anti-FLAG M2 Research Articles

Suppression of the Ligand-mediated Down-regulation of Epidermal Growth Factor Receptor by Ymer, a Novel Tyrosine-phosphorylated and Ubiquitinated Protein

The ligand-mediated down-regulation of the growth factor receptors is preceded by the involvement of various other factors. In particular, a ubiquitin ligase, Cbl, plays a central role in this event. Several candidates that have potential effects on the negative control of the epidermal growth factor (EGF) receptor have now been identified by our recent studies in phospho-proteomics. Among these molecules, we focus on characterizing a novel protein, Ymer, which is a tyrosine-phosphorylated and ubiquitinated protein. Ymer is found to be phosphorylated at tyrosine 145 and 146 upon EGF stimulation, and lysine 129 of Ymer has been identified as a ubiquitination site. Ymer has two motifs interacting with the ubiquitin (MIU) domains that might function as a binding site for the ubiquitinated EGF receptor. Although Ymer and EGF receptors are associated in an EGF-dependent manner, their interaction is required not only for MIU domains but also for the tyrosine phosphorylation of Ymer. Phosphorylated Ymer is mainly located at the plasma membrane with EGF receptor and functions in its endocytosis and degradation. Furthermore, EGF-mediated secondary modifications of an activated-EGF receptor are inhibited by overexpressing Ymer in COS7 cells. Therefore, Ymer may have competitive effects on the activation of the EGF receptor. Our findings suggest that Ymer functions as a novel inhibitor for the down-regulation of the EGF receptor and plays a crucial role for regulating the amount of the EGF receptor on the cell surface membrane.

Different Domains of the AMPA Receptor Direct Stargazin-mediated Trafficking and Stargazin-mediated Modulation of Kinetics

Stargazin is an accessory protein of AMPA receptors that enhances surface expression and also affects the biophysical properties of the receptor. AMPA receptor domains necessary for either of these two processes have not yet been identified. Here, we used confocal imaging and electrophysiology of heterologously expressed, fluorophore-tagged GluR1, GluR2, and stargazin to study surface expression and desensitization kinetics. Stargazin-mediated trafficking was sensitive to the nature of the AMPA receptor cytoplasmic domain. The insertion of YFP after residue 15 of the truncated cytoplasmic tail of GluR1i perturbed stargazin-mediated trafficking of the receptor but not its modulation of desensitization kinetics. This construct also failed to permit fluorescence resonance energy transfer (FRET) with stargazin in the endoplasmic reticulum (ER), whereas FRET between fluorophore-tagged stargazin and non-truncated AMPA receptors demonstrated a specific interaction between these proteins, both in the ER and the plasma membrane. Rather than encoding a specific binding site, the fluorophore-tagged C terminus may restrict access to one or more ER retention sites. Although perturbations of the C terminus impeded stargazin-mediated trafficking to the plasma membrane, the effects of stargazin on the biophysical properties of AMPA receptors (i.e. modulation of desensitization) remained intact. These data provide strong evidence that the AMPA receptor domains required for stargazin modulation of gating and trafficking are separable.

Hyperactivation of Mammalian Target of Rapamycin (mTOR) Signaling by a Gain-of-Function Mutant of the Rheb GTPase

Gain-of-function mutants of Ras and Rho family small GTPases have proven to be important tools in analyzing signaling downstream of these small GTPases. The Ras-related GTPase Rheb has emerged as a key player downstream of TSC1-2 in activating signaling to mammalian target of rapamycin (mTOR) effectors of cell growth such as S6K and 4E-BP1. The TSC1-2 tumor suppressor complex has been shown to act as a RhebGAP, converting Rheb from a GTP-bound to a GDP-bound form. Here we report the identification of a mutant Rheb (S16HRheb) that exhibits gain-of-function properties. At endogenous levels of expression S16HRheb exhibits increased GTP loading in vivo and is resistant to TSC1-2 GAP in vitro. Compared with wild-type Rheb, S16HRheb is more active at promoting the phosphorylation of the mTOR effectors S6K1 and 4E-BP1. Thus S16HRheb will help to identify proximal signaling events downstream of Rheb and allow potential Rheb-independent functions downstream of TSC1-2 to be investigated.

