Large-scale Functional Screen Research Articles

Large-scale functional screen identifies genetic variants with splicing effects in modern and archaic humans

Humans coexisted and interbred with other hominins which later became extinct. These archaic hominins are known to us only through fossil records and for two cases, genome sequences. Here, we engineer Neanderthal and Denisovan sequences into thousands of artificial genes to reconstruct the pre-mRNA processing patterns of these extinct populations. Of the 5,169 alleles tested in this massively parallel splicing reporter assay (MaPSy), we report 962 exonic splicing mutations that correspond to differences in exon recognition between extant and extinct hominins. Using MaPSy splicing variants, predicted splicing variants, and splicing quantitative trait loci, we show that splice-disrupting variants experienced greater purifying selection in anatomically modern humans than that in Neanderthals. Adaptively introgressed variants were enriched for moderate-effect splicing variants, consistent with positive selection for alternative spliced alleles following introgression. As particularly compelling examples, we characterized a unique tissue-specific alternative splicing variant at the adaptively introgressed innate immunity gene TLR1, as well as a unique Neanderthal introgressed alternative splicing variant in the gene HSPG2 that encodes perlecan. We further identified potentially pathogenic splicing variants found only in Neanderthals and Denisovans in genes related to sperm maturation and immunity. Finally, we found splicing variants that may contribute to variation among modern humans in total bilirubin, balding, hemoglobin levels, and lung capacity. Our findings provide unique insights into natural selection acting on splicing in human evolution and demonstrate how functional assays can be used to identify candidate causal variants underlying differences in gene regulation and phenotype.

Time-Dependent Odorant Sensitivity Modulation in Insects.

Simple SummaryInsects, including blood-feeding female mosquitoes, can transmit deadly diseases, such as malaria, encephalitis, dengue, and yellow fever. Insects use olfaction to locate food sources, mates, and hosts. The nature of odorant plumes poses a challenge for insects in locating odorant sources in the environment. In order to modulate the system for the detection of fresh stimuli or changes in odorant concentrations, the olfaction system desensitizes to different concentrations and durations of stimuli. Without this ability, the chemotaxis behaviors of insects are defective. Thus, understanding how insects adjust their olfactory response dynamics to parse the chemical language of the external environment is not only a basic biology question but also has far-reaching implications for repellents and pest control. Insects use olfaction to detect ecologically relevant chemicals in their environment. To maintain useful responses over a variety of stimuli, olfactory receptor neurons are desensitized to prolonged or high concentrations of stimuli. Depending on the timescale, the desensitization is classified as short-term, which typically spans a few seconds; or long-term, which spans from minutes to hours. Compared with the well-studied mechanisms of desensitization in vertebrate olfactory neurons, the mechanisms underlying invertebrate olfactory sensitivity regulation remain poorly understood. Recently, using a large-scale functional screen, a conserved critical receptor phosphorylation site has been identified in the model insect Drosophila melanogaster, providing new insight into the molecular basis of desensitization in insects. Here, we summarize the progress in this area and provide perspectives on future directions to determine the molecular mechanisms that orchestrate the desensitization in insect olfaction.

Functional patient-derived organoid screenings identify MCLA-158 as a therapeutic EGFR × LGR5 bispecific antibody with efficacy in epithelial tumors.

Patient-derived organoids (PDOs) recapitulate tumor architecture, contain cancer stem cells and have predictive value supporting personalized medicine. Here we describe a large-scale functional screen of dual-targeting bispecific antibodies (bAbs) on a heterogeneous colorectal cancer PDO biobank and paired healthy colonic mucosa samples. More than 500 therapeutic bAbs generated against Wingless-related integration site (WNT) and receptor tyrosine kinase (RTK) targets were functionally evaluated by high-content imaging to capture the complexity of PDO responses. Our drug discovery strategy resulted in the generation of MCLA-158, a bAb that specifically triggers epidermal growth factor receptor degradation in leucine-rich repeat-containing G-protein-coupled receptor 5-positive (LGR5+) cancer stem cells but shows minimal toxicity toward healthy LGR5+ colon stem cells. MCLA-158 exhibits therapeutic properties such as growth inhibition of KRAS-mutant colorectal cancers, blockade of metastasis initiation and suppression of tumor outgrowth in preclinical models for several epithelial cancer types.

