Evidence of a tendency to self-association of the transmembrane domain of ErbB-2 in fluid phospholipid bilayers

The transmembrane domains of receptor tyrosine kinases are single-span helical structures suggested to participate directly in the formation of side-to-side receptor homodimers/homooligomers that modulate signal transduction. Transmembrane peptides from the class I receptor tyrosine kinase, ErbB-2, were examined directly by 2H NMR spectroscopy as a means of following such phenomena under the dynamic conditions that characterize fluid, fully hydrated bilayers of natural phospholipids. Appropriate peptides were expressed as 50-mers, containing the transmembrane domain of ErbB-2 plus contiguous stretches of amino acids from the cytoplasmic and extracellular domains. Deuterium probes were incorporated in place of 1H at a site within the helical intramembranous portion (the -CH3 side chain of Ala657), and the peptides were assembled into bilayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) for study. An analogous peptide corresponding to the oncogenic variant characterized by a Val659-->Glu point mutation was also examined. At high peptide concentration, prominent spectral features could be assigned to rapidly rotating transmembrane monomers and to large oligomers rotating very slowly relative to a time scale of 10(-5) s. As peptide concentration was lowered, the latter feature was greatly reduced, and an additional population of mobile species became identifiable, consistent with the presence of homodimers and/or small oligomers. The defined nature of these latter spectral features suggests that preferred interaction sites exist on the peptides. The appearance of similar phenomena in the case of transmembrane peptides from both wild-type ErbB-2 and the transforming mutant argues for the involvement of additional factors in signal modulation, such as limitations normally imposed by the missing extramembranous portions.     

Structural implications of a Val-->Glu mutation in transmembrane peptides from the EGF receptor.

Certain specific point mutations within the transmembrane domains of class I receptor tyrosine kinases are known to induce altered behavior in the host cell. An internally controlled pair of peptides containing the transmembrane portion of the human epidermal growth factor (EGF) receptor (ErbB-1) was examined in fluid, fully hydrated lipid bilayers by wide-line 2H-NMR for insight into the physical basis of this effect. One member of the pair encompassed the native transmembrane sequence from ErbB-1, while in the other the valine residue at position 627 was replaced by glutamic acid to mimic a substitution that produces a transformed phenotype in cells. Heteronuclear probes having a defined relationship to the peptide backbone were incorporated by deuteration of the methyl side chains of natural alanine residues. 2H-NMR spectra were recorded in the range 35 degrees C to 65 degrees C in membranes composed of 1-palmitoyl-2-oleoyl phosphatidylcholine. Narrowed spectral components arising from species rotating rapidly and symmetrically within the membrane persisted to very high temperature and appeared to represent monomeric peptide. Probes at positions 623 and 629 within the EGF receptor displayed changes in quadrupole splitting when Val(627) was replaced by Glu, while probes downstream at position 637 were relatively unaffected. The results demonstrate a measurable spatial reorientation in the region of the 5-amino acid motif (residues 624-628) often suggested to be involved in side-to-side interactions of the receptor transmembrane domain. Spectral changes induced by the Val-->Glu mutation in ErbB-1 were smaller than those induced by the analogous oncogenic mutation in the homologous human receptor, ErbB-2 (Sharpe, S., K. R. Barber, and C. W. M. Grant. 2000. Biochemistry. 39:6572-6580). Quadrupole splittings at probe sites examined were only modestly sensitive to temperature, suggesting that each transmembrane peptide behaved as a motionally ordered unit possessing considerable conformational stability.     

The transmembrane domain of the oncogenic mutant ErbB-2 receptor: a structure obtained from site-specific infrared dichroism and molecular dynamics

ErbB-2 is a member of the family of epidermal growth factor receptors, which shows an oncogenic mutation in the rat gene neu, Val664Glu in the transmembrane domain that causes permanent dimerisation and subsequently leads to uncontrollable cell division and tumour formation. We have obtained the alpha-helical structure of the mutant transmembrane domain dimer experimentally with site-specific infrared dichroism (SSID) based on six transmembrane peptides with 13C18O carbonyl group-labelled residues. The derived orientational data indicate a local helix tilt ranging from 28(+/-6) degrees to 22(+/-4) degrees. Altogether using orientational constraints from SSID and experimental alpha-helical constraints while performing a systematic conformational search including molecular dynamics simulation in a lipid bilayer, we have obtained a unique experimentally defined atomic structure. The resulting structure consists of a right handed alpha-helical bundle with the residues Ile659, Val663, Leu667, Ile671, Val674 and Leu679 in the dimerisation interface. The right-handed bundle is in contrast to the left-handed structures obtained in previous modelling efforts. In order to facilitate tight helical packing, the spacious Glu664 residues do not interact directly but with water molecules that enter the bilayer.     

