Transmembrane mutations to FcgammaRIIA alter its association with lipid rafts: implications for receptor signaling

Many immunoreceptors have been reported to associate with lipid rafts upon ligand binding. The way in which this association is regulated is still obscure. We investigated the roles for various domains of the human immunoreceptor FcgammaRIIA in regulating its association with lipid rafts by determining the resistance of unligated, or ligated and cross-linked, receptors to solubilization by the nonionic detergent Triton X-100, when expressed in RBL-2H3 cells. Deletion of the cytoplasmic domain, or destruction of the cytoplasmic palmitoylation site, had no effect on the association of the receptor with lipid rafts. A transmembrane mutant, A224S, lost the ability to associate with lipid rafts upon receptor cross-linking, whereas transmembrane mutants VA231-2MM and VVAL234-7GISF showed constitutive lipid raft association. Wild-type (WT) FcgammaRIIA and all transmembrane mutants activated Syk, regardless of their association with lipid rafts. WT FcgammaRIIA and mutants that associated with lipid rafts efficiently activated NF-kappaB, in an ERK-dependent manner. In contrast, WT FcgammaRIIA and the A224S mutant both presented efficient phagocytosis, while VA231-2MM and VVAL234-7GISF mutants presented lower phagocytosis, suggesting that phagocytosis may proceed independently of lipid raft association. These data identify the transmembrane domain of FcgammaRIIA as responsible for regulating its inducible association with lipid rafts and suggest that FcgammaRIIA-mediated responses, like NF-kappaB activation or phagocytosis, can be modulated by lipid raft association of the ligated receptor.     

Src-dependent phosphorylation of Scar1 promotes its association with the Arp2/3 complex

The WAVE/Scar proteins regulate actin polymerisation at the leading edge of motile cells via activation of the Arp2/3 complex in response to extracellular cues. Within cells they form part of a pentameric complex that is thought to regulate their ability to interact and activate the Arp2/3 complex. However, the exact mechanism for this is not known. We set out to assess whether phosphorylation of Scar1 by the non-receptor tyrosine kinase Src may influence the function of Scar1 and its ability to regulate Arp2/3-mediated actin polymerisation. We show that Scar1 is phosphorylated by Src in vitro and in vivo and identify tyrosine 125 as the major site in Scar1 to be phosphorylated in cells. Src-dependent phosphorylation of Scar1 on tyrosine 125 enhances its ability to bind to the Arp2/3 complex and regulates its ability to control actin polymerisation in cells. Thus, Src may act as an intermediary to regulate the activity of the Arp2/3 complex in response to external stimuli, via modulation of its interaction with WAVE/Scar proteins.     

p53 SUMOylation promotes its nuclear export by facilitating its release from the nuclear export receptor CRM1

Chromosomal region maintenance 1 (CRM1) mediates p53 nuclear export. Although p53 SUMOylation promotes its nuclear export, the underlying mechanism is unclear. Here we show that tethering of a small, ubiquitin-like modifier (SUMO) moiety to p53 markedly increases its cytoplasmic localization. SUMO attachment to p53 does not affect its oligomerization, suggesting that subunit dissociation required for exposing p53’s nuclear export signal (NES) is unnecessary for p53 nuclear export. Surprisingly, SUMO-mediated p53 nuclear export depends on the SUMO-interacting motif (SIM)-binding pocket of SUMO-1. The CRM1 C-terminal domain lacking the NES-binding groove interacts with tetrameric p53, and the proper folding of the p53 core domain, rather than the presence of the N- or C-terminal tails, appears to be important for p53–CRM1 interaction. The CRM1 Huntington, EF3, a subunit of PP2A, and TOR1 9 (HEAT9) loop, which regulates GTP-binding nuclear protein Ran binding and cargo release, contains a prototypical SIM. Remarkably, disruption of this SIM in conjunction with a mutated SIM-binding groove of SUMO-1 markedly enhances the binding of CRM1 to p53-SUMO-1 and their accumulation in the nuclear pore complexes (NPCs), as well as their persistent association in the cytoplasm. We propose that SUMOylation of a CRM1 cargo such as p53 at the NPCs unlocks the HEAT9 loop of CRM1 to facilitate the disassembly of the transporting complex and cargo release to the cytoplasm.     