Identification of genes required for eye development by high-throughput screening of mouse knockouts

Despite advances in next generation sequencing technologies, determining the genetic basis of ocular disease remains a major challenge due to the limited access and prohibitive cost of human forward genetics. Thus, less than 4,000 genes currently have available phenotype information for any organ system. Here we report the ophthalmic findings from the International Mouse Phenotyping Consortium, a large-scale functional genetic screen with the goal of generating and phenotyping a null mutant for every mouse gene. Of 4364 genes evaluated, 347 were identified to influence ocular phenotypes, 75% of which are entirely novel in ocular pathology. This discovery greatly increases the current number of genes known to contribute to ophthalmic disease, and it is likely that many of the genes will subsequently prove to be important in human ocular development and disease.

Zinc Detoxification: A Functional Genomics and Transcriptomics Analysis in Drosophila melanogaster Cultured Cells.

Cells require some metals, such as zinc and manganese, but excess levels of these metals can be toxic. As a result, cells have evolved complex mechanisms for maintaining metal homeostasis and surviving metal intoxication. Here, we present the results of a large-scale functional genomic screen in Drosophila cultured cells for modifiers of zinc chloride toxicity, together with transcriptomics data for wild-type or genetically zinc-sensitized cells challenged with mild zinc chloride supplementation. Altogether, we identified 47 genes for which knockdown conferred sensitivity or resistance to toxic zinc or manganese chloride treatment, and >1800 putative zinc-responsive genes. Analysis of the ‘omics data points to the relevance of ion transporters, glutathione (GSH)-related factors, and conserved disease-associated genes in zinc detoxification. Specific genes identified in the zinc screen include orthologs of human disease-associated genes CTNS, PTPRN (also known as IA-2), and ATP13A2 (also known as PARK9). We show that knockdown of red dog mine (rdog; CG11897), a candidate zinc detoxification gene encoding an ABCC-type transporter family protein related to yeast cadmium factor (YCF1), confers sensitivity to zinc intoxication in cultured cells, and that rdog is transcriptionally upregulated in response to zinc stress. As there are many links between the biology of zinc and other metals and human health, the ‘omics data sets presented here provide a resource that will allow researchers to explore metal biology in the context of diverse health-relevant processes.

Abstract NTOC-093: SYNTHETIC LETHAL APPROACHES TO TARGET ARID1A DEFICIENT OVARIAN CANCERS

Abstract Epithelial ovarian cancer (EOC) remains the most lethal gynaecological malignancy in the Western world. Ovarian clear cell carcinoma (OCCC), a distinct histological subtype, has a notably poor prognosis in the advanced setting compared to patients with high-grade serous ovarian cancer (HGSOC). Understanding why these patients have a poor outcome may be due to the underlying genetic drivers and their response to treatment. Dysregulation of the SWI/SNF complex is one of the most commonly occurring defects in solid cancers. Mutations in ARID1A (AT-rich interactive domain-containing protein 1A), a gene that encodes for BAF250A, forming part of the SWI/SNF chromatin remodeling complex, rarely occur in HGSOC but are common in ovarian clear cell carcinomas. The vast majority of these are loss of function frameshift or nonsense mutations, resulting in loss of protein function. In addition, loss of ARID1A expression in tumour specimens has been associated with a shorter progression free survival and chemoresistance in ovarian clear cell carcinoma (OCCC). Despite the understanding that ARID1A defects are associated with tumourigenesis, targeted therapy approaches that exploit this deficiency have not as yet been developed. Our aims were to identify ways of targeting ARID1A deficient tumours by performing a large-scale functional genomics screen to identify actionable synthetic lethal effects. Using a high-throughput combination drug screen with a plate library of 80 compounds and a phase 1 compound, in isogenic ARID1A null and wild type HCT116 cells, we have identified candidate therapeutic approaches to targeting ARID1A mutant tumours that could be assessed in proof of concept clinical trials. We have undertaken subsequent high throughput drug screens in isogenic ARID1A null and wild type MCF10A cells that in we have identified a series of novel synthetic lethal effects. Assessment of this combinatorial approach in in vivo models of ARID1A mutant cancers is now underway. In conclusion, we have identified clinically actionable combinatorial approaches that may provide additional therapeutic benefit for ARID1A deficient patients. Citation Format: Saira Khalique, Chris T. Williamson, Helen Pemberton, Patty T. Wai, Malini Menon, Rachel Brough, Andri Leonidou, Barrie Peck, Susana Banerjee, Rachael C. Natrajan and Christopher J. Lord,. SYNTHETIC LETHAL APPROACHES TO TARGET ARID1A DEFICIENT OVARIAN CANCERS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-093.