A novel homodimeric geranyl diphosphate synthase from the orchid Phalaenopsis bellina lacking a DD(X)2-4D motif

Geranyl diphosphate (GDP) is the precursor of monoterpenes, which are the major floral scent compounds in Phalaenopsis bellina . The cDNA of P. bellina GDP synthase ( PbGDPS ) was cloned, and its sequence corresponds to the second Asp-rich motif (SARM), but not to any aspartate-rich (Asp-rich) motif. The recombinant PbGDPS enzyme exhibits dual prenyltransferase activity, producing both GDP and farnesyl diphosphate (FDP), and a yeast two-hybrid assay and gel filtration revealed that PbGDPS was able to form a homodimer. Spatial and temporal expression analyses showed that the expression of PbGDPS was flower specific, and that maximal PbGDPS expression was concomitant with maximal emission of monoterpenes on day 5 post-anthesis. Homology modelling of PbGDPS indicated that the Glu-rich motif might provide a binding site for Mg²⁺ and catalyze the formation of prenyl products in a similar way to SARM. Replacement of the key Glu residues with alanine totally abolished enzyme activity, whereas their mutation to Asp resulted in a mutant with two-thirds of the activity of the wild-type protein. Phylogenetic analysis indicated that plant GDPS proteins formed four clades: members of both GDPS-a and GDPS-b clades contain Asp-rich motifs, and function as homodimers. In contrast, proteins in the GDPS-c and GDPS-d clades do not contain Asp-rich motifs, but although members of the GDPS-c clade function as heterodimers, PbGDPS, which is more closely related to the GDPS-c clade proteins than to GDPS-a and GDPS-b proteins, and is currently the sole member of the GDPS-d clade, functions as a homodimer.     

Conformation of the transmembrane domain of the c-erbB-2 oncogene-encoded protein in its monomeric and dimeric states

The human c-erbB-2 oncogene is homologous to the ratneu oncogene, both encoding transmembrane growth factor receptors. Overexpression and point mutations in the transmembrane domain of the encoded proteins in both cases have been implicated in cell transformation and carcinogenesis. In the case of theneu protein, it has been proposed that these effects are mediated by conformational preferences for anα-helix in the transmembrane domain, which facilitates receptor dimerization, an important step in the signal transduction process. To examine whether this is the case for c-erbB-2 as well, we have used conformational energy analysis to determine the preferred three-dimensional structures for the transmembrane domain of the c-erbB-2 protein from residues 650 to 668 with Val (nontransforming) and Glu (transforming) at position 659. The global minimum energy conformation for the Val-659 peptide from the normal, nontransforming protein was found to contain several bends, whereas the global minimum energy conformation for Glu-659 peptide from the mutant, transforming protein was found to beα-helical. Thus, the difference in conformational preferences for these transmembrane domains may explain the difference in transforming ability of these proteins. The presence of higher-energyα-helical conformations for the transmembrane domain from the normal Val-659 protein may provide an explanation for the presence of a transforming effect from overexpression of c-erbB-2. In addition, docking of the oncogenic sequences in theirα-helical and bend conformations shows that the all-α-helical dimer is clearly favored energetically over the bend dimer.     

Structure prediction of the dimeric neu/ErbB-2 transmembrane domain from multi-nanosecond molecular dynamics simulations

Dimerization of the neu/ErbB-2 receptor tyrosine kinase is a necessary but not a sufficient step for signaling. Despite the efforts expended to identify the molecular interactions responsible for receptor-receptor contacts and particularly those involving the transmembrane domain, structural details are still unknown. In this work, molecular dynamics simulations of the helical transmembrane domain (TM) of neu and ErbB-2 receptors are used to predict their dimer structure both in the wild and oncogenic forms. A global conformational search method, applied to define the best orientations of parallel helices, showed an energetically favorable configuration with the specific mutation site within the interface, common for both the nontransforming and the transforming neu/ErbB-2 TM dimers. Starting from this configuration, a total of 10 simulations, about 1.4 ns each, performed in vacuum, without any constraints, show that the two helices preferentially wrap in left-handed interactions with a packing angle at about 20°. The resulting structures are nonsymmetric and the hydrogen bond network analysis shows that helices experience π local distortions that facilitate inter-helix hydrogen bond interactions and may result in a change in the helix packing, leading to a symmetric interface. For the mutated sequences, we show that the Glu side chain interacts directly with its cognate or with carbonyl groups of the facing backbone. We show that the connectivity between interfacial residues conforms to the knobs-into-holes packing mode of transmembrane helices. The dimeric interface described in our models is discussed with respect to mutagenesis studies. Received: 12 March 1999 / Revised version: 23 August 1999 / Accepted: 23 August 1999     

