Andrey S. Shaw, MD - US grants

The T cell membrane glycoproteins, CD4 and CD8, interact with the tyrosine protein kinase, p56lck, in T cells. This interaction supports a transmembrane signalling function for CD4 and CD8 that could play an important role in T cell development and activation. It is the goal of this proposal to analyze the structure of these protein complexes and their function in the normal and diseased immune system. Specifically, we hope to reconstitute various CD8 complexes composed of CD8alpha (lyt2) CD8beta (lyt3), CD1a and class I histocompatibility antigens and test each type of complex for its ability to bind p56lck. Expression of these different forms of CD8 are developmentally regulated and could modulate signalling by p56lck. We would also like to define domains of p56lck which interact with the cytoskeleton. Such an interaction could play a role in the assembly of protein complexes containing p56lck or could play a role in the reorganization of membrane proteins that occurs during T cell activation. Lastly, we would like to determine the requirements for reconstitution of the CD8alpha/p56lck complex in vitro using peptides that correspond to the binding domains. Reconstitution in vitro would allow us to test whether a metal ion might be involved in the interaction as suggested by our earlier data. To study the function of p56lck during T cell activation, we propose to 1) determine if the kinase activity of p56lck is altered by T cell activation and what the substrates of p56lck might be, and 2) determine the effect of downregulating p56lck expression on T cell activation using anti-sense RNA strategies. Because signalling mediated by CD4 may play a role in the immune dysfunction that accompanies infection with HIV, we hope to investigate whether HIV gp120 binding to CD4 is sufficient to generate a signal transduced by p56lck. Aberrant signalling through CD4 could play a role in HIV pathogenesis.

The T cell membrane glycoproteins, CD4 and CD8, interact with the tyrosine protein kinase, p56lck, in T cells. This interaction supports a transmembrane signalling function for CD4 and CD8 that could play an important role in T cell development and activation. It is the goal of this proposal to analyze the structure of these protein complexes and their function in the normal and diseased immune system. Specifically, we hope to reconstitute various CD8 complexes composed of CD8alpha (lyt2) CD8beta (lyt3), CD1a and class I histocompatibility antigens and test each type of complex for its ability to bind p56lck. Expression of these different forms of CD8 are developmentally regulated and could modulate signalling by p56lck. We would also like to define domains of p56lck which interact with the cytoskeleton. Such an interaction could play a role in the assembly of protein complexes containing p56lck or could play a role in the reorganization of membrane proteins that occurs during T cell activation. Lastly, we would like to determine the requirements for reconstitution of the CD8alpha/p56lck complex in vitro using peptides that correspond to the binding domains. Reconstitution in vitro would allow us to test whether a metal ion might be involved in the interaction as suggested by our earlier data. To study the function of p56lck during T cell activation, we propose to 1) determine if the kinase activity of p56lck is altered by T cell activation and what the substrates of p56lck might be, and 2) determine the effect of downregulating p56lck expression on T cell activation using anti-sense RNA strategies. Because signalling mediated by CD4 may play a role in the immune dysfunction that accompanies infection with HIV, we hope to investigate whether HIV gp120 binding to CD4 is sufficient to generate a signal transduced by p56lck. Aberrant signalling through CD4 could play a role in HIV pathogenesis.

Engagement of the T cell receptor by ligand results in a cascade of biochemical events that result in T cell proliferation and T cell effector responses such as cytolysis and cytokine secretion. The broad goals of this proposal are to define some of these early T cell signaling events and to determine how these events are initiated. As these signaling events play critical roles in almost all immune responses, a greater understanding of these events have clear practical relevance. Early signaling molecules would be excellent targets for therapies that aim to either enhance or suppress a specific immune response. Activation of a tyrosine kinase is thought to be the primary signaling event transduced by the T cell receptor. Although the identity of the tyrosine kinase activated is unknown, one candidate is the tyrosine kinase, p59fyn. It is associated with the T cell receptor, and can enhance signaling of T cells when it is overexpressed in transgenic mice. The specific goals of this proposal are to perform a structural characterization of the p59fyn/T cell receptor complex and to investigate the mechanism(s) used by p59fyn to signal in T cells. We propose to continue our previous work defining important structural features of the p59fYn/T cell receptor interaction (Aim #1). We will then determine the functional significance of this interaction by determining whether p59fyn is required for T cell receptor signaling or signaling by chimeric proteins that contain only single components of the T cell receptor (Aim #2). To define the specific roles of p56lck and p59fyn in T cell activation, we will define the domains of both proteins that give specificity to their actions (Aim #3). The function of these domains will then be examined by trying to identify proteins that interact with these domains (Aim #4).