Abstract P3-03-08: A large-scale functional screen to identify resistance mechanisms to selective estrogen receptor degraders fulvestrant and GDC-810 in ER+ breast cancer

Abstract Therapies that target the estrogen receptor provide clinical benefit and improved survival for patients with estrogen receptor-positive (ER+) breast cancer, yet drug resistance remains a challenging problem, leading to disease relapse and mortality. In recent years, the selective estrogen receptor degrader (SERD) fulvestrant has become an important therapeutic option for patients with resistant ER+ metastatic breast cancer, and newer oral SERDs such as GDC-810 are currently being tested in clinical trials. The mechanisms of intrinsic and acquired resistance to SERDs remain to be fully elucidated. We conducted a large-scale lentiviral open reading frame (ORF) screen to identify genes whose overexpression confers drug resistance to either fulvestrant or GDC-810 in the ER+ breast cancer cell line T47D. The lentivral ORF expression library used in this study consists of 16,544 barcoded ORFs, including 2,767 ORFs with mutations. The initial screen yielded 72 genes resulting in resistance to fulvestrant and 85 genes resulting in resistance to GDC-0810, with 44 genes overlapping. The top ranked-genes included multiple genes belonging to the PI3K/Akt, ERbB/HER, and FGF/FGFR pathways as well as genes involved in cell cycle progression. Fibroblast growth factor receptor 1 (FGFR1) amplifications are frequently observed in patients with ER+ breast cancer, and have previously been implicated in resistance to endocrine therapies. Several FGFs (FGF3, FGF6, FGF10, and FGF22) were among the top-ranked resistance genes for both fulvestrant and GDC-0810, suggesting that activation of the FGFR signaling pathway may render cells resistant to fulvestrant and GDC-810. In the presence of FGF2, overexpression of FGFR1 in ER+ breast cancer cells resulted in resistance to both fulvestrant and GDC-0810. The ability of an FGFR inhibitor to overcome FGFR-mediated resistance to SERDs is being tested. Additional potential resistance genes identified in the ORF screen are also being validated. In summary, a whole-genome functional resistance screen has identified several candidate genes and pathways that may cause resistance to fulvestrant and GDC-810. Several of these candidates, such as FGFR1, are also found in patients who develop resistance to SERDs, suggesting rational combination therapies to overcome or preempt SERD resistance. Citation Format: Mao P, Quartey Q, Cohen O, Piccioni F, Wagle N. A large-scale functional screen to identify resistance mechanisms to selective estrogen receptor degraders fulvestrant and GDC-810 in ER+ breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-03-08.

RAC-LATS1/2 signaling regulates YAP activity by switching between the YAP-binding partners TEAD4 and RUNX3.

