Reassembly of JIP1 Scaffold Complex in JNK MAP Kinase Pathway Using Heterologous Protein Interactions

Formation of signaling protein complexes is crucial for proper signal transduction. Scaffold proteins in MAP kinase pathways are thought to facilitate complex assembly, thereby promoting efficient and specific signaling. To elucidate the assembly mechanism of scaffold complexes in mammals, we attempted to rationally rewire JIP1-dependent JNK MAP kinase pathway via alternative assembly of JIP1 complex. When JIP1-JNK docking interaction in the complex was replaced with heterologous protein interaction domains, such as PDZ domains and JNK-binding domains, a functional scaffold complex was reconstituted, and JNK signaling was rescued. Reassembly of JIP1 complex using heterologous protein interactions was sufficient for restoring of JNK MAP kinase pathway to induce signaling responses, including JNK activation and cell death. These results suggest a simple yet modular mechanism for JIP1 scaffold assembly in mammals.     

Assembly of oligomeric death domain complexes during Toll receptor signaling

The Drosophila Toll receptor is activated by the endogenous protein ligand Sp?tzle in response to microbial stimuli in immunity and spatial cues during embryonic development. Downstream signaling is mediated by the adaptor proteins Tube, the kinase Pelle, and the Drosophila homologue of myeloid differentiation primary response protein (dMyD88). Here we have characterized heterodimeric (dMyD88-Tube) and heterotrimeric (dMyD88-Tube-Pelle) death domain complexes. We show that both the heterodimeric and heterotrimeric complexes form kidney-shaped structures and that Tube is bivalent and has separate high affinity binding sites for dMyD88 and Pelle. Additionally we found no interaction between the isolated death domains of Pelle and dMyD88. These results indicate that the mode of assembly of the heterotrimeric dMyD88-Tube-Pelle complex downstream of the activated Toll receptor is unique. The measured dissociation constants for the interaction between the death domains of dMyD88 and Tube and of Pelle and a preformed dMyD88-Tube complex are used to propose a model of the early postreceptor events in Drosophila Toll receptor signaling.     

The mode of bone morphogenetic protein (BMP) receptor oligomerization determines different BMP-2 signaling pathways.

Bone morphogenetic proteins (BMPs) are multifunctional proteins regulating cell growth, differentiation, and apoptosis. BMP-2 signals via two types of receptors (BRI and BRII) that are expressed at the cell surface as homomeric as well as heteromeric complexes. Prior to ligand binding, a low but measurable level of BMP-receptors is found in preformed hetero-oligomeric complexes. The major fraction of the receptors is recruited into hetero-oligomeric complexes only after ligand addition. For this, BMP-2 binds first to the high affinity receptor BRI and then recruits BRII into the signaling complex. However, ligand binding to the preformed complex composed of BRII and BRI is still required for signaling, suggesting that it may mediate activating conformational changes. Using several approaches we have addressed the following questions: (i) Are preformed complexes incompetent of signaling in the absence of BMP-2? (ii) Which domains of the BRII receptors are essential for this complex formation? (iii) Are there differences in signals sent from BMP-induced versus preformed receptor complexes? By measuring the activation of Smads, of p38 MAPK and of alkaline phosphatase, we show that the ability of kinase-deficient BRII receptor mutants to inhibit BMP signaling depends on their ability to form heteromeric complexes with BRI. Importantly, a BRII mutant that is incapable in forming preassembled receptor complexes but recruits into a BMP-induced receptor complex does not interfere with the Smad pathway but does inhibit the induction of alkaline phosphatase as well as p38 phosphorylation. These results indicate that signals induced by binding of BMP-2 to preformed receptor complexes activate the Smad pathway, whereas BMP-2-induced recruitment of receptors activates a different, Smad-independent pathway resulting in the induction of alkaline phosphatase activity via p38 MAPK.     

