Systems analysis of RhoGEF and RhoGAP regulatory proteins reveals spatially organized RAC1 signalling from integrin adhesions.

Rho GTPases are central regulators of the cytoskeleton and, in humans, are controlled by 145 multidomain guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs). How Rho signalling patterns are established in dynamic cell spaces to control cellular morphogenesis is unclear. Through a family-wide characterization of substrate specificities, interactomes and localization, we reveal at the systems level how RhoGEFs and RhoGAPs contextualize and spatiotemporally control Rho signalling.

Multivalent nephrin-Nck interactions define a threshold for clustering and tyrosine-dependent nephrin endocytosis.

Assembly of signaling molecules into micrometer-sized clusters is driven by multivalent protein-protein interactions, such as those found within the nephrin-Nck (Nck1 or Nck2) complex. Phosphorylation on multiple tyrosine residues within the tail of the nephrin transmembrane receptor induces recruitment of the cytoplasmic adaptor protein Nck, which binds via its triple SH3 domains to various effectors, leading to actin assembly. The physiological consequences of nephrin clustering are not well understood.

Targeting LIF-mediated paracrine interaction for pancreatic cancer therapy and monitoring.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis largely owing to inefficient diagnosis and tenacious drug resistance. Activation of pancreatic stellate cells (PSCs) and consequent development of dense stroma are prominent features accounting for this aggressive biology. The reciprocal interplay between PSCs and pancreatic cancer cells (PCCs) not only enhances tumour progression and metastasis but also sustains their own activation, facilitating a vicious cycle to exacerbate tumorigenesis and drug resistance.

Interplay between ShcA Signaling and PGC-1α Triggers Targetable Metabolic Vulnerabilities in Breast Cancer.

The ShcA adaptor protein transduces oncogenic signals downstream of receptor tyrosine kinases. We show here that breast tumors engage the ShcA pathway to increase their metabolism. ShcA signaling enhanced glucose catabolism through glycolysis and oxidative phosphorylation, rendering breast cancer cells critically dependent on glucose.

Structural basis for the interaction between the cell polarity proteins Par3 and Par6.

Polarity is a fundamental property of most cell types. The Par protein complex is a major driving force in generating asymmetrically localized protein networks and consists of atypical protein kinase C (aPKC), Par3, and Par6. Dysfunction of this complex causes developmental abnormalities and diseases such as cancer.

Native KCC2 interactome reveals PACSIN1 as a critical regulator of synaptic inhibition.

KCC2 is a neuron-specific K-Cl cotransporter essential for establishing the Cl gradient required for hyperpolarizing inhibition in the central nervous system (CNS). KCC2 is highly localized to excitatory synapses where it regulates spine morphogenesis and AMPA receptor confinement. Aberrant KCC2 function contributes to human neurological disorders including epilepsy and neuropathic pain.

Grb2 regulates the proliferation of hematopoietic stem and progenitors cells.

Although Hematopoietic Stem and Progenitor Cell (HSPC) proliferation, survival and expansion have been shown to be supported by the cooperative action of different cytokines, little is known about the intracellular signaling pathways that are activated by cytokines upon binding to their receptors. Our study showed that Growth factor receptor-bound protein 2 (Grb2) mRNAs are preferentially expressed in HSC compared to progenitors and differentiated cells of the myeloid and erythroid lineages. Conditional deletion of Grb2 induced a rapid decline of erythroid and myeloid progenitors and a progressive decline of HSC numbers in steady state conditions.

The Shc1 adaptor simultaneously balances Stat1 and Stat3 activity to promote breast cancer immune suppression.

Tyrosine kinase signalling within cancer cells is central to the establishment of an immunosuppressive microenvironment. Although tyrosine kinase inhibitors act, in part, to augment adaptive immunity, the increased heterogeneity and functional redundancy of the tyrosine kinome is a hurdle to achieving durable responses to immunotherapies. We previously identified the Shc1 (ShcA) scaffold, a central regulator of tyrosine kinase signalling, as essential for promoting breast cancer immune suppression.

