David A. Calderwood - US grants

DESCRIPTION (provided by applicant): Support is requested to analyze the interaction between the actin crosslinking protein filamin and integrin beta subunit cytoplasmic tails; the effect this interaction has on integrin signaling; the mechanisms by which integrin-filamin interactions inhibit cell migration; and how the interaction is regulated in cells. Heterodimeric integrin adhesion receptors mediate cell-extracellular matrix and cell-cell adhesion events throughout development, during hemostasis and in the response to injury and infection. The binding of specific signaling and cytoskeletal proteins to integrin beta tails controls cell adhesion, bidirectional integrin signaling and regulates cell motility. Integrin-mediated cell migration is essential for development, the immune response, and for tissue remodeling and wound healing. Integrin-mediated cell migration also contributes to a number of disease states and is required for tumor metastasis, inflammation and the formation of atherosclerotic plaques. The increased association of filamin with integrin beta tails inhibits cell migration. The applicant hypothesizes that regulated association of filamin with integrin beta tails mediates distinct integrin functions and acts as a brake on cell migration. To test this hypothesis he aims to map the integrin binding site in filamin and identify mutations that selectively inhibit integrin binding. He will use these mutants and previously identified integrin mutants that up- or down-regulate filamin binding to characterize the effect of integrin-filamin interactions on integrin-mediated signal transduction. He will then investigate whether the inhibition of cell migration following increased filamin binding to integrins is due to the observed effects on integrin signaling, to local filamin-mediated effects on actin filament crosslinking or to a combination of both processes. Finally he will assess effect of filamin proteolysis, filamin or integrin phosphorylation and competition between integrins and other filamin-binding proteins on the association of filamin with integrin beta tails. These studies will characterize the regulation and mechanism of action of a pathway that controls cell migration. Cell migration is a tightly controlled process that plays a central role in many biological phenomena. Therefore these studies will provide insight into a process essential during health and disease and may identify novel therapeutic targets for treatment of cancers, arthritis and atherosclerosis.

[unreadable] DESCRIPTION (provided by applicant): The purpose of this exploratory and developmental research project application is to identify proteins involved in the regulation of [unreadable]1 integrin activation. Integrin adhesion receptors are heterodimers formed from ? and [unreadable] subunits - each of which is a type I transmembrane protein with a large extracellular portion, a single transmembrane domain and a short cytoplasmic tail. The ability of integrin adhesion receptors to undergo activation (rapid regulated changes in affinity for their extracellular ligands) is essential for the development and functioning of the cardiovascular system. Inappropriate integrin activation contributes to thrombosis, atherosclerosis, myocardial infarction, stroke and reperfusion injury, and integrins are targets for therapeutic regulation of these conditions and for both inhibition and stimulation of angiogenesis. Detailed insight into the mechanisms controlling integrin activation is therefore directly relevant to strategies aimed at understanding and controlling cardiovascular disease and stroke. Our long-term aim is to understand the molecular mechanisms regulating integrin activation. We previously showed that binding of the cytoskeletal protein talin to the [unreadable]3 subunit cytoplasmic tail is necessary and sufficient for activation of the platelet integrin ?IIb[unreadable]3. Our new data show that talin is also required but not sufficient for [unreadable]1 integrin activation and indicate that other [unreadable]1 tail binding factors are also required. We aim to identify and characterize those additional factors. Specifically we aim to: 1) Use expression cloning to identify proteins that cooperate with talin to activate [unreadable]1 integrins: 2) Find proteins required for [unreadable]1 integrin activation using a siRNA screen: 3) Identify proteins whose binding to [unreadable]1 cytoplasmic tails is altered by mutations which we suggest impair binding of a 2nd activating factor: and 4) Characterize identified proteins. To achieve these aims we will rely on our ability to assess integrin activation in live cells in FACS based assays. We will transfect cells with talin and a cDNA expression library and collect cells where [unreadable]1 integrins have become activated; the transfected library cDNA will be recovered, re- screened and sequenced to identify genes that cooperate with