed2005

SHIP2 interaction with the cytoskeletal protein Vinexin.

The src homology 2 (SH2) domain-containing inositol 5-phosphatase 2 (SHIP2) catalyses the dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] to phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2]. We report the identification of the cytoskeletal protein Vinexin as a protein interacting with SHIP2. This was achieved by yeast two-hybrid screening using the C-terminal region of SHIP2 as bait. Vinexin has previously been identified as a vinculin-binding protein that plays a key role in cell spreading and cytoskeletal organization. The interaction between SHIP2 and Vinexin was confirmed in lysates of both COS-7 cells and mouse embryonic fibroblasts (MEF). The C-terminus was involved in the interaction, as shown by the transfection of a truncated C-terminus mutant of SHIP2. In addition, we showed the colocalization between Vinexin alpha and SHIP2 at the periphery of transfected COS-7 cells. When added in vitro to SHIP2, Vinexin did not affect the PtdIns(3,4,5)P3 5-phosphatase activity of SHIP2. Enhanced cell adhesion to collagen-I-coated dishes was shown upon transfection of either SHIP2 or Vinexin to COS-7 cells. This effect was no longer observed with either a catalytic mutant or the C-terminus mutant of SHIP2. It also appears SHIP2 specific; this was not seen with SHIP1. Adhesion to the same matrix was decreased in SHIP2-/- MEF cells compared with MEF+/+ cells. Our data suggest that SHIP2 interaction with Vinexin promotes the localization of SHIP2 at the periphery of the cells leaving its catalytic site intact. The complex formation between Vinexin and SHIP2 may increase cellular adhesion. The data reinforce the concept that SHIP2 is active both as a PtdIns(3,4,5)P3 5-phosphatase and as a modulator of focal contact formation.

Amino Acid Sequence↗
PubMed2005

Molecular pathogenesis of mucosa-associated lymphoid tissue lymphoma.

Extranodal marginal zone B-cell lymphomas of mucosa-associated lymphoid tissue (MALT) type occur in a number of anatomic sites, but share overlapping morphologic and immunophenotypic features. Helicobacter pylori infection has been identified as an etiologic factor in gastric MALT lymphoma, and a growing list of other infectious organisms have recently been shown to be associated with MALT lymphomas at other anatomic sites. Although cause and effect has not been established for most of these infectious agents, our understanding of the biology has significantly improved, in part through the application of standard cytogenetic analyses. The common karyotypic alterations that characterize MALT lymphomas include the trisomies 3 and 18, the translocations t(11;18)(q21;q21), t(1;14)(p22;q32), t(14;18)(q32;q21), t(3;14)(q27;q32), and the recently described t(3;14)(p14.1;q32). This apparent complexity of cytogenetic alterations that have now been implicated in the pathogenesis of extranodal MALT lymphoma serves as a paradigm for molecular cross talk in neoplastic disease. Recent data have shown that at least three of the disparate translocations affect a common signaling mechanism, and thus unify all three under a common pathogenesis, resulting in the constitutive activation of the nuclear factor kappa B (NF-kappaB) pathway. It may be that the new MALT-related translocation involving the FOXP1 gene and other as yet undiscovered translocations may all have in common increased NF-kappaB signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2007

Kinetic analysis of GTP hydrolysis catalysed by the Arf1-GTP-ASAP1 complex.

Arf (ADP-ribosylation factor) GAPs (GTPase-activating proteins) are enzymes that catalyse the hydrolysis of GTP bound to the small GTP-binding protein Arf. They have also been proposed to function as Arf effectors and oncogenes. We have set out to characterize the kinetics of the GAP-induced GTP hydrolysis using a truncated form of ASAP1 [Arf GAP with SH3 (Src homology 3) domain, ankyrin repeats and PH (pleckstrin homology) domains 1] as a model. We found that ASAP1 used Arf1-GTP as a substrate with a k(cat) of 57+/-5 s(-1) and a K(m) of 2.2+/-0.5 microM determined by steady-state kinetics and a kcat of 56+/-7 s(-1) determined by single-turnover kinetics. Tetrafluoroaluminate (AlF4-), which stabilizes complexes of other Ras family members with their cognate GAPs, also stabilized a complex of Arf1-GDP with ASAP1. As anticipated, mutation of Arg-497 to a lysine residue affected kcat to a much greater extent than K(m). Changing Trp-479, Iso-490, Arg-505, Leu-511 or Asp-512 was predicted, based on previous studies, to affect affinity for Arf1-GTP. Instead, these mutations primarily affected the k(cat). Mutants that lacked activity in vitro similarly lacked activity in an in vivo assay of ASAP1 function, the inhibition of dorsal ruffle formation. Our results support the conclusion that the Arf GAP ASAP1 functions in binary complex with Arf1-GTP to induce a transition state towards GTP hydrolysis. The results have led us to speculate that Arf1-GTP-ASAP1 undergoes a significant conformational change when transitioning from the ground to catalytically active state. The ramifications for the putative effector function of ASAP1 are discussed.

