Signaling In Mammalian Cells Research Articles

Mammalian Mitochondrial Inorganic Polyphosphate (polyP) and Cell Signaling: Crosstalk Between PolyP and the Activity of AMPK.

Inorganic polyphosphate (polyP) is an evolutionary ancient polymer composed by orthophosphate units linked by phosphoanhydride bonds. In mammalian cells, polyP shows a high localization in mammalian mitochondria, and its regulatory role in various aspects of bioenergetics has already been demonstrated, via molecular mechanism(s) yet to be fully elucidated. In recent years, a role for polyP in signal transduction, from brain physiology to bloodstream, has also emerged. The intriguing possibility is that the effects of polyP on signal transduction could be mechanistically linked to those exerted on bioenergetics. Here, using a combination of cellular and animal models, we demonstrate for the first time the intimate crosstalk between the levels of polyP and the activation status of the AMPK signaling pathway, via a mechanism involving free phosphate homeostasis. AMPK is a key player in mammalian cell signaling, and a crucial regulator of cellular and mitochondrial homeostasis. Our results show that the depletion of mitochondrial polyP in mammalian cells downregulates the activity of AMPK. Moreover, increased levels of polyP activate AMPK. Accordingly, the genetic downregulation of AMPKα1 impairs polyP levels in both SH-SY5Y cells and in the brains of female mice. Our findings shed new light on the regulation of AMPK and position polyP as a potent regulator of mammalian cell physiology beyond mere bioenergetics, paving the road for using its metabolism as an innovative pharmacological target in pathologies characterized by dysregulated bioenergetics.

Interrogation of the interplay between DNA N6-methyladenosine (6mA) and hypoxia-induced chromatin accessibility by a randomized empirical model (EnrichShuf).

N 6-Methyladenosine (6mA) is an epigenetic mark in eukaryotes regulating development, stress response and tumor progression. METTL4 has been reported as a 6mA methyltransferase induced by hypoxia. The detection and annotation of 6mA signals in mammalian cells have been hampered by the techniques and analytical methods developed so far. Here we developed a 6mA-ChIP-exo-5.1-seq to improve the sensitivity of detecting 6mAs in human cell lines. Furthermore, an EnrichShuf analysis tool for comprehensively comparing 6mA-ChIP-exo-5.1-seq, ATAC-seq, ChIP-seq and RNA-seq has been developed to annotate the functional relevance of 6mA in relation to chromatin accessibility and histone marks. Using a hypoxia-induced 6mA induction system as a model, we showed that hypoxic 6mA signals positively correlated with accessible chromatin regions. These 6mA signals correlate with their regulation by METTL4 under hypoxia, consistent with previous results. 6mAs also co-exist with H3K4me1, a histone mark for enhancers. Further analysis of enhancers using an ABC (active-by-contact) model shows that hypoxia-inducible factor-1α-induced H3K4me3 surrounds the 6mA/H3K4me1 site to augment active enhancers. These results suggest that correlation between 6mA and accessible chromatin regions plays a significant role in enhancer-promoter interactions during hypoxia-induced gene expression.

Open-ended molecular recording of sequential cellular events into DNA.

Genetically encoded DNA recorders noninvasively convert transient biological events into durable mutations in a cell's genome, allowing for the later reconstruction of cellular experiences by DNA sequencing. We present a DNA recorder, peCHYRON, that achieves high-information, durable, and temporally resolved multiplexed recording of multiple cellular signals in mammalian cells. In each step of recording, prime editor, a Cas9-reverse transcriptase fusion protein, inserts a variable triplet DNA sequence alongside a constant propagator sequence that deactivates the previous and activates the next step of insertion. Insertions accumulate sequentially in a unidirectional order, editing can continue indefinitely, and high information is achieved by coexpressing a variety of prime editing guide RNAs (pegRNAs), each harboring unique triplet DNA sequences. We demonstrate that the constitutive expression of pegRNA collections generates insertion patterns for the straightforward reconstruction of cell lineage relationships and that the inducible expression of specific pegRNAs results in the accurate recording of exposures to biological stimuli.

