Biology subjects

Ladbury, J. E.

Publications and source records attributed to Ladbury, J. E..

9 recordsLinked to original sources

bioRxiv2024

Affinity purification mass spectrometry characterization of the interactome of receptor tyrosine kinase proline rich motifs in cancer

Receptor tyrosine kinase (RTK) overexpression is linked to the development and progression of multiple cancers. RTKs are classically considered to initiate cytoplasmic signalling pathways via ligand-induced tyrosine phosphorylation, however recent evidence points to a second tier of signalling contingent on interactions mediated by the proline-rich motif (PRM) regions of non-activated RTKs. The presence of PRMs on the C-termini of >40% of all RTKs and the abundance of PRM-binding proteins encoded by the human genome suggests that there is likely to be a large number of previously unexplored interactions which add to the RTK intracellular interactome. Here, we explore the RTK PRM interactome and its potential significance using affinity purification mass spectrometry and in silico enrichment analyses. Peptides comprising PRM-containing C-terminal tail regions of EGFR, FGFR2 and HER2 were used as bait to affinity purify bound proteins from different cancer cell line lysates. 490 unique interactors were identified, amongst which proteins with metabolic, homeostatic and migratory functions were overrepresented. This suggests that PRMs from RTKs may sustain a diverse interactome in cancer cells. Since RTK overexpression is common in cancer RTK PRM-derived signalling may be an important, but as yet underexplored, contributor to negative cancer outcomes including resistance to kinase inhibitors.

cell biology↗

bioRxiv2024

Functional selection in SH3-mediated activation of the PI3 kinase

The phosphoinositide-3 kinase (PI3K), a heterodimeric enzyme, plays a pivotal role in cellular metabolism and survival. Its deregulation is associated with major human diseases, particularly cancer. The p85 regulatory subunit of PI3K binds to the catalytic p110 subunit via its C-terminal domains, stabilising it in an inhibited state. Certain Src homology 3 (SH3) domains can activate p110 by binding to the proline-rich (PR) 1 motif located at the N-terminus of p85. However, the mechanism by which this N-terminal interaction activates the C-terminally bound p110 remains elusive. Moreover, the intrinsically poor ligand selectivity of SH3 domains raises the question of how they can control PI3K. Combining structural, biophysical, and functional methods, we demonstrate that the answers to both these unknown issues are linked: PI3K-activating SH3 domains engage in additional "tertiary" interactions with the C-terminal domains of p85, thereby relieving their inhibition of p110. SH3 domains lacking these tertiary interactions may still bind to p85 but cannot activate PI3K. Thus, p85 uses a functional selection mechanism that precludes nonspecific activation rather than nonspecific binding. This separation of binding and activation may provide a general mechanism for how biological activities can be controlled by promiscuous protein-protein interaction domains.

biochemistry↗

bioRxiv2024

E7-mediated repression of miR-203 promotes LASP1-dependent proliferation in HPV-positive cervical cancer

Human papillomaviruses (HPV) are a major cause of malignancy, contributing to [~]5% of all human cancers worldwide, including most cervical cancer cases and a growing number of ano-genital and oral cancers. The major HPV viral oncogenes, E6 and E7, manipulate many host cellular pathways that promote cell proliferation and survival, predisposing infected cells to malignant transformation. Despite the availability of highly effective vaccines, there are still no specific anti-viral therapies targeting HPV or treatments for HPV-associated cancers. As such, a better understanding of viral-host interactions may allow the identification of novel therapeutic targets. Here, we demonstrate that the actin-binding protein LASP1 is upregulated in cervical cancer and significantly correlates with a poorer overall survival. In HPV positive cervical cancer, LASP1 depletion significantly inhibited proliferation in vitro, whilst having minimal effects in HPV negative cervical cancer cells. Furthermore, we show that the LASP1 SH3 domain is essential for LASP1-mediated proliferation in these cells. Mechanistically, we show that HPV E7 regulates LASP1 at the post-transcriptional level by repressing the expression of miR-203, which negatively regulated LASP1 mRNA levels by binding to its 3UTR. Finally, we demonstrated that LASP1 expression is required for the growth of HPV positive cervical cancer cells in an in vivo tumourigenicity model. Together, these data demonstrate that HPV induces LASP1 expression to promote proliferation and survival role in cervical cancer, thus identifying a potential therapeutic target in these cancers.

