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Results for “Adaptor Protein Complex 1”

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PubMed2003

Novel human homologues of p47phox and p67phox participate in activation of superoxide-producing NADPH oxidases.

The catalytic core of a superoxide-producing NADPH oxidase (Nox) in phagocytes is gp91phox/Nox2, a membrane-integrated protein that forms a heterodimer with p22phox to constitute flavocytochrome b558. The cytochrome becomes activated by interacting with the adaptor proteins p47phox and p67phox as well as the small GTPase Rac. Here we describe the cloning of human cDNAs for novel proteins homologous to p47phox and p67phox, designated p41nox and p51nox, respectively; the former is encoded by NOXO1 (Nox organizer 1), and the latter is encoded by NOXA1 (Nox activator 1). The novel homologue p41nox interacts with p22phox via the two tandem SH3 domains, as does p47phox. The protein p51nox as well as p67phox can form a complex with p47phox and with p41nox via the C-terminal SH3 domain and binds to GTP-bound Rac via the N-terminal domain containing four tetratricopeptide repeat motifs. These bindings seem to play important roles, since p47phox and p67phox activate the phagocyte oxidase via the same interactions. Indeed, p41nox and p51nox are capable of replacing the corresponding classical homologue in activation of gp91phox. Nox1, a homologue of gp91phox, also can be activated in cells, when it is coexpressed with p41nox and p51nox, with p41nox and p67phox, or with p47phox and p51nox; in the former two cases, Nox1 is partially activated without any stimulants added, suggesting that p41nox is normally in an active state. Thus, the novel homologues p41nox and p51nox likely function together or in combination with a classical one, thereby activating the two Nox family oxidases.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Dual roles of the A kinase-anchoring protein Yotiao in the modulation of a cardiac potassium channel: a passive adaptor versus an active regulator.

Cardiac function is regulated critically by the autonomic nervous system to adapt to the physical activity and emotional stress. A slowly activating cardiac potassium channel (I(Ks)) is modulated by stimulation of the sympathetic nervous system (SNS) and contributes to cardiac action potential shortening in the face of concomitant increases in heart rate. Activation of beta-adrenergic receptors in response to SNS stimulation results in protein kinase A (PKA)-mediated phosphorylation of I(Ks) channels. We have found that the functional regulation of the I(Ks) channel by PKA requires the A kinase-anchoring protein (AKAP) Yotiao. Yotiao forms a macromolecular complex with the channel and recruits key enzymes such as PKA and protein phosphatase 1 (PP1) to control the phosphorylation state of I(Ks). Our recent findings revealed a more active role of Yotiao in the PKA modulation of I(Ks). We found that Yotiao participates actively in translating the phosphorylation-induced change into altered channel activity. Moreover Yotiao itself can be phosphorylated by PKA upon beta-adrenergic stimulation. Ablation of Yotiao phosphorylation impairs PKA-induced changes in I(Ks) voltage-dependent activation and current kinetics. Taken together we have evidence to suggest that Yotiao plays dual roles in the PKA modulation of the I(Ks) channel. It acts not only as an adaptor protein to coordinate enzymatic reactions but also as an active regulator that directly affects channel function.

A Kinase Anchor Proteins↗

PubMed2005

Membrane recruitment of effector proteins by Arf and Rab GTPases.

In their GTP-bound form, Arf and Rab family GTPases associate with distinct organelle membranes, to which they recruit specific sets of effector proteins that regulate vesicular transport. The Arf GTPases are involved in the formation of coated carrier vesicles by recruiting coat proteins. On the other hand, the Rab GTPases are involved in the tethering, docking and fusion of transport vesicles with target organelles, acting in concert with the tethering and fusion machineries. Recent structural studies of the Arf1-GGA and Rab5-Rabaptin-5 complexes, as well as other effector structures in complex with the Arf and Rab GTPases, have shed light on the mechanisms underlying the GTP-dependent membrane recruitment of these effector proteins.

ADP-Ribosylation Factor 1↗

PubMed2000

GAKIN, a novel kinesin-like protein associates with the human homologue of the Drosophila discs large tumor suppressor in T lymphocytes.