Hitting the target: cell signaling pathways modulation by extracellular vesicles.

Extracellular vesicles (EVs) are lipid bilayer-enclosed nanoparticles released outside the cell. EVs have drawn attention not only for their role in cell waste disposal, but also as additional tools for cell-to-cell communication. Their complex contents include not only lipids, but also proteins, nucleic acids (RNA, DNA), and metabolites. A large part of these molecules are involved in mediating or influencing signal transduction in target cells. In multicellular organisms, EVs have been suggested to modulate signals in cells localized either in the neighboring tissue or in distant regions of the body by interacting with the cell surface or by entering the cells via endocytosis or membrane fusion. Most of the EV-modulated cell signaling pathways have drawn considerable attention because they affect morphogenetic signaling pathways, as well as pathways activated by cytokines and growth factors. Therefore, they are implicated in relevant biological processes, such as embryonic development, cancer initiation and spreading, tissue differentiation and repair, and immune response. Furthermore, it has recently emerged that multicellular organisms interact with and receive signals through EVs released by their microbiota as well as by edible plants. This review reports studies investigating EV-mediated signaling in target mammalian cells, with a focus on key pathways for organism development, organ homeostasis, cell differentiation and immune response.

The dishevelled associated activator of morphogenesis protein 2 (Daam2) regulates neural tube closure.

The Wnt signaling pathway is highly conserved in metazoans and regulates a large array of cellular processes including motility, polarity and fate determination, and stem cell homeostasis. Modulation of the actin cytoskeleton via the non-canonical Wnt pathway regulate cell polarity and cell migration that are required for proper vertebrate gastrulation and subsequent neurulation. However, the mechanism(s) of how the non-canonical pathway mediates actin cytoskeleton modulation is not fully understood. Herein, we characterize the role of the Formin-homology protein; dishevelled associated activator of morphogenesis 2 (Daam2) protein in the Wnt signaling pathway. Co-immunoprecipitation assays confirm the binding of Daam2 to dishevelled2 (Dvl2) as well as the domains within these proteins required for interaction; additionally, the interaction between Daam2 and Dvl2 was Wnt-regulated. Sub-cellular localization studies reveal Daam2 is cytoplasmic and regulates the cellular actin cytoskeleton by modulating actin filament formation. During Xenopus development, a knockdown or loss of Daam2 specifically produces neural tube closure defects indicative of a role in non-canonical signaling. Additionally, our studies did not identify any role for Daam2 in canonical Wnt signaling in mammalian culture cells or the Xenopus embryo. Our studies together identify Daam2 as a component of the non-canonical Wnt pathway and Daam2 is a regulator of neural tube morphogenesis during vertebrate development.

The E3/E4 ubiquitin ligase UFD-2 suppresses normal and oncogenic signaling mediated by a Raf ortholog in Caenorhabditis elegans.

Signaling by the kinase cascade composed of Raf, MEK, and ERK is critical for animal development and is often inappropriately activated in human malignancies. We sought to identify factors that control signaling mediated by the Caenorhabditis elegans Raf ortholog LIN-45. A genetic screen showed that the degradation of LIN-45 required the E3/E4 ubiquitin ligase UFD-2. Both UFD-2 and its partner, the ATP-dependent segregase CDC-48, were required for the developmental regulation of LIN-45 protein abundance. We showed that UFD-2 acted in the same pathway as the E3 ubiquitin ligase SCFSEL-10 to decrease LIN-45 abundance in cells in which Raf-MEK-ERK signaling was most highly active. UFD-2 also reduced the protein abundance of activated LIN-45 carrying a mutation equivalent to the cancer-associated BRAF(V600E) variant. Our structure-function studies showed that the disruption of LIN-45 domains that mediate protein-protein interactions, including the conserved cysteine-rich domain and 14-3-3 binding motifs, were required for UFD-2-independent degradation of LIN-45. We propose a model in which UFD-2 and CDC-48 act downstream of SCFSEL-10 to remove LIN-45 from its protein interaction partners and facilitate proteasomal targeting and degradation. These findings imply that UFD-2 and CDC-48 may be important for Raf degradation during normal and oncogenic Ras and MAPK signaling in mammalian cells.