The tumor-suppressor RUNX3 has a critical role in a lineage determination, cell cycle arrest and apoptosis. Lozenge (Lz), a Drosophila homolog of mammalian RUNX family members, has integral roles in these processes and specifically in eye cell fate determination. To elucidate the genetic modifiers of Lz/RUNX3, we performed a large-scale functional screen in a fly mutant library. The screen revealed genetic interactions between the Lz, Rac and Hippo pathways. Analysis of interactions among these genes revealed that the defective phenotype resulting from activation of Yki, an end point effector of the Hippo pathway, was suppressed by Lz and enhanced by Rac-Trio. Molecular biological analysis using mammalian homologs reveled that LATS1/2-mediated YAP phosphorylation-facilitated dissociation of the YAP-TEAD4 complex and association of the YAP-RUNX3 complex. When cells were stimulated to proliferate, activated RAC-TRIO signaling inhibited LATS1/2-mediated YAP phosphorylation; consequently, YAP dissociated from RUNX3 and associated with TEAD, thereby replacing the YAP-RUNX3 complex with YAP-TEAD. RUNX3 contributed to both association and dissociation of YAP-TEAD complex, most likely through the formation of the YAP-TEAD-RUNX3 ternary complex. Ectopic expression of RUNX3 in MKN28 gastric cancer cells reduced tumorigenicity, and the tumor-suppressive activity of RUNX3 was associated with its ability to interact with YAP. These results identify a novel regulatory mechanism, mediated by the Hippo and RAC-TRIO pathways, that changes the binding partner of YAP.

Large-Scale Functional RNAi Screen in C. elegans Identifies TGF-β and Notch Signaling Pathways as Modifiers of CACNA1A.

Variants in CACNA1A that encodes the pore-forming α1-subunit of human voltage-gated Cav2.1 (P/Q-type) Ca2+ channels cause several autosomal-dominant neurologic disorders, including familial hemiplegic migraine type 1, episodic ataxia type 2, and spinocerebellar ataxia type 6. To identify modifiers of incoordination in movement disorders, we performed a large-scale functional RNAi screen, using the Caenorhabditis elegans strain CB55, which carries a truncating mutation in the unc-2 gene, the worm ortholog for the human CACNA1A. The screen was carried out by the feeding method in 96-well liquid culture format, using the ORFeome v1.1 feeding library, and time-lapse imaging of worms in liquid culture was used to assess changes in thrashing behavior. We looked for genes that, when silenced, either ameliorated the slow and uncoordinated phenotype of unc-2, or interacted to produce a more severe phenotype. Of the 350 putative hits from the primary screen, 37 genes consistently showed reproducible results. At least 75% of these are specifically expressed in the C. elegans neurons. Functional network analysis and gene ontology revealed overrepresentation of genes involved in development, growth, locomotion, signal transduction, and vesicle-mediated transport. We have expanded the functional network of genes involved in neurodegeneration leading to cerebellar ataxia related to unc-2/CACNA1A, further confirming the involvement of the transforming growth factor β pathway and adding a novel signaling cascade, the Notch pathway.

Kinase Inhibitor Profiling Reveals Unexpected Opportunities to Inhibit Disease-Associated Mutant Kinases.

Small-molecule kinase inhibitors have typically been designed to inhibit wild-type kinases rather than the mutant forms that frequently arise in diseases such as cancer. Mutations can have serious clinical implications by increasing kinase catalytic activity or conferring therapeutic resistance. To identify opportunities to repurpose inhibitors against disease-associated mutant kinases, we conducted a large-scale functional screen of 183 known kinase inhibitors against 76 recombinant mutant kinases. The results revealed lead compounds with activity against clinically important mutant kinases, including ALK, LRRK2, RET, and EGFR, as well as unexpected opportunities for repurposing FDA-approved kinase inhibitors as leads for additional indications. Furthermore, using T674I PDGFRα as an example, we show how single-dose screening data can provide predictive structure-activity data to guide subsequent inhibitor optimization. This study provides a resource for the development of inhibitors against numerous disease-associated mutant kinases and illustrates the potential of unbiased profiling as an approach to compound-centric inhibitor development.

A Large-Scale Functional Screen to Identify Epigenetic Repressors of Retrotransposon Expression.