Reorganization of the cortical cytoskeleton is a hallmark of T lymphocyte activation. Upon binding to antigen presenting cells, the T cells rapidly undergo cytoskeletal re-organization thus forming a cap at the cell-cell contact site leading to receptor clustering, protein segregation, and cellular polarization. Previously, we reported cloning of the human lymphocyte homologue of the Drosophila Discs Large tumor suppressor protein (hDlg). Here we show that a novel protein termed GAKIN binds to the guanylate kinase-like domain of hDlg. Affinity protein purification, peptide sequencing, and cloning of GAKIN cDNA from Jurkat J77 lymphocytes identified GAKIN as a novel member of the kinesin superfamily of motor proteins. GAKIN mRNA is ubiquitously expressed, and the predicted amino acid sequence shares significant sequence similarity with the Drosophila kinesin-73 motor protein. GAKIN sequence contains a motor domain at the NH(2) terminus, a central stalk domain, and a putative microtubule-interacting sequence called the CAP-Gly domain at the COOH terminus. Among the MAGUK superfamily of proteins examined, GAKIN binds to the guanylate kinase-like domain of PSD-95 but not of p55. The hDlg and GAKIN are localized mainly in the cytoplasm of resting T lymphocytes, however, upon CD2 receptor cross-linking the hDlg can translocate to the lymphocyte cap. We propose that the GAKIN-hDlg interaction lays the foundation for a general paradigm of coupling MAGUKs to the microtubule-based cytoskeleton, and that this interaction may be functionally important for the intracellular trafficking of MAGUKs and associated protein complexes in vivo.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Molecular cloning of LILRC1 and LILRC2 in the mouse and the rat, two novel immunoglobulin-like receptors encoded by the leukocyte receptor gene complex.

We report the molecular cloning of two novel single-member receptor families with homology to LILR/CD85, PIR, and gp49: LILRC1 in the rat and the mouse, and LILRC2 in the rat. LILRC1 and LILRC2 both have two extracellular Ig-like domains and a cytoplasmic tail devoid of any known signaling motifs. The transmembrane regions of LILRC1 and LILRC2 contain an arginine residue, a common feature in receptors that associate with activating adaptor proteins. Rat and mouse LILRC1 are orthologs sharing 81.5% amino acid identity. LILRC2 represents a distinct receptor family, 47.9% identical to LILRC1. No murine LILRC2 ortholog was detected in genome or expressed sequence tag sequence databases. By radiation hybrid mapping, the rat Lilrc1 and Lilrc2 loci were localized to the leukocyte receptor gene complex (LRC) on chromosome 1, and the mouse Lilrc1 locus was mapped to the LRC on chromosome 7. Moreover, the mouse and rat Lilrc1 loci were localized to similar positions within the LRC. As shown by RT-PCR, rat LILRC1 was expressed by B cells, neutrophils, and a macrophage cell line. Transcription of LILRC2 was detected in T cells, B cells, neutrophils, and macrophages.

Amino Acid Sequence↗

PubMed2003

Endocytosis of synaptotagmin 1 is mediated by a novel, tryptophan-containing motif.

The rate at which a membrane protein is internalized from the plasma membrane can be regulated by revealing a latent internalization signal in response to an appropriate stimulus. Internalization of the synaptic vesicle membrane protein, synaptotagmin 1, is controlled by two distinct regions of its intracytoplasmic C2B domain, an internalization signal present in the 29 carboxyterminal (CT) amino acids and a separate regulatory region. We have now characterized the internalization motif by mutagenesis and found that it involves an essential tryptophan in the last beta strand of the C2B domain, a region that is distinct from the AP2-binding site previously described. Internalization through the tryptophan-based motif is sensitive to eps15 and dynamin mutants and is therefore likely to be clathrin mediated. A tryptophan-to-phenylalanine mutation had no effect on internalization of the CT domain alone, but completely inhibited endocytosis of the folded C2B domain. This result suggests that recognition of sorting motifs can be influenced by their structural context. We conclude that endocytosis of synaptotagmin 1 requires a novel type of internalization signal that is subject to regulation by the rest of the C2B domain.