Multiple ErbB-2/Neu Phosphorylation Sites Mediate Transformation through Distinct Effector Proteins

Amplification of the type I receptor tyrosine kinase ErbB-2 (HER2/Neu) is observed in 20-30% of human mammary carcinomas, correlating with a poor clinical prognosis. We have previously demonstrated that four (Tyr(1144), Tyr(1201), Tyr(1226/1227), or Tyr(1253)) of the five known Neu/ErbB-2 autophosphorylation sites can independently mediate transforming signals. The transforming potential of at least two of these autophosphorylation sites (Tyr(1144) and Tyr(1226/1227)) has been further correlated with their ability to associate with Grb2 and Shc adapter proteins, respectively. To confirm the specificity of these interactions, we have created a series of second site mutants in these phosphorylation sites. The results showed that Grb2 recruitment to site 1144 is absolutely required for transforming signal from this autophosphorylation site, whereas association of Shc-mediated transformation is dependent on conservation of the NPXY motif spanning Tyr(1227). A stretch of amino acid identity around tyrosines 1201 (ENPEYLTP)and 1253 (ENPEYLDL) exists, and mutation of key residues within this motif reveals distinct requirements for an intact protein tyrosine-binding protein (NPXY). We show that DOK-R, a protein tyrosine-binding site-containing protein implicated in Ras signaling, interacts with Neu/ErbB-2 at Tyr(1253) as do two unidentified proteins, p150 and p34, the latter correlating with transformation. Together these data argue that ErbB-2/Neu is capable of mediating transformation through distinct effector pathways.     

Molecular modeling of c-erbB2 receptor dimerization: Coiled-coil structure of wild and oncogenic transmembrane domains—Stabilization by interhelical hydrogen bonds in the oncogenic form

Dimerization models of c-erbB2 transmembrane domains (Leu651-Ile675) are studied by molecular mechanics and molecular dynamics simulations. Both wild and Glu mutated transmembrane helices exhibit the same relative orientation for favorable associations and dimerize preferentially in left-handed coiled-coil structures. The mutation point 659 belongs to the interfacing residues, and in the transforming domain, symmetric hydrogen bonds between Glu carboxylic groups stabilize the dimeric structure. The same helix packing found for the wild dimers, except side-chain—side-chain hydrogen bonds, suggests that the transmembrane domains dimerize according to similar process. Structural and energetical characterization of the models are presented. © 1997 John Wiley & Sons, Inc. Biopoly 42: 157–168, 1997     

Ectodomain cleavage of ErbB-4: characterization of the cleavage site and m80 fragment

Ectodomain cleavage of the ErbB-4 receptor tyrosine kinase generates a membrane-associated fragment of 80 kDa (m80) that has been subjected to N-terminal sequencing. The sequence obtained shows that the N terminus of this fragment begins with Ser-652 of ErbB-4. When a 12-residue peptide corresponding to ErbB-4 residues 646-657 was incubated with recombinant tumor necrosis factor-alpha-converting enzyme, fragments representing residues 646-651 and 652-657 were obtained. These data indicate that ectodomain cleavage of ErbB-4 occurs between His-651 and Ser-652, placing the cleavage site within the ectodomain stalk region approximately 8 residues prior to the transmembrane domain. Several experiments have characterized other aspects of the m80 ErbB-4 fragment. Inhibition of ErbB-4 tyrosine kinase activity with pan-ErbB tyrosine kinase inhibitors indicates that kinase activity is stringently required for heregulin-dependent, but not 12-O-tetradecanoylphorbol-13-acetate-induced, ErbB-4 ectodomain cleavage and formation of the m80 fragment. When the m80 ErbB-4 fragment is generated by cell treatment with heregulin or 12-O-tetradecanoylphorbol-13-acetate, the fragment associates with intact ErbB-2. However, this fragment does not associate with the intact ErbB-4 molecule.     