Thyroxine promotes association of mitogen-activated protein kinase and nuclear thyroid hormone receptor (TR) and causes serine phosphorylation of TR

Activated nongenomically by l-thyroxine (T(4)), mitogen-activated protein kinase (MAPK) complexed in 10-20 min with endogenous nuclear thyroid hormone receptor (TRbeta1 or TR) in nuclear fractions of 293T cells, resulting in serine phosphorylation of TR. Treatment of cells with the MAPK kinase inhibitor, PD 98059, prevented both T(4)-induced nuclear MAPK-TR co-immunoprecipitation and serine phosphorylation of TR. T(4) treatment caused dissociation of TR and SMRT (silencing mediator of retinoid and thyroid hormone receptor), an effect also inhibited by PD 98059 and presumptively a result of association of nuclear MAPK with TR. Transfection into CV-1 cells of TR gene constructs in which one or both zinc fingers in the TR DNA-binding domain were replaced with those from the glucocorticoid receptor localized the site of TR phosphorylation by T(4)-activated MAPK to a serine in the second zinc finger of the TR DNA-binding domain. In an in vitro cell- and hormone-free system, purified activated MAPK phosphorylated recombinant human TRbeta1 (). Thus, T(4) activates MAPK and causes MAPK-mediated serine phosphorylation of TRbeta1 and dissociation of TR and the co-repressor SMRT.     

CSF-1 stimulated multiubiquitination of the CSF-1 receptor and of Cbl follows their tyrosine phosphorylation and association with other signaling proteins

Addition of colony stimulating factor-1 (CSF-1) to macrophages stimulates the rapid, transient tyrosine phosphorylation, membrane association and multiubiquitination of Cbl (Wang et al. [1996] J. Biol. Chem. 271:17-20). Kinetic analysis reveals that the tyrosine phosphorylation of Cbl is coincident with its plasma membrane translocation and association with the activated tyrosine phosphorylated CSF-1 R, p85, Grb2, and tyrosine phosphorylated p58Shc and that these events precede the simultaneous multiubiquitination of Cbl and the CSF-1 R. Tyrosine phosphorylation and multiubiquitination of the cell surface CSF-1 R are stoichiometric and the multiubiquitinated CSF-1 R is degraded. Similarly, the membrane associated Cbl is almost stoichiometrically ubiquitinated, but the ubiquitinated Cbl is not degraded, being recovered, deubiquitinated, in the cytosol 3-10 min after stimulation at 37 degrees C. In the membrane fraction of cells stimulated at 4 degrees C, the association of p58Shc and Grb2 with Cbl is stable, whereas its association with Sos and p85 is transient and their dissociation occurs at the time CSF-1 R and Cbl multiubiquitination commence. The membrane translocation and the pattern of association of Sos with the CSF-1R, p85, Grb2, and p58Shc resemble those of Cbl but Sos is not tyrosine phosphorylated, nor multiubiquitinated and the coprecipitation of these proteins, other than Grb2, with Sos is much less. Complexes formed by Sos and Cbl are largely independent and membrane complexes of Cbl with other tyrosine phosphorylated proteins, p85 and Grb2 also contain CSF-1 R. These data raise the possibility that the predicted negative regulatory role of Cbl in macrophages is its enhancement of ligand-induced CSF-1 R internalization/degradation.     

An algebra of dimerization and its implications for G-protein coupled receptor signaling

Many species of receptors form dimers, but how can we use this information to make predictions about signal transduction? This problem is particularly difficult when receptors dimerize with many different species, leading to a combinatoric increase in the possible number of dimer pairs. As an example system, we focus on receptors in the G-protein coupled receptor (GPCR) family. GPCRs have been shown to reversibly form dimers, but this dimerization does not directly affect signal transduction. Here we present a new theoretical framework called a dimerization algebra. This algebra provides a systematic and rational way to represent, manipulate, and in some cases simplify large and often complicated networks of dimerization interactions. To compliment this algebra, Monte Carlo simulations are used to predict dimerization's effect on receptor organization on the membrane, signal transduction, and internalization. These simulation results are directly comparable to various experimental measures such as fluorescence resonance energy transfer (FRET), and as such provide a link between the dimerization algebra and experimental data. As an example, we show how the algebra and computational results can be used to predict the effects of dimerization on the dopamine D2 and somatastatin SSTR1 receptors. When these predictions were compared to experimental findings from the literature, good agreement was found, demonstrating the utility of our approach. Applications of this work to the development of a novel class of dimerization-modulating drugs are also discussed.     