Adaptor Protein Complex 2↗

PubMed2003

The Grb10/Nedd4 complex regulates ligand-induced ubiquitination and stability of the insulin-like growth factor I receptor.

The adapter protein Grb10 belongs to a superfamily of related proteins, including Grb7, -10, and -14 and Caenorhabditis elegans Mig10. Grb10 is an interacting partner of the insulin-like growth factor I receptor (IGF-IR) and the insulin receptor (IR). Previous work showed an inhibitory effect of mouse Grb10 (mGrb10alpha) on IGF-I-mediated mitogenesis (A. Morrione et al., J. Biol. Chem. 272:26382-26387, 1997). With mGrb10alpha as bait in a yeast two-hybrid screen, mouse Nedd4 (mNedd4-1), a ubiquitin protein ligase, was previously isolated as an interacting protein of Grb10 (A. Morrione et al., J. Biol. Chem. 274:24094-24099, 1999). However, Grb10 is not ubiquitinated by Nedd4 in cells. Here we show that in mouse embryo fibroblasts overexpressing Grb10 and the IGF-IR (p6/Grb10), there is a strong ligand-dependent increase in ubiquitination of the IGF-IR compared with that in parental cells (p6). This increased ubiquitination is associated with a shorter half-life and increased internalization of the IGF-IR. The IGF-IR is stabilized following treatment with both MG132 and chloroquine, indicating that both the proteasome and lysosomal pathways mediate degradation of the receptor. Ubiquitination of the IGF-IR likely occurs at the plasma membrane, prior to the formation of endocytic vesicles, as it is insensitive to dansylcadaverine, an inhibitor of early endosome formation in IGF-IR endocytosis. Grb10 coimmunoprecipitates with the IGF-IR and endogenous Nedd4 in p6/Grb10 cells, suggesting the presence of a Grb10/Nedd4/IGF-IR complex. Ubiquitination of the IGF-IR in p6/Grb10 cells is severely impaired by overexpression of a catalytically inactive Nedd4 mutant (Nedd4-CS), which also stabilizes the receptor. Likewise, overexpression of a Grb10 mutant lacking the Src homology 2 (SH2) domain impaired ubiquitination of the IGF-IR in parental p6 and p6/Grb10 cells, indicating that Grb10 binding to Nedd4 is critical for ubiquitination of the receptor. These results suggest a role for the Grb10/Nedd4 complex in regulating ubiquitination and stability of the IGF-IR, and they suggest that Grb10 serves as an adapter to form a bridge between Nedd4 and the IGF-IR. This is the first demonstration of regulation of stability of a tyrosine kinase receptor by the Nedd4 (HECT) family of E3 ligases.

Animals↗

PubMed2006

Oxidative and electrophilic stresses activate Nrf2 through inhibition of ubiquitination activity of Keap1.

The Keap1-Nrf2 system is the major regulatory pathway of cytoprotective gene expression against oxidative and/or electrophilic stresses. Keap1 acts as a stress sensor protein in this system. While Keap1 constitutively suppresses Nrf2 activity under unstressed conditions, oxidants or electrophiles provoke the repression of Keap1 activity, inducing the Nrf2 activation. However, the precise molecular mechanisms behind the liberation of Nrf2 from Keap1 repression in the presence of stress remain to be elucidated. We hypothesized that oxidative and electrophilic stresses induce the nuclear accumulation of Nrf2 by affecting the Keap1-mediated rapid turnover of Nrf2, since such accumulation was diminished by the protein synthesis inhibitor cycloheximide. While both the Cys273 and Cys288 residues of Keap1 are required for suppressing Nrf2 nuclear accumulation, treatment of cells with electrophiles or mutation of these cysteine residues to alanine did not affect the association of Keap1 with Nrf2 either in vivo or in vitro. Rather, these treatments impaired the Keap1-mediated proteasomal degradation of Nrf2. These results support the contention that Nrf2 protein synthesized de novo after exposure to stress accumulates in the nucleus by bypassing the Keap1 gate and that the sensory mechanism of oxidative and electrophilic stresses is closely linked to the degradation mechanism of Nrf2.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Receptor-interacting protein 140 binds c-Jun and inhibits estradiol-induced activator protein-1 activity by reversing glucocorticoid receptor-interacting protein 1 effect.