CD44 acts as a coreceptor for cell-specific enhancement of signaling and regulatory T cell induction by TGM1, a parasite TGF-β mimic

Long-lived parasites evade host immunity through highly evolved molecular strategies. The murine intestinal helminth, Heligmosomoides polygyrus, down-modulates the host immune system through release of an immunosuppressive TGF-β mimic, TGM1, which is a divergent member of the CCP (Sushi) protein family. TGM1 comprises 5 domains, of which domains 1-3 (D1/2/3) bind mammalian TGF-β receptors, acting on T cells to induce Foxp3+ regulatory T cells; however, the roles of domains 4 and 5 (D4/5) remain unknown. We noted that truncated TGM1, lacking D4/5, showed reduced potency. Combination of D1/2/3 and D4/5 as separate proteins did not alter potency, suggesting that a physical linkage is required and that these domains do not deliver an independent signal. Coprecipitation from cells treated with biotinylated D4/5, followed by mass spectrometry, identified the cell surface protein CD44 as a coreceptor for TGM1. Both full-length and D4/5 bound strongly to a range of primary cells and cell lines, to a greater degree than D1/2/3 alone, although some cell lines did not respond to TGM1. Ectopic expression of CD44 in nonresponding cells conferred responsiveness, while genetic depletion of CD44 abolished enhancement by D4/5 and ablated the ability of full-length TGM1 to bind to cell surfaces. Moreover, CD44-deficient T cells showed attenuated induction of Foxp3 by full-length TGM1, to levels similar to those induced by D1/2/3. Hence, a parasite protein known to bind two host cytokine receptor subunits has evolved a third receptor specificity, which serves to raise the avidity and cell type-specific potency of TGF-β signaling in mammalian cells.

Shedding light on ORAI1 channel with genetic code expansion

Genetic code expansion technology has been widely applied to control protein activity and biological systems by taking advantage of an amber stop codon suppressor tRNA and orthogonal aminoacyl-tRNA synthetase pair. With this chemical biology approach, Maltan et al. incorporated photocrosslinking unnatural amino acids (UAAs) into the transmembrane domains of ORAI1 to enable UV light-inducible calcium influx across the plasma membrane, mechanistic interrogation of the calcium release-activated calcium (CRAC) channel at the single amino acid level, and remote control of downstream calcium-modulated signaling in mammalian cells.

Rate thresholds in cell signaling have functional and phenotypic consequences in non-linear time-dependent environments.

All cells employ signal transduction pathways to respond to physiologically relevant extracellular cytokines, stressors, nutrient levels, hormones, morphogens, and other stimuli that vary in concentration and rate in healthy and diseased states. A central unsolved fundamental question in cell signaling is whether and how cells sense and integrate information conveyed by changes in the rate of extracellular stimuli concentrations, in addition to the absolute difference in concentration. We propose that different environmental changes over time influence cell behavior in addition to different signaling molecules or different genetic backgrounds. However, most current biomedical research focuses on acute environmental changes and does not consider how cells respond to environments that change slowly over time. As an example of such environmental change, we review cell sensitivity to environmental rate changes, including the novel mechanism of rate threshold. A rate threshold is defined as a threshold in the rate of change in the environment in which a rate value below the threshold does not activate signaling and a rate value above the threshold leads to signal activation. We reviewed p38/Hog1 osmotic stress signaling in yeast, chemotaxis and stress response in bacteria, cyclic adenosine monophosphate signaling in Amoebae, growth factors signaling in mammalian cells, morphogen dynamics during development, temporal dynamics of glucose and insulin signaling, and spatio-temproral stressors in the kidney. These reviewed examples from the literature indicate that rate thresholds are widespread and an underappreciated fundamental property of cell signaling. Finally, by studying cells in non-linear environments, we outline future directions to understand cell physiology better in normal and pathophysiological conditions.