Deposition of epigenetic marks is an important layer of the transcriptional control of retrotransposons, especially during early embryogenesis. Krüppel-associated box domain zinc finger proteins (KRAB-ZFPs) are one of the largest families of transcription factors, and collectively partake in this process by tethering to thousands of retroelement-containing genomic loci their cofactor KAP1, which acts as a scaffold for a heterochromatin-inducing machinery. However, while the sequence-specific DNA binding potential of the poly-zinc finger-containing KRAB-ZFPs is recognized, very few members of the family have been assigned specific targets. In this chapter, we describe a large-scale functional screen to identify the retroelements bound by individual murine KRAB-ZFPs. Our method is based on the automated transfection of a library of mouse KRAB-ZFP-containing vectors into 293T cells modified to express GFP from a PGK promoter harboring in its immediate vicinity a KAP1-recruiting retroelement-derived sequence. Analysis is then performed by plate reader and flow cytometry fluorescence readout. Such large-scale DNA-centered functional approach can not only help to identify the trans-acting factors responsible for silencing retrotransposons, but also serve as a model for dissecting the transcriptional networks influenced by retroelement-derived cis-acting sequences.

Chemogenetic profiling identifies RAD17 as synthetically lethal with checkpoint kinase inhibition.

Chemical inhibitors of the checkpoint kinases have shown promise in the treatment of cancer, yet their clinical utility may be limited by a lack of molecular biomarkers to identify specific patients most likely to respond to therapy. To this end, we screened 112 known tumor suppressor genes for synthetic lethal interactions with inhibitors of the CHEK1 and CHEK2 checkpoint kinases. We identified eight interactions, including the Replication Factor C (RFC)-related protein RAD17. Clonogenic assays in RAD17 knockdown cell lines identified a substantial shift in sensitivity to checkpoint kinase inhibition (3.5-fold) as compared to RAD17 wild-type. Additional evidence for this interaction was found in a large-scale functional shRNA screen of over 100 genotyped cancer cell lines, in which CHEK1/2 mutant cell lines were unexpectedly sensitive to RAD17 knockdown. This interaction was widely conserved, as we found that RAD17 interacts strongly with checkpoint kinases in the budding yeast Saccharomyces cerevisiae. In the setting of RAD17 knockdown, CHEK1/2 inhibition was found to be synergistic with inhibition of WEE1, another pharmacologically relevant checkpoint kinase. Accumulation of the DNA damage marker γH2AX following chemical inhibition or transient knockdown of CHEK1, CHEK2 or WEE1 was magnified by knockdown of RAD17. Taken together, our data suggest that CHEK1 or WEE1 inhibitors are likely to have greater clinical efficacy in tumors with RAD17 loss-of-function.

A large‐scale functional screen identifies N ova1 and N coa3 as regulators of neuronal mi RNA function

MicroRNAs (miRNAs) are important regulators of neuronal development, network connectivity, and synaptic plasticity. While many neuronal miRNAs were previously shown to modulate neuronal morphogenesis, little is known regarding the regulation of miRNA function. In a large-scale functional screen, we identified two novel regulators of neuronal miRNA function, Nova1 and Ncoa3. Both proteins are expressed in the nucleus and the cytoplasm of developing hippocampal neurons. We found that Nova1 and Ncoa3 stimulate miRNA function by different mechanisms that converge on Argonaute (Ago) proteins, core components of the miRNA-induced silencing complex (miRISC). While Nova1 physically interacts with Ago proteins, Ncoa3 selectively promotes the expression of Ago2 at the transcriptional level. We further show that Ncoa3 regulates dendritic complexity and dendritic spine maturation of hippocampal neurons in a miRNA-dependent fashion. Importantly, both the loss of miRNA activity and increased dendrite complexity upon Ncoa3 knockdown were rescued by Ago2 overexpression. Together, we uncovered two novel factors that control neuronal miRISC function at the level of Ago proteins, with possible implications for the regulation of synapse development and plasticity.

RUNX family members are covalently modified and regulated by PIAS1-mediated sumoylation.