In the presence of estradiol, estrogen receptor-alpha (ERalpha) increases transcription triggered by activator protein-1 (AP-1). We have previously shown that induction is mediated by the direct interaction between c-Jun and ERalpha, which stabilizes a multiprotein complex containing the coactivator GRIP1 (glucocorticoid receptor interacting protein 1). The effect of receptor-interacting protein 140 (RIP140) in this regulation was assessed in the present study. We report that overexpression of RIP140 inhibits estradiol-induced AP-1-dependent transcription in a dose-dependent manner. Inhibition is not affected by trichostatin A, suggesting that histone deacetylase recruitment is not implicated. RIP140, which binds Jun proteins in pull-down assays and in intact cells, as shown by coimmunoprecipitation analysis and a mammalian one-hybrid system, participates in a multiprotein complex containing c-Jun and ERalpha. Moreover, the negative effect of RIP140 on AP-1-mediated transcription is relieved by GRIP1 overexpression and, conversely, RIP140 inhibits the stimulatory effect of GRIP1. The two cofactors compete for binding to c-Jun and ERalpha both in vitro and in intact cells, and GRIP1 interaction with both ERalpha and c-Jun is required for an efficient competition. These overall results suggest that the ratio between RIP140 and GRIP1 could determine, as proposed for hormone response element-mediated responses, the efficacy of estradiol in stimulating transcription of genes under AP-1 control.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Male mouse recombination maps for each autosome identified by chromosome painting.

Linkage maps constructed from genetic analysis of gene order and crossover frequency provide few clues to the basis of genomewide distribution of meiotic recombination, such as chromosome structure, that influences meiotic recombination. To bridge this gap, we have generated the first cytological recombination map that identifies individual autosomes in the male mouse. We prepared meiotic chromosome (synaptonemal complex [SC]) spreads from 110 mouse spermatocytes, identified each autosome by multicolor fluorescence in situ hybridization of chromosome-specific DNA libraries, and mapped >2,000 sites of recombination along individual autosomes, using immunolocalization of MLH1, a mismatch repair protein that marks crossover sites. We show that SC length is strongly correlated with crossover frequency and distribution. Although the length of most SCs corresponds to that predicted from their mitotic chromosome length rank, several SCs are longer or shorter than expected, with corresponding increases and decreases in MLH1 frequency. Although all bivalents share certain general recombination features, such as few crossovers near the centromeres and a high rate of distal recombination, individual bivalents have unique patterns of crossover distribution along their length. In addition to SC length, other, as-yet-unidentified, factors influence crossover distribution leading to hot regions on individual chromosomes, with recombination frequencies as much as six times higher than average, as well as cold spots with no recombination. By reprobing the SC spreads with genetically mapped BACs, we demonstrate a robust strategy for integrating genetic linkage and physical contig maps with mitotic and meiotic chromosome structure.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Orientation of peptide fragments from Sos proteins bound to the N-terminal SH3 domain of Grb2 determined by NMR spectroscopy.

NMR spectroscopy has been used to characterize the protein-protein interactions between the mouse Grb2 (mGrb2) N-terminal SH3 domain complexed with a 15-residue peptide (SPLLPKLPP-KTYKRE) corresponding to residues 1264-1278 of the mouse Sos-2 (mSos-2) protein. Intermolecular interactions between the peptide and 13C-15N-labeled SH3 domain were identified in half-reverse-filtered 2D and 3D NOESY experiments. Assignments for the protons involved in interactions between the peptide and the SH3 domain were confirmed in a series of NOESY experiments using a set of peptides in which different leucine positions were fully deuterated. The peptide ligand-binding site of the mGrb2 N-terminal SH3 domain is defined by the side chains of specific aromatic residues (Tyr7, Phe9, Trp36, Tyr52) that form two hydrophobic subsites contacting the side chains of the peptide Leu4 and Leu7 residues. An adjacent negatively charged subsite on the SH3 surface is likely to interact with the side chain of a basic residue at peptide position 10 that we show to be involved in binding. The peptide-binding site of the SH3 is characterized by large perturbations of amide chemical shifts when the peptide is added to the SH3 domain. The mGrb2 N-terminal SH3 domain structure in the complex is well-defined (backbone RMSD of 0.56 +/- 0.21 calculated over the backbone N, C alpha, and C atoms of residues 1-54). The structure of the peptide in the complex is less well-defined but displays a distinct orientation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptor Proteins, Signal Transducing↗