ADP-Ribosylation Factor 1↗
PubMed2006

Toll-like receptor-dependent and -independent viperin gene expression and counter-regulation by PRDI-binding factor-1/BLIMP1.

Here we identify Viperin as a highly inducible gene in response to lipopolysaccharide (LPS), double-stranded RNA (poly(I-C)) or Sendai virus (SV). The only known function of Viperin relates to its ability to inhibit human Cytomegalovirus replication. Very little data are available on the regulation of this gene. In silico analysis of the promoter identified two interferon (IFN)-stimulated response elements (ISRE), which in other genes bind IRF3 or the IFN-stimulated gene factor-3 (ISGF3) complex. LPS and poly(I-C) induce very high levels of Viperin in wild type cells but not in cells deficient in TRIF, TBK1, IRF3, or the type I IFNalpha/betaR. SV-induced Viperin gene expression was mediated independently of Toll-like receptor (TLR) signaling by retinoic acid-inducible gene (RIG-I) and the downstream adapter, mitochondrial anti-viral signaling (MAVS). Virus-induced Viperin expression was not attenuated in macrophages deficient in either TBK1 or IKKepsilon alone. Moreover, IRF3-deficient, but not IFNalpha/betaR deficient, macrophages still induced Viperin in response to SV. Promoter reporter studies combined with DNA immunoprecipitation assays identified the ISGF3 complex as the key regulator of Viperin gene expression. Moreover, positive regulatory domain I-binding factor 1 (PRDI-BF1, also called BLIMP1) binds the ISRE sites and competes with ISGF3 binding in a virus inducible manner to inhibit Viperin transcription. Collectively, these studies identify Viperin as a tightly regulated ISGF3 target gene, which is counter-regulated by PRDI-BF1.

Adaptor Proteins, Vesicular Transport↗
PubMed2005

ERK phosphorylates p66shcA on Ser36 and subsequently regulates p27kip1 expression via the Akt-FOXO3a pathway: implication of p27kip1 in cell response to oxidative stress.

Mice deficient for p66shcA represent an animal model to link oxidative stress and aging. p66shcA is implicated in oxidative stress response and mitogenic signaling. Phosphorylation of p66shcA on Ser36 is critical for its function in oxidative stress response. Here we report the identification of ERK as the kinase phosphorylating p66shcA on Ser36. Activation of ERKs was necessary and sufficient for Ser36 phosphorylation. p66shcA interacted with ERK and was demonstrated to be a substrate for ERK, with Ser36 being the major phosphorylation site. Furthermore, in response to H2O2, inhibition of ERK activation repressed p66shcA-dependent phosphorylation of FOXO3a and the down-regulation of its target gene p27kip1. Down-regulation of p27 might promote cell survival, as p27 played a proapoptotic role in oxidative stress response. As a feedback regulation, Ser36 phosphorylated p66shcA attenuated H2O2-induced ERK activation, whereas p52/46shcA facilitated ERK activation, which required tyrosine phosphorylation of CH1 domain. p66shcA formed a complex with p52/46ShcA, which may provide a platform for efficient signal propagation. Taken together, the data suggest there exists an interplay between ERK and ShcA proteins, which modulates the expression of p27 and cell response to oxidative stress.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Regulation of the MST1 kinase by autophosphorylation, by the growth inhibitory proteins, RASSF1 and NORE1, and by Ras.