Medicinal plant-derived mtDNA via nanovesicles induces the cGAS-STING pathway to remold tumor-associated macrophages for tumor regression

Plant-derived nanovesicles (PDNVs) have been proposed as a major mechanism for the inter-kingdom interaction and communication, but the effector components enclosed in the vesicles and the mechanisms involved are largely unknown. The plant Artemisia annua is known as an anti-malaria agent that also exhibits a wide range of biological activities including the immunoregulatory and anti-tumor properties with the mechanisms to be further addressed. Here, we isolated and purified the exosome-like particles from A. annua, which were characterized by nano-scaled and membrane-bound shape and hence termed artemisia-derived nanovesicles (ADNVs). Remarkably, the vesicles demonstrated to inhibit tumor growth and boost anti-tumor immunity in a mouse model of lung cancer, primarily through remolding the tumor microenvironment and reprogramming tumor-associated macrophages (TAMs). We identified plant-derived mitochondrial DNA (mtDNA), upon internalized into TAMs via the vesicles, as a major effector molecule to induce the cGAS-STING pathway driving the shift of pro-tumor macrophages to anti-tumor phenotype. Furthermore, our data showed that administration of ADNVs greatly improved the efficacy of PD-L1 inhibitor, a prototypic immune checkpoint inhibitor, in tumor-bearing mice. Together, the present study, for the first time, to our knowledge, unravels an inter-kingdom interaction wherein the medical plant-derived mtDNA, via the nanovesicles, induces the immunostimulatory signaling in mammalian immune cells for resetting anti-tumor immunity and promoting tumor eradication.Graphical

Bioluminescent imaging systems boosting near-infrared signals in mammalian cells.

Bioluminescence (BL) is broadly used as an optical readout in bioassays and molecular imaging. In this study, the near-infrared (NIR) BL imaging systems were developed. The system was harnessed by prototype copepod luciferases, artificial luciferase 30 (ALuc30) and its miniaturized version picALuc, and were characterized with 17 kinds of coelenterazine (CTZ) analogues carrying bulky functional groups or cyanine 5 (Cy5). They were analyzed of BL spectral peaks and enzymatic kinetics, and explained with computational modeling. The results showed that (1) the picALuc-based system surprisingly boosts the BL intensities predominantly in the red and NIR region with its specific CTZ analogues; (2) both ALuc30- and picALuc-based systems develop unique through-bond energy transfer (TBET)-driven spectral bands in the NIR region with a Cy5-conjugated CTZ analogue (Cy5-CTZ); and (3) according to the computational modeling, the miniaturized version, picALuc, has a large binding pocket, which can accommodate CTZ analogues containing bulky functional groups and thus allowing NIR BL. This study is an important addition to the BL imaging toolbox with respect to the development of orthogonal NIR reporter systems applicable to physiological samples, together with the understanding of the BL-emitting chemistry of marine luciferases.

DBP7 and YRF1-6 Are Involved in Cell Sensitivity to LiCl by Regulating the Translation of PGM2 mRNA.

Lithium chloride (LiCl) has been widely researched and utilized as a therapeutic option for bipolar disorder (BD). Several pathways, including cell signaling and signal transduction pathways in mammalian cells, are shown to be regulated by LiCl. LiCl can negatively control the expression and activity of PGM2, a phosphoglucomutase that influences sugar metabolism in yeast. In the presence of galactose, when yeast cells are challenged by LiCl, the phosphoglucomutase activity of PGM2p is decreased, causing an increase in the concentration of toxic galactose metabolism intermediates that result in cell sensitivity. Here, we report that the null yeast mutant strains DBP7∆ and YRF1-6∆ exhibit increased LiCl sensitivity on galactose-containing media. Additionally, we demonstrate that DBP7 and YRF1-6 modulate the translational level of PGM2 mRNA, and the observed alteration in translation seems to be associated with the 5'-untranslated region (UTR) of PGM2 mRNA. Furthermore, we observe that DBP7 and YRF1-6 influence, to varying degrees, the translation of other mRNAs that carry different 5'-UTR secondary structures.