Tumor necrosis factor-alpha induces Bax-Bak interaction and apoptosis, which is inhibited by adenovirus E1B 19K

Tumor necrosis factor (TNF)-alpha-mediated death signaling induces oligomerization of proapoptotic Bcl-2 family member Bax into a high molecular mass protein complex in mitochondrial membranes. Bax complex formation is associated with the release of cytochrome c, which propagates death signaling by acting as a cofactor for caspase-9 activation. The adenovirus Bcl-2 homologue E1B 19K blocks TNF-alpha-mediated apoptosis by preventing cytochrome c release, caspase-9 activation, and apoptosis of virus-infected cells. TNF-alpha induces E1B 19K-Bax interaction and inhibits Bax oligomerization. Oligomerized Bax may form a pore to release mitochondrial proteins, analogous to the homologous pore-forming domains of bacterial toxins. E1B 19K can also bind to proapoptotic Bak, but the functional significance is not known. TNF-alpha signaling induced Bak-Bax interaction and both Bak and Bax oligomerization. E1B 19K was constitutively in a complex with Bak, and blocked the Bak-Bax interaction and oligomerization of both. The TNF-alpha-mediated cytochrome c and Smac/DIABLO release from mitochondria was inhibited by E1B 19K expression in adenovirus-infected cells. Since either Bax or Bak is essential for death signaling by TNF-alpha, the interaction between E1B 19K and both Bak and Bax may be required to inhibit their cooperative or independent oligomerization to release proteins from mitochondria which promote caspase activation and cell death.     

Dynamics of the gp130 cytokine complex: a model for assembly on the cellular membrane

Cytokines of the interleukin-6 (IL-6)-type family all bind to the glycoprotein gp130 on the cell surface and require interaction with two gp130 or one gp130 and another related signal transducing receptor subunit. In addition, some cytokines of this family, such as IL-6, interleukin-11, ciliary neurotrophic factor, neuropoietin, cardiotrophin-1, and cardiotrophin-1-like-cytokine, interact with specific ligand binding receptor proteins. High- and low-affinity binding sites have been determined for these cytokines. So far, however, the stoichiometry of the signaling receptor complexes has remained unclear, because the formation of the cytokine/cytokine-receptor complexes has been analyzed with soluble receptor components in solution, which do not necessarily reflect the situation on the cellular membrane. Consequently, the binding affinities measured in solution have been orders of magnitude below the values obtained with whole cells. We have expressed two gp130 extracellular domains in the context of a Fc-fusion protein, which fixes the receptors within one dimension and thereby restricts the flexibility of the proteins in a fashion similar to that within the plasma membrane. We measured binding of IL-6 and interleukin-b receptor (IL-6R) by means of fluorescence-correlation spectroscopy. For the first time we have succeeded in recapitulating in a cell-free condition the binding affinities and dynamics of IL-6 and IL-6R to the gp130 receptor proteins, which have been determined on whole cells. Our results demonstrate that a dimer of gp130 first binds one IL-6/IL-6R complex and only at higher ligand concentrations does it bind a second IL-6/IL-6R complex. This view contrasts with the current perception of IL-6 receptor activation and reveals an alternative receptor activation mechanism.     

Protein kinase C modulates tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by targeting the apical events of death receptor signaling

