Phosphorylation Signatures Research Articles

Heptad-Specific Phosphorylation of RNA Polymerase II CTD

The carboxy-terminal domain (CTD) of RNA polymerase II (Pol II) consists of heptad repeats with the consensus motif Y1-S2-P3-T4-S5-P6-S7. Dynamic phosphorylation of the CTD coordinates Pol II progression through the transcription cycle. Here, we use genetic and mass spectrometric approaches to directly detect and map phosphosites along the entire CTD. We confirm phosphorylation of CTD residues Y1, S2, T4, S5, and S7 in mammalian and yeast cells. Although specific phosphorylation signatures dominate, adjacent CTD repeats can be differently phosphorylated, leading to a high variation of coexisting phosphosites in mono- and di-heptad CTD repeats. Inhibition of CDK9 kinase specifically reduces S2 phosphorylation levels within the CTD.

Leptin, BMI, and a Metabolic Gene Expression Signature Associated with Clinical Outcome to VEGF Inhibition in Colorectal Cancer

VEGF (vascular endothelial growth factor) signaling inhibitors are widely used in different cancer types; however, patient selection remains a challenge. Analyses of samples from a phase III clinical trial in metastatic colorectal cancer testing chemotherapy versus chemotherapy with the small molecule VEGF receptors inhibitor cediranib identified circulating leptin levels, BMI, and a tumor metabolic and angiogenic gene expression signature associated with improved clinical outcome in patients treated with cediranib. Patients with a glycolytic and hypoxic/angiogenic profile were associated with increased benefit from cediranib, whereas patients with a high lipogenic, oxidative phosphorylation and serine biosynthesis signature did not gain benefit. These findings translated to pre-clinical tumor xenograft models where the same metabolic gene expression profiles were associated with in vivo sensitivity to cediranib as monotherapy. These findings suggest a link between patient physiology, tumor biology, and response to antiangiogenics, which may guide patient selection for VEGF therapy in the future.

Abstract 4977: Dramatic differences in the extents and patterns of PKC delta modification among the different members of the NCI-60 cancer cell line panel

Abstract Protein kinase Cs (PKCs) are validated targets for cancer therapy with many agents in clinical trials which affect their activity and/or expression. PKC delta is one of the most highly expressed PKC isoforms and typically functions biologically to promote apoptosis. Multiple phosphorylation sites, both on serine/threonine and on tyrosine, have been identified on PKC delta and various sites have been shown to be differentially important for the various biological activities of PKC delta. Unfortunately, phosphosite specific antibodies are only available for a few of these phosphorylation sites. Capillary isoelectric focusing on a nanoscale with immunoassay readout, using pan-reactive antibodies that detect all modified states, reveals signatures of the differently modified states of PKC delta in cells. In LNCaP cells, the phosphorylation pattern of PKC delta was modestly complex under basal conditions but it became highly complex after ligand treatment. This phosphorylation signature depended on the ligand and the subsequent subcellular localization of PKC delta. To assess the generality of the findings in the LNCaP cells, we have extended our analysis to assess the phosphorylation signatures of PKC delta in different cell lines of the NCI-60 cancer cell line panel. Under basal conditions, the various cell lines showed dramatic differences. Some of them (SR leukemia, HT-29 colon cancer, OVCAR-5 ovarian cancer, many lung cancer cell lines, T-47D breast cancer) showed a pattern similar to that of LNCaP cells. Many others (most renal cell lines, OVCAR-8 ovarian cancer, MDA-MB-468 breast cancer, SK-MEL5 melanoma) displayed a complex pattern of highly phosphorylated peaks in the PKC delta signature. The extents of PKC delta phosphorylation did not correlate with the sensitivity of the cell lines to growth inhibition by the PKC ligand mezerein. Current efforts with selected cell lines from the NCI-60 cell line panel are directed at 1) characterizing in detail the PKC delta signature using different phosphosite specific antibodies to deconvolute the modifications responsible for the multiplicity of the peaks, 2) exploring what factors influence the PKC delta signature, and 3) evaluating whether the basal PKC delta phosphorylation signature is a measure of overall DAG signaling or is specific to the PKC delta isoform. We conclude that capillary isoelectric focusing with immunoassay is a powerful tool for exploring the complexity of PKC delta activation/modification in different cells and for identifying those cancer types where dysregulation of the DAG signaling pathway is most prominent. While the current studies focus on PKC delta, this same methodology should be similarly informative for the many other proteins with an intermediate level of complexity in their post-translational modifications. Citation Format: Noemi Kedei, Jin-Qiu Chen, Michelle Herrmann, Tiffany Hu, Karina Chang, Peter M. Blumberg. Dramatic differences in the extents and patterns of PKC delta modification among the different members of the NCI-60 cancer cell line panel. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4977. doi:10.1158/1538-7445.AM2015-4977