MST1 (mammalian Sterile20-like 1) and MST2 are closely related Class II GC (protein Ser/Thr) kinases that initiate apoptosis when transiently overexpressed in mammalian cells. In the present study, we show that recombinant MST1/2 undergo a robust autoactivation in vitro, mediated by an intramolecular autophosphorylation of a single site [MST1(Thr183)/MST2(Thr180)] on the activation loop of an MST dimer. Endogenous full-length MST1 is activated by a variety of stressful stimuli, accompanied by the secondary appearance of a 36 kDa Thr183-phosphorylated, caspase-cleaved catalytic fragment. Recombinant MST1 exhibits only 2-5% activation during transient expression; endogenous MST1 in the cycling HeLa or KB cells has a similar low fractional activation, but 2 h incubation with okadaic acid (1 mM) results in 100% activation. Endogenous MST1 immunoprecipitated from KB cells is specifically associated with substoichiometric amounts of the growth inhibitory polypeptides RASSF1A and NORE1A (novel Ras effector 1A; a Ras-GTP-binding protein). Co-expression of RASSF1A, RASSF1C, NORE1A and NORE1B with MST1 markedly suppresses MST1(Thr183) phosphorylation in vivo and abolishes the ability of MST1 to undergo Mg-ATP-mediated autoactivation in vitro; direct addition of purified NORE1A in vitro also inhibits MST1 activation. In contrast, co-transfection of MST1 with NORE1A modified by the addition of a C-terminal CAAX motif results in a substantial increase in MST1(Thr183) phosphorylation, as does fusion of a myristoylation motif directly on to the MST1 N-terminus. Moreover, MST1 polypeptides, bound via wild-type NORE1A to Ras(G12V) (where G12V stands for Gly12Val), exhibit higher Thr183 phosphorylation compared with MST1 bound to NORE1A alone. Nevertheless, serum stimulation of KB cells does not detectably increase the activation state of endogenous MST1 or MST2 despite promoting the recruitment of the endogenous NORE1-MST1 complex to endogenous Ras. We propose that the NORE1/RASSF1 polypeptides, in addition to their role in maintaining the low activity of MST1 in vivo, direct MST1 to sites of activation and perhaps co-localization with endogenous substrates.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Chitin synthase III: synthetic lethal mutants and "stress related" chitin synthesis that bypasses the CSD3/CHS6 localization pathway.

We screened Saccharomyces strains for mutants that are synthetically lethal with deletion of the major chitin synthase gene CHS3. In addition to finding, not surprisingly, that mutations in major cell wall-related genes such as FKS1 (glucan synthase) and mutations in any of the Golgi glycosylation complex genes (MNN9 family) are lethal in combination with chs3Delta, we found that a mutation in Srv2p, a bifunctional regulatory gene, is notably lethal in the chs3 deletion. In extending studies of fks1-chitin synthase 3 interactions, we made the surprising discovery that deletion of CSD3/CHS6, a gene normally required for Chs3p delivery and activity in vivo, was not lethal with fks1 and, in fact, that lack of Csd3p/Chs6p did not decrease the high level of stress-related chitin made in the fks1 mutant. This finding suggests that "stress response" chitin synthesis proceeds through an alternate Chs3p targeting pathway.

Adaptor Proteins, Signal Transducing↗
PubMed2001

CD2AP and p130Cas localize to different F-actin structures in podocytes.

Mice lacking the 80-kDa CD2-associated protein (CD2AP) develop progressive renal failure that starts soon after birth with proteinuria and foot process effacement by unknown mechanisms. CD2AP has been identified and cloned independently by virtue of its interaction with the T cell protein CD2 and with the docking protein p130Cas. In the present study we examined the localization of CD2AP and p130Cas in the mouse glomerulus and in cultured podocytes. In glomeruli, CD2AP and p130Cas immunofluorescence were observed in podocytes, where they colocalized with F-actin in foot processes. In addition, p130Cas was strongly expressed in mesangial cells. Immunoelectron microscopy demonstrated that CD2AP was present in podocyte foot processes without a prevailing localization. In cultured podocytes, p130Cas was enriched at sites of focal adhesions, where it colocalized like vinculin with F-actin at stress fiber ends. In contrast, CD2AP colocalized with F-actin at the leading edge of lamellipodia and in small spots, which were unevenly distributed in the cytoplasm. The spot-shaped F-actin structures were also stained by antibodies against the actin nucleation Arp2/3 complex and cortactin, both contributing to dynamic actin assembly. Moreover, CD2AP spots in cultured podocytes were in close spatial association with actinin-4, but not actinin-1. Our results suggest that CD2AP and p130Cas, which both colocalize with F-actin in podocytes in situ, possess different functions. Whereas p130Cas is found in focal adhesions, CD2AP seems to be involved in the regulation of highly dynamic F-actin structures in podocyte foot processes.

Actin-Related Protein 2↗
PubMed1999

Syndapin I, a synaptic dynamin-binding protein that associates with the neural Wiskott-Aldrich syndrome protein.