Human ErbB-2 (Her-2) transgenic mice: a model system for testing Her-2 based vaccines

Her-2 transgenic (Tg) mice were generated with wild-type human c-ErbB-2 (Her-2) under the whey acidic protein promoter. They are tolerant to Her-2 and appropriate for testing Her-2 vaccines. The expression of transmembrane ErbB-2 from the whey acidic protein-Her-2 cassette and its up-regulation by insulin and hydrocortisone was verified by in vitro transfection. The transgene cassette was microinjected into fertilized eggs from B6C3 (C3H x C57BL/6) females mated with B6C3 males. Transgene-positive mice were backcrossed onto C57BL/6 mice. Human ErbB-2 was expressed in the secretory mammary epithelia during pregnancy and lactation and expressed constitutively in the Bergman glia cells within the molecular layer of the cerebellum. Overt, neoplastic transformation was not detected in any tissue examined. Tolerance to Her-2 was demonstrated by inoculating mice with a syngenic tumor expressing high levels of human ErbB-2. Tumors grew exclusively in Her-2 Tg mice without inducing an Ab response, while the nontransgenic littermates remained tumor free for 10 mo and mounted a robust anti-ErbB-2 Ab response. When immunized five times with plasmid DNA encoding secErbB-2 and GM-CSF, respectively, approximately 33% of the Her-2 Tg mice rejected a lethal challenge of EL-4/E2 tumor cells, whereas all immunized littermates rejected the tumor. Therefore, Her-2 Tg mice express human ErbB-2 in the brain and mammary gland and demonstrated tolerance to ErbB-2 which was partially overcome by DNA vaccination. The breakable tolerance of Her-2 Tg mice resembles that in human and these mice are particularly suited for testing human ErbB-2 based vaccines.     

Influence of a Mutation in the Transmembrane Domain of the pl85c-erbB2 Oncogene-Encoded Protein Studied by Molecular Dynamics Simulations

Abstract

The c-erbB2 proto-oncogene encodes for a protein of 185kDa (p 185) which becomes transforming upon the Val→-Glu transmembrane amino acid substitution. The transforming ability seems to be due to a substitution-resulting constitutive activation of the tyrosine kinase cytosolic domain of the protein. These observations prompted us to evaluate the structural and dynamical behavior of the transmembrane region of the wild and transforming p 185 protein in order to understand the role of this region in the transduction mechanism. 160 ps molecular dynamics simulations in vacuo have been performed on two peptides corresponding to the sequence [651-679] of p 185^c-erbB2 protein and its transforming mutant Val⁶⁵⁹→Glu⁶⁵⁹. These two sequences include the transmembrane domain and are initially postulated to be in an α- helix conformation. Noticeable differences in the flexibility of the two peptides are shown. The nontransforming sequence seems rather flexible and several conformational changes are detected at the junction of the mutation point [658-659] and at position Val⁶⁶⁵-Val⁶⁶⁶ during the 160 ps simulations. On the contrary, no transitions were observed for the mutated sequence which adopts a stable α-helix conformation. This difference in flexibility could be hypothesized as a factor involved in the regulation of the tyrosine kinase activity of 185^c-erbB2     

特超强毒648株马立克氏病病毒的囊膜糖蛋白gE基因的克隆和序列比较

鸡马立克氏病毒特超强毒 (vv+MDV) 648株的囊膜糖蛋白 gE基因经PCR扩增并克隆入pUC1 8载体。 648株gE基因经核酸序列分析测定 ,全长为 1 494碱基。所编码的蛋白具有跨膜糖蛋白的一些特征。它含有 8个潜在的糖基化位点、N端有一段疏水区 (1～ 1 9aa)所构成的信号肽、C端有一段疏水区 (391～ 41 9aa)所构成的膜锚着序列。经 648株 (vv+MDV)和马立克氏病毒强毒 (vMDV)GA株的 gE相比较 ,在MDV血清 1型中 gE是较为保守的 ,二者仅有 2个核苷酸的差异 (第 51 2位、第 1 472位 ) ,并导致了有二个相应的氨基酸的改变 (第1 71位Leu/Pro、第 491位Arg/Lys)。     