PubMed1996

Insulin stimulation of a MEK-dependent but ERK-independent SOS protein kinase.

The Ras guanylnucleotide exchange protein SOS undergoes feedback phosphorylation and dissociation from Grb2 following insulin receptor kinase activation of Ras. To determine the serine/threonine kinase(s) responsible for SOS phosphorylation in vivo, we assessed the role of mitogen-activated, extracellular-signal-regulated protein kinase kinase (MEK), extracellular-signal-regulated protein kinase (ERK), and the c-JUN protein kinase (JNK) in this phosphorylation event. Expression of a dominant-interfering MEK mutant, in which lysine 97 was replaced with arginine (MEK/K97R), resulted in an inhibition of insulin-stimulated SOS and ERK phosphorylation, whereas expression of a constitutively active MEK mutant, in which serines 218 and 222 were replaced with glutamic acid (MEK/EE), induced basal phosphorylation of both SOS and ERK. Although expression of the mitogen-activated protein kinase-specific phosphatase (MKP-1) completely inhibited the insulin stimulation of ERK activity both in vitro and in vivo, SOS phosphorylation and the dissociation of the Grb2-SOS complex were unaffected. In addition, insulin did not activate the related protein kinase JNK, demonstrating the specificity of insulin for the ERK pathway. The insulin-stimulated and MKP-1-insensitive SOS-phosphorylating activity was reconstituted in whole-cell extracts and did not bind to a MonoQ anion-exchange column. In contrast, ERK1/2 protein was retained by the MonoQ column, eluted with approximately 200 mM NaCl, and was MKP-1 sensitive. Although MEK also does not bind to MonoQ, immunodepletion analysis demonstrated that MEK is not the insulin-stimulated SOS-phosphorylating activity. Together, these data demonstrate that at least one of the kinases responsible for SOS phosphorylation and functional dissociation of the Grb2-SOS complex is an ERK-independent but MEK-dependent insulin-stimulated protein kinase.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes.

The function of the ATR (ataxia-telangiectasia mutated- and Rad3-related)-ATRIP (ATR-interacting protein) protein kinase complex is crucial for the cellular response to replication stress and DNA damage. Here, we show that replication protein A (RPA), a protein complex that associates with single-stranded DNA (ssDNA), is required for the recruitment of ATR to sites of DNA damage and for ATR-mediated Chk1 activation in human cells. In vitro, RPA stimulates the binding of ATRIP to ssDNA. The binding of ATRIP to RPA-coated ssDNA enables the ATR-ATRIP complex to associate with DNA and stimulates phosphorylation of the Rad17 protein that is bound to DNA. Furthermore, Ddc2, the budding yeast homolog of ATRIP, is specifically recruited to double-strand DNA breaks in an RPA-dependent manner. A checkpoint-deficient mutant of RPA, rfa1-t11, is defective for recruiting Ddc2 to ssDNA both in vivo and in vitro. Our data suggest that RPA-coated ssDNA is the critical structure at sites of DNA damage that recruits the ATR-ATRIP complex and facilitates its recognition of substrates for phosphorylation and the initiation of checkpoint signaling.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Comparative activity of ADP-ribosylation factor family members in the early steps of coated vesicle formation on rat liver Golgi membranes.