Engineering receptors in the secretory pathway for orthogonal signalling control

Synthetic receptors targeted to the secretory pathway often fail to exhibit the expected activity due to post-translational modifications (PTMs) and/or improper folding. Here, we engineered synthetic receptors that reside in the cytoplasm, inside the endoplasmic reticulum (ER), or on the plasma membrane through orientation adjustment of the receptor parts and by elimination of dysfunctional PTMs sites. The cytoplasmic receptors consist of split-TEVp domains that reconstitute an active protease through chemically-induced dimerization (CID) that is triggered by rapamycin, abscisic acid, or gibberellin. Inside the ER, however, some of these receptors were non-functional, but their activity was restored by mutagenesis of cysteine and asparagine, residues that are typically associated with PTMs. Finally, we engineered orthogonal chemically activated cell-surface receptors (OCARs) consisting of the Notch1 transmembrane domain fused to cytoplasmic tTA and extracellular CID domains. Mutagenesis of cysteine residues in CID domains afforded functional OCARs which enabled fine-tuning of orthogonal signalling in mammalian cells.

ER Stress-Induced Sphingosine-1-Phosphate Lyase Phosphorylation Potentiates the Mitochondrial Unfolded Protein Response

The unfolded protein response (UPR) is an elaborate signaling network that evolved to maintain proteostasis in the endoplasmic reticulum (ER) and mitochondria (mt). These organelles are functionally and physically associated, and consequently, their stress responses are often intertwined. It is unclear how these two adaptive stress responses are coordinated during ER stress. The inositol-requiring enzyme-1 (IRE1), a central ER stress sensor and proximal regulator of the UPRER, harbors dual kinase and endoribonuclease (RNase) activities. IRE1 RNase activity initiates the transcriptional layer of the UPRER, but IRE1’s kinase substrate(s) and their functions are largely unknown. Here, we discovered that sphingosine 1-phosphate (S1P) lyase (SPL), the enzyme that degrades S1P, is a substrate for the mammalian IRE1 kinase. Our data show that IRE1-dependent SPL phosphorylation inhibits SPL’s enzymatic activity, resulting in increased intracellular S1P levels. S1P has previously been shown to induce the activation of mitochondrial UPR (UPRmt) in nematodes. We determined that IRE1 kinase-dependent S1P induction during ER stress potentiates UPRmt signaling in mammalian cells. Phosphorylation of eukaryotic translation initiation factor 2α (eif2α) is recognized as a critical molecular event for UPRmt activation in mammalian cells. Our data further demonstrate that inhibition of the IRE1-SPL axis abrogates the activation of two eif2α kinases, namely double-stranded RNA-activated protein kinase (PKR) and PKR–like ER kinase upon ER stress. These findings show that the IRE1-SPL axis plays a central role in coordinating the adaptive responses of ER and mitochondria to ER stress in mammalian cells.

The organotin triphenyltin disrupts cholesterol signaling in mammalian ovarian steroidogenic cells through a combination of LXR and RXR modulation

Organotins, a chemical family with over 30 congeners to which humans are directly exposed to through food consumption, are a chemical class widely used as stabilizers in polyvinyl chloride, and biocides in antifouling products. Aside from tributyltin (TBT), toxicological information on other organotin congeners, such as triphenyltin (TPT), remains scarce. Our previous work has demonstrated that TBT can interfere with cholesterol trafficking in steroidogenic cells. Given their structural similarities, we hypothesized that TPT, similar to TBT, disrupts intracellular cholesterol transport and impairs steroidogenesis in ovarian theca cells. To test this, human and ovine primary ovarian theca cells were isolated, purified and exposed to TPT at environmentally relevant doses (1 or 10 ng/ml) in pre-luteinized (48 h exposure) or luteinizing cells (72 h exposure). Intracellular cholesterol levels, progesterone, and testosterone secretion and gene expression of nuclear receptors, cholesterol transporters, and steroidogenic enzymes were evaluated. In ovine cells, TPT upregulated StAR, ABCA1, and SREBF1 mRNA and ABCA1 protein in both pre-luteinized and luteinized stages. TPT did not alter intracellular cholesterol or testosterone synthesis, but upregulated progesterone production. Inhibitor and shRNA knockdown approaches were then used to evaluate the role of retinoid X receptor (RXR) and liver X receptor (LXR) on TPT's effects. TPT upregulated ABCA1 and StAR expression was blocked by both LXR and RXR antagonists. TPT's effect on ABCA1 expression was reduced in LXRβ and RXRβ knockdown theca cells. Similar findings were obtained with primary human theca cells. No synergistic effect of TBT and TPT was observed. In conclusion, at an environmentally relevant dose, TPT upregulates theca cell cholesterol transporter ABCA1 expression via RXR and LXR pathways. Similar effects of TPT on human and sheep theca cells supports its conserved mechanism across mammalian theca cells.