Suppressed Disassembly of Autolyzing p94/CAPN3 by N2A Connectin/Titin in a Genetic Reporter System

p94/calpain 3 is a skeletal muscle-specific member of the Ca(2+)-regulated cytosolic cysteine protease family, the calpains. Defective p94 protease activity originating from gene mutations causes a muscular dystrophy called calpainopathy, indicating the indispensability of p94 for muscle survival. Because of the existence of the p94-specific regions IS1 and IS2, p94 undergoes very rapid and exhaustive autolysis. To elucidate the physiological relevance of this unique activity, the autolytic profiles of p94 and the effect of the p94 binding protein, connectin/titin, on this process were investigated. In vitro analysis of p94 autolysis showed that autolysis in IS1 proceeds without immediate disassembly into fragments and that the newly identified cryptic autolytic site in IS2 is critical for disassembling autolyzed fragments. As a genetic system to assay p94 autolysis semiquantitatively, p94 was expressed in yeast as a hybrid protein between the DNA binding and activation domains of the yeast transcriptional activator Gal4. Transcriptional activation by the Gal4-p94:WT hybrid protein is precluded by p94 autolysis. Complete or partial loss of autolytic activity by C129S active site mutation, limb girdle muscular dystrophy type 2A pathogenic missense mutations, or PCR-based random mutagenesis could be detected by semiquantitative restoration of Gal4-dependent beta-galactosidase gene expression. Using this system, the N2A connectin fragment that binds to p94 was shown to suppress p94 autolytic disassembly. The proximity of the IS2 autolytic and connectin-binding sites in p94 suggested that N2A connectin suppresses IS2 autolysis. These data indicate the importance of p94-connectin interaction in the control of p94 functions by regulating autolytic decay of p94.

N-Linked Oligosaccharides on Chondroitin 6-Sulfotransferase-1 Are Required for Production of the Active Enzyme, Golgi Localization, and Sulfotransferase Activity toward Keratan Sulfate

We have shown previously that purified chondroitin 6-sulfotransferase-1 (C6ST-1) was a glycoprotein abundant in N-linked oligosaccharides and could sulfate both chondroitin (C6ST activity) and keratan sulfate (KSST activity); however, functional roles of the N-glycans have remained unclear. In the present study, we show essential roles of N-glycans attached to C6ST-1 in the generation of the active enzyme and in its KSST activity. Treatment with tunicamycin of COS-7 cells transfected with C6ST-1 cDNA totally abolished production of the active C6ST-1. A nearly complete removal of N-glycans of the recombinant C6ST-1 by peptide N-glycosidase F increased the C6ST activity but decreased the KSST activity. Among six potential N-glycosylation sites, deletion of the fourth or sixth site from the amino terminus inhibited production of the active C6ST-1, whereas deletion of the fifth site resulted in a marked loss of the KSST activity. Wild-type recombinant C6ST-1 showed a typical Golgi localization, whereas M-4 recombinant C6ST-1, in which the fourth N-glycosylation site was deleted, colocalized with calnexin, an endoplasmic reticulum-resident protein. Unlike wildtype recombinant C6ST-1, M-4 recombinant C6ST-1 showed a weak affinity toward wheat germ agglutinin and was converted completely to the nonglycosylated form by endoglycosidase H. These observations suggest that N-glycan attached to the fourth N-glycosylation site may function in the proper processing of N-glycans required for the Golgi localization, thereby causing the production of the active C6ST-1, and that N-glycan attached to the fifth N-glycosylation site may contribute to the KSST activity of C6ST-1.

REV1 Protein Interacts with PCNA: Significance of the REV1 BRCT Domain In Vitro and In Vivo

REV1 protein, a eukaryotic member of the Y family of DNA polymerases, is involved in the tolerance of DNA damage by translesion DNA synthesis. It is unclear how REV1 is recruited to replication foci in cells. Here, we report that mouse REV1 can bind directly to PCNA and that monoubiquitylation of PCNA enhances this interaction. The interaction between REV1 protein and PCNA requires a functional BRCT domain located near the N terminus of the former protein. Deletion or mutational inactivation of the BRCT domain abolishes the targeting of REV1 to replication foci in unirradiated cells, but not in UV-irradiated cells. In vivo studies in both chicken DT40 cells and yeast directly support the requirement of the BRCT domain of REV1 for cell survival and DNA damage-induced mutagenesis.