Mutational Analysis of Glycogen Synthase Kinase 3β Protein Kinase Together with Kinome-Wide Binding and Stability Studies Suggests Context-Dependent Recognition of Kinases by the Chaperone Heat Shock Protein 90.

The heat shock protein 90 (HSP90) and cell division cycle 37 (CDC37) chaperones are key regulators of protein kinase folding and maturation. Recent evidence suggests that thermodynamic properties of kinases, rather than primary sequences, are recognized by the chaperones. In concordance, we observed a striking difference in HSP90 binding between wild-type (WT) and kinase-dead (KD) glycogen synthase kinase 3β (GSK3β) forms.

SMN and symmetric arginine dimethylation of RNA polymerase II C-terminal domain control termination.

The carboxy-terminal domain (CTD) of the RNA polymerase II (RNAP II) subunit POLR2A is a platform for modifications specifying the recruitment of factors that regulate transcription, mRNA processing, and chromatin remodelling. Here we show that a CTD arginine residue (R1810 in human) that is conserved across vertebrates is symmetrically dimethylated (me2s). This R1810me2s modification requires protein arginine methyltransferase 5 (PRMT5) and recruits the Tudor domain of the survival of motor neuron (SMN, also known as GEMIN1) protein, which is mutated in spinal muscular atrophy.

Phosphoinositide 3-kinase enables phagocytosis of large particles by terminating actin assembly through Rac/Cdc42 GTPase-activating proteins.

Phagocytosis is responsible for the elimination of particles of widely disparate sizes, from large fungi or effete cells to small bacteria. Though superficially similar, the molecular mechanisms involved differ: engulfment of large targets requires phosphoinositide 3-kinase (PI3K), while that of small ones does not. Here, we report that inactivation of Rac and Cdc42 at phagocytic cups is essential to complete internalization of large particles.

Selection of recombinant anti-SH3 domain antibodies by high-throughput phage display.

Antibodies are indispensable tools in biochemical research and play an expanding role as therapeutics. While hybridoma technology is the dominant method for antibody production, phage display is an emerging technology. Here, we developed and employed a high-throughput pipeline that enables selection of antibodies against hundreds of antigens in parallel.

A Rac/Cdc42 exchange factor complex promotes formation of lateral filopodia and blood vessel lumen morphogenesis.

During angiogenesis, Rho-GTPases influence endothelial cell migration and cell-cell adhesion; however it is not known whether they control formation of vessel lumens, which are essential for blood flow. Here, using an organotypic system that recapitulates distinct stages of VEGF-dependent angiogenesis, we show that lumen formation requires early cytoskeletal remodelling and lateral cell-cell contacts, mediated through the RAC1 guanine nucleotide exchange factor (GEF) DOCK4 (dedicator of cytokinesis 4). DOCK4 signalling is necessary for lateral filopodial protrusions and tubule remodelling prior to lumen formation, whereas proximal, tip filopodia persist in the absence of DOCK4.

Twisted amide electrophiles enable cyclic peptide sequencing.

There is an ever-increasing interest in synthetic methods that not only enable peptide macrocyclization, but also facilitate downstream application of the synthesized molecules. We have found that aziridine amides are stereoelectronically attenuated in a macrocyclic environment such that non-specific interactions with biological nucleophiles are reduced or even shut down. The electrophilic reactivity, revealed at high pH, enables peptide sequencing by mass spectrometry, which will further broaden the utility of aziridine amide-containing libraries of macrocycles.

Nck adaptor proteins modulate differentiation and effector function of T cells.

Understanding the molecular mechanisms regulating T cell reactivity is required for successful reprogramming of immune responses in medical conditions, characterized by dysfunctions of the immune system. Nck proteins are cytoplasmic adaptors mediating diverse cellular functions, including TCR signaling. By enhancing TCR signal strength, Nck proteins influence thymic selection and regulate the size and sensitivity of the peripheral T cell repertoire.

SELPHI: correlation-based identification of kinase-associated networks from global phospho-proteomics data sets.

