A predictive computational model reveals that GIV/girdin serves as a tunable valve for EGFR-stimulated cyclic AMP signals.

Cellular levels of the versatile second messenger cyclic (c)AMP are regulated by the antagonistic actions of the canonical G protein → adenylyl cyclase pathway that is initiated by G-protein-coupled receptors (GPCRs) and attenuated by phosphodiesterases (PDEs). Dysregulated cAMP signaling drives many diseases; for example, its low levels facilitate numerous sinister properties of cancer cells. Recently, an alternative paradigm for cAMP signaling has emerged in which growth factor-receptor tyrosine kinases (RTKs; e.g., EGFR) access and modulate G proteins via a cytosolic guanine-nucleotide exchange modulator (GEM), GIV/girdin; dysregulation of this pathway is frequently encountered in cancers. In this study, we present a network-based compartmental model for the paradigm of GEM-facilitated cross-talk between RTKs and G proteins and how that impacts cellular cAMP. Our model predicts that cross-talk between GIV, Gαs, and Gαi proteins dampens ligand-stimulated cAMP dynamics. This prediction was experimentally verified by measuring cAMP levels in cells under different conditions. We further predict that the direct proportionality of cAMP concentration as a function of receptor number and the inverse proportionality of cAMP concentration as a function of PDE concentration are both altered by GIV levels. Taking these results together, our model reveals that GIV acts as a tunable control valve that regulates cAMP flux after growth factor stimulation. For a given stimulus, when GIV levels are high, cAMP levels are low, and vice versa. In doing so, GIV modulates cAMP via mechanisms distinct from the two most often targeted classes of cAMP modulators, GPCRs and PDEs

Activation of G proteins by GIV-GEF is a pivot point for insulin resistance and sensitivity.

Insulin resistance (IR) is a metabolic disorder characterized by impaired insulin signaling and cellular glucose uptake. The current paradigm for insulin signaling centers upon the insulin receptor (InsR) and its substrate IRS1; the latter is believed to be the sole conduit for postreceptor signaling. Here we challenge that paradigm and show that GIV/Girdin, a guanidine exchange factor (GEF) for the trimeric G protein Gαi, is another major hierarchical conduit for the metabolic insulin response. By virtue of its ability to directly bind InsR, IRS1, and phosphoinositide 3-kinase, GIV serves as a key hub in the immediate postreceptor level, which coordinately enhances the metabolic insulin response and glucose uptake in myotubes via its GEF function. Site-directed mutagenesis or phosphoinhibition of GIV-GEF by the fatty acid/protein kinase C-theta pathway triggers IR. Insulin sensitizers reverse phosphoinhibition of GIV and reinstate insulin sensitivity. We also provide evidence for such reversible regulation of GIV-GEF in skeletal muscles from patients with IR. Thus GIV is an essential upstream component that couples InsR to G-protein signaling to enhance the metabolic insulin response, and impairment of such coupling triggers IR. We also provide evidence that GIV-GEF serves as therapeutic target for exogenous manipulation of physiological insulin response and reversal of IR in skeletal muscles

Structural basis for activation of trimeric Gi proteins by multiple growth factor receptors via GIV/Girdin.

A long-standing issue in the field of signal transduction is to understand the cross-talk between receptor tyrosine kinases (RTKs) and heterotrimeric G proteins, two major and distinct signaling hubs that control eukaryotic cell behavior. Although stimulation of many RTKs leads to activation of trimeric G proteins, the molecular mechanisms behind this phenomenon remain elusive. We discovered a unifying mechanism that allows GIV/Girdin, a bona fide metastasis-related protein and a guanine-nucleotide exchange factor (GEF) for Gαi, to serve as a direct platform for multiple RTKs to activate Gαi proteins. Using a combination of homology modeling, protein-protein interaction, and kinase assays, we demonstrate that a stretch of ∼110 amino acids within GIV C-terminus displays structural plasticity that allows folding into a SH2-like domain in the presence of phosphotyrosine ligands. Using protein-protein interaction assays, we demonstrated that both SH2 and GEF domains of GIV are required for the formation of a ligand-activated ternary complex between GIV, Gαi, and growth factor receptors and for activation of Gαi after growth factor stimulation. Expression of a SH2-deficient GIV mutant (Arg 1745→Leu) that cannot bind RTKs impaired all previously demonstrated functions of GIV-Akt enhancement, actin remodeling, and cell migration. The mechanistic and structural insights gained here shed light on the long-standing questions surrounding RTK/G protein cross-talk, set a novel paradigm, and characterize a unique pharmacological target for uncoupling GIV-dependent signaling downstream of multiple oncogenic RTKs

Modulation of Rab GTPase function by a protein phosphocholine transferase.

The intracellular pathogen Legionella pneumophila modulates the activity of host GTPases to direct the transport and assembly of the membrane-bound compartment in which it resides. In vitro studies have indicated that the Legionella protein DrrA post-translationally modifies the GTPase Rab1 by a process called AMPylation. Here we used mass spectrometry to investigate post-translational modifications to Rab1 that occur during infection of host cells by Legionella. Consistent with in vitro studies, DrrA-mediated AMPylation of a conserved tyrosine residue in the switch II region of Rab1 was detected during infection. In addition, a modification to an adjacent serine residue in Rab1 was discovered, which was independent of DrrA. The Legionella effector protein AnkX was required for this modification. Biochemical studies determined that AnkX directly mediates the covalent attachment of a phosphocholine moiety to Rab1. This phosphocholine transferase activity used CDP-choline as a substrate and required a conserved histidine residue located in the FIC domain of the AnkX protein. During infection, AnkX modified both Rab1 and Rab35, which explains how this protein modulates membrane transport through both the endocytic and exocytic pathways of the host cell. Thus, phosphocholination of Rab GTPases represents a mechanism by which bacterial FIC-domain-containing proteins can alter host-cell functions

NOTCH3 inactivation increases triple negative breast cancer sensitivity to gefitinib by promoting EGFR tyrosine dephosphorylation and its intracellular arrest.