SHAP Potentiates the CD44-mediated Leukocyte Adhesion to the Hyaluronan Substratum

CD44-hyaluronan (HA) interaction is involved in diverse physiological and pathological processes. Regulation of interacting avidity is well studied on CD44 but rarely on HA. We discovered a unique covalent modification of HA with a protein, SHAP, that corresponds to the heavy chains of inter-alpha-trypsin inhibitor family molecules circulating in blood. Formation of the SHAP.HA complex is often associated with inflammation, a well known process involving the CD44-HA interaction. We therefore examined the effect of SHAP on the CD44-HA interaction-mediated lymphocyte adhesion. Under both static and flowing conditions, Hut78 cells (CD44-positive) and CD44-transfected Jurkat cells (originally CD44-negative) adhered preferentially to the immobilized SHAP.HA complex than to HA. The enhanced adhesion is exclusively mediated by the CD44-HA interaction, because it was inhibited by HA, but not IalphaI, and was completely abolished by pretreating the cells with anti-CD44 antibodies. SHAP appears to potentiate the interaction by increasing the avidity of HA to CD44 and altering their distribution on cell surfaces. Large amounts of the SHAP.HA complex accumulate in the hyperplastic synovium of rheumatoid arthritis patients. Leukocytes infiltrated to the synovium were strongly positive for HA, SHAP, and CD44 on their surfaces, suggesting a role for the adhesion-enhancing effect of SHAP in pathogenesis.

Expression and Characterization of Full-length Human Huntingtin, an Elongated HEAT Repeat Protein

Huntington disease is an inherited neurodegenerative disorder that is caused by expanded CAG trinucleotide repeats, resulting in a polyglutamine stretch of >37 on the N terminus of the protein huntingtin (htt). htt is a large (347 kDa), ubiquitously expressed protein. The precise functions of htt are not clear, but its importance is underscored by the embryonic lethal phenotype in htt knock-out mice. Despite the fact that the htt gene was cloned 13 years ago, little is known about the properties of the full-length protein. Here we report the expression and preliminary characterization of recombinant full-length wild-type human htt. Our results support a model of htt composed entirely of HEAT repeats that stack to form an elongated superhelix.

ADP-ribosyltransferase-specific Modification of Human Neutrophil Peptide-1

Epithelial cells lining human airways and cells recruited to airways participate in the innate immune response in part by releasing human neutrophil peptides (HNP). Arginine-specific ADP-ribosyltransferases (ART) on the surface of these cells can catalyze the transfer of ADP-ribose from NAD to proteins. We reported that ART1, a mammalian ADP-ribosyltransferase, present in epithelial cells lining the human airway, modified HNP-1, altering its function. ADP-ribosylated HNP-1 was identified in bronchoalveolar lavage fluid (BALF) from patients with asthma, idiopathic pulmonary fibrosis, or a history of smoking (and having two common polymorphic forms of ART1 that differ in activity), but not in normal volunteers or patients with lymphangioleiomyomatosis. Modified HNP-1 was not found in the sputum of cystic fibrosis patients or in leukocyte granules of normal volunteers. The finding of ADP-ribosyl-HNP-1 in BALF but not in leukocyte granules suggests that the modification occurred in the airway. Most of the HNP-1 in the BALF from individuals with a history of smoking was, in fact, mono- or di-ADP-ribosylated. ART1 synthesized in Escherichia coli, glycosylphosphatidylinositol-anchored ART1 released with phosphatidylinositol-specific phospholipase C from transfected NMU cells, or ART1 expressed endogenously on C2C12 myotubes modified arginine 14 on HNP-1 with a secondary site on arginine 24. ADP-ribosylation of HNP-1 by ART1 was substantially greater than that by ART3, ART4, ART5, Pseudomonas aeruginosa exoenzyme S, or cholera toxin A subunit. Mouse ART2, which is an NAD:arginine ADP-ribosyltransferase, was able to modify HNP-1, but to a lesser extent than ART1. Although HNP-1 was not modified to a significant degree by ART5, it inhibited ART5 as well as ART1 activities. Human beta-defensin-1 (HBD1) was a poor transferase substrate. Reduction of the cysteine-rich defensins enhanced their ability to serve as ADP-ribose acceptors. We conclude that ADP-ribosylation of HNP-1 appears to be primarily an activity of ART1 and occurs in inflammatory conditions and disease.