The GTPase dynamin has been clearly implicated in clathrin-mediated endocytosis of synaptic vesicle membranes at the presynaptic nerve terminal. Here we describe a novel 52-kDa protein in rat brain that binds the proline-rich C terminus of dynamin. Syndapin I (synaptic, dynamin-associated protein I) is highly enriched in brain where it exists in a high molecular weight complex. Syndapin I can be involved in multiple protein-protein interactions via a src homology 3 (SH3) domain at the C terminus and two predicted coiled-coil stretches. Coprecipitation studies and blot overlay analyses revealed that syndapin I binds the brain-specific proteins dynamin I, synaptojanin, and synapsin I via an SH3 domain-specific interaction. Coimmunoprecipitation of dynamin I with antibodies recognizing syndapin I and colocalization of syndapin I with dynamin I at vesicular structures in primary neurons indicate that syndapin I associates with dynamin I in vivo and may play a role in synaptic vesicle endocytosis. Furthermore, syndapin I associates with the neural Wiskott-Aldrich syndrome protein, an actin-depolymerizing protein that regulates cytoskeletal rearrangement. These characteristics of syndapin I suggest a molecular link between cytoskeletal dynamics and synaptic vesicle recycling in the nerve terminal.

Adaptor Proteins, Signal Transducing↗
PubMed2006

The adaptor protein soc-1/Gab1 modifies growth factor receptor output in Caenorhabditis elegans.

Previous genetic analysis has shown that dos/soc-1/Gab1 functions positively in receptor tyrosine kinase (RTK)-stimulated Ras/Map kinase signaling through the recruitment of csw/ptp-2/Shp2. Using sensitized assays in Caenorhabditis elegans for let-23/Egfr and daf-2/InsR (insulin receptor-like) signaling, it is shown that soc-1/Gab1 inhibits phospholipase C-gamma (PLCgamma) and phosphatidylinositol 3'-kinase (PI3K)-mediated signaling. Furthermore, as well as stimulating Ras/Map kinase signaling, soc-1/Gab1 stimulates a poorly defined signaling pathway that represses class 2 daf-2 phenotypes. In addition, it is shown that SOC-1 binds the C-terminal SH3 domain of SEM-5. This binding is likely to be functional as the sem-5(n2195)G201R mutation, which disrupts SOC-1 binding, behaves in a qualitatively similar manner to a soc-1 null allele in all assays for let-23/Egfr and daf-2/InsR signaling that were examined. Further genetic analysis suggests that ptp-2/Shp2 mediates the negative function of soc-1/Gab1 in PI3K-mediated signaling, as well as the positive function in Ras/Map kinase signaling. Other effectors of soc-1/Gab1 are likely to inhibit PLCgamma-mediated signaling and stimulate the poorly defined signaling pathway that represses class 2 daf-2 phenotypes. Thus, the recruitment of soc-1/Gab1, and its effectors, into the RTK-signaling complex modifies the cellular response by enhancing Ras/Map kinase signaling while inhibiting PI3K and PLCgamma-mediated signaling.

Aging↗
PubMed2003

Proteome analysis reveals caspase activation in hyporesponsive CD4 T lymphocytes induced in vivo by the oral administration of antigen.

The oral administration of antigen can lead to systemic antigen-specific hyporesponsiveness, also known as oral tolerance. This phenomenon is a representative form of immune tolerance to exogenous antigen under physiological conditions. We have previously reported that long term feeding of dietary antigen to ovalbumin-specific T cell receptor (TCR) transgenic mice induced oral tolerance of peripheral T cells with impairment in their TCR-induced calcium-signaling pathway. In this study, we utilized two-dimensional electrophoresis to compare intracellular protein expression patterns of orally tolerant and unsensitized CD4 T cells. We detected 26 increased and 16 decreased protein spots and identified 35 of these by mass spectrometry. The results indicated that the expression of caspases was up-regulated and that the protein levels of intact proteins susceptible to caspase cleavage, such as Grb2-related adaptor downstream of Shc (GADS), were decreased in orally tolerant CD4 T cells. Western blotting experiments confirmed that expression of the active form of caspase-3 and the antiapoptotic factor, X-linked inhibitor of apoptosis, were both up-regulated in orally tolerant CD4 T cells, which were found to be nonapoptotic. We further demonstrated that orally tolerant CD4 T cells could not form normal TCR signaling complexes associated with GADS and showed down-regulated phospholipase C-gamma1 activation, which is likely to contribute to the impairment of TCR-induced calcium signaling. Our findings indicate that orally tolerant CD4 T cells up-regulate caspase activation and show decreased levels of caspase-targeted proteins, including TCR signaling-associated molecules, while up-regulating antiapoptotic factors, all of which appear to contribute to their unique tolerant characteristics.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Quantitative analysis of TIP47-receptor cytoplasmic domain interactions: implications for endosome-to-trans Golgi network trafficking.