The src-family of tyrosine kinases are ubiquitously expressed proteins that have been implicated in the regulation of growth, differentiation and transformation. As these proteins are kinases, understanding their mechanism of action will necessitate the identification of the proteins that are their substrates. Recently, we and others, identified a protein that binds to and is phosphorylated by src-family tyrosine kinases. This protein, p62, was first noted as a major tyrosine phosphorylated protein in transformed and growth factor treated cells. Although its function is unknown, it is known to bind ras-GAP and has therefore been implicated in the regulation of ras. The cloning of p62 in 1992 showed that p62 was related to RNA binding proteins, that it had a tyrosine-rich C-terminus and that it contains at least 5 proline-rich motifs. As phosphorylated p62 binds src-family kinases, phospholipase C and grb2 as well as ras-GAP via both SH2 and SH3 mediated interactions, we have proposed that p62 serves as a scaffolding for the recruitment of SH2 and SH3 containing signalling proteins in src- mediated signalling pathways. We propose to test this model as well as try and understand the role of the RNA binding domain of p62. Recently, a 68kD protein was described that is closely related to p62. As we have reagents that distinguish between the two, our first goal is to determine the relationship between p62 and p68. Our second goal, is to determine the function of p62/p68 in signal transduction pathways. Experiments are proposed to study the role of p62 in transformation as well as cell cycle regulation. Lastly, we propose to study the structural features of the p62 RNA binding domain and its regulation by tyrosine phosphorylation. We will then attempt to identify the cognate RNAs which bind to p62/p68 in vivo. As the phosphorylation of p62 has been demonstrated to be an almost ubiquitous feature of transformation and growth factor treatment, the findings generated in these studies will have immediate relevance to our understanding and possibly the treatment of human cancers.

The src-family of tyrosine kinases are ubiquitously expressed proteins that have been implicated in the regulation of growth, differentiation and transformation. As these proteins are kinases, understanding their mechanism of action will necessitate the identification of the proteins that are their substrates. Recently, we and others, identified a protein that binds to and is phosphorylated by src-family tyrosine kinases. This protein, p62, was first noted as a major tyrosine phosphorylated protein in transformed and growth factor treated cells. Although its function is unknown, it is known to bind ras-GAP and has therefore been implicated in the regulation of ras. The cloning of p62 in 1992 showed that p62 was related to RNA binding proteins, that it had a tyrosine-rich C-terminus and that it contains at least 5 proline-rich motifs. As phosphorylated p62 binds src-family kinases, phospholipase C and grb2 as well as ras-GAP via both SH2 and SH3 mediated interactions, we have proposed that p62 serves as a scaffolding for the recruitment of SH2 and SH3 containing signalling proteins in src- mediated signalling pathways. We propose to test this model as well as try and understand the role of the RNA binding domain of p62. Recently, a 68kD protein was described that is closely related to p62. As we have reagents that distinguish between the two, our first goal is to determine the relationship between p62 and p68. Our second goal, is to determine the function of p62/p68 in signal transduction pathways. Experiments are proposed to study the role of p62 in transformation as well as cell cycle regulation. Lastly, we propose to study the structural features of the p62 RNA binding domain and its regulation by tyrosine phosphorylation. We will then attempt to identify the cognate RNAs which bind to p62/p68 in vivo. As the phosphorylation of p62 has been demonstrated to be an almost ubiquitous feature of transformation and growth factor treatment, the findings generated in these studies will have immediate relevance to our understanding and possibly the treatment of human cancers.