Additional Sex Comb-like 1 (ASXL1), in Cooperation with SRC-1, Acts as a Ligand-dependent Coactivator for Retinoic Acid Receptor

Additional sex comb-like 1 (ASXL1, 170 kDa), a mammalian homolog of Drosophila ASX, was identified as a protein that interacts with retinoic acid receptor (RAR) in the presence of retinoic acid (RA). Systematic binding assays showed that the C-terminal nuclear receptor box (LVMQLL) of ASXL1 and the activation function-2 activation domain (AF-2 AD) core of the RAR are critical for ligand-dependent interaction. The interaction was confirmed using in vitro glutathione S-transferase pulldown and in vivo immunoprecipitation (IP) assays. Confocal microscopy revealed that ASXL1 localizes in the nucleus. In addition to the intrinsic transactivation function of ASXL1, its cotransfection together with an RA-responsive luciferase reporter increased the RAR activity. This ASXL1 activity appears to be mediated through the functional cooperation with SRC-1, as shown by GST pulldown, IP, chromatin IP, and transcription assays. In the presence of ASXL1, more acetylated histone H3 was accumulated on the RA-responsive promoter in response to RA. Finally, stable expression of ASXL1 increased the expression of endogenous RA-regulated genes and enhanced the antiproliferative potential of RA. Overall, these results suggest that ASXL1 is a novel coactivator of RAR that cooperates with SRC-1 and implicates it as a potential antitumor target of RA in RA-resistant cancer cells.

Hdm2 Nuclear Export, Regulated by Insulin-like Growth Factor-I/MAPK/p90Rsk Signaling, Mediates the Transformation of Human Cells

Insulin-like growth factor (IGF)-I receptor activation leads to enhanced proliferation and cell survival via the MAP kinase and phosphatidylinositol 3-kinase-signaling pathways. Upon stimulation by IGF-I, the Hdm2 oncoprotein is phosphorylated by AKT, leading to its rapid nuclear translocation and subsequent inhibition of p53. We now show that IGF-I stimulation regulates the nuclear export of Hdm2 and p53 via the MAP kinase pathway. Inhibition of p38 MAPK or MEK via pharmacological means or expression of dominant negative proteins inhibited the cytoplasmic accumulation of Hdm2 and increased Hdm2 and p53 protein levels, whereas constitutively active p90Rsk promoted the nuclear export of Hdm2. Expression of constitutively active p90Rsk with E1A, oncogenic H-Ras, and hTERT resulted in the anchorage-independent growth of normal human fibroblasts. Our findings link p90Rsk-mediated modulation of Hdm2 nuclear to cytoplasmic shuttling with the diminished ability of p53 to regulate cell cycle checkpoints that ultimately leads to transformation.

Characterization of the Residues in Helix 8 of the Human β1-Adrenergic Receptor That Are Involved in Coupling the Receptor to G Proteins