Insulin receptor structure and its implications for the IGF-1 receptor

The insulin receptor (isoforms IR-A and IR-B) and the type-I insulin-like growth factor receptor (IGF-1R) are homologous, multi-domain tyrosine kinases that bind insulin and IGF-1 with differing specificity. IR is involved in metabolic regulation and IGF-1R in normal growth and development. IR-A also binds IGF-2 with an affinity comparable to IGF-1R and, like the latter, is implicated in a range of cancers. The recent structure of the IR ectodomain dimer explains many features of ligand-receptor binding and provides insight into the structure of the intact ligand-binding site in both receptors. The structures of the L1-CR-L2 fragments of IR and IGF-1R reveal major differences in the regions that govern ligand specificity. The IR ectodomain X-ray structure raises doubts about that obtained by STEM reconstruction.     

Crystal structure of the ephrin-B1 ectodomain: implications for receptor recognition and signaling

Eph receptors and their ephrin ligands are involved in various aspects of cell-cell communication during development, including axonal pathfinding in the nervous system and cell-cell interactions of the vascular endothelial cells. Recent structural studies revealed unique molecular features, not previously seen in any other receptor-ligand families, and explained many of the biochemical and signaling properties of Ephs and ephrins. However, unresolved questions remain regarding the potential oligomerization and clustering of these important signaling molecules. In this study, the biophysical properties and receptor-binding preferences of the extracellular domain of ephrin-B1 were investigated and its crystal structure was determined at 2.65 A resolution. Ephrin-B1 is a monomer both in solution and in the crystals, while it was previously shown that the closely related ephrin-B2 forms homodimers. The main structural difference between ephrin-B1 and ephrin-B2 is the conformation of the receptor-binding G-H loop and the partially disordered N-terminal tetramerization region of ephrin-B1. The G-H loop is structurally rigid in ephrin-B2 and adopts the same conformation in both the receptor-bound and unbound ligand, where it mediates receptor-independent homodimerization. In the ephrin-B1 structure, on the other hand, the G-H loop is not involved in any homotypic interactions and adopts a new, distinct conformation. The implications of the ephrin-B1 structure, in context of available ephrin-B1 mutagenesis data, for the mechanism of Eph-ephrin recognition and signaling initiation are discussed.     

ULK1 inhibits mTORC1 signaling,promotes multisite Raptor phosphorylation and hinders substrate binding

Protein synthesis and autophagy work as two opposing processes to control cell growth in response to nutrient supply. The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) pathway, which acts as a master regulator to control protein synthesis, has recently been shown to inhibit autophagy by phosphorylating and inactivating ULK1, an autophagy regulatory protein. ULK1 also inhibits phosphorylation of a mTORC1 substrate, S6K1, indicating that a complex signaling interplay exists between mTORC1 and ULK1. Here, we demonstrate that ULK1 induces multisite phosphorylation of Raptor in vivo and in vitro. Using phospho-specific antibodies we identify Ser855 and Ser859 as being strongly phosphorylated by ULK1, with moderate phosphorylation of Ser792 also observed. Interestingly, ULK1 overexpression also increases phosphorylation of Raptor Ser863 and the mTOR autophosphorylation site, Ser2481 in a mTORC1-dependent manner. Despite this evidence for heightened mTORC1 kinase activity following ULK1 overexpresssion, mTORC1-mediated phosphorylation of S6K1 and 4E-BP1 is significantly inhibited. ULK1 expression has no effect on protein-protein interactions between the components of mTORC1, but does reduce the ability of Raptor to bind to the substrate 4E-BP1. Furthermore, shRNA knockdown of ULK1 leads to increased phosphorylation of mTORC1 substrates and decreased phosphorylation of Raptor at Ser859 and Ser792. We propose a new mechanism whereby ULK1 contributes to mTORC1 inhibition through hindrance of substrate docking to Raptor. This is a novel negative feedback loop that occurs upon activation of autophagy to maintain mTORC1 inhibition when nutrient supplies are limiting.     