While phospho-proteomics studies have shed light on the dynamics of cellular signaling, they mainly describe global effects and rarely explore mechanistic details, such as kinase/substrate relationships. Tools and databases, such as NetworKIN and PhosphoSitePlus, provide valuable regulatory details on signaling networks but rely on prior knowledge. They therefore provide limited information on less studied kinases and fewer unexpected relationships given that better studied signaling events can mask condition- or cell-specific 'network wiring'.

Combinatorial proteomic analysis of intercellular signaling applied to the CD28 T-cell costimulatory receptor.

Systematic characterization of intercellular signaling approximating the physiological conditions of stimulation that involve direct cell-cell contact is challenging. We describe a proteomic strategy to analyze physiological signaling mediated by the T-cell costimulatory receptor CD28. We identified signaling pathways activated by CD28 during direct cell-cell contact by global analysis of protein phosphorylation.

Ankyrin repeats of ANKRA2 recognize a PxLPxL motif on the 3M syndrome protein CCDC8.

Peptide motifs are often used for protein-protein interactions. We have recently demonstrated that ankyrin repeats of ANKRA2 and the paralogous bare lymphocyte syndrome transcription factor RFXANK recognize PxLPxL/I motifs shared by megalin, three histone deacetylases, and RFX5. We show here that that CCDC8 is a major partner of ANKRA2 but not RFXANK in cells.

Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles.

Cells chemically isolate molecules in compartments to both facilitate and regulate their interactions. In addition to membrane-encapsulated compartments, cells can form proteinaceous and membraneless organelles, including nucleoli, Cajal and PML bodies, and stress granules. The principles that determine when and why these structures form have remained elusive.

SYK interaction with ITGβ4 suppressed by Epstein-Barr virus LMP2A modulates migration and invasion of nasopharyngeal carcinoma cells.

Epstein-Barr virus (EBV)-encoded Latent Membrane Protein 2A (LMP2A) is an EBV latency-associated protein regularly expressed in nasopharyngeal carcinoma (NPC). In B cells, LMP2A activity resembles that of a constitutively activated antigen receptor, which recruits the Syk tyrosine kinase to activate a set of downstream signaling pathways. LMP2A also downregulates cellular Syk levels.

Src homology 2 domain containing protein 5 (SH2D5) binds the breakpoint cluster region protein, BCR, and regulates levels of Rac1-GTP.

SH2D5 is a mammalian-specific, uncharacterized adaptor-like protein that contains an N-terminal phosphotyrosine-binding domain and a C-terminal Src homology 2 (SH2) domain. We show that SH2D5 is highly enriched in adult mouse brain, particularly in Purkinjie cells in the cerebellum and the cornu ammonis of the hippocampus. Despite harboring two potential phosphotyrosine (Tyr(P)) recognition domains, SH2D5 binds minimally to Tyr(P) ligands, consistent with the absence of a conserved Tyr(P)-binding arginine residue in the SH2 domain.

Albumin decrease is associated with spontaneous preterm delivery within 48 h in women with threatened preterm labor.

Threatened preterm labor (TPTL) accounts for ∼30% of pregnancy-related hospital admissions. Maternal peripheral leukocytes can be used to monitor a variety of physiological processes occurring in the body. Two high-throughput mass spectrometry methodologies, SWATH and iTRAQ, were used to study differentially expressed peripheral blood leukocyte lysate proteins in symptomatic women admitted for TPTL who had a preterm birth within 48 h (n = 16) and those who did not (n = 24).

Cell cycle-regulated oscillator coordinates core histone gene transcription through histone acetylation.

DNA replication occurs during the synthetic (S) phase of the eukaryotic cell cycle and features a dramatic induction of histone gene expression for concomitant chromatin assembly. Ectopic production of core histones outside of S phase is toxic, underscoring the critical importance of regulatory pathways that ensure proper expression of histone genes. Several regulators of histone gene expression in the budding yeast Saccharomyces cerevisiae are known, yet the key oscillator responsible for restricting gene expression to S phase has remained elusive.