Immunity against cancerous cells requires the recognition of tumor specific antigens by T lymphocytes. Although the specificity of such responses are determined, in part, by the T cell antigen receptors, the successful recognition of tumor cells requires the cooperation of other proteins on the surface of the T cell. In the studies proposed here, we will focus on one of these accessory proteins, CD2. Interactions between CD2 and its ligands CD48 and CD58 are known to enhance tumor cell recognition by both T and NK cells but the exact function of CD2 is not known. Recently, we cloned a novel protein, CD2AP, that binds to CD2 during T cell activation. This allowed us to propose that the clustering of CD2 regulates the affinity of CD2 for its ligands to facilitate recognition of target cells by T and NK cells. In specific aim#1, we propose to determine how CD2AP is regulated and how it functions. In specific aim #2, we propose to analyze the role of CD2 clustering in T cell activation. Lastly, we plan to study the role for CD2 in tumor immunity. The results of our work may lead to a better understanding of how tumor evade the immune system.

T lymphocytes play a critical role in mediating and regulating immune responses. Constantly surveying the body, T cells must be able to distinguish self from non-self. Although T cell development in the thymus plays a critical role in "teaching" T cells to distinguish between self and non self, peripheral mechanisms also play an important role. One of the mechanisms is co-stimulation. T cells are thought to require two signals to become activated. Engagement of the TCR without the second "co-stimulatory" signal leads to T cell unresponsiveness. Extensive studies over the last five years, however, have yet to reveal the biochemical nature of the co-stimulatory signal. Here, the functions of CD28 and CD2 as co-stimulatory molecules will be analyzed. One notable feature of the cytoplasmic domains of CD28 and CD2 is the presence of proline-rich sequences which could serve as ligands for SH3 domains. The recently solved crystal structures of the Src- kinases, lck and hck, suggest that an important mechanism for src-kinase activation will be via SH3 engagement. Preliminary data demonstrate that peptides based on the sequences of CD28 and CD2 can serve as potent activators of lck, fyn and ITK. This appears to be specific as distinct peptides activate different kinases. In specific aim #1, we will determine the structural basis for this effect. In specific aim #2, we propose to delineate critical structural features of CD28 and CD2 in primary T cells. It is still not known which sequences of CD28 and CD2 are critical for its ability to regulate T cell anergy. All of the CD28 structure-function studies to date were performed in transformed T cells which cannot be anergized. Experiments are proposed using retroviral and transgenic approaches to reconstitute CD2/CD28 knockout animals with wild-type and mutated forms of CD28 and CD2. Lastly, it was recently proposed that fyn activation of the small G- protein, Rap1, is responsible for the maintenance of T cell unresponsiveness. Experiments (specific aim #3) are planned to test this hypothesis using transgenic mice expressing B-Ray, and mutated forms of Rap1. These experiments are designed to definitively test the role of Rap1 in the process of anergy.

DESCRIPTION (provided by applicant): This is the second renewal application for this project to understand the role of genetic changes in the pathogenesis of glomerular diseases like focal segmental glomerulosclerosis (FSGS). In this application, we propose two specific aims. In the first, we propose to characterize a gene that we discovered that is highly expressed in podocytes. This gene, ARHGAP24, is a known regulator of the actin cytoskeleton and the high expression of this gene implicates a specific actin regulatory pathway in the normal function of podocytes. To test the role of this gene in the glomerulus, we propose to generate and characterize a mouse that lacks expression of ARHGAP24. In the second aim, we will set-up a genetic screen that combines RNAi technology and state of the art mouse genetic methods to perform a genetic screen in mouse to identify genes that when mutated contribute with CD2AP and another podocyte specific gene, synaptopodin, in the pathogenesis of glomerular dysfunction. Our long-term goal is the identification of all genes that, when mutated, contribute to the pathogenesis of human FSGS. PUBLIC HEALTH RELEVANCE: The goals of this project are to better understand the genetic causes of kidney disease. We will test the role of specific genes in the development of kidney diseases.