Function conservation and disparities of zebrafish and human LGP2 genes in fish and mammalian cells responsive to poly(I:C).

Retinoic acid inducible gene-I (RIG-I)-like receptors (RLRs) are viral RNA sensors that regulate host interferon (IFN)-mediated antiviral signaling. LGP2 (laboratory genetics and physiology 2) lacks the N-terminal caspase activation and recruitment domains (CARDs) responsible for signaling transduction in the other two RLR proteins, RIG-I and melanoma differentiation associated gene-5 (MDA5). How LGP2 regulates IFN signaling is controversial, and inconsistent results have often been obtained in overexpression assays when performed in fish cells and mammalian cells. Here we report that the differential sensitivity of fish cells and mammalian cells to poly(I:C) transfection conceals the function conservation of zebrafish and human LGP2. In fish cells, overexpression of zebrafish or human LGP2 initially activates IFN signaling in a dose-dependent manner, followed by inhibition at a critical threshold of LGP2 expression. A similar trend exists for LGP2-dependent IFN induction in response to stimulation by low and high concentrations of poly(I:C). In contrast, overexpression of zebrafish or human LGP2 alone in mammalian cells does not activate IFN signaling, but co-stimulation with very low or very high concentrations of poly(I:C) shows LGP2-dependent enhancement or inhibition of IFN signaling, respectively. Titration assays show that LGP2 promotes MDA5 signaling in mammalian cells mainly under low concentration of poly(I:C) and inhibits RIG-I/MDA5 signaling mainly under high concentration of poly(I:C). Our results suggest that fish and human LGP2s switch regulatory roles from a positive one to a negative one in increasing concentrations of poly(I:C)-triggered IFN response.

A Novel Antiviral Protein Derived from Oenanthe javanica: Type I Interferon-Dependent Antiviral Signaling and Its Pharmacological Potential.

Pathogenesis-related (PR) proteins produced in plants play a crucial role in self-defense against microbial attacks. Previously, we have identified a novel PR-1-like protein (OPRP) from Oenanthe javanica and examined its pharmacologic relevance and cell signaling in mammalian cells. Purified full-length OPRP protein significantly increased toll-like receptor 4 (TLR4)-dependent expression levels of genes such as inducible nitric oxide synthase (iNOS), tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and CD80. We also found that small peptides (OPRP2 and OPRP3) designed from OPRP remarkably upregulated myxovirus resistance (Mx1), 2′-5′ oligoadenylate sythetase (OAS), and interferon (IFN) α/β genes in mouse splenocytes as well as human epithelial cells. Notably, OPRP protein distinctively activated STAT1 phosphorylation and ISGF-3γ. Interestingly, OPRP2 and OPRP3 were internalized to the cytoplasm and triggered dimerization of STAT1/STAT2, followed by upregulation of type I IFN-dependent antiviral cytokines. Moreover, OPRP1 successfully inhibited viral (Pseudo SARS-CoV-2) entry into host cells. Taken together, we conclude that OPRP and its small peptides (OPRP1 to 3) present a new therapeutic intervention for modulating innate immune activity through type I IFN-dependent antiviral signaling and a new therapeutic approach that drives an antiviral state in non-immune cells by producing antiviral cytokines.