Abstract 5404: PAK4 allosteric modulators (PAMs) repress the Wnt/β-catenin signaling pathway and tumor growth

Abstract Wnt/β-catenin signaling is a ubiquitous pathway conserved throughout evolution and plays a role in embryonic and cancer development. This pathway has proven to be difficult to target therapeutically. Another intractable target is oncogenic Ras which is highly mutated in many cancer types, including pancreatic, colon and lung. An available target at the intersection of both of these genes is p21-Activated Kinase 4 (PAK4). PAK4 (1 of 6 PAK proteins) that is downstream of the Ras oncogene and a direct kinase that stabilizes transcriptional activity of β-catenin. Therefore therapeutically targeting PAK4 in cancer could be beneficial in a broad range of cancer types. PAK4 Allosteric Modulator (PAM) represents a novel and selective class of compounds that inhibit PAK4 allosterically. Methods: Flow cytometry and CellTiter AQueous One assay (MTS) were used to determine compound effects on cell cycle distribution and proliferation. CCLE, COSMIC and other databases were used for bioinformatics analysis of mutations in cancer genes. Deep sequencing and qPCR were used to analyze mRNA expression profiles. Proteomic platforms - KinomeScan, PTScan (Cell Signaling) and immunoblots were used to study phosphorylation signature of whole cell proteins and total protein steady state levels. Results: We identified a selective, orally bioavailable small molecule PAM, KPT-9274, which demonstrated anti-tumor activity in a variety of cancer cell lines (IC50 values from 0.005 - 1 μM). Bioinformatics revealed that sensitivity to KPT-9274 was directly correlated with mutations in APC or N-ras and inversely correlated with mutations in β-catenin, K-ras, or PI3K. KPT-9274 and other PAMs reduced phosphorylation and steady state levels of PAK4 protein while reducing Phospho-S675 and total β-catenin, Wnt5, Phospho- and total LRP6, Dvl2, Phospho-Akt, and Phospho-ERK. PAMs also reduced β-catenin transcriptional activity (i.e. cyclin D1, MM7, and S100A4). PAMs arrested cancer cell cycle at the G1 and G2 phases and induced apoptosis through Caspase and PARP cleavage. KPT-9274 (∼100 mg/kg BIDx5 orally) has demonstrated potent anti-tumor activity against hematological (Z-138, Molt-4, MM1S) and solid (MDA-MB-468 and Colo-205) xenograft models in mice. PAM-treated xenografts showed reduction of PAK4, β-catenin and cyclin D1 proteins. Conclusions: PAK4 represents a novel anti-cancer target at the crossroads of Ras and Wnt/β-catenin signaling. We have identified selective small molecule PAMs with anti-tumor activity both in vitro and in vivo. These allosteric modulators induce tumor cell growth arrest and apoptosis. Bioinformatics helped identify potential predictive markers in the Wnt signaling pathway while deep sequencing and proteiomics revealed possible PDn markers. Based on the in vitro and in vivo activity, KPT-9274 may be beneficial for the treatment of a wide variety of cancers and preclinical toxicology studies are ongoing. Citation Format: William T. Senapedis, Scott Donovan, Gali Golan, Dilara McCauley, Joel Ellis, Marsha Crochiere, Trinayan Kashyap, Boris Klebanov, Sharon Shacham, Yosef Landesman, Erkan Baloglu. PAK4 allosteric modulators (PAMs) repress the Wnt/β-catenin signaling pathway and tumor growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5404. doi:10.1158/1538-7445.AM2015-5404

Uncoupling Stress-Inducible Phosphorylation of Heat Shock Factor 1 from Its Activation.