ULK1 inhibits mTORC1 signaling, promotes multisite Raptor phosphorylation and hinders substrate binding

Protein synthesis and autophagy work as two opposing processes to control cell growth in response to nutrient supply. The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) pathway, which acts as a master regulator to control protein synthesis, has recently been shown to inhibit autophagy by phosphorylating and inactivating ULK1, an autophagy regulatory protein. ULK1 also inhibits phosphorylation of a mTORC1 substrate, S6K1, indicating that a complex signaling interplay exists between mTORC1 and ULK1. Here, we demonstrate that ULK1 induces multisite phosphorylation of Raptor in vivo and in vitro. Using phospho-specific antibodies we identify Ser855 and Ser859 as being strongly phosphorylated by ULK1, with moderate phosphorylation of Ser792 also observed. Interestingly, ULK1 overexpression also increases phosphorylation of Raptor Ser863 and the mTOR autophosphorylation site, Ser2481 in a mTORC1-dependent manner. Despite this evidence for heightened mTORC1 kinase activity following ULK1 overexpresssion, mTORC1-mediated phosphorylation of S6K1 and 4E-BP1 is significantly inhibited. ULK1 expression has no effect on protein-protein interactions between the components of mTORC1, but does reduce the ability of Raptor to bind to the substrate 4E-BP1. Furthermore, shRNA knockdown of ULK1 leads to increased phosphorylation of mTORC1 substrates and decreased phosphorylation of Raptor at Ser859 and Ser792. We propose a new mechanism whereby ULK1 contributes to mTORC1 inhibition through hindrance of substrate docking to Raptor. This is a novel negative feedback loop that occurs upon activation of autophagy to maintain mTORC1 inhibition when nutrient supplies are limiting.     

Mitotic phosphorylation of chromosomal protein HMGN1 inhibits nuclear import and promotes interaction with 14.3.3 proteins

Progression through mitosis is associated with reversible phosphorylation of many nuclear proteins including that of the high-mobility group N (HMGN) nucleosomal binding protein family. Here we use immunofluorescence and in vitro nuclear import studies to demonstrate that mitotic phosphorylation of the nucleosomal binding domain (NBD) of the HMGN1 protein prevents its reentry into the newly formed nucleus in late telophase. By microinjecting wild-type and mutant proteins into the cytoplasm of HeLa cells and expressing these proteins in HmgN1(-/-) cells, we demonstrate that the inability to enter the nucleus is a consequence of phosphorylation and is not due to the presence of negative charges. Using affinity chromatography with recombinant proteins and nuclear extracts prepared from logarithmically growing or mitotically arrested cells, we demonstrate that phosphorylation of the NBD of HMGN1 promotes interaction with specific 14.3.3 isotypes. We conclude that mitotic phosphorylation of HMGN1 protein promotes interaction with 14.3.3 proteins and suggest that this interaction impedes the reentry of the proteins into the nucleus during telophase. Taken together with the results of previous studies, our results suggest a dual role for mitotic phosphorylation of HMGN1: abolishment of chromatin binding and inhibition of nuclear import.     

Tesk1 interacts with Spry2 to abrogate its inhibition of ERK phosphorylation downstream of receptor tyrosine kinase signaling

The Sprouty (Spry) proteins function as inhibitors of the Ras-ERK pathway downstream of various receptor tyrosine kinases. In this study, we have identified Tesk1 (testicular protein kinase 1) as a novel regulator of Spry2 function. Endogenous Tesk1 and Spry2 exist in a complex in cell lines and mouse tissues. Tesk1 coexpression relocalizes Spry2 to vesicles including endosomes, inhibiting its translocation to membrane ruffles upon growth factor stimulation. Independent of its kinase activity, Tesk1 binding leads to a loss of Spry2 function as an inhibitor of ERK phosphorylation and reverses inhibition of basic fibroblast growth factor (bFGF)- and nerve growth factor-induced neurite outgrowth in PC12 cells by Spry2. Furthermore, depletion of endogenous Tesk1 in PC12 cells leads to a reduction in neurite outgrowth induced by bFGF. Tesk1 nullifies the inhibitory effect of Spry2 by abrogating its interaction with the adaptor protein Grb2 and interfering with its serine dephosphorylation upon bFGF and FGF receptor 1 stimulation by impeding its binding to the catalytic subunit of protein phosphatase 2A. A construct of Tesk1 that binds to Spry2 but does not localize to the vesicles does not interfere with its function, highlighting the importance of subcellular localization of Tesk1 in this context. Conversely, Tesk1 does not affect interaction of Spry2 with the E3 ubiquitin ligase, c-Cbl, and consequently, does not affect its inhibition of Cbl-mediated ubiquitination of the epidermal growth factor receptor. By selectively modulating the downstream effects of Spry2, Tesk1 may thus serve as a molecular determinant of the signaling outcome.     