A general approach for engineering RTKs optically controlled with far-red light

Regulation of receptor tyrosine kinase (RTK) activity is necessary for studying cell signaling pathways in health and disease. We developed a generalized approach for engineering RTKs optically controlled with far-red light. We targeted the bacterial phytochrome DrBphP to the cell surface and allowed its light-induced conformational changes to be transmitted across the plasma membrane via transmembrane helices to intracellular RTK domains. Systematic optimization of these constructs has resulted in optically regulated epidermal growth factor receptor, HER2, TrkA, TrkB, FGFR1, IR1, cKIT and cMet, named eDrRTKs. eDrRTKs induced downstream signaling in mammalian cells in tens of seconds. The ability to activate eDrRTKs with far-red light enabled spectral multiplexing with fluorescent probes operating in a shorter spectral range, allowing for all-optical assays. We validated eDrTrkB performance in mice and found that minimally invasive stimulation in the neocortex with penetrating via skull far-red light-induced neural activity, early immediate gene expression and affected sleep patterns.

Inhibition of SHP2 and SHP1 Protein Tyrosine Phosphatase Activity by Chemically Induced Dimerization.

Protein tyrosine phosphatases (PTPs), the enzymes that catalyze the dephosphorylation of phosphotyrosine residues, are important regulators of mammalian cell signaling, whose activity is misregulated in numerous human diseases. PTPs are also notoriously difficult to selectively modulate with small molecules, and relatively few small-molecule tools for controlling their activities in the context of complex signaling pathways have been developed. Here, we show that a chemical inducer of dimerization (CID) can be used to selectively and potently inhibit constructs of Src-homology-2-containing PTP 2 (SHP2) that have been engineered to contain dimerization domains. Our strategy was inspired by the naturally occurring mechanism of SHP2 regulation, in which the PTP activity of SHP2’s catalytic domain is autoinhibited through an intramolecular interaction with the protein’s N-terminal SH2 (N-SH2) domain. We have re-engineered this inhibitory interaction to function intermolecularly by independently fusing the SHP2 catalytic and N-SH2 domains to protein domains that heterodimerize upon the introduction of the CID rapamycin. We show that rapamycin-induced protein dimerization leads to potent inhibition of SHP2’s catalytic activity, which is driven by increased proximity of the SHP2 catalytic and N-SH2 domains. We also demonstrate that CID-based inhibition of PTP activity can be applied to an oncogenic gain-of-function SHP2 mutant (E76K SHP2) and to the catalytic domain of the SHP2’s closest homologue, SHP1. In sum, CID-driven inhibition of PTP activity provides a broadly applicable tool for inhibiting dimerizable forms of the SHP PTPs and represents a novel paradigm for selective PTP inhibition through inducible protein–protein interactions.

Determinants of IGF-II influencing stability, receptor binding and activation

Insulin like growth factor II (IGF-II) is involved in metabolic and mitogenic signalling in mammalian cells and plays important roles in normal fetal development and postnatal growth. It is structurally similar to insulin and binds not only with high affinity to the type 1 insulin-like growth factor receptor (IGF-1R) but also to the insulin receptor isoform A (IR-A). As IGF-II expression is commonly upregulated in cancer and its signalling promotes cancer cell survival, an antagonist that blocks IGF-II action without perturbing insulin signalling would be invaluable. The high degree of structural homology between the IR and IGF-1R makes selectively targeting either receptor in the treatment of IGF-II-dependent cancers very challenging. However, there are sequence differences between insulin and IGF-II that convey receptor selectivity and influence binding affinity and signalling outcome. Insulin residue YB16 is a key residue involved in maintaining insulin stability, dimer formation and IR binding. Mutation of this residue to glutamine (as found in IGF-II) results in reduced binding affinity. In this study we sought to determine if the equivalent residue Q18 in IGF-II plays a similar role. We show through site-directed mutagenesis of Q18 that this residue contributes to IGF-II structural integrity, selectivity of IGF-1R/IR binding, but surprisingly does not influence IR-A signalling activation. These findings provide insights into a unique IGF-II residue that can influence receptor binding specificity whilst having little influence on signalling outcome.