We have compared the abilities of mammalian ADP-ribosylation factors (ARFs) 1, 5, and 6 and Saccharomyces cerevisiae ARF2 to serve as substrates for the rat liver Golgi membrane guanine nucleotide exchange factor and to initiate the formation of clathrin- and coatomer protein (COP) I-coated vesicles on these membranes. While Golgi membranes stimulated the exchange of GTPgammaS for GDP on all of the ARFs tested, mammalian ARF1 was the best substrate, with an apparent Km of 5 microM. In all cases myristoylation of ARF was required for stimulation. Agents that inhibit the Golgi membrane guanine nucleotide exchange factor (the fungal metabolite brefeldin A and trypsin treatment) selectively inhibited the guanine nucleotide exchange on mammalian ARF1. Taken together, these data indicate that of the ARFs tested, only mammalian ARF1 is activated efficiently by the Golgi guanine nucleotide exchange factor. The other ARFs are activated mainly by another mechanism, possibly phospholipid-mediated. Once activated, all of the membrane-associated, myristoylated ARFs promoted the recruitment of coatomer to about the same extent. Mammalian ARFs 1 and 5 were the most effective in promoting the recruitment of the AP-1 adaptor complex, whereas yeast ARF2 was the least active. These data indicate that the specificity for ARF action on the Golgi membranes is primarily determined by the Golgi guanine nucleotide exchange factor, which has a strong preference for myristoylated mammalian ARF1.

ADP-Ribosylation Factor 1↗

PubMed2004

Direct cell cycle regulation by the fibroblast growth factor receptor (FGFR) kinase through phosphorylation-dependent release of Cks1 from FGFR substrate 2.

Fibroblast growth factors (FGFs) are upstream activators of the mitogen-activated protein kinase pathway and mitogens in a wide variety of cells. However, whether the mitogen-activated protein kinase pathway solely accounts for the induction of cell cycle or antiapoptotic activity of the FGF receptor (FGFR) tyrosine kinase is not clear. Here we report that cell cycle inducer Cks1, which triggers ubiquitination and degradation of p27(Kip1), associates with the unphosphorylated form of FGFR substrate 2 (FRS2), an adaptor protein that is phosphorylated by FGFR kinases and recruits downstream signaling molecules. FGF-dependent activation of FGFR tyrosine kinases induces FRS2 phosphorylation, causes release of Cks1 from FRS2, and promotes degradation of p27(Kip1) in 3T3 cells. Since degradation of p27(Kip1) is a key regulatory step in activation of the cyclin E/A-Cdk complex during the G(1)/S transition of the cell cycle, the results suggest a novel mitogenic pathway whereby FGF and other growth factors that activate FRS2 directly activate cyclin-dependent kinases.

3T3 Cells↗

PubMed2006

Dynamic regulation of pro- and anti-inflammatory cytokines by MAPK phosphatase 1 (MKP-1) in innate immune responses.

Engagement of Toll-like receptors (TLRs) on macrophages leads to activation of the mitogen-activated protein kinases (MAPKs), which contribute to innate immune responses. MAPK activity is regulated negatively by MAPK phosphatases (MKPs). MKP-1, the founding member of this family of dual-specificity phosphatases, has been implicated in regulating lipopolysaccharide (LPS) responses, but its role in TLR-mediated immune responses in vivo has not been defined. Here, we show that mice deficient in MKP-1 were highly susceptible to endotoxic shock in vivo, associated with enhanced production of proinflammatory cytokines TNF-alpha and IL-6 and an anti-inflammatory cytokine, IL-10. We further examined the regulation and function of MKP-1 in macrophages, a major cell type involved in endotoxic shock. MKP-1 was transiently induced by TLR stimulation through pathways mediated by both myeloid differentiation factor 88 (MyD88) and TIR domain-containing adaptor inducing IFN-beta (TRIF). MKP-1 deficiency led to sustained activation of p38 MAPK and c-Jun N-terminal kinase (JNK) in LPS-treated macrophages. In response to TLR signals, MKP-1-deficient macrophages produced 5- to 10-fold higher IL-10, which could be blocked by a p38 MAPK inhibitor. Thus, p38 MAPK plays a critical role in mediating IL-10 synthesis in TLR signaling. TNF-alpha was found to be more abundant in MKP-1-deficient macrophages within 2 hours of TLR stimulation, but its production was rapidly down-regulated by IL-10. Our studies demonstrate that MKP-1 attenuates the activities of p38 MAPK and JNK to regulate both pro- and anti-inflammatory cytokines in TLR signaling. These results highlight the complex mechanisms by which the MAPKs regulate innate immunity.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Structure-activity relationships of small phosphopeptides, inhibitors of Grb2 SH2 domain, and their prodrugs.