Microsomal receptor for steroid hormones: functional implications for nuclear activity

Target tissues for steroid hormones are responsive by virtue of and to the extent of their content of functional intracellular receptors. Recent years have seen a shift in considerations of the cellular dynamics and distribution of these receptors, with current views favoring predominant intranuclear localization in the intact cell. This paper summarizes our analyses of the microsomal estrogen and androgen binding capability of rat uterine and ventral prostate tissue, respectively; these studies have revealed a set of high affinity sites that may act as a conduit for estrogen traversing the cell en route to the nucleus. These sites have many properties in common with cytosolic receptors, with the salient difference of a failure to activate to a more avid DNA-binding form under conditions which permit such activation of cytosolic receptors. The microsomal estrogen-binding proteins also have appreciable affinity for progesterone, another distinction from other known cellular estrogen receptor species. Various experimental approaches were employed to demonstrate that the microsomal receptors were not simply cytosol contaminants; the most convincing evidence is the recent successful separation of the cytosolic and microsomal forms by differential ammonium sulfate precipitation. Discrete subfractionation of subcellular components on successive sucrose gradients, with simultaneous assessments of binding capability and marker enzyme concentrations, indicates that the major portion of the binding is localized within the vesicles of the endoplasmic reticulum free of significant plasma membrane contamination. The microsomal receptors are readily solubilized by extraction with high- or low-salt-containing buffers or with steroid. The residual microsomes following such extraction have the characteristics of saturable acceptor sites for cytosolic estrogen-receptor complexes. The extent to which these sites will accept the cytosolic complexes is equal to the concentration of microsomal binding sites extracted. These observations suggest three possible roles for the microsomal receptor-like proteins: (a) modulation of estrogen access to nuclear binding sites; (b) formation of functional complexes which diffuse to other extranuclear sites to alter non-genomic cellular processes; (c) regulation of nuclear concentration of estrogen-receptor complexes by virtue of producing microsomal acceptor sites for uptake of free or loosely associated nuclear complexes, previously thought to exist in the cytoplasm.     

Cytoskeletal control of CD36 diffusion promotes its receptor and signaling function

The mechanisms that govern receptor coalescence into functional clusters--often a critical step in their stimulation by ligand--are poorly understood. We used single-molecule tracking to investigate the dynamics of CD36, a clustering-responsive receptor that mediates oxidized LDL uptake by macrophages. We found that CD36 motion in the membrane was spatially structured by the cortical cytoskeleton. A subpopulation of receptors diffused within linear confinement regions whose unique geometry simultaneously facilitated freedom of movement along one axis while increasing the effective receptor density. Co-confinement within troughs enhanced the probability of collisions between unligated receptors and promoted their clustering. Cytoskeleton perturbations that inhibited diffusion in linear confinement regions reduced receptor clustering in the absence of ligand and, following ligand addition, suppressed CD36-mediated signaling and internalization. These observations demonstrate a role for the cytoskeleton in controlling signal transduction by structuring receptor diffusion within membrane regions that increase their collision frequency.     

A CSF-1 receptor phosphotyrosine 559 signaling pathway regulates receptor ubiquitination and tyrosine phosphorylation

Receptor tyrosine kinase (RTK) activation involves ligand-induced receptor dimerization and transphosphorylation on tyrosine residues. Colony-stimulating factor-1 (CSF-1)-induced CSF-1 receptor (CSF-1R) tyrosine phosphorylation and ubiquitination were studied in mouse macrophages. Phosphorylation of CSF-1R Tyr-559, required for the binding of Src family kinases (SFKs), was both necessary and sufficient for these responses and for c-Cbl tyrosine phosphorylation and all three responses were inhibited by SFK inhibitors. In c-Cbl-deficient macrophages, CSF-1R ubiquitination and tyrosine phosphorylation were substantially inhibited. Reconstitution with wild-type, but not ubiquitin ligase-defective C381A c-Cbl rescued these responses, while expression of C381A c-Cbl in wild-type macrophages suppressed them. Analysis of site-directed mutations in the CSF-1R further suggests that activated c-Cbl-mediated CSF-1R ubiquitination is required for a conformational change in the major kinase domain that allows amplification of receptor tyrosine phosphorylation and full receptor activation. Thus the results indicate that CSF-1-mediated receptor dimerization leads to a Tyr-559/SFK/c-Cbl pathway resulting in receptor ubiquitination that permits full receptor tyrosine phosphorylation of this class III RTK in macrophages.