In mammals the stress-inducible expression of genes encoding heat shock proteins is under the control of the heat shock transcription factor 1 (HSF1). Activation of HSF1 is a multistep process, involving trimerization, acquisition of DNA-binding and transcriptional activities, which coincide with several posttranslational modifications. Stress-inducible phosphorylation of HSF1, or hyperphosphorylation, which occurs mainly within the regulatory domain (RD), has been proposed as a requirement for HSF-driven transcription and is widely used for assessing HSF1 activation. Nonetheless, the contribution of hyperphosphorylation to the activity of HSF1 remains unknown. In this study, we generated a phosphorylation-deficient HSF1 mutant (HSF1Δ∼PRD), where the 15 known phosphorylation sites within the RD were disrupted. Our results show that the phosphorylation status of the RD does not affect the subcellular localization and DNA-binding activity of HSF1. Surprisingly, under stress conditions, HSF1Δ∼PRD is a potent transactivator of both endogenous targets and a reporter gene, and HSF1Δ∼PRD has a reduced activation threshold. Our results provide the first direct evidence for uncoupling stress-inducible phosphorylation of HSF1 from its activation, and we propose that the phosphorylation signature alone is not an appropriate marker for HSF1 activity.

Pareto Optimization Identifies Diverse Set of Phosphorylation Signatures Predicting Response to Treatment with Dasatinib.

Multivariate biomarkers that can predict the effectiveness of targeted therapy in individual patients are highly desired. Previous biomarker discovery studies have largely focused on the identification of single biomarker signatures, aimed at maximizing prediction accuracy. Here, we present a different approach that identifies multiple biomarkers by simultaneously optimizing their predictive power, number of features, and proximity to the drug target in a protein-protein interaction network. To this end, we incorporated NSGA-II, a fast and elitist multi-objective optimization algorithm that is based on the principle of Pareto optimality, into the biomarker discovery workflow. The method was applied to quantitative phosphoproteome data of 19 non-small cell lung cancer (NSCLC) cell lines from a previous biomarker study. The algorithm successfully identified a total of 77 candidate biomarker signatures predicting response to treatment with dasatinib. Through filtering and similarity clustering, this set was trimmed to four final biomarker signatures, which then were validated on an independent set of breast cancer cell lines. All four candidates reached the same good prediction accuracy (83%) as the originally published biomarker. Although the newly discovered signatures were diverse in their composition and in their size, the central protein of the originally published signature — integrin β4 (ITGB4) — was also present in all four Pareto signatures, confirming its pivotal role in predicting dasatinib response in NSCLC cell lines. In summary, the method presented here allows for a robust and simultaneous identification of multiple multivariate biomarkers that are optimized for prediction performance, size, and relevance.

Unraveling the molecular signatures of oxidative phosphorylation to cope with the nutritionally changing metabolic capabilities of liver and muscle tissues in farmed fish.

Mitochondrial oxidative phosphorylation provides over 90% of the energy produced by aerobic organisms, therefore the regulation of mitochondrial activity is a major issue for coping with the changing environment and energy needs. In fish, there is a large body of evidence of adaptive changes in enzymatic activities of the OXPHOS pathway, but less is known at the transcriptional level and the first aim of the present study was to define the molecular identity of the actively transcribed subunits of the mitochondrial respiratory chain of a livestock animal, using gilthead sea bream as a model of farmed fish with a high added value for European aquaculture. Extensive BLAST searches in our transcriptomic database (www.nutrigroup-iats.org/seabreamdb) yielded 97 new sequences with a high coverage of catalytic, regulatory and assembly factors of Complex I to V. This was the basis for the development of a PCR array for the simultaneous profiling of 88 selected genes. This new genomic resource allowed the differential gene expression of liver and muscle tissues in a model of 10 fasting days. A consistent down-regulated response involving 72 genes was made by the liver, whereas an up-regulated response with 29 and 10 differentially expressed genes was found in white skeletal muscle and heart, respectively. This differential regulation was mostly mediated by nuclear-encoded genes (skeletal muscle) or both mitochondrial- and nuclear-encoded genes (liver, heart), which is indicative of a complex and differential regulation of mitochondrial and nuclear genomes, according to the changes in the lipogenic activity of liver and the oxidative capacity of glycolytic and highly oxidative muscle tissues. These insights contribute to the identification of the most responsive elements of OXPHOS in each tissue, which is of relevance for the appropriate gene targeting of nutritional and/or environmental metabolic disturbances in livestock animals.