DESCRIPTION (provided by applicant): The Ras/MAP kinase signaling pathway plays a central role in cell responses to growth factors as well as stress responses. It also plays a critical role in tumor progression. While it has been intensively studied over the last decade, many important questions remain unanswered. For example, while it is clear that Ras is activated at the plasma membrane, where and when Raf, MEK and ERK are activated is not known. A second issue concerns the architecture of the signaling pathway. While it has been suggested that the three-kinase cascade is designed for signal amplification or for "ultrasensitive signaling", these ideas are largely untested. Lastly, progress in signal transduction has seen the identification of scaffolds involved in organizing signaling pathways, but their effects on the magnitude of signaling responses and the quality of signal transduction is unknown. We have recently reported the phenotype of a knock-out mouse of what we believe to be the first scaffold in the mammalian MAP kinase signaling pathway. Here we propose to use this knockout mouse to explore the role of scaffolds in the Ras/MAP kinase signaling pathway. Because our understanding of these signaling pathways will rely on biophysical parameters that are not known, it will be important to measure and establish these parameters for each step of the pathway. Our first aim is to study the role of KSR in the interactions as well as on the kinetic enzymatic rate constants for each kinase in the cascade. This project mainly involves purification of proteins and biophysical measurements of affinity as well as enzymatic activity. Our second aim is to study the role of KSR in vivo. First, we focus on determining whether KSR effects the localization of MAP kinase signaling proteins in vivo as well as where MAP kinase components are activated. Secondly, we focus on effects of KSR on the kinetic of MAP kinase signaling at the single cell level. This includes measurements of the lag times between each step, the level of amplification as well as on the digital/analog quality of signaling. We hope that our studies will lead to a more comprehensive understanding of signaling pathways. Since these pathways are so critical to normal cell homeostasis, a better understanding of these pathways should also lead to a better understanding of what is happening when these pathways go awry in cancer, diabetes and autoimmune diseases.

DESCRIPTION (provided by applicant): CD2AP is an 80 KD protein that was cloned as a protein involved in T cell activation. CD2AP also plays a major role in the kidney. CD2AP knockout mice are born with congenital nephritic syndrome and CD2AP is expressed in the glomerular epithelial cell or podocyte. Our previous work suggests that CD2AP plays a critical role in maintaining the integrity of the slit diaphragm, a structure that is critical in the glomerular filtration apparatus. Recently, we discovered that our CD2AP heterozygous mice demonstrate an increased susceptibility to renal injury caused by nephrotoxic antibodies or when bred to the NZB mouse. This suggested that CD2AP heterozygosity might play a role in human glomerular disease. In our preliminary work, we have identified human patients with the diagnosis of focal segmental glomerulosclerosis who are heterozygous for CD2AP. In this grant application, we propose to extend these studies by analyzing a larger population of patients with FSGS for mutations in CD2AP. In the first two aims, we propose to identify genetic variants of CD2AP and determine their prevalence in the population. In aim #3, we propose a series of experiments to determine whether these mutations are disease causing, by testing mutated forms of CD2AP biochemically, by their ability to reconstitute function after transfection in CD2AP deficient cell lines and by their ability to rescue the renal phenotype of the knockout mouse. We hope that these studies lead to new insights into diseases of the glomerulus.

DESCRIPTION (provided by applicant): The specific arrangement of proteins in the contact surface between the T cell and the APC is known as the immunological synapse. The exact function of the synapse is not clear but it has been proposed to function in the process of TCR signaling and also for TCR downregulation. Here we propose a model that suggests that there is a complex relationship between signaling, full receptor phosphorylation and down-regulation. Recruitment to the center of the synapse facilitates full receptor phosphorylation, and fully phosphorylated receptors are targeted for degradation. The model also suggests that receptors that are unable to be recruited to the center of the synapse become only partially phosphorylated and are recycled to the plasma membrane after dephosphorylation. In this application, we propose a series of experiments to test this hypothesis using state of the art imaging techniques. Specifically, in Specific Aim # 1, we propose to analyze the relationship between synapse formation, receptor phosphorylation and receptor degradation. In Specific Aim #2, we propose to determine the site of phosphorylation of fully phosphorylated versus partially phosphorylated receptors. In Specific Aim #3, we propose to examine the role of receptor downregulation in thymocyte signaling. Lastly, in Specific Aim #4, we propose to examine the role of CD28 and CD2 in TCR recycling and degradation.