Several key amino acids within amphipathic helix 8 of the human beta1-adrenergic receptor (beta1-AR) were mutagenized to characterize their role in signaling by G protein-coupled receptors. Mutagenesis of phenylalanine at position 383 in the hydrophobic interface to histidine (F383H) prevented the biosynthesis of the receptor, indicating that the orientation of helix 8 is important for receptor biosynthesis. Mutagenesis of aspartic acid at position 382 in the hydrophilic interface to leucine (D382L) reduced the binding and uncoupled the receptor from G protein activation. Mutagenesis of the basic arginine residue at position 384 to glutamine (R384Q) or to glutamic acid (R384E) increased basal and agonist-stimulated adenylyl cyclase activities. R384Q and R384E displayed features associated with constitutively active receptors because inverse agonists markedly reduced their elevated basal adenylyl cyclase activities. Isoproterenol increased the phosphorylation and promoted the desensitization of the Gly389 or Arg389 allelic variants of the wild type beta1-AR but failed to produce these effects in R384Q and R384E, because these receptors were maximally phosphorylated and desensitized under basal conditions. In contrast to the membranous distribution of the wild type beta1-AR, R384Q and R384E were localized mostly within intracellular punctate structures. Inverse agonists restored the membranous distribution of R384Q and R384E, indicating that they recycled normally when their constitutive internalization was blocked by inverse agonists. These data combined with computer modeling of the putative three-dimensional organization of helix 8 indicated that the amphipathic character of helix 8 and side chain projections of Asp382 and Arg384 within the hydrophilic interface might serve as a tethering site for the G protein.

Direct Binding of Translation Initiation Factor eIF2γ-G Domain to Its GTPase-activating and GDP-GTP Exchange Factors eIF5 and eIF2Bϵ

The GTP-binding eukaryotic translation initiation factor eIF2 delivers initiator methionyl-tRNA to the 40 S ribosomal subunit. The factor eIF5 stimulates hydrolysis of GTP by eIF2 upon AUG codon recognition, whereas the factor eIF2B promotes guanine nucleotide exchange on eIF2 to recycle the factor for additional rounds of translation initiation. The GTP-binding (G) domain resides in the gamma subunit of the heterotrimeric eIF2; however, only eIF2beta, and not eIF2gamma, has been reported to directly bind to eIF5 or eIF2B. Using proteins expressed in yeast or recombinant systems we show that full-length yeast eIF2gamma, as well as its isolated G domain, binds directly to eIF5 and the epsilon subunit of eIF2B, and we map the interaction sites to the catalytically important regions of these factors. Consistently, an internal deletion of residues 50-100 of yeast eIF5 impairs the interaction with recombinant eIF2gamma-G domain and abolishes the ability of eIF5 to stimulate eIF2 GTPase activity in translation initiation complexes in vitro. Thus, rather than allosterically regulating eIF2gamma-G domain function via eIF2beta, our data support a model in which the GTPase-activating factor eIF5 and the guanine-nucleotide exchange factor eIF2B modulate eIF2 function through direct interactions with the eIF2gamma-G domain.

Equimolar Production of Amyloid β-Protein and Amyloid Precursor Protein Intracellular Domain from β-Carboxyl-terminal Fragment by γ-Secretase

We showed previously that cells expressing wild-type (WT) beta-amyloid precursor protein (APP) or coexpressing WTAPP and WT presenilin (PS) 1/2 produced APP intracellular domains (AICD) 49-99 and 50-99, with the latter predominating. On the other hand, the cells expressing mutant (MT) APP or coexpressing WTAPP and MTPS1/2 produced a greater proportion of AICD-(49-99) than AICD-(50-99). In addition, the expression of amyloid beta-protein (Abeta) 49 in cells resulted in predominant production of Abeta40 and that of Abeta48 leads to preferential production of Abeta42. These observations suggest that epsilon-cleavage and gamma-cleavage are interrelated. To determine the stoichiometry between Abeta and AICD, we have established a 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid-solubilized gamma-secretase assay system that exhibits high specific activity. By using this assay system, we have shown that equal amounts of Abeta and AICD are produced from beta-carboxyl-terminal fragment (C99) by gamma-secretase, irrespective of WT or MTAPP and PS1/2. Although various Abeta species, including Abeta40, Abeta42, Abeta43, Abeta45, Abeta48, and Abeta49, are generated, only two species of AICD, AICD-(49-99) and AICD-(50-99), are detected. We also have found that M233T MTPS1 produced only one species of AICD, AICD-(49-99), and only one for its counterpart, Abeta48, in contrast to WT and other MTPS1s. These strongly suggest that epsilon-cleavage is the primary event, and the produced Abeta48 and Abeta49 rapidly undergo gamma-cleavage, resulting in generation of various Abeta species.