TIP47 (tail-interacting protein of 47 kDa) binds to the cytoplasmic domains of the cation-independent and cation-dependent mannose 6-phosphate receptors and is required for their transport from late endosomes to the trans Golgi network in vitro and in vivo. We report here a quantitative analysis of the interaction of recombinant TIP47 with mannose 6-phosphate receptor cytoplasmic domains. Recombinant TIP47 binds more tightly to the cation-independent mannose 6-phosphate receptor (K(D) = 1 microm) than to the cation-dependent mannose 6-phosphate receptor (K(D) = 3 microm). In addition, TIP47 fails to interact with the cytoplasmic domains of the hormone-processing enzymes, furin, phosphorylated furin, and metallocarboxypeptidase D, as well as the cytoplasmic domain of TGN38, proteins that are also transported from endosomes to the trans Golgi network. Although these proteins failed to bind TIP47, furin and TGN38 were readily recognized by the clathrin adaptor, AP-2. These data suggest that TIP47 recognizes a very select set of cargo molecules. Moreover, our data suggest unexpectedly that furin, TGN38, and carboxypeptidase D may use a distinct vesicular carrier and perhaps a distinct route for transport between endosomes and the trans Golgi network.

Adaptor Protein Complex alpha Subunits↗
PubMed2002

Endophilin-1: a multifunctional protein.

Endophilin-1, a cytoplasmic Src homology 3 (SH3) domain-containing protein, localises in brain presynaptic nerve termini. Endophilin dimerises through its N-terminus, and participates at multiple stages in clathrin-coated endocytosis, from early membrane invagination to synaptic vesicle uncoating. Both its C-terminal SH3 domain and N-terminus are required for endocytosis. Through its SH3 domain, endophilin bound to proline-rich domains (PRDs) in other endocytic proteins, including synaptojanin and dynamin. The N-terminal region possesses unique functions affecting lipid membrane curvature, through lysophosphatidic acid acyl transferase (LPAAT) activity and direct binding and tubulating activity. In addition to synaptic vesicle formation, endophilin-1 complexes with signalling molecules, including cell surface receptors, metalloprotease disintegrins and germinal centre kinase-like kinase (GLK). Therefore, endophilin-1 may serve to couple vesicle biogenesis with intracellular signalling cascades.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Outside-in integrin signal transduction. Alpha IIb beta 3-(GP IIb IIIa) tyrosine phosphorylation induced by platelet aggregation.

alpha IIb beta 3-(GP IIb IIIa) is the most abundant integrin expressed on platelets and plays a critical role in platelet aggregation and normal hemostasis. In response to platelet stimulation by agonists such as thrombin, alpha IIb beta 3 becomes a receptor for the adhesive proteins fibrinogen, von Willebrand factor, vitronectin, and fibronectin. Binding of extracellular matrix ligands allows the integrin to transmit a signal to the inside of the cell, but the exact mechanisms whereby integrins transduce these signals remains unclear. In this paper we demonstrate that the beta 3 subunit of alpha IIb beta 3 was phosphorylated on tyrosine residues in response to thrombin-induced platelet aggregation. However, tyrosine phosphorylation was not observed when platelets were stimulated by thrombin in the presence of an inhibitor of aggregation. Phosphotyrosine was only detected when platelets were solubilized under protein-denaturing conditions. A peptide corresponding to residues 740-762 of the beta 3 cytoplasmic domain was capable of binding the signaling proteins SHC and GRB2. GRB2 binding occurred only when both tyrosine residues (Tyr-747 and Tyr-759) were phosphorylated. SHC binding also occurred to a peptide monophosphorylated at Tyr-759. The data suggest that tyrosine phosphorylation of an integrin beta subunit may be important in initiating outside-in signaling cascades by inducing association of signaling components directly with the integrin.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Tra1p is a component of the yeast Ada.Spt transcriptional regulatory complexes.