DESCRIPTION (provided by applicant): CD2AP is an 80 KD protein that was cloned as a protein involved in T cell activation. CD2AP also plays a major role in the kidney. CD2AP knockout mice are born with congenital nephritic syndrome and CD2AP is expressed in the glomerular epithelial cell or podocyte. Our previous work suggests that CD2AP plays a critical role in maintaining the integrity of the slit diaphragm, a structure that is critical in the glomerular filtration apparatus. Recently, we discovered that our CD2AP heterozygous mice demonstrate an increased susceptibility to renal injury caused by nephrotoxic antibodies or when bred to the NZB mouse. This suggested that CD2AP heterozygosity might play a role in human glomerular disease. In our preliminary work, we have identified human patients with the diagnosis of focal segmental glomerulosclerosis who are heterozygous for CD2AP. In this grant application, we propose to extend these studies by analyzing a larger population of patients with FSGS for mutations in CD2AP. In the first two aims, we propose to identify genetic variants of CD2AP and determine their prevalence in the population. In aim #3, we propose a series of experiments to determine whether these mutations are disease causing, by testing mutated forms of CD2AP biochemically, by their ability to reconstitute function after transfection in CD2AP deficient cell lines and by their ability to rescue the renal phenotype of the knockout mouse. We hope that these studies lead to new insights into diseases of the glomerulus.

Despite extensive large population analysis, genome-wide association studies of diseases with single nucleotide polymorphisms (SNPs) have been disappointing thus far because SNPs account for a small fraction of the genetic risk for disease. We hypothesize that rare genetic variants giving rise to functional mutations may account for susceptibility to certain rheumatic illnesses. Moreover, we propose a strategy to further accelerate decreases in costs of exome sequencing by establishing a high throughput platform to enrich analysis of immune related genes. These sequences will then be analyzed for potential disease causing mutations by computational approaches.

DESCRIPTION (provided by applicant): This application seeks partial funding for a FASEB Summer Conference on Signal Transduction in the Immune System, to be held June 19-24, 2013 Nassau, Bahamas, with future meetings to be held in 2015 and 2017. The major goal of this conference is to provide a comprehensive focus on recent advances regarding the signaling mechanisms involved in the response of immune cells in both health and disease. The meeting in 2013 will be the 7th occurrence of this FASEB conference. This conference has garnered widespread interest in research institutes, academia, and industry because of the outstanding speaker rosters, the cutting edge nature of the work presented and the relevance of many presentations to immunologic diseases. Additionally, since most mainstream immunology meetings do not provide an in-depth focus on signal transduction mechanisms, this dedicated FASEB conference has attracted leaders in immune signal transduction world-wide, as well as new investigators and students. As a result, this FASEB conference has been one of the most highly attended FASEB Summer conferences. The meeting planned for 2013 includes a joint emphasis on adaptive and innate immune signaling mechanisms. The 2013 conference includes a strong focus on cytoskeletal changes that regulate leukocyte activation and migration, the importance of metabolic pathways during lymphocyte differentiation, and the use of microscopy and mathematical modeling to uncover detailed biochemical mechanisms of leukocyte receptor signaling as well as the application of recent breakthroughs to the treatment of human disease. These topics represent the cutting edge of research at the intersection of immune signaling, biology and translational to human disease. The conference has a backbone of regular attendees and speakers, but we will ensure that new ideas and areas are addressed in 2013 by featuring 25 new speakers. In addition, each session has space for several short talks to be selected from abstracts, with priority given to new investigators or post-docs from groups in which PIs are not presenting. This will ensure a diversity of topics at the meeting. The overall objectives of the meeting are: 1. To provide a critical and intensive overview of the recent advances in immune signaling; 2. To bring together senior scientists and young investigators in a convivial atmosphere with informal discussions to promote the skills of students and young investigators; 3. To advance research on the molecular mechanisms in the immune system by broadly disseminating information and recent breakthroughs. We anticipate that this conference will build upon the success of past conferences in influencing the direction of immune signaling research.