Highlights of This Issue

Epigenetic events like methylation are critically important to normal tissue homeostasis. As such, epigenetic changes often occur during tumorigenesis. Jumonji domain-containing protein 3 (JMJD3) demethylates tri- and dimethylated histone H3 at lysine 27 (H3K27) and is central in controlling the chromatin state as well as numerous biologic programs such as cellular senescence. Here, Perrigue and colleagues assessed the status of JMJD3 in the context of glioma and found intrinsic differences that reflected both phenotypes and variations associated with senescence. Mechanistic study demonstrated that JMJD3 activated the senescence-associated secretory phenotype (SASP) and, based on RNA-seq analysis, regulated cancer-related and inflammatory-related transcripts. Combined, the epigenetic reprogramming and secretory phenotype induced by JMJD3 may stimulate homing of nearby cells in the tumor microenvironment to the tumor.Little is known about the molecular drivers of phyllodes tumors, which are rare stromal breast tumors with malignant potential. In the current Rapid Impact article, Cani and colleagues report on a study in which they performed next-generation sequencing on routine clinical specimens to shed light on the mutational and copy number alteration landscape of phyllodes tumors. Importantly, MED12, a RNA polymerase II transcriptional machinery component that has been reported in other estrogen-driven stromal tumors, was mutated with high frequency, suggesting a candidate driver. Phyllodes tumors with aggressive behavior were characterized by additional alterations in tumor suppressors and oncogenes, including a high level of EGFR and IGF1R amplifications, presenting opportunities for targeted therapy.To date, there are no effective treatments for metastatic prostate cancer, and a better understanding of the metastatic process is urgently needed. Lymphoid enhancer-binding factor-1 (LEF1) modulates epithelial–mesenchymal transition (EMT), promoting invasive ability in cancer cells and metastasis. Liang and colleagues integrated miRNA microarray, luciferase, and chromatin immunoprecipitation (ChIP) analyses to identify that miR-34a directly and negatively targets LEF1 and its effects on EMT, reducing cell migration and invasion. Consistently, miR-34a is also significantly downregulated in human prostate cancer specimens. Together, these results indicate that the miR-34a/LEF1 axis is a potential molecular target for novel therapeutic strategies in prostate cancer.EGFR is often mutated in various cancers and therefore is an important therapeutic target. Erlotinib and gefitinib are FDA- approved drugs targeting EGFR. Understanding differences in autophosphorylation kinetics and drug sensitivity between wild- type and oncogenic EGFR signaling is important for correlating specific phosphorylation signatures to drug responsiveness. By comparing drug responsiveness in wild-type and oncogenic EGFR expressing cells, Kim and colleagues observed dramatic differences in potency of these drugs for inhibiting downstream effectors, Cbl and STAT5. This study provides a new understanding of the EGFR signaling environment and reveals useful paradigms for predicting patient response to EGFR targeted therapy as well as combination treatments.

Combining Genetic Perturbations and Proteomics to Examine Kinase-Phosphatase Networks in Drosophila Embryos

Connecting phosphorylation events to kinases and phosphatases is key to understanding the molecular organization and signaling dynamics of networks. We have generated a validated set of transgenic RNA-interference reagents for knockdown and characterization of all protein kinases and phosphatases present during early Drosophila melanogaster development. These genetic tools enable collection of sufficient quantities of embryos depleted of single gene products for proteomics. As a demonstration of an application of the collection, we have used multiplexed isobaric labeling for quantitative proteomics to derive global phosphorylation signatures associated with kinase-depleted embryos to systematically link phosphosites with relevant kinases. We demonstrate how this strategy uncovers kinase consensus motifs and prioritizes phosphoproteins for kinase target validation. We validate this approach by providing auxiliary evidence for Wee kinase-directed regulation of the chromatin regulator Stonewall. Further, we show how correlative phosphorylation at the site level can indicate function, as exemplified by Sterile20-like kinase-dependent regulation of Stat92E.

Abstract 5309: Phospho-reactome profiling reveals the heterogenic, targetable kinase signature of breast cancer

Abstract Background: Improper control of phosphorylation networks represents a powerful regulator and biomarker of cell dys-function leading to malignancy. Discovering which active mechanisms of disease progression can be efficiently targeted, and knowing whether kinase networks circumvent therapeutic interventions, are challenges researchers and clinicians face. Surprisingly