Migfilin Interacts with Vasodilator-stimulated Phosphoprotein (VASP) and Regulates VASP Localization to Cell-Matrix Adhesions and Migration

Cell migration is a complex process that is coordinately regulated by cell-matrix adhesion and actin cytoskeleton. We report here that migfilin, a recently identified component of cell-matrix adhesions, is a biphasic regulator of cell migration. Loss of migfilin impairs cell migration. Surprisingly, overexpression of migfilin also reduces cell migration. Molecularly, we have identified vasodilator-stimulated phosphoprotein (VASP) as a new migfilin-binding protein. The interaction is mediated by the VASP EVH1 domain and a single L104PPPPP site located within the migfilin proline-rich domain. Migfilin and VASP form a complex in both suspended and adhered cells, and in the latter, they co-localize in cell-matrix adhesions. Functionally, migfilin facilitates VASP localization to cell-matrix adhesions. Using two different approaches (VASP-binding defective migfilin mutants and small interfering RNA-mediated VASP knockdown), we show that the interaction with VASP is crucially involved in migfilin-mediated regulation of cell migration. Our results identify migfilin as an important regulator of cell migration and provide new information on the mechanism by which migfilin regulates this process.

Asf1 Is Required for Viability and Chromatin Assembly during DNA Replication in Vertebrate Cells

Asf1 (anti-silencing function 1), a well conserved protein from yeast to humans, acts as a histone chaperone and is predicted to participate in a variety of chromatin-mediated cellular processes. To investigate the physiological role of vertebrate Asf1 in vivo, we generated a conditional Asf1-deficient mutant from chicken DT40 cells. Induction of Asf1 depletion resulted in the accumulation of cells in S phase with decreased DNA replication and increased mitotic aberrancy forming multipolar spindles, leading to cell death. In addition, nascent chromatin in Asf1-depleted cells showed increased nuclease sensitivity, indicating impaired nucleosome assembly during DNA replication. Complementation analyses revealed that the functional domain of Asf1 for cell viability was confined to the N-terminal core domain (amino acids 1-155) that is a binding platform for histones H3/H4, CAF-1p60, and HIRA, whereas Asf1 mutant proteins, abolishing binding abilities with both p60 and HIRA, exhibit no effect on viability. These results together indicate that the vertebrate Asf1 plays a crucial role in replication-coupled chromatin assembly, cell cycle progression, and cellular viability and provide a clue of a possible role in a CAF-1- and HIRA-independent chromatin-modulating process for cell proliferation.

BTB Domain-containing Speckle-type POZ Protein (SPOP) Serves as an Adaptor of Daxx for Ubiquitination by Cul3-based Ubiquitin Ligase

Daxx is a multifunctional protein that regulates a variety of cellular processes, including transcription, cell cycle, and apoptosis. SPOP is a BTB (Bric-a-brac/Tramtrack/Broad complex) protein that constitutes Cul3-based ubiquitin ligases. Here we show that SPOP serves as an adaptor of Daxx for the ubiquitination by Cul3-based ubiquitin ligase and subsequent degradation by the proteasome. Expression of SPOP with Cul3 markedly reduced Daxx level, and this degradation was blocked by SPOP-specific short hairpin RNAs. Inhibition of the proteasome by MG132 caused the prevention of Daxx degradation in parallel with the accumulation of ubiquitinated Daxx. Expression of SPOP with Cul3 reversed Daxx-mediated repression of ETS1- and p53-dependent transcription, and short hairpin RNA-mediated knock down of SPOP blocked the recovery of their transcriptional activation. Furthermore, Daxx degradation led to the cleavage of poly(ADP-ribose) polymerase and the increase in the number of terminal deoxynucleotidyltransferase-mediated dUTP-fluorescein nick end-labeling-positive apoptotic cells. These results suggest that SPOP/Cul3-ubiquitin ligase plays an essential role in the control of Daxx level and, thus, in the regulation of Daxx-mediated cellular processes, including transcriptional regulation and apoptosis.

