Ross SH, Rollings CM, Cantrell DA Immunology

Metabolic and nutrient-sensing pathways play an important role in controlling the efficacy of effector T cells. Oxygen is a critical regulator of cellular metabolism. However, during immune responses T cells must function in oxygen-deficient, or hypoxic, environments. Here, we used high resolution mass spectrometry to investigate how the proteome of primary murine CD8 cytotoxic T lymphocytes (CTLs) is reconfigured in response to hypoxia . We identified and quantified over 7,600 proteins and discovered that hypoxia increased the abundance of a selected number of proteins in CTLs. This included glucose transporters, metabolic enzymes, transcription factors, cytolytic effector molecules, checkpoint receptors and adhesion molecules. While some of these proteins may augment the effector functions of CTLs, others may limit their cytotoxicity. Moreover, we determined that hypoxia could inhibit IL-2-induced proliferation cues and antigen-induced pro-inflammatory cytokine production in CTLs. These data provide a comprehensive resource for understanding the magnitude of the CTL response to hypoxia and emphasise the importance of oxygen-sensing pathways for controlling CD8 T cells. Additionally, this study provides new understanding about how hypoxia may promote the effector function of CTLs, while contributing to their dysfunction in some contexts.

+view abstract Frontiers in immunology, PMID: 34603285 2021

Ross SH, Cantrell DA Immunology

Personalized medicines require understanding the molecular causes of disease. In this issue of Immunity, Gruber et al. reveal that a gain-of-function JAK1 genetic variant results in a mutant protein with mosaic expression that drives multi-organ immune dysregulation via kinase dependent and independent mechanisms. The work highlights how biochemistry can inform therapies to resolve complex immune disorders.

+view abstract Immunity, PMID: 32937149 15 Sep 2020

Ross SH, Cantrell DA ,

The discovery of interleukin-2 (IL-2) changed the molecular understanding of how the immune system is controlled. IL-2 is a pleiotropic cytokine, and dissecting the signaling pathways that allow IL-2 to control the differentiation and homeostasis of both pro- and anti-inflammatory T cells is fundamental to determining the molecular details of immune regulation. The IL-2 receptor couples to JAK tyrosine kinases and activates the STAT5 transcription factors. However, IL-2 does much more than control transcriptional programs; it is a key regulator of T cell metabolic programs. The development of global phosphoproteomic approaches has expanded the understanding of IL-2 signaling further, revealing the diversity of phosphoproteins that may be influenced by IL-2 in T cells. However, it is increasingly clear that within each T cell subset, IL-2 will signal within a framework of other signal transduction networks that together will shape the transcriptional and metabolic programs that determine T cell fate.

+view abstract Annual review of immunology, PMID: 29677473 2018

Rollings CM, Sinclair LV, Brady HJM, Cantrell DA, Ross SH Immunology,

Interleukin-2 (IL-2) and Janus kinases (JAKs) regulate transcriptional programs and protein synthesis to promote the differentiation of effector CD8 cytotoxic T lymphocytes (CTLs). Using high-resolution mass spectrometry, we generated an in-depth characterization of how IL-2 and JAKs configure the CTL proteome to control CTL function. We found that IL-2 signaling through JAK1 and JAK3 (JAK1/3) increased the abundance of a key subset of proteins to induce the accumulation of critical cytokines and effector molecules in T cells. Moreover, IL-2 maintained the concentration of proteins that support core metabolic processes essential for cellular fitness. One fundamental insight was the dominant role for IL-2 in stimulating effector T cells to detect microenvironmental cues. IL-2-JAK1/3 signaling pathways thus increased the abundance of nutrient transporters, nutrient sensors, and critical oxygen-sensing molecules. These data provide key insights into how IL-2 promotes T cell function and highlight signaling mechanisms and transcription factors that integrate oxygen sensing to transcriptional control of CD8 T cell differentiation.

+view abstract Science signaling, PMID: 29666307 2018

Ross SH, Rollings C, Anderson KE, Hawkins PT, Stephens LR, Cantrell DA Signalling,

Interleukin-2 (IL-2) is a fundamental cytokine that controls proliferation and differentiation of T cells. Here, we used high-resolution mass spectrometry to generate a comprehensive and detailed map of IL-2 protein phosphorylations in cytotoxic T cells (CTL). The data revealed that Janus kinases (JAKs) couple IL-2 receptors to the coordinated phosphorylation of transcription factors, regulators of chromatin, mRNA translation, GTPases, vesicle trafficking, and the actin and microtubule cytoskeleton. We identified an IL-2-JAK-independent SRC family Tyr-kinase-controlled signaling network that regulates ∼10% of the CTL phosphoproteome, the production of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), and the activity of the serine/threonine kinase AKT. These data reveal a signaling framework wherein IL-2-JAK-controlled pathways coordinate with IL-2-independent networks of kinase activity and provide a resource toward the further understanding of the networks of protein phosphorylation that program CTL fate.