To develop potential antitumor agents directed toward HER2/ErbB2 overexpression in cancer, we have designed inhibitors of the recognition between the phosphotyrosine of the receptor and the SH2 domain of the adaptor protein Grb2. In the first part of the paper, we report the synthesis of mimetics of the constrained (alpha-Me)phosphotyrosine residue such as (alpha-Me)-4-phosphonomethylphenylalanine (-CH2PO3H2), (alpha-Me) 4-phosphonodifluoromethylphenylalanine (-CF2PO3H2), and (alpha-Me)-4-phosphonophenylalanine (-PO3H2). The incorporation of these residues in the mAZ-pTyr-Xaa-Asn-NH2 series provided compounds with very high affinity for the Grb2 SH2 domain, in the 10(-8)-10(-9) range of Kd values. These compounds behave as potent antagonists of the Grb2-Shc interaction. Our results highlight the importance of the doubly negative charge borne by the pY + 1 amino acid in accordance with the interactions observed in the complex crystallized between mAZ-pTyr-(alphaMe)pTyr-Asn-NH2 and the Grb2 SH2 domain. mAZ-pTyr-(alphaMe)pTyr-Asn-NH2 was derivatized as the S-acetyl thioester (SATE) of the phosphotyrosine residues, and its surrogates provided prodrugs with very potent antiproliferative activity on cells overexpressing HER2/ErbB2, with ED50 values amounting to 0.1 microM. Finally a new prodrug is put forth under the form of a monobenzyl ester of phosphate group that is as active as and much easier to synthesize than SATE prodrugs. These compounds show promising activity for further testing on in vivo models.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Regulatory effects of mammalian target of rapamycin-activated pathways in type I and II interferon signaling.

The mechanisms regulating initiation of mRNA translation for the generation of protein products that mediate interferon (IFN) responses are largely unknown. We have previously shown that both Type I and II IFNs engage the mammalian target of rapamycin (mTOR), resulting in downstream phosphorylation and deactivation of the translational repressor 4E-BP1 (eIF4E-binding protein 1). In the current study, we provide direct evidence that such regulation of 4E-BP1 by IFNalpha or IFNgamma results in sequential dissociation of 4E-BP1 from eukaryotic initiation factor-4E and subsequent formation of a functional complex between eukaryotic initiation factor-4E and eukaryotic initiation factor-4G, to allow initiation of mRNA translation. We also demonstrate that the induction of key IFNalpha- or IFNgamma-inducible proteins (ISG15 (interferon-stimulated gene 15) and CXCL10) that mediate IFN responses are enhanced in 4E-BP1 (4E-BP1(-/-)) knockout MEFs, as compared with wild-type 4E-BP1(+/+) MEFs. On the other hand, IFN-dependent transcriptional regulation of the Isg15 and Cxcl10 genes is intact in the absence of 4E-BP1, as determined by real time reverse transcriptase-PCR assays and promoter assays for ISRE and GAS, establishing that 4E-BP1 plays a selective negative regulatory role in IFN-induced mRNA translation. Interestingly, the induction of expression of ISG15 and CXCL10 proteins by IFNs was also strongly enhanced in cells lacking expression of the tuberin (TSC2(-/-)) or hamartin (TSC1(-/-)) genes, consistent with the known negative regulatory effect of the TSC1-TSC2 complex on mTOR activation. In other work, we demonstrate that the induction of an IFN-dependent antiviral response is strongly enhanced in cells lacking expression of 4E-BP1 and TSC2, demonstrating that these elements of the IFN-activated mTOR pathway exhibit important regulatory effects in the generation of IFN responses. Taken altogether, our data suggest an important role for mTOR-dependent pathways in IFN signaling and identify 4E-BP1 and TSC1-TSC2 as key components in the generation of IFN-dependent biological responses.

Adaptor Proteins, Signal Transducing↗