Cell-surface Processing of Pro-ADAMTS9 by Furin

Processing of polypeptide precursors by proprotein convertases (PCs) such as furin typically occurs within the trans-Golgi network. Here, we show in a variety of cell types that the propeptide of ADAMTS9 is not excised intracellularly. Pulse-chase analysis in HEK293F cells indicated that the intact zymogen was secreted to the cell surface and was subsequently processed there before release into the medium. The processing occurred via a furin-dependent mechanism as shown using PC inhibitors, lack of processing in furin-deficient cells, and rescue by furin in these cells. Moreover, down-regulation of furin by small interference RNA reduced ADAMTS9 processing in HEK293F cells. PC5A could also process pro-ADAMTS9, but similarly to furin, processed forms were absent intracellularly. Cell-surface, furin-dependent processing of pro-ADAMTS9 creates a precedent for extracellular maturation of endogenously produced secreted proproteins. It also indicates the existence of a variety of mechanisms for processing of ADAMTS proteases.

Interacting JNK-docking Sites in MKK7 Promote Binding and Activation of JNK Mitogen-activated Protein Kinases

D-sites are a class of MAPK-docking sites that have been found in many MAPK regulators and substrates. A single functional, high affinity D-site has been identified near the N terminus of each of the MAPK kinases (MKKs or MEKs) MEK1, MEK2, MKK3, MKK4, and MKK6. Here we demonstrated that MKK7 recognizes its target JNK by a novel mechanism involving a partially cooperative interaction of three low affinity D-sites in the N-terminal domain of MKK7. Mutations of the conserved residues within any one of the three docking sites (D1, D2, and D3) disrupted the ability of the N-terminal domain of MKK7beta to bind JNK1 by about 50-70%. Moreover, mutation of any two of the three D-sites reduced binding by about 80-90%, and mutation of all three reduced binding by 95%. Full-length MKK7 containing combined D1/D2 mutations was compromised for binding to JNK1 and exhibited reduced JNK1 kinase activity when compared with wild-type MKK7. Peptide versions of the D-sites from MKK4 or the JIP-1 scaffold protein inhibited MKK7-JNK binding, suggesting that all three JNK regulators bind to the same region of JNK. Moreover, peptide versions of any of the three D-sites of MKK7 inhibited the ability of JNK1 and JNK2 to phosphorylate their transcription factor substrates c-Jun and ATF2, suggesting that D-site-containing substrates also compete with MKK7 for docking to JNK. Finally, MKK7-derived D-site peptides exhibited selective inhibition of JNK1 versus ERK2. We conclude that MKK7 contains three JNK-docking sites that interact to selectively bind JNK and contribute to JNK signal transmission and specificity.

A Mutation Linked with Autism Reveals a Common Mechanism of Endoplasmic Reticulum Retention for the α,β-Hydrolase Fold Protein Family

A mutation linked to autistic spectrum disorders encodes an Arg to Cys replacement in the C-terminal portion of the extracellular domain of neuroligin-3. The solvent-exposed Cys causes virtually complete retention of the protein in the endoplasmic reticulum when the protein is expressed in transfected cells. An identical Cys substitution was reported for butyrylcholinesterase through genotyping patients with post-succinylcholine apnea. Neuroligin, butyrylcholinesterase, and acetylcholinesterase are members of the alpha,beta-hydrolase fold family of proteins sharing sequence similarity and common tertiary structures. Although these proteins have distinct oligomeric assemblies and cellular dispositions, homologous Arg residues in neuroligin-3 (Arg-451), in butyrylcholinesterase (Arg-386), and in acetylcholinesterase (Arg-395) are conserved in all studied mammalian species. To examine whether an homologous Arg to Cys mutation affects related proteins similarly despite their differing capacities to oligomerize, we inserted homologous mutations in the acetylcholinesterase and butyrylcholinesterase cDNAs. Using confocal fluorescence microscopy and analysis of oligosaccharide processing, we find that the homologous Arg to Cys mutation also results in endoplasmic reticulum retention of the two cholinesterases. Small quantities of mutated acetylcholinesterase exported from the cell retain activity but show a greater K(m), a much smaller k(cat), and altered substrate inhibition. The nascent proteins associate with chaperones during processing, but the mutation presumably restricts processing through the endoplasmic reticulum and Golgi apparatus, because of local protein misfolding and inability to oligomerize. The mutation may alter the capacity of these proteins to dissociate from their chaperone prior to oligomerization and processing for export.