+view abstract Immunity, PMID: 27566939 2016

Post A, Pannekoek WJ, Ross SH, Verlaan I, Brouwer PM, Bos JL ,

Rap1 is a small GTPase regulating cell-cell adhesion, cell-matrix adhesion, and actin rearrangements, all processes dynamically coordinated during cell spreading and endothelial barrier function. Here, we identify the adaptor protein ras-interacting protein 1 (Rasip1) as a Rap1-effector involved in cell spreading and endothelial barrier function. Using Förster resonance energy transfer, we show that Rasip1 interacts with active Rap1 in a cellular context. Rasip1 mediates Rap1-induced cell spreading through its interaction partner Rho GTPase-activating protein 29 (ArhGAP29), a GTPase activating protein for Rho proteins. Accordingly, the Rap1-Rasip1 complex induces cell spreading by inhibiting Rho signaling. The Rasip1-ArhGAP29 pathway also functions in Rap1-mediated regulation of endothelial junctions, which controls endothelial barrier function. In this process, Rasip1 cooperates with its close relative ras-association and dilute domain-containing protein (Radil) to inhibit Rho-mediated stress fiber formation and induces junctional tightening. These results reveal an effector pathway for Rap1 in the modulation of Rho signaling and actin dynamics, through which Rap1 modulates endothelial barrier function.

+view abstract Proceedings of the National Academy of Sciences of the United States of America, PMID: 23798437 2013

Ross SH, Post A, Raaijmakers JH, Verlaan I, Gloerich M, Bos JL ,

The Rap family of small GTPases regulate the adhesion of cells to extracellular matrices. Several Rap-binding proteins have been shown to function as effectors that mediate Rap-induced adhesion. However, little is known regarding the relationships between these effectors, or about other proteins that are downstream of or act in parallel to the effectors. To establish whether an array of effectors was required for Rap-induced cell adhesion and spreading, and to find new components involved in Rap-signal transduction, we performed a small-scale siRNA screen in A549 lung epithelial cells. Of the Rap effectors tested, only Radil blocked Rap-induced spreading. Additionally, we identified a novel role for Ezrin downstream of Rap1. Ezrin was necessary for Rap-induced cell spreading, but not Rap-induced cell adhesion or basal adhesion processes. Furthermore, Ezrin depletion inhibited Rap-induced cell spreading in several cell lines, including primary human umbilical vein endothelial cells. Interestingly, Radixin and Moesin, two proteins with high homology to Ezrin, are not required for Rap-induced cell spreading and cannot compensate for loss of Ezrin to rescue Rap-induced cell spreading. Here, we present a novel function for Ezrin in Rap1-induced cell spreading and evidence of a non-redundant role of an ERM family member.

+view abstract Journal of cell science, PMID: 21540295 2011

Ross SH, Lindsay Y, Safrany ST, Lorenzo O, Villa F, Toth R, Clague MJ, Downes CP, Leslie NR ,

The Protein Tyrosine Phosphatase (PTP) family comprises a large and diverse group of enzymes, regulating a range of biological processes through de-phosphorylation of many proteins and lipids. These enzymes share a catalytic mechanism that requires a reduced and reactive cysteine nucleophile, making them potentially sensitive to inactivation and regulation by oxidation. Analysis of ten PTPs identified substantial differences in the sensitivity of these enzymes to oxidation in vitro. More detailed experiments confirmed the following rank order of sensitivity: PTEN and Sac1>PTPL1/FAP-1>myotubularins. When the apparent sensitivity to oxidation of these PTPs in cells treated with hydrogen peroxide was analysed, this correlated well with the observed sensitivities to oxidation in vitro. These data suggested that different PTPs may fall into at least three different classes with respect to mechanisms of cellular redox regulation. 1. PTEN and Sac1 were readily and reversibly oxidised in vitro and in cells treated with hydrogen peroxide 2. PTPL1 appeared to be resistant to oxidation in cells, correlating with its sensitivity to reduction by glutathione in vitro 3. The myotubularin family of lipid phosphatases was almost completely resistant to oxidation in vitro and in cells. Our results show that sensitivity to reversible oxidation is not a necessary characteristic of the PTPs and imply that such sensitivity has evolved as a regulatory mechanism for some of this large family, but not others.

+view abstract Cellular signalling, PMID: 17346927 2007

Downes CP, Ross S, Maccario H, Perera N, Davidson L, Leslie NR ,

+view abstract Advances in enzyme regulation, PMID: 17343901 2007

Downes CP, Perera N, Ross S, Leslie NR ,

PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a tumour suppressor that functions as a PtdIns(3,4,5)P3 3-phosphatase to inhibit cell proliferation, survival and growth by antagonizing PI3K (phosphoinositide 3-kinase)-dependent signalling. Recent work has begun to focus attention on potential biological functions of the protein phosphatase activity of PTEN and on the possibility that some of its functions are phosphatase-independent. We discuss here the structural and regulatory mechanisms that account for the remarkable specificity of PTEN with respect to its PtdIns substrates and how it avoids the soluble headgroups of PtdIns that occur commonly in cells. Secondly we discuss the concept of PTEN as a constitutively active enzyme that is subject to negative regulation both physiologically and pathologically. Thirdly, we review the evidence that PTEN functions as a dual specificity phosphatase with discrete lipid and protein substrates. Lastly we present a current model of how PTEN may participate in the control of cell migration.

+view abstract Biochemical Society symposium, PMID: 17233581 2007

Leslie NR, Lindsay Y, Ross SH, Downes CP ,

Although reactive oxygen species play important roles in cellular physiology as signalling molecules, their molecular targets are largely unknown. A probable group of targets for mediating many of the effects of reactive oxygen species on cell signalling is the large diverse family of cysteine-dependent phosphatases, which includes the protein tyrosine phosphatases. Our work and that of others suggest that the oxidative inactivation of protein and lipid phosphatases plays an important part in signalling, downstream of many cellular stimuli. Future studies should give us a clearer picture of the role of phosphatase inactivation in cellular behaviour and explain how specificity is achieved in redox signalling.

+view abstract Biochemical Society transactions, PMID: 15506952 2004