We have further examined the mechanism by which phorbol ester-mediated protein kinase C (PKC) activation protects against tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced cytotoxicity. We now report that activation of PKC targets death receptor signaling complex formation. Pre-treatment with 12-O-tetradecanoylphorbol-13-acetate (PMA) led to inhibition of TRAIL-induced apoptosis in HeLa cells, which was characterized by a reduction in phosphatidylserine (PS) externalization, decreased caspase-8 processing, and incomplete maturation and activation of caspase-3. These effects of PMA were completely abrogated by the PKC inhibitor, bisindolylmaleimide I (Bis I), clearly implicating PKC in the protective effect of PMA. TRAIL-induced mitochondrial release of the apoptosis mediators cytochrome c and Smac was blocked by PMA. This, together with the observed decrease in Bid cleavage, suggested that PKC activation modulates apical events in TRAIL signaling upstream of mitochondria. This was confirmed by analysis of TRAIL death-inducing signaling complex formation, which was disrupted in PMA-treated cells as evidenced by a marked reduction in Fas-associated death domain protein (FADD) recruitment, an effect that could not be explained by any change in FADD phosphorylation state. In an in vitro binding assay, the intracellular domains of both TRAIL-R1 and TRAIL-R2 bound FADD: activation of PKC significantly inhibited this interaction suggesting that PKC may be targeting key apical components of death receptor signaling. Significantly, this effect was not confined to TRAIL, because isolation of the native TNF receptor signaling complex revealed that PKC activation also inhibited TNF receptor-associated death domain protein recruitment to TNF-R1 and TNF-induced phosphorylation of IkappaB-alpha. Taken together, these results show that PKC activation specifically inhibits the recruitment of key obligatory death domain-containing adaptor proteins to their respective membrane-associated signaling complexes, thereby modulating TRAIL-induced apoptosis and TNF-induced NF-kappaB activation, respectively.     

Organization and synergistic binding of copine I and annexin A1 on supported lipid bilayers observed by atomic force microscopy

The transduction of signals across the plasma membrane of cells after receptor activation frequently involves the assembly of interacting protein molecules on the cytoplasmic face of the membrane. However, the structural organization and dynamics of the formation of such complexes has not been well defined. In this study atomic force microscopy was used to monitor the assemblies formed in vitro by two classes of calcium-dependent, membrane-binding proteins that participate in the formation of signaling complexes on membranes - the annexins and the copines. When applied to supported lipid bilayers composed of 25% brain phosphatidylserine and 75% dioleyl phosphatidylcholine in the presence of 1 mM Ca²⁺ both human annexin A1 and human copine I bound only to specialized domains that appeared to be 0.5 to 1.0 nm lower than the rest of the bilayer. These domains may be enriched in phosphatidylserine and have a more disordered structure allowing probe penetration. Confinement of the binding of the proteins to these domains may be important in the process of concentrating other signaling proteins bound to the copine or annexin. The binding of the annexin promoted the growth of the domains and created additional binding space for the copine. This may reflect a general ability of annexins to alter membrane structure in such a way that C2 domain-containing proteins like copine can bind. Copine I formed a reticular lattice composed of linear elements approximately 45 nm long on the specialized domains. This lattice might provide a scaffold for the assembly and interaction of copine target proteins in signaling complexes.     

WAVE2 signaling mediates invasion of polarized epithelial cells by Salmonella typhimurium

The bacterial pathogen Salmonella penetrates the intestinal epithelium by inducing its own phagocytosis into epithelial cells. The dramatic reorganization of the actin cytoskeleton required for internalization is driven by bacterial manipulation of host signaling pathways, including activation of the Rho family GTPase Rac1 and subsequent activation of the Arp2/3 complex. However, the mechanisms linking these two events remain poorly understood. Rac1 is thought to promote activation of the Arp2/3 complex through its interaction with suppressor of cAMP receptor/WASP family verprolin-homologous (SCAR/WAVE) family proteins, but this interaction is apparently indirect. Two different Rac1 effectors have been shown to bind WAVE2: IRSp53, the SH3 domain of which binds the WAVE2 proline-rich domain, and PIR121/Sra-1, which forms a pentameric complex containing WAVE, Abi1, Nap1, and HSPC300. However, the extent to which each of these complexes contributes to Arp2/3 complex activation in the context of Salmonella infection is unclear. Here, we show that WAVE2 is necessary for efficient invasion of epithelial cells by Salmonella typhimurium. We found that although Salmonella infection strongly promotes the formation of an IRSp53/WAVE2 complex, IRSp53 is not necessary for bacterial internalization. In contrast, disruption of the PIR121/Nap1/Abi1/WAVE2/HSPC300 complex potently inhibits bacterial uptake. These results indicate that WAVE2 is an important component in signaling pathways leading to Salmonella invasion. Although infection leads to the formation of an IRSp53/WAVE2 complex, it is the association of WAVE2 with the Abi1/Nap1/PIR121/HSPC300 complex that regulates bacterial internalization.     