cancer biology↗

bioRxiv2022

Expansion microscopy reveals subdomains in C. elegans germ granules

Many membraneless organelles (MLOs) have been shown to form via liquid-liquid phase separation (LLPS). Light and electron microscopy techniques have been indispensable in the identification and characterization of LLPS MLOs. However, for complex MLOs such as the perinuclear germ granule in C. elegans, our understanding of how the organelle as a whole is regulated is hampered by 1) the technical limitations in confocal fluorescence imaging in which only a few granule protein markers can be examined at a time and 2) the inaccessibility of electron microscopy. In this study, we take advantage of the newly developed super-resolution method of expansion microscopy (ExM) and in-situ staining of the whole proteome to examine the C. elegans germ granule, the P granule. We show that in small RNA pathway mutants, the P granule is smaller compared with wild type animals. Furthermore, we investigate the relationship between the P granule and two other germ granules, mutator foci and Z granule, and show that they are located within the same protein-dense regions while occupying distinct subdomains within this ultrastructure. The experimental workflow developed here will serve as an important tool in our understanding of germ granule biology as well as the biological role of LLPS.

cell biology↗

bioRxiv2021

Calcium-binding protein S100A6 interaction with VEGF receptors integrates signaling and trafficking pathways

The mammalian endothelium which lines all blood vessels responds to soluble factors which control vascular development and sprouting. Endothelial cells bind to vascular endothelial growth factor A via two different receptor tyrosine kinases (VEGFR1, VEGFR2) which regulate such cellular responses. The integration of VEGFR signal transduction and membrane trafficking is not well understood. Here, we used a yeast-based membrane protein screen to identify VEGFR-interacting factor(s) which modulate endothelial cell function. By screening a human endothelial cDNA library, we identified a calcium-binding protein, S100A6, which can interact with either VEGFR. We found that S100A6 binds in a calcium-dependent manner to either VEGFR1 or VEGFR2. S100A6 binding was mapped to the VEGFR2 tyrosine kinase domain. Depletion of S100A6 impacts on VEGF-A-regulated signaling through the canonical mitogen-activated protein kinase (MAPK) pathway. Furthermore, S100A6 depletion caused contrasting effects on biosynthetic VEGFR delivery to the plasma membrane. Co-distribution of S100A6 and VEGFRs on tubular profiles suggest the presence of transport carriers that facilitate VEGFR trafficking. We propose a mechanism whereby S100A6 acts as a calcium-regulated switch which facilitates biosynthetic VEGFR trafficking from the TGN-to-plasma membrane. VEGFR-S100A6 interactions thus enable integration of signaling and trafficking pathways in controlling the endothelial response to VEGF-A.

cell biology↗

bioRxiv2020

Regulation of kinase activity by combined action of juxtamembrane and C-terminal regions of receptors

Despite the kinetically-favorable, ATP-rich intracellular environment, the mechanism by which receptor tyrosine kinases (RTKs) repress activation prior to extracellular stimulation is poorly understood. RTKs are activated through a precise sequence of phosphorylation reactions starting with a tyrosine on the activation loop (A-loop) of the intracellular kinase domain (KD). This forms an essential mono-phosphorylated active intermediate state on the path to further phosphorylation of the receptor. We show that this state is subjected to stringent control imposed by the peripheral juxtamembrane (JM) and C-terminal tail (CT) regions. This entails interplay between the intermolecular interaction between JM with KD, which stabilizes the asymmetric active KD dimer, and the opposing intramolecular binding of CT to KD. A further control step is provided by the previously unobserved direct binding between JM and CT. Mutations in JM and CT sites that perturb regulation are found in numerous pathologies, revealing novel sites for potential pharmaceutical intervention.