talin to activate [unreadable]1 integrins. Target cell populations will also be transduced with siRNA libraries and cells expressing inactivated integrins collected; siRNA sequences will be recovered and sequences enriched in the inactivated cell population identified by microarray analysis. This should reveal genes, and so proteins, essential to maintain integrin activation. We have identified point mutations in the [unreadable]1 tail which seem to inhibit binding of a [unreadable]1 activating factor. We will apply protein profiling and identification techniques to identify proteins ca[able of binding to the wild-type but not mutant [unreadable] tails. Finally, we will characterize identified proteins biochemically and attempt to place them in integrin activation pathways. Tight control of the adhesive interactions between cells and between cells and the extracellular matrix which surrounds them is essential for the development and functioning of the cardiovascular system and inappropriate cell adhesion contributes to cardiovascular disease. Cell surface adhesion receptors called integrins mediate many of the cell's adhesive interactions and their activity is normally tightly regulated. We aim to identify and characterize the proteins that regulate integrin activity with a view towards understanding the molecular mechanisms regulating integrin function. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The integrin-linked kinase (ILK) is critical for anchorage-dependent cell growth and survival, cell cycle progression, epithelial to mesenchymal transition, cell motility, contractility and early development. ILK is also required for cardiac, vascular, brain, kidney, muscle, skin, platelet, chondrocyte and T cell function and plays important roles in tumor angiogenesis. There are multiple signaling pathways downstream of integrins but many of these pathways require the formation of a heterotrimeric complex between ILK, PINCH and parvin (IPP). This IPP complex serves as a hub in integrin-actin and integrin-signaling networks, and in mammalian systems IPP complex formation precedes and is required for its correct targeting to adhesions. There are currently significant deficiencies in our understanding of how the IPP complex forms, how it interacts with integrins, and whether it is enzymatically competent. We aim to improve the understanding of integrin signaling by the IPP complex using a structure-directed functional approach and to resolve the key functional question of whether ILK is catalytically active. In Aim 1 we will determine crystal structures and conduct binding studies to provide a comprehensive molecular description of the interaction between ILK and PINCH. These biophysical studies will allow us to rationally investigate the cellular effects of targeted interruption of this interaction. In Aim 2 we will provide structural, enzymatic and functional analyses of the ILK kinase domain. The catalytic competence of this pseudokinase remains the subject of much controversy, but is reported to be critical for IPP-mediated integrin signaling. We will solve crystal structures of the ILK kinase domain and assess its catalytic activity to provide structural evidence for the molecular basis of ILK catalytic competence. Potential substrate specificities will be studied as will the role of intramolecular interactions on kinase activity. Using structure-guided site-directed mutagenesis we will investigate the functional role of ILK catalytic activity in cells. In Aim 3 we will determine the molecular basis for ILK interactions with parvin and whether ILK directly interacts with integrin 2 subunit cytoplasmic tails. If ILK binds integrin 2 tails we will determine the structural basis for this interaction and investigate the functional effects of its targeted disruption. In Aim 4 we will determine the molecular architecture of the complete IPP complex. This long-term goal will provide a structural description of how the IPP complex forms and will allow targeted functional analysis of its cellular role. The studies proposed will provide answers to some of the critical unresolved questions regarding integrin signaling by the IPP complex. They may also facilitate the design of targeted anti-ILK or anti-IPP therapeutics relevant to the treatment of cancer, cardiovascular and inflammatory and kidney diseases. Our results will significantly enhance the molecular, enzymatic and functional understanding of a critical integrin signaling complex. PUBLIC HEALTH RELEVANCE: The project aims to enhance our molecular and functional understanding of integrin-linked kinase-PINCH- parvin (IPP) complex. This complex is essential for embryonic development, tissue maintenance and repair, host defense and hemostasis. We will determine crystal structures, conduct functional studies and describe the effects of mutations in cells. The proposed studies will help us understand how the IPP complex mediates its critical cellular roles.