Genetic Characterization of Tube and Pelle, Genes Required for Signaling between Toll and Dorsal in the Specification of the Dorsal-Ventral Pattern of the Drosophila Embryo

tube and pelle are two of the maternally transcribed genes required for dorsal-ventral patterning of the Drosophila embryo. Females homozygous for strong alleles of tube or pelle produce embryos that lack all ventral and lateral embryonic pattern elements. By analyzing the phenotypes caused by 24 pelle and 9 tube alleles, we have defined characteristic features of the two genes, including the extremely variable phenotypes of a number of tube alleles and the antimorphic character of a number of pelle alleles. Double mutant females carrying dominant ventralizing alleles of Toll and dorsalizing alleles of tube or pelle produce dorsalized embryos, suggesting that tube and pelle act downstream of the membrane protein Toll in the signaling pathway that defines the embryonic dorsal-ventral pattern. Both tube and pelle are also important zygotically for survival: at least 30% of the zygotes lacking either tube or pelle die before adult stages, while 90-95% of tube(-) pelle(-) double mutant zygotes die. We discuss the phenotypes of tube-pelle double mutants in the context of whether the two proteins interact directly.     

Apoptotic cell death influences the signaling activity of the amyloid precursor protein through ShcA and Grb2 adaptor proteins in neuroblastoma SH-SY5Y cells

The amyloid precursor protein (APP) is an ubiquitous receptor-like molecule involved in the pathogenesis of Alzheimer's disease (AD). APP and some of its C-terminal proteolytic fragments (CTFs) have been shown to be phosphorylated and to interact with cytosolic phosphotyrosine binding (PTB) domain containing proteins involved in cell signaling and vesicular transport. Among others, the interaction between tyrosine-phosphorylated CTFs and ShcA-Grb2 adaptors is highly enhanced in AD brain. Here we have identified in SH-SY5Y neuroblastoma cells an interaction between APP holoprotein and the adaptor Grb2. Upon activation of apoptotic cell death this interaction is rapidly degraded, APP is partially cleaved and the complex APP/Grb2 is replaced by a new complex between CTFs and ShcA that still involves Grb2. The formation of these complexes is regulated by beta-site APP-cleaving enzyme 1 and influences the phosphorylation of mitogen-activated protein kinase p44/42 extracellular signal-regulated kinase as well as the level of apoptotic death of the cells. These data suggest a dual role in cell signaling for APP and its CTFs in neuroblastoma cells, in a manner similar to that previously reported for other tyrosine kinase receptor, through a tightly regulated coupling with alternative intracellular adaptors to control the signaling of the cell.     

Oligomerization of NHERF-1 and NHERF-2 PDZ domains: differential regulation by association with receptor carboxyl-termini and by phosphorylation.