Role of the ErbB-4 carboxyl terminus in gamma-secretase cleavage

The ErbB-4 receptor tyrosine kinase has a PDZ domain recognition motif at its carboxyl terminus. The first step in ErbB-4 proteolytic processing is a metalloprotease-dependent cleavage of the receptor ectodomain, which is not influenced by deletion of this motif. Metalloprotease cleavage of ErbB-4 produces a membrane-associated 80-kDa fragment that is a substrate for subsequent gamma-secretase cleavage, which releases the cytoplasmic domain from the membrane and allows nuclear translocation of this fragment. Deletion of the PDZ domain recognition motif does abrogate the gamma-secretase cleavage of ErbB-4. The wild-type 80-kDa ErbB-4 fragment forms an association complex with presenilin, thought to be the catalytic moiety of gamma-secretase activity. However, this association is significantly impaired by loss of the PDZ domain recognition motif from ErbB-4. Deletion of this ErbB-4 motif prevents the nuclear localization of the ErbB-4 cytoplasmic domain. Data also show that the basal cleavage of wild-type ErbB-4 by this proteolytic system can produce a sufficient level of ErbB-4 processing to negatively influence cell growth and that loss of the PDZ domain recognition motif abrogates this response.     

Two motifs within a transmembrane domain, one for homodimerization and the other for heterodimerization

Protein assembly is a critical process involved in a wide range of cellular events and occurs through extracellular and/or transmembrane domains (TMs). Previous studies demonstrated that a GXXXG motif is crucial for homodimer formation. Here we selected the TMs of ErbB1 and ErbB2 as a model since these receptors function both as homodimers and as heterodimers. Both TMs contain two GXXXG-like motifs located at the C and N termini. The C-terminal motifs were implicated previously in homodimer formation, but the role of the N-terminal motifs was not clear. We used the ToxR system and expressed the TMs of both ErbB1 and ErbB2 containing only the N-terminal GXXXG motifs. The data revealed that the ErbB2 but not the ErbB1 construct formed homodimers. Importantly, a synthetic ErbB1 TM peptide was able to form a heterodimer with ErbB2, by displacing the ErbB2 TM homodimer. The specificity of the interaction was demonstrated by using three controls: (i) Two single mutations within the GXXXG-like motif of the ErbB1 peptide reduced or preserved its activity, in agreement with similar mutations in glycophorin A. (ii) A TM peptide of the bacterial Tar receptor did not assemble with the ErbB2 construct. (iii) The ErbB1 peptide had no effect on the dimerization of a construct containing the TM-1 domain of the Tar receptor. Fluorescence microscopy demonstrated that all the peptides localized on the membrane. Furthermore, incubation with the peptides had no effect on bacterial growth and protein expression levels. Our results suggest that the N-terminal GXXXG-like motif of the ErbB1 TM plays a role in heterodimerization with the ErbB2 transmembrane domain. To our knowledge, this is the first demonstration of a transmembrane domain with two distinct recognition motifs, one for homodimerization and the other for heterodimerization.     

Specific locations of hydrophilic amino acids in constructed transmembrane ligands of the platelet-derived growth factor beta receptor

The 44 amino acid E5 transmembrane protein is the primary oncogene product of bovine papillomavirus. Homodimers of the E5 protein activate the cellular PDGF beta receptor tyrosine kinase by binding to its transmembrane domain and inducing receptor dimerization, resulting in cellular transformation. To investigate the role of transmembrane hydrophilic amino acids in receptor activation, we constructed a library of dimeric small transmembrane proteins in which 16 transmembrane amino acids of the E5 protein were replaced with random, predominantly hydrophobic amino acids. A low level of hydrophilic amino acids was encoded at each of the randomized positions, including position 17, which is an essential glutamine in the wild-type E5 protein. Library proteins that induced transformation in mouse C127 cells stably bound and activated the PDGF beta receptor. Strikingly, 35% of the transforming clones had a hydrophilic amino acid at position 17, highlighting the importance of this position in activation of the PDGF beta receptor. Hydrophilic amino acids in other transforming proteins were found adjacent to position 17 or at position 14 or 21, which are in the E5 homodimer interface. Approximately 22% of the transforming proteins lacked hydrophilic amino acids. The hydrophilic amino acids in the transforming clones appear to be important for driving homodimerization, binding to the PDGF beta receptor, or both. Interestingly, several of the library proteins bound and activated PDGF beta receptor transmembrane mutants that were not activated by the wild-type E5 protein. These experiments identified transmembrane proteins that activate the PDGF beta receptor and revealed the importance of hydrophilic amino acids at specific positions in the transmembrane sequence. Our identification of transformation-competent transmembrane proteins with altered specificity suggests that this approach may allow the creation and identification of transmembrane proteins that modulate the activity of a variety of receptor tyrosine kinases.     