biochemistry↗

bioRxiv2019

Grb2 binding induces phosphorylation-independent activation of Shp2

The regulation of phosphatase activity is fundamental to the control of intracellular signalling and in particular the tyrosine kinase-mediated mitogen-activated protein kinase (MAPK) pathway. Shp2 is a ubiquitously expressed protein tyrosine phosphatase and its kinase-induced hyperactivity is associated with many cancer types. In non-stimulated cells we find that binding of the adaptor protein, Grb2, in its monomeric state initiates Shp2 activity independent of phosphatase phosphorylation. Grb2 forms a bidentate interaction with both the N-terminal SH2 and the catalytic domains of Shp2, releasing the phosphatase from its auto-inhibited conformation. Grb2 typically exists as a dimer in the cytoplasm. However, its monomeric state prevails under basal conditions when it is expressed at low concentration, or when it is constitutively phosphorylated on a specific tyrosine residue (Y160). Thus, Grb2 can activate Shp2 and downstream signal transduction, in the absence of extracellular growth factor stimulation or kinase-activating mutations, in response to defined cellular conditions. We identify a polypeptide biotool capable of blocking the Grb2-Shp2 interaction. This peptide down-regulates Shp2 activity in vitro and MAPK signalling in a cancer cell line.

cancer biology↗

bioRxiv2019

Receptor tyrosine kinases regulate signal transduction through a liquid-liquid phase separated state

Receptor tyrosine kinases (RTKs), the largest class of transmembrane cell surface receptors, initiate signalling pathways which regulate diverse cellular processes. On activation these receptors rapidly recruit multiple downstream effector proteins to moderate affinity tyrosyl phosphate (pY) binding sites. However, the mechanism for expedient downstream effector protein recruitment via random molecular diffusion through the cytoplasm is not fully understood. One way in which the probabilistic outcome associated with random diffusion could be alleviated is through localized accumulation of high effective concentrations of signalling proteins in discrete pools in the cell (1). The inclusion of interacting proteins into liquid-liquid phase-separated (LLPS), membraneless protein droplets maintains functionally relevant proteins at high concentrations in a liquid phase at the required point of action, enhancing equilibrium binding and enzyme activity (2-6). These LLPS states have been associated with a wide range of cellular functions including regulation of signalling through, for example, nephrin (7, 8), the T-cell receptor (9), mTOR (10), and Sos-Ras (11), however, whether LLPS extends to RTK-mediated signal transduction has not been investigated. Here, we show that an RTK, fibroblast growth factor receptor 2 (FGFR2), forms a signalling competent LLPS state with two downstream effectors, a tandem Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 2 (Shp2), and 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma 1 (Plc{gamma}1). We show that these proteins assemble into a ternary complex which exploits LLPS condensation to simultaneously modulate kinase, phosphatase and phospholipase activities. Therefore, LLPS formation ensures that the requirement for prolonged, high-fidelity signalling is achieved. Additional RTKs also form LLPS with their downstream effectors, suggesting that formation of biological condensates is a key organising principle of RTK-mediated signalling, with broad implications for further mechanistic studies as well as therapeutic intervention.

cell biology↗

bioRxiv2019

Direct interaction of PIWI and DEPS-1 is essential for piRNA function and condensate ultrastructure in Caenorhabditis elegans

Membraneless organelles are platforms for many aspects of RNA biology including small non-coding RNA (ncRNA) mediated gene silencing. How small ncRNAs utilise phase separated environments for their function is unclear. To address this question, we investigated how the PIWI-interacting RNA (piRNA) pathway engages with the membraneless organelle P granule in Caenorhabditis elegans. Proteomic analysis of the PIWI protein PRG-1 revealed an interaction with the constitutive P granule protein DEPS-1. Furthermore we identified a novel motif on DEPS-1, PBS, which interacts directly with the Piwi domain of PRG-1. This protein complex forms intertwining ultrastructures to build elongated condensates in vivo. These sub-organelle ultrastructures depend on the Piwi-interacting motif of DEPS-1 and mediate piRNA function. Additionally, we identify a novel interactor of DEPS-1, EDG-1, which is required for DEPS-1 condensates to form correctly. We show that DEPS-1 is not required for piRNA biogenesis but piRNA function: deps-1 mutants fail to produce the secondary endo-siRNAs required for the silencing of piRNA targets. Our study reveals how specific protein-protein interactions drive the spatial organisation and function of small RNA pathways within membraneless organelles.

cell biology↗