PDZ domains bind to the carboxyl-termini of target proteins, and some PDZ domains are capable of oligomerization to facilitate the formation of intracellular signaling complexes. The Na(+)/H(+) exchanger regulatory factor (NHERF-1; also called "EBP50") and its relative NHERF-2 (also called "E3KARP", "SIP-1", and "TKA-1") both have two PDZ domains. We report here that the PDZ domains of NHERF-1 and NHERF-2 bind specifically to each other but not to other PDZ domains. Purified NHERF-2 PDZ domains associate with each other robustly in the absence of any associated proteins, but purified NHERF-1 PDZ domains associate with each other only weakly when examined alone. The oligomerization of the NHERF-1 PDZ domains is greatly facilitated when they are bound with carboxyl-terminal ligands, such as the carboxyl-termini of the beta(2)-adrenergic receptor or the platelet-derived growth factor receptor. Oligomerization of full-length NHERF-1 is also enhanced by mutation of serine 289 to aspartate (S289D), which mimics the phosphorylated form of NHERF-1. Co-immunoprecipitation experiments with differentially tagged versions of the NHERF proteins reveal that NHERF-1 and NHERF-2 form homo- and hetero-oligomers in a cellular context. A point-mutated version of NHERF-1 (S289A), which cannot be phosphorylated on serine 289, exhibits a reduced capacity for co-immunoprecipitation from cells. These studies reveal that both NHERF-1 and NHERF-2 can oligomerize, which may facilitate NHERF-mediated formation of cellular signaling complexes. These studies furthermore reveal that oligomerization of NHERF-1, but not NHERF-2, is highly regulated by association with other proteins and by phosphorylation.     

BLNK: molecular scaffolding through 'cis'-mediated organization of signaling proteins

Assembly of intracellular macromolecular complexes is thought to provide an important mechanism to coordinate the generation of second messengers upon receptor activation. We have previously identified a B cell linker protein, termed BLNK, which serves such a scaffolding function in B cells. We demonstrate here that phosphorylation of five tyrosine residues within human BLNK nucleates distinct signaling effectors following B cell antigen receptor activation. The phosphorylation of multiple tyrosine residues not only amplifies PLCgamma-mediated signaling but also supports 'cis'-mediated interaction between distinct signaling effectors within a large molecular complex. These data demonstrate the importance of coordinate phosphorylation of molecular scaffolds, and provide insights into how assembly of macromolecular complexes is required for normal receptor function.     

Functional roles for the cytoplasmic domain of the type III transforming growth factor beta receptor in regulating transforming growth factor beta signaling

Transforming growth factor beta (TGF-beta) signals through three high affinity cell surface receptors, TGF-beta type I, type II, and type III receptors. The type III receptor, also known as betaglycan, binds to the type II receptor and is thought to act solely by "presenting" the TGF-beta ligand to the type II receptor. The short cytoplasmic domain of the type III receptor is thought to have no role in TGF-beta signaling because deletion of this domain has no effect on association with the type II receptor, or with the presentation role of the type III receptor. Here we demonstrate that the cytoplasmic domains of the type III and type II receptors interact specifically in a manner dependent on the kinase activity of the type II receptor and the ability of the type II receptor to autophosphorylate. This interaction results in the phosphorylation of the cytoplasmic domain of the type III receptor by the type II receptor. The type III receptor with the cytoplasmic domain deleted is able to bind TGF-beta, to bind the type II receptor, and to enhance TGF-beta binding to the type II receptor but is unable to enhance TGF-beta2 signaling, determining that the cytoplasmic domain is essential for some functions of the type III receptor. The type III receptor functions by selectively binding the autophosphorylated type II receptor via its cytoplasmic domain, thus promoting the preferential formation of a complex between the autophosphorylated type II receptor and the type I receptor and then dissociating from this active signaling complex. These studies, for the first time, elucidate important functional roles of the cytoplasmic domain of the type III receptor and demonstrate that these roles are essential for regulating TGF-beta signaling.     

Cooperative assembly of TGF-beta superfamily signaling complexes is mediated by two disparate mechanisms and distinct modes of receptor binding

Dimeric ligands of the transforming growth factor-beta (TGF-beta) superfamily signal across cell membranes in a distinctive manner by assembling heterotetrameric complexes of structurally related serine/threonine-kinase receptor pairs. Unlike complexes of the bone morphogenetic protein (BMP) branch that apparently form due to avidity from membrane localization, TGF-beta complexes assemble cooperatively through recruitment of the low-affinity (type I) receptor by the ligand-bound high-affinity (type II) pair. Here we report the crystal structure of TGF-beta3 in complex with the extracellular domains of both pairs of receptors, revealing that the type I docks and becomes tethered via unique extensions at a composite ligand-type II interface. Disrupting the receptor-receptor interactions conferred by these extensions abolishes assembly of the signaling complex and signal transduction (Smad activation). Although structurally similar, BMP and TGF-beta receptors bind in dramatically different modes, mediating graded and switch-like assembly mechanisms that may have coevolved with branch-specific groups of cytoplasmic effectors.     