Transcription factors of the RUNX family (RUNXs), which play pivotal roles in normal development and neoplasia, are regulated by various post-translational modifications. To understand the molecular mechanisms underlying the regulation of RUNXs, we performed a large-scale functional genetic screen of a fly mutant library. The screen identified dPias (the fly ortholog of mammalian PIASs), an E3 ligase for the SUMO (small ubiquitin-like modifier) modification, as a novel genetic modifier of lz (the fly ortholog of mammalian RUNX3). Molecular biological analysis revealed that lz/RUNXs are sumoylated by dPias/PIAS1 at an evolutionarily conserved lysine residue (K372 of lz, K144 of RUNX1, K181 of RUNX2 and K148 of RUNX3). PIAS1-mediated sumoylation inhibited RUNX3 transactivation activity, and this modification was promoted by the AKT1 kinase. Importantly, PIAS1 failed to sumoylate some RUNX1 mutants associated with breast cancer. In nude mice, tumorigenicity was promoted by RUNX3 bearing a mutation in the sumoylation site, but suppressed by wild-type RUNX3. Our results suggest that RUNXs are sumoylated by PIAS1, and that this modification could play a critical role in the regulation of the tumor-suppressive activity of these proteins.

Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes

The quality of genetically encoded calcium indicators (GECIs) has improved dramatically in recent years, but high-performing ratiometric indicators are still rare. Here we describe a series of fluorescence resonance energy transfer (FRET)-based calcium biosensors with a reduced number of calcium binding sites per sensor. These 'Twitch' sensors are based on the C-terminal domain of Opsanus troponin C. Their FRET responses were optimized by a large-scale functional screen in bacterial colonies, refined by a secondary screen in rat hippocampal neuron cultures. We tested the in vivo performance of the most sensitive variants in the brain and lymph nodes of mice. The sensitivity of the Twitch sensors matched that of synthetic calcium dyes and allowed visualization of tonic action potential firing in neurons and high resolution functional tracking of T lymphocytes. Given their ratiometric readout, their brightness, large dynamic range and linear response properties, Twitch sensors represent versatile tools for neuroscience and immunology.

A Design Principle for a Posttranslational Biochemical Oscillator

Multisite phosphorylation plays an important role in biological oscillators such as the circadian clock. Its general role, however, has been elusive. In this theoretical study, we show that a simple substrate with two modification sites acted upon by two opposing enzymes (e.g., a kinase and a phosphatase) can show oscillations in its modification state. An unbiased computational analysis of this oscillator reveals two common characteristics: a unidirectional modification cycle and sequestering of an enzyme by a specific modification state. These two motifs cause a substrate to act as a coupled system in whicha unidirectional cycle generates single-molecule oscillators, whereas sequestration synchronizes the population by limiting the available enzyme under conditions in which substrate is in excess. We also demonstrate the conditions under which the oscillation period is temperature compensated, an important feature of the circadian clock. This theoretical model will provide a framework for analyzing and synthesizing posttranslational oscillators.

Large-scale functional RNAi screen in C. elegans identifies genes that regulate the dysfunction of mutant polyglutamine neurons

BackgroundA central goal in Huntington's disease (HD) research is to identify and prioritize candidate targets for neuroprotective intervention, which requires genome-scale information on the modifiers of early-stage neuron injury in HD.ResultsHere, we performed a large-scale RNA interference screen in C. elegans strains that express N-terminal huntingtin (htt) in touch receptor neurons. These neurons control the response to light touch. Their function is strongly impaired by expanded polyglutamines (128Q) as shown by the nearly complete loss of touch response in adult animals, providing an in vivo model in which to manipulate the early phases of expanded-polyQ neurotoxicity. In total, 6034 genes were examined, revealing 662 gene inactivations that either reduce or aggravate defective touch response in 128Q animals. Several genes were previously implicated in HD or neurodegenerative disease, suggesting that this screen has effectively identified candidate targets for HD. Network-based analysis emphasized a subset of high-confidence modifier genes in pathways of interest in HD including metabolic, neurodevelopmental and pro-survival pathways. Finally, 49 modifiers of 128Q-neuron dysfunction that are dysregulated in the striatum of either R/2 or CHL2 HD mice, or both, were identified.ConclusionsCollectively, these results highlight the relevance to HD pathogenesis, providing novel information on the potential therapeutic targets for neuroprotection in HD.