Epidermal growth factor contains both positive and negative determinants for interaction with ErbB-2/ErbB-3 heterodimers

Epidermal growth factor (EGF) and transforming growth factor (TGF)-alpha are potent activators of the ErbB-1 receptor, but, unlike TGF-alpha, EGF is also a weak activator of ErbB-2/ErbB-3 heterodimers. To understand the specificity of EGF-like growth factors for binding to distinct ErbB members, we used EGF/TGF-alpha chimeras to examine the requirements for ErbB-2/ErbB-3 activation. Here we show that in contrast to these two wild-type ligands, distinct EGF/TGF-alpha chimeras are potent activators of ErbB-2/ErbB-3 heterodimers. On the basis of differences in the potency of these various chimeras, specific residues in the linear N-terminal region and the so-called B-loop of these ligands were identified to be involved in interaction with ErbB-2/ErbB-3. A chimera consisting of human EGF sequences with the linear N-terminal region of human TGF-alpha was found to be almost as potent as the natural ligand neuregulin (NRG)-1beta in activating 32D cells expressing ErbB-2/ErbB-3 and human breast cancer cells. Binding studies revealed that this chimera, designated T1E, has high affinity for ErbB-2/ErbB-3 heterodimers, but not for ErbB-3 alone. Subsequent exchange studies revealed that introduction of both His2 and Phe3 into the linear N-terminal region was already sufficient to make EGF a potent activator of ErbB-2/ErbB-3 heterodimers, indicating that these two amino acids contribute positively to this receptor binding. Analysis of the B-loop revealed that Leu26 in EGF facilitates interaction with ErbB-2/ErbB-3 heterodimers, while the equivalent Glu residue in TGF-alpha impairs binding. Since all EGF/TGF-alpha chimeras tested have maintained high binding affinity for ErbB-1, it is concluded that the diversity of the ErbB signaling network is determined by specific amino acids that facilitate binding to one receptor member, in addition to residues that impede binding to other ErbB family members.     

mTOR complex 2 (mTORC2) controls hydrophobic motif phosphorylation and activation of serum- and glucocorticoid-induced protein kinase 1 (SGK1)

SGK1 (serum- and glucocorticoid-induced protein kinase 1) is a member of the AGC (protein kinase A/protein kinase G/protein kinase C) family of protein kinases and is activated by agonists including growth factors. SGK1 regulates diverse effects of extracellular agonists by phosphorylating regulatory proteins that control cellular processes such as ion transport and growth. Like other AGC family kinases, activation of SGK1 is triggered by phosphorylation of a threonine residue within the T-loop of the kinase domain and a serine residue lying within the C-terminal hydrophobic motif (Ser(422) in SGK1). PDK1 (phosphoinositide-dependent kinase 1) phosphorylates the T-loop of SGK1. The identity of the hydrophobic motif kinase is unclear. Recent work has established that mTORC1 [mTOR (mammalian target of rapamycin) complex 1] phosphorylates the hydrophobic motif of S6K (S6 kinase), whereas mTORC2 (mTOR complex 2) phosphorylates the hydrophobic motif of Akt (also known as protein kinase B). In the present study we demonstrate that SGK1 hydrophobic motif phosphorylation and activity is ablated in knockout fibroblasts possessing mTORC1 activity, but lacking the mTORC2 subunits rictor (rapamycin-insensitive companion of mTOR), Sin1 (stress-activated-protein-kinase-interacting protein 1) or mLST8 (mammalian lethal with SEC13 protein 8). Furthermore, phosphorylation of NDRG1 (N-myc downstream regulated gene 1), a physiological substrate of SGK1, was also abolished in rictor-, Sin1- or mLST8-deficient fibroblasts. mTORC2 immunoprecipitated from wild-type, but not from mLST8- or rictor-knockout cells, phosphorylated SGK1 at Ser(422). Consistent with mTORC1 not regulating SGK1, immunoprecipitated mTORC1 failed to phosphorylate SGK1 at Ser(422), under conditions which it phosphorylated the hydrophobic motif of S6K. Moreover, rapamycin treatment of HEK (human embryonic kidney)-293, MCF-7 or HeLa cells suppressed phosphorylation of S6K, without affecting SGK1 phosphorylation or activation. The findings of the present study indicate that mTORC2, but not mTORC1, plays a vital role in controlling the hydrophobic motif phosphorylation and activity of SGK1. Our findings may explain why in previous studies phosphorylation of substrates, such as FOXO (forkhead box O), that could be regulated by SGK, are reduced in mTORC2-deficient cells. The results of the present study indicate that NDRG1 phosphorylation represents an excellent biomarker for mTORC2 activity.     