DESCRIPTION (provided by applicant): Continued support is requested for our investigation of the roles of filamin in cell differentiation, invasion and disease. Filamins are essential actin crosslinking proteins composed of an N-terminal actin-binding domain followed by 24 immunoglobulin-like domains which interact with numerous cytosolic signaling proteins and transmembrane receptors. Humans have three filamin genes, encoding the widely expressed filamin A and B and largely muscle specific filamin C. Missense point mutations in filamins cause a variety of human diseases, ranging from altered neuronal migration, to cardiac and skeletal muscle defects, and a spectrum of congenital malformations generally characterized by skeletal dysplasias but also including extra-skeletal malformations such as cleft palate, cardiac defects and obstructive uropathy. The actin-binding domain is a hotspot for filamin mutations but, despite dramatic progress in understanding filamin structure and function, how filamin point mutations cause disease remains poorly understood. Furthermore, reduced filamin A expression correlates with increased breast cancer invasion and metastasis, and we recently discovered that loss of filamin increases extracellular matrix (ECM) remodeling and cell invasion. How filamin controls ECM degradation and invasion is unknown. In addition, we have shown that ASB2 (ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2), part of an E3 ubiquitin ligase complex, targets filamins for rapid proteasomal degradation and we suggest that the resultant transient loss of filamin contributes to retinoic acid-induced differentiation of acute promyelocytic leukemia cells. However, the molecular basis for ASB2 function and how loss of filamin influences cell differentiation have not been elaborated. To address the important unanswered questions highlighted above we propose three specific aims which draw on our extensive experience using biochemical, cellular and structural techniques to investigate filamins. Specifically, we will: 1) Characterize the mechanism of ASB2-mediated filamin-degradation and test its role in cell differentiation; 2) Assess the role of filamins in EC remodeling and cell invasion; and 3) Identify cellular phenotypes associated with disease-associated filamin point mutations, revealing potential molecular mechanisms of disease.

DESCRIPTION (provided by applicant): Loss of function of the three proteins, KRIT1 (Krev/Rap1 Interacting Trapped 1; CCM1, cerebral cavernous malformation 1), CCM2 (cerebral cavernous malformation 2; OSM, osmosensing scaffold for MEKK3) and CCM3 (cerebral cavernous malformation 2; PDCD10, programmed cell death 10), cause the familial form of the devastating Cerebral Cavernous Malformations (CCM) disease. Loss of function of these proteins is therefore directly linked with stroke, focal neurological defects, seizures and vascula abnormalities. The goal of this application is to understand the molecular underpinnings for normal function of these proteins. To do this we will conduct cell-based, biochemical and structural studies that will address our two central hypotheses: Molecular-level organization of the CCM complex regulates key signaling events and Intra- or inter- molecular head-tail KRIT1 interactions regulate KRIT1 function. In our preliminary studies we have determined the first crystal structures of each of the CCM proteins, KRIT1, CCM2 and CCM3, and have found each of these proteins to contain previously unpredicted protein interaction scaffold domains. Furthermore, our functional studies of the CCM proteins have highlighted important new aspects of their cellular function, particularly with regards to the regulation of integrin activation stat and signaling. Therefore, in Aim 1 we will use our advantaged position to assemble the CCM complex crystallographically and to investigate its functional roles in cells. Our previous studies also investigated the direct interactions of CCM proteins with partners, including ICAP1 and Rap1. These proteins bind KRIT1 and may impact its conformational status, which in turn is suggested to impact formation of the CCM complex. Therefore, in Aim 2 we will discover the molecular mechanisms that regulate KRIT1 conformation and the impact of KRIT1 conformational state on signaling via the CCM complex. In this Multi-Investigator proposal, the Boggon and Calderwood laboratories will conduct a highly collaborative structure-directed functional study of these proteins to better understand their normal functions, with particular attention to CCM disease-related cellular functions. Furthermore, as the CCM proteins are each widely expressed and have high sequence conservation through evolution, we expect that the improved understanding of the CCM proteins obtained from this study will also highlight further roles for the CCM proteins outside of the neurovasculature.