A network analysis of changes in molecular interactions in cellular signaling

Multiprotein complexes play an essential role in the propagation and integration of cellular signals. However, systems level analyses of signaling-dependent changes in the pattern of molecular interactions are still missing. Signaling in T-lymphocytes is one prominent example in which multiprotein complexes orchestrate signal transduction. We implemented peptide microarrays comprising a set of interaction motifs of signaling proteins for network-based analyses of signaling-dependent changes in molecular interactions. Lysates of resting or stimulated cells were incubated on these arrays, and the binding of signaling proteins was detected by immunofluorescence. Signaling-dependent complex formation led to changes of signals on the microarrays in two ways. 1) Masking of a binding site of a signaling protein for a peptide on the array resulted in a signal decrease. 2) Interaction of a protein with a second protein, which in turn binds to a peptide on the array, resulted in a signal increase for the first protein. Dissipation of complexes led to the reverse changes. Competition with peptides corresponding to interaction motifs provided detailed information on the architecture of complexes; lack of individual signaling proteins revealed the functional interdependence of interactions in the network. We show that complex formation through phosphorylation of the scaffolding protein LAT (linker for activation of T-cells) acted as a signal amplifier. PLCgamma1 deficiency increased the resting state levels of LAT-dependent complexes and augmented the recruitment of the phosphatase SHPTP2 into complexes. For the analysis of signaling networks, the parallel detection of changes in interactions enabled the identification of functional interdependencies with minimum a priori knowledge.     

Increased stability of the TM helix oligomer abrogates the apoptotic activity of the human Fas receptor

Human death receptors control apoptotic events during cell differentiation, cell homeostasis and the elimination of damaged or infected cells. Receptor activation involves ligand-induced structural reorganizations of preformed receptor trimers. Here we show that the death receptor transmembrane domains only have a weak intrinsic tendency to homo-oligomerize within a membrane, and thus these domains potentially do not significantly contribute to receptor trimerization. However, mutation of Pro183 in the human CD95/Fas receptor transmembrane helix results in a dramatically increased interaction propensity, as shown by genetic assays. The increased interaction of the transmembrane domain is coupled with a decreased ligand-sensitivity of cells expressing the Fas receptor, and thus in a decreased number of apoptotic events. Mutation of Pro183 likely results in a substantial rearrangement of the self-associated Fas receptor transmembrane trimer, which likely abolishes further signaling of the apoptotic signal but may activate other signaling pathways. Our study shows that formation of a stable Fas receptor transmembrane helix oligomer does not per se result in receptor activation.     

Dynamics of RASSF1A/MOAP-1 association with death receptors

RASSF1A is a tumor suppressor protein involved in death receptor-dependent apoptosis utilizing the Bax-interacting protein MOAP-1 (previously referred to as MAP-1). However, the dynamics of death receptor recruitment of RASSF1A and MOAP-1 are still not understood. We have now detailed recruitment to death receptors (tumor necrosis factor receptor 1 [TNF-R1] and TRAIL-R1/DR4) and identified domains of RASSF1A and MOAP-1 that are required for death receptor interaction. Upon TNF-alpha stimulation, the C-terminal region of MOAP-1 associated with the death domain of TNF-R1; subsequently, RASSF1A was recruited to MOAP-1/TNF-R1 complexes. Prior to recruitment to TNF-R1/MOAP-1 complexes, RASSF1A homodimerization was lost. RASSF1A associated with the TNF-R1/MOAP-1 or TRAIL-R1/MOAP-1 complex via its N-terminal cysteine-rich (C1) domain containing a potential zinc finger binding motif. Importantly, TNF-R1 association domains on both MOAP-1 and RASSF1A were essential for death receptor-dependent apoptosis. The association of RASSF1A and MOAP-1 with death receptors involves an ordered recruitment to receptor complexes to promote cell death and inhibit tumor formation.     