Characterization of the GXXXG motif in the first transmembrane segment of Japanese encephalitis virus precursor membrane (prM) protein

The interaction between prM and E proteins in flavivirus-infected cells is a major driving force for the assembly of flavivirus particles. We used site-directed mutagenesis to study the potential role of the transmembrane domains of the prM proteins of Japanese encephalitis virus (JEV) in prM-E heterodimerization as well as subviral particle formation. Alanine insertion scanning mutagenesis within the GXXXG motif in the first transmembrane segment of JEV prM protein affected the prM-E heterodimerization; its specificity was confirmed by replacing the two glycines of the GXXXG motif with alanine, leucine and valine. The GXXXG motif was found to be conserved in the JEV serocomplex viruses but not other flavivirus groups. These mutants with alanine inserted in the two prM transmembrane segments all impaired subviral particle formation in cell cultures. The prM transmembrane domains of JEV may play importation roles in prM-E heterodimerization and viral particle assembly.     

Conformation of the transmembrane domain of the c-erbB-2 oncogene-encoded protein in its monomeric and dimeric states

The human c-erbB-2 oncogene is homologous to the ratneu oncogene, both encoding transmembrane growth factor receptors. Overexpression and point mutations in the transmembrane domain of the encoded proteins in both cases have been implicated in cell transformation and carcinogenesis. In the case of theneu protein, it has been proposed that these effects are mediated by conformational preferences for an-helix in the transmembrane domain, which facilitates receptor dimerization, an important step in the signal transduction process. To examine whether this is the case for c-erbB-2 as well, we have used conformational energy analysis to determine the preferred three-dimensional structures for the transmembrane domain of the c-erbB-2 protein from residues 650 to 668 with Val (nontransforming) and Glu (transforming) at position 659. The global minimum energy conformation for the Val-659 peptide from the normal, nontransforming protein was found to contain several bends, whereas the global minimum energy conformation for Glu-659 peptide from the mutant, transforming protein was found to be-helical. Thus, the difference in conformational preferences for these transmembrane domains may explain the difference in transforming ability of these proteins. The presence of higher-energy-helical conformations for the transmembrane domain from the normal Val-659 protein may provide an explanation for the presence of a transforming effect from overexpression of c-erbB-2. In addition, docking of the oncogenic sequences in their-helical and bend conformations shows that the all--helical dimer is clearly favored energetically over the bend dimer.     

Dimer interface of transmembrane domains for neu/erbB-2 receptor dimerization and transforming activation: a model revealed by molecular dynamics simulations

The specific point mutation Val-->Glu664 within the transmembrane domain of the neu/erbB-2 receptor is associated with increased receptor dimerization and increased receptor tyrosine kinase activity resulting in malignant transformation of cells. It is well established that Glu and residues in proximity are necessary for receptor dimerization but many studies suggest that other intramembrane constraints, not yet elucidated, are determinant for transformation. In this work, we investigated dimer models both to understand the structural role of the Glu mutation in the transmembrane domain association and to determine helix-helix contacts required for oncogenic transformation. Different types of helix-helix association based on data resulting from Cys mutational studies of the full wild receptor and spectroscopic data of transmembrane neu peptides have been explored by molecular dynamics simulations. The study leads to propose a model for the dimeric association of the transmembrane domains of the oncogenic neu receptor showing left-handed interactions of the two helices stabilized by symmetrical hydrogen bonding interactions involving the Glu side chain on one helix and the facing carbonyl of Ala661 on the second helix. Contacting residues observed in the symmetric interface explain the transforming activity or the non transforming activity of many neu mutants. Moreover the left-handed coiled coil structure is fully consistent with recent results proving the role of rotational linkage of the transmembrane domain with the kinase domain. Comparison between the predicted dimer model and those presumed from experiments strongly suggests helix flexibility in the extracellular juxtamembrane region.