? DESCRIPTION (provided by applicant): This proposal will test the hypothesis that decreasing Filamin A (FLNA) levels or function in tuberous sclerosis complex (TSC) prevents cortical malformations and associated seizure activity. TSC is caused by mutations in TSC1 or TSC2 leading to mTOR complex 1 (mTORC1) hyperactivity and cortical malformations associated with seizures and worsening of cognitive and psychiatric deficits. The mTORC1 inhibitor rapamycin is the only therapeutic option, does not rescue all defects, and has mild to life-threatening complications emphasizing the need to find novel drug targets. We recently reported that the level of the actin cross-linking molecule FLNA is increased in Tsc1null neurons and that this increase is responsible for dendrite abnormalities. In addition, FLNA regulates the migration of cortical neurons during development. These findings are attractive with respect to TSC for the following reasons: (1) Neuronal dysmorphogenesis (including dendritic abnormality) and stalled migration are hallmarks of TSC-related cortical malformations. (2) FLNA increase in Tsc1null neurons as well as in cells expressing a constitutively active Rheb (the mTORC1 canonical activator) was not mTORC1-dependent but rather ERK1/2-dependent, opening a novel pharmacological option for a possible combination therapy, and (3) Our preliminary data show that normalizing FLNA levels using shRNA or administrating the new FLNA modulator, PTI-125, during development prevents neuronal misplacement and dysmorphogenesis in our new model of TSC-related cortical malformations. To address our hypothesis we have the following three aims. In Aim 1, we will determine whether FLNA controls development of cortical pyramidal neurons in vivo and whether decreasing FLNA levels during development prevents cortical malformations. In Aim 2, we will examine whether there is a time-window in neonates for treatments aimed at preventing cortical malformations and reducing or eliminating associated seizure activity. Our new murine model of focal cortical malformations is associated with a high rate of daily convulsive seizures. Finally in Aim 3, we will investigate how increased FLNA levels leads to dysmorphogenesis and stalled migration, which may identify novel FLNA binding partners involved in cortical defects in TSC. Most experiments will use in utero electroporation to selectively manipulate the development of layer 2/3 cortical pyramidal neurons. This is a 2 PIs grant. The Bordey lab will be in charge of Aim 1 and 2, and the Calderwood lab will be in charge of Aim 3. Both labs will heavily interact on a weekly basis due to the need for tool (plasmid) development and the need for cell biology and in vivo experiments in Aim 2 and 3, respectively. Dr. Bordey is an expert on neuronal development and TSC, and Dr. Calderwood is a cell biologist with extensive expertise on FLNA.