The prevalence and significance of PDZ domain-phosphoinositide interactions

PDZ domains predominate in multi-cellular organisms. They are ubiquitous protein-interaction modules recognizing short peptide sequences generally situated at the C-terminal end of plasma membrane proteins. They contribute to the formation and spatial confinement of protein complexes and thereby play an essential role in the control of cell signaling. Recent studies indicate that PDZ domains can also interact with phosphoinositides (PIPs), signaling lipids with key-roles in receptor signal transduction, membrane trafficking, cytoskeleton remodeling and nuclear processes. In particular the PDZ domains of syntenin-1 and syntenin-2 bind to phosphatidylinositol 4, 5-bisphosphate (PIP2) with high-affinity. Syntenin-1/PIP2 interaction is important for receptor cargo recycling and syntenin-2 plays a role in the organization of nuclear PIP2. In addition, other lower-affinity PDZ domain/PIPs interactions are documented. Here, we summarize and discuss the present knowledge about the occurrence, the biochemistry and the biology of PDZ domain-lipid interactions.     

Receptor-mediated protein kinase activation and the mechanism of transmembrane signaling in bacterial chemotaxis.

Chemotaxis responses of Escherichia coli and Salmonella are mediated by type I membrane receptors with N-terminal extracytoplasmic sensing domains connected by transmembrane helices to C-terminal signaling domains in the cytoplasm. Receptor signaling involves regulation of an associated protein kinase, CheA. Here we show that kinase activation by a soluble signaling domain construct involves the formation of a large complex, with approximately 14 receptor signaling domains per CheA dimer. Electron microscopic examination of these active complexes indicates a well defined bundle composed of numerous receptor filaments. Our findings suggest a mechanism for transmembrane signaling whereby stimulus-induced changes in lateral packing interactions within an array of receptor-sensing domains at the cell surface perturb an equilibrium between active and inactive receptor-kinase complexes within the cytoplasm.     

Modeling and simulation of aggregation of membrane protein LAT with molecular variability in the number of binding sites for cytosolic Grb2-SOS1-Grb2

The linker for activation of T cells (LAT), the linker for activation of B cells (LAB), and the linker for activation of X cells (LAX) form a family of transmembrane adaptor proteins widely expressed in lymphocytes. These scaffolding proteins have multiple binding motifs that, when phosphorylated, bind the SH2 domain of the cytosolic adaptor Grb2. Thus, the valence of LAT, LAB and LAX for Grb2 is variable, depending on the strength of receptor activation that initiates phosphorylation. During signaling, the LAT population will exhibit a time-varying distribution of Grb2 valences from zero to three. In the cytosol, Grb2 forms 1:1 and 2:1 complexes with the guanine nucleotide exchange factor SOS1. The 2:1 complex can bridge two LAT molecules when each Grb2, through their SH2 domains, binds to a phosphorylated site on a separate LAT. In T cells and mast cells, after receptor engagement, receptor phosphoyrlation is rapidly followed by LAT phosphorylation and aggregation. In mast cells, aggregates containing more than one hundred LAT molecules have been detected. Previously we considered a homogeneous population of trivalent LAT molecules and showed that for a range of Grb2, SOS1 and LAT concentrations, an equilibrium theory for LAT aggregation predicts the formation of a gel-like phase comprising a very large aggregate (superaggregate). We now extend this theory to investigate the effects of a distribution of Grb2 valence in the LAT population on the formation of LAT aggregates and superaggregate and use stochastic simulations to calculate the fraction of the total LAT population in the superaggregate.