Notch dysregulation has been implicated in numerous tumors, including triple-negative breast cancer (TNBC), which is the breast cancer subtype with the worst clinical outcome. However, the importance of individual receptors in TNBC and their specific mechanism of action remain to be elucidated, even if recent findings suggested a specific role of activated-Notch3 in a subset of TNBCs. Epidermal growth factor receptor (EGFR) is overexpressed in TNBCs but the use of anti-EGFR agents (including tyrosine kinase inhibitors, TKIs) has not been approved for the treatment of these patients, as clinical trials have shown disappointing results. Resistance to EGFR blockers is commonly reported. Here we show that Notch3-specific inhibition increases TNBC sensitivity to the TKI-gefitinib in TNBC-resistant cells. Mechanistically, we demonstrate that Notch3 is able to regulate the activated EGFR membrane localization into lipid rafts microdomains, as Notch3 inhibition, such as rafts depletion, induces the EGFR internalization and its intracellular arrest, without involving receptor degradation. Interestingly, these events are associated with the EGFR tyrosine dephosphorylation at Y1173 residue (but not at Y1068) by the protein tyrosine phosphatase H1 (PTPH1), thus suggesting its possible involvement in the observed Notch3-dependent TNBC sensitivity response to gefitinib. Consistent with this notion, a nuclear localization defect of phospho-EGFR is observed after combined blockade of EGFR and Notch3, which results in a decreased TNBC cell survival. Notably, we observed a significant correlation between EGFR and NOTCH3 expression levels by in silico gene expression and immunohistochemical analysis of human TNBC primary samples. Our findings strongly suggest that combined therapies of TKI-gefitinib with Notch3-specific suppression may be exploited as a drug combination advantage in TNBC treatment

The arabidopsis RCC1 family protein TCF1 regulates freezing tolerance and cold acclimation through modulating lignin biosynthesis

Cell water permeability and cell wall properties are critical to survival of plant cells during freezing, however the underlying molecular mechanisms remain elusive. Here, we report that a specifically cold-induced nuclear protein, Tolerant to Chilling and Freezing 1 (TCF1), interacts with histones H3 and H4 and associates with chromatin containing a target gene, BLUE-COPPER-BINDING PROTEIN (BCB), encoding a glycosylphosphatidylinositol-anchored protein that regulates lignin biosynthesis. Loss of TCF1 function leads to reduced BCB transcription through affecting H3K4me2 and H3K27me3 levels within the BCB gene, resulting in reduced lignin content and enhanced freezing tolerance. Furthermore, plants with knocked-down BCB expression (amiRNA-BCB) under cold acclimation had reduced lignin accumulation and increased freezing tolerance. The pal1pal2 double mutant (lignin content reduced by 30% compared with WT) also showed the freezing tolerant phenotype, and TCF1 and BCB act upstream of PALs to regulate lignin content. In addition, TCF1 acts independently of the CBF (C-repeat binding factor) pathway. Our findings delineate a novel molecular pathway linking the TCF1-mediated cold-specific transcriptional program to lignin biosynthesis, thus achieving cell wall remodeling with increased freezing tolerance

PAKing up to the endothelium

Angiogenesis recapitulates the growth of blood vessels that progressively expand and remodel into a highly organized and stereotyped vascular network. During adulthood, endothelial cells that formed the vascular wall retain their plasticity and can be engaged in neo-vascularization in response to physiological stimuli, such as hypoxia, wound healing and tissue repair, ovarian cycle and pregnancy. In addition, numerous human diseases and pathological conditions are characterized by an excessive, uncontrolled and aberrant angiogenesis. The signalling pathways involving the small Rho GTPase, Rac and its downstream effector the p21-activated serine/threonine kinase (PAK) had recently emerged as pleiotropic modulators in these processes. Indeed, Rac and PAK were found to modulate endothelial cell biology, such as sprouting, migration, polarity, proliferation, lumen formation, and maturation. Elucidating the Rac/PAK molecular circuitry will provide essential information for the development of new therapeutic agents designed to normalize the blood vasculature in human diseases.Comment: Cell Signal (2009) epub ahead of prin

Paradoxical signaling regulates structural plasticity in dendritic spines

Transient spine enlargement (3-5 min timescale) is an important event associated with the structural plasticity of dendritic spines. Many of the molecular mechanisms associated with transient spine enlargement have been identified experimentally. Here, we use a systems biology approach to construct a mathematical model of biochemical signaling and actin-mediated transient spine expansion in response to calcium-influx due to NMDA receptor activation. We have identified that a key feature of this signaling network is the paradoxical signaling loop. Paradoxical components act bifunctionally in signaling networks and their role is to control both the activation and inhibition of a desired response function (protein activity or spine volume). Using ordinary differential equation (ODE)-based modeling, we show that the dynamics of different regulators of transient spine expansion including CaMKII, RhoA, and Cdc42 and the spine volume can be described using paradoxical signaling loops. Our model is able to capture the experimentally observed dynamics of transient spine volume. Furthermore, we show that actin remodeling events provide a robustness to spine volume dynamics. We also generate experimentally testable predictions about the role of different components and parameters of the network on spine dynamics

Daple is a novel non-receptor GEF required for trimeric G protein activation in Wnt signaling

Peer reviewedPublisher PD