Abstract Tuberous sclerosis complex (TSC) and focal cortical dysplasia type II (FCDII) are caused by mutations in mTOR pathway genes leading to mTOR hyperactivity, focal malformations of cortical development (fMCD), and seizures in 80-90% of the patients. The current definitive treatments for epilepsy are surgical resection or treatment with everolimus, which inhibits mTOR activity (only approved for TSC). Because both options have severe limitations, there is a major need to better understand the mechanisms leading to seizures to improve life-long epilepsy treatment in TSC and FCDII. To investigate such mechanisms, we recently developed a murine model of fMCD-associated epilepsy that recapitulates the human TSC and FCDII disorders. fMCD are defined by the presence of misplaced, dysmorphic cortical neurons expressing hyperactive mTOR ? for simplicity we will refer to these as ?mutant? neurons. In our model and in human TSC tissue, we made a surprising finding that mutant neurons express HCN4 channels, which are not normally functionally expressed in cortical neurons. These data led us to ask several important questions based on the known biology of HCN4 channels: (1) As HCN4 channels are responsible for the pacemaking activity of the heart, can HCN4 channel expression lead to repetitive firing of mutant neurons resulting in seizures? (2) HCN4 is the most cAMP-sensitive of the four HCN isoforms. Do coincident increases in cAMP (e.g., ?-adrenergic receptors) and hyperpolarization or depolarizations drive HCN4 channel opening and neuronal firing? (3) HCN4 channel mRNA is expressed in cortical neurons. Is the abnormal HCN4 expression in mutant neurons due to increased translation via mTOR? (4) Seizures can start at any age in patients that have been seizure-free for decades, but we do not know why. Can this be explained by worsening of mTOR hyperactivity with age leading to a progressive increase in HCN4 expression until there is enough HCN4 channels to depolarize cells and reach firing threshold upon activation? (5) There is no selective blocker of the HCN4 channel and blocking other HCN channels would have serious central and peripheral side-effects. Identifying the mechanisms responsible for functional HCN4 expression may therefore provide alternative therapeutic targets. Do binding partners and/or post-translational modifications contribute to HCN4 abnormal expression in mutant neurons? We will address these questions in three aims testing our central hypothesis that abnormal mTOR- and translation- dependent expression of HCN4 channels leads to repetitive neuronal firing and seizures in TSC and FCDII. Aim 1: Test the hypothesis that abnormal HCN4 channel expression in murine TSC/FCDII mutant neurons contribute to neuron excitability and seizure activity. Aim2: Test the hypothesis that abnormal HCN4 expression is mTOR- and translation-dependent and increases with age and seizures. Aim 3: Test the hypothesis that HCN4 binding partners and posttranslational modifications are necessary for its functional expression and function. The proposed studies will be performed through a collaborative effort between the Bordey and Calderwood labs that together combine unique and extensive expertise in in vivo neurobiology, and biochemical and protein science.

ABSTRACT Tousled-like kinases (TLKs) are poorly studied nuclear serine-threonine kinases essential to proper cell division and overall viability in animals. Humans have two TLK isozymes, TLK1 and TLK2 (TLK1/2), that are closely related and thought to have redundant roles in genome maintenance. In addition to having key roles in normal cell physiology, dysregulation of TLKs has also been implicated in human disease: TLK2 haploinsufficiency causes a distinct neurodevelopmental disorder, and TLK1/2 upregulation drives cancer cell proliferation. Though TLKs have ascribed roles during DNA synthesis, other critical functions of the kinases in the cell cycle and in responses to DNA damage are poorly understood. A more complete understanding of TLK1/2 function has been hampered by 1) an absence of potent and specific small molecule inhibitors for use as tool compounds to allow temporal control of kinase activity and 2) limited knowledge of direct in vivo substrates of the kinases. The aim of this pilot project is to address these deficiencies through solution of X-ray crystal structures of TLK2-inhibitor and TLK2-substrate complexes, and by identification of new TLK substrates through unbiased screens. Screening a focused kinase inhibitor library, we have identified a set of small molecule TLK2 inhibitors. We will optimize established conditions for growing obtain crystals of TLK2-inhibitor complexes, and solve their high resolution structures. To understand the structural basis for selective substrate targeting, we will map interactions between TLK2 and its best-characterized substrate, ASF1a, and solve structures of TLK2 in complex with synthetic and ASF1a-derivied peptide substrates. We will use a chemical genetic approach to identify new TLK2 substrates. An analog-sensitive TLK2 allele will be used with N6- substituted analogs of ATP-?-S to thiophosphorylate its direct substrates in intact cell nuclei. Tryptic thiophosphorylated peptides will be isolated by covalent capture and release, and then identified by LC-MS/MS analysis. These studies will set the stage for future studies investigations of the cellular function of newly identified substrates. and provide a basis for structure-guided elaboration of potent and specific inhibitors.