The yeast Ada and TBP class of Spt proteins interact in multiple complexes that are required for transcriptional regulation. We have identified Tra1p as a component of these complexes through tandem mass spectrometry analysis of proteins that associate with Ngg1p/Ada3p. TRA1 is an essential gene and encodes a 3744-amino acid protein that is a member of a group of proteins including the catalytic subunit of DNA-dependent protein kinase, ATM and TRRAP, with carboxyl-terminal regions related to phosphatidylinositol 3-kinases. The interaction between Tra1p and Ada/Spt components was verified by the reciprocal coimmunoprecipitation of Ada2p and Tra1p from whole cell extracts in one or more complexes containing Spt7p. Tra1p cofractionated with Ngg1p and Spt7p through consecutive chromatography on Mono Q, DNA-cellulose, and Superose 6 columns. Binding of Tra1p to DNA-cellulose required Ada components. The association of Tra1p with two Ada.Spt complexes was suggested by its cofractionation with Ngg1p and Spt7p in two peaks on the Mono Q column. In the absence of Ada2p, the elution profile of Tra1p shifted to a distinct peak. Despite the similarity of Tra1p to a group of putative protein kinases, we have not detected protein kinase activity within immunoprecipitates of Tra1p or the Ada.Spt complexes.

Adaptor Proteins, Signal Transducing↗
PubMed2007

RET/papillary thyroid carcinoma oncogenic signaling through the Rap1 small GTPase.

RET/papillary thyroid carcinoma (PTC) oncoproteins result from the in-frame fusion of the RET receptor tyrosine kinase with protein dimerization motifs encoded by heterologous genes. Here, we show that RET/PTC1 activates the Rap1 small GTPase. The activation of Rap1 was dependent on the phosphorylation of RET Tyr(1062). RET/PTC1 recruited a complex containing growth factor receptor binding protein 2-associated binding protein 1 (Gab1), CrkII (v-crk sarcoma virus CT10 oncogene homologue II), and C3G (Rap guanine nucleotide exchange factor 1). By using dominant-negative and small interfering duplex (small interfering RNA) oligonucleotides, we show that RET/PTC1-mediated Rap1 activation was dependent on CrkII, C3G, and Gab1. Activation of Rap1 was involved in the RET/PTC1-mediated stimulation of the BRAF kinase and the p42/p44 mitogen-activated protein kinases. Proliferation and stress fiber formation of RET/PTC1-expressing PC Cl 3 thyroid follicular cells were inhibited by the dominant-negative Rap1(N17) and by Rap1-specific GTPase-activating protein. Thus, Rap1 is a downstream effector of RET/PTC and may contribute to the transformed phenotype of RET/PTC-expressing thyrocytes.

Actins↗
PubMed2006

The NYN domains: novel predicted RNAses with a PIN domain-like fold.

Using sensitive sequence profile searches and contextual information gleaned from domain architectures and predicted operons we identify a novel family of protein domains with predicted ribonuclease activity. These domains are found in the eukaryotic proteins typified by the Nedd4-binding protein 1 and the bacterial YacP-like proteins (Nedd4-BP1, YacP nucleases; NYN domains). We show that the NYN domain shares a common protein fold with two other previously characterized groups of nucleases, namely the PIN (PilT N-terminal) and FLAP/5' --> 3' exonuclease superfamilies. We also show that all these proteins share a common set of 4 acidic conserved residues that are predicted to constitute their active site. Based on the conservation of the acidic residues and structural elements we suggest that PIN and NYN domains are likely to bind only a single metal ion, unlike the FLAP/5' --> 3' exonuclease superfamily, which binds two metal ions. We also present evidence that the other conserved residues shared by all these three domains are likely to play critical roles in sensing the substrate and positioning the catalytic residues in the right conformation. Based on conserved gene neighborhoods we infer that the bacterial members are likely to be components of the processome/degradsome that process tRNAs or ribosomal RNAs. Eukaryotic versions appear to have undergone extensive functional diversification as suggested by the several distinctive multi-domain architectures showing fusions with various other RNA-binding domains like CCCH, PPR and KH domains. Interestingly, the eukaryotic NYN domains also show multiple fusions to the UBA domain, an ubiquitin-binding adaptor domain. This observation, together with the monoubiquitination of Nedd4-BP1 by the ubiquitin ligase Nedd4 suggests that the NYN domain proteins of eukaryotes are regulated by monoubiquitination. Given the localization of Nedd4-BP1 to punctuate nuclear bodies, it is likely that they are parts of nuclear RNA-processing complexes that are dependent on monoubiquitination for their assembly.

Amino Acid Sequence↗