A large-scale functional RNAi screen reveals a role for CK2 in the mammalian circadian clock

Post-translational processes are essential for the generation and dynamics of mammalian circadian rhythms. In particular, phosphorylation of the key circadian protein PER2 precisely controls the period and phase of circadian oscillations. However, the mechanisms underlying that control are poorly understood. Here, we identified in a high-throughput RNAi-based genetic screen casein kinase 2 (CK2) as a PER2-phosphorylating kinase and novel component of the mammalian circadian clock. When CK2 subunits are silenced by RNAi or when CK2 activity is inhibited pharmacologically, circadian rhythms are disrupted. CK2 binds to PER2 in vivo, phosphorylates PER2 specifically at N-terminal residues in vitro, and supports normal nuclear PER2 accumulation. Mutation of CK2 phosphorylation sites decreases PER2 stability and copies CK2 inhibition regarding oscillation dynamics. We propose a new concept of how PER2 phosphorylation and stabilization can set the clock speed in opposite directions, dependent on the phase of action.

A novel role for p21-activated protein kinase 2 in T cell activation.

To identify novel components of the TCR signaling pathway, a large-scale retroviral-based functional screen was performed using CD69 expression as a marker for T cell activation. In addition to known regulators, two truncated forms of p21-activated kinase 2 (PAK2), PAK2DeltaL(1-224) and PAK2DeltaS(1-113), both lacking the kinase domain, were isolated in the T cell screen. The PAK2 truncation, PAK2DeltaL, blocked Ag receptor-induced NFAT activation and TCR-mediated calcium flux in Jurkat T cells. However, it had minimal effect on PMA/ionomycin-induced CD69 up-regulation in Jurkat cells, on anti-IgM-mediated CD69 up-regulation in B cells, or on the migratory responses of resting T cells to chemoattractants. We show that PAK2 kinase activity is increased in response to TCR stimulation. Furthermore, a full-length kinase-inactive form of PAK2 blocked both TCR-induced CD69 up-regulation and NFAT activity in Jurkat cells, demonstrating that kinase activity is required for PAK2 function downstream of the TCR. We also generated a GFP-fused PAK2 truncation lacking the Cdc42/Rac interactive binding region domain, GFP-PAK2(83-149). We show that this construct binds directly to the kinase domain of PAK2 and inhibits anti-TCR-stimulated T cell activation. Finally, we demonstrate that, in primary T cells, dominant-negative PAK2 prevented anti-CD3/CD28-induced IL-2 production, and TCR-induced CD40 ligand expression, both key functions of activated T cells. Taken together, these results suggest a novel role for PAK2 as a positive regulator of T cell activation.

Functional cloning of Src-like adapter protein-2 (SLAP-2), a novel inhibitor of antigen receptor signaling.

In an effort to identify novel therapeutic targets for autoimmunity and transplant rejection, we developed and performed a large-scale retroviral-based functional screen to select for proteins that inhibit antigen receptor-mediated activation of lymphocytes. In addition to known regulators of antigen receptor signaling, we identified a novel adaptor protein, SLAP-2 which shares 36% sequence similarity with the known Src-like adaptor protein, SLAP. Similar to SLAP, SLAP-2 is predominantly expressed in hematopoietic cells. Overexpression of SLAP-2 in B and T cell lines specifically impaired antigen receptor-mediated signaling events, including CD69 surface marker upregulation, nuclear factor of activated T cells (NFAT) promoter activation and calcium influx. Signaling induced by phorbol myristate acetate (PMA) and ionomycin was not significantly reduced, suggesting SLAP-2 functions proximally in the antigen receptor signaling cascade. The SLAP-2 protein contains an NH2-terminal myristoylation consensus sequence and SH3 and SH2 Src homology domains, but lacks a tyrosine kinase domain. In antigen receptor-stimulated cells, SLAP-2 associated with several tyrosine phosphorylated proteins, including the ubiquitin ligase Cbl. Deletion of the COOH terminus of SLAP-2 blocked function and abrogated its association with Cbl. Mutation of the putative myristoylation site of SLAP-2 compromised its inhibitory activity and impaired its localization to the membrane compartment. Our identification of the negative regulator SLAP-2 demonstrates that a retroviral-based screening strategy may be an efficient way to identify and characterize the function of key components of many signal transduction systems.