ABSTRACT The ability of cells to assemble, adhere to and dynamically sense the extracellular matrix (ECM) is essential for multicellular life. Integrins, a family of heterodimeric ?? transmembrane adhesion receptors, bind specific ECM ligands via their ectodomains and permit bidirectional communication vital for cell adhesion, migration, differentiation, and survival. Proper integrin function is paramount for tissue morphogenesis, and is perturbed in cancer, skin disorders, musculoskeletal, cardiovascular and inflammatory diseases. A major site of integrin- matrix engagement is in dynamic micron-sized signaling platforms, called focal adhesions (FA). Integrins can laterally exchange into and out of FA, but also traffic to and from the cell surface and this trafficking influences cell migration, invasion and cancer metastasis. However, despite its importance, understanding at the cellular and mechanistic level of precisely where and how integrins undergo exocytic and endocytic traffic has been challenging due to difficulties directly visualizing integrin exo-endocytosis in live cells. In response to this challenge we recently generated `ecto-tagged' integrins containing the pH-sensitive fluorophore pHluorin or a chemical-genetic Halo-tag inserted into an extracellular loop. These ecto-tagged integrins provided the first direct views of integrin exocytosis and revealed that, contrary to initial expectations, integrin exocytosis occurs at a subset of FA. Drawing on our expertise in integrins (Calderwood) and live-cell imaging of membrane trafficking (Toomre), this multi-investigator R01 proposal seeks to test major new hypotheses arising from these results. In Aim 1 we test the hypothesis that integrins are selectively delivered to growing FA in a subunit-specific, ECM-regulated process. In Aim 2 we test the hypotheses that integrin endocytosis occurs at a distinct set of FA that are turning over and that endocytosed integrins are recycled to growing FA. Furthermore, we probe molecular mechanisms involved in recycling to FA. Finally, in Aim 3 we test the hypothesis that integrin-dependent fibronectin (FN) exocytosis also occurs at growing FA, while FN endocytosis occurs at FA that are turning over. Our experimental approaches combine novel ecto-tagged integrins and matrix constructs with new cleavable Halo dyes and pH-switching to follow exo-endocytosis in live cells so as to test new hypothesis about where integrin is delivered and the underlying mechanisms.

P21-activated kinases in cell-cell and cell-matrix adhesion signaling ABSTRACT The development and functioning of multicellular organisms, tissue formation, and responses to injury and infection rely on tightly coordinated adhesion of cells to one another, and of cells to the extracellular matrix. These processes are mediated in large part through the action of two families of adhesion receptors: the integrins which are principally responsible for cell-matrix adhesion, and the cadherins which are central to cell-cell adhesion. Our preliminary data suggest that the type-II p21-activated kinases (PAKs), a group of serine- threonine kinases, use a range of mechanisms to influence cell-matrix adhesion and cell-cell adhesion. The ability to regulate both adhesion systems places the PAKs as central players in coordination of cell adhesion dynamics. This proposal aims to understand the functional, cellular and molecular basis for this regulation. To address how they control cell-matrix and cell-cell adhesions we propose a combination of structural, biochemical and cellular approaches. In Aim 1 we test the hypothesis that Direct binding of PAK to cytoplasmic tails of integrin adhesion receptors regulates matrix adhesion and/or PAK signaling. We will conduct an extensive study employing structural, biophysical, biochemical and cell biological approaches. This will allow us to comprehensively understand how integrin adhesion receptors bind PAK serine-threonine kinases, and the functional consequences of such interactions on cell signaling, adhesion, motility and invasion. In Aim 2 we test the hypothesis that PAK targeted to cell-cell contacts phosphorylates b-catenin, triggering adhesion turnover and escape of individual cells from epithelial islands. We will determine the mechanisms by which PAKs drive colony escape, and the structural basis for PAK regulation of ?-catenin. Finally, we will test whether the roles of PAKs in cell-cell and cell-ECM adhesion are linked. Our proposed work will define how PAKs regulate both integrin- mediated cell-matrix adhesion, and ?-catenin-associated cell-cell adhesion, and therefore will provide new understanding of interconnections between cell-matrix and cell-cell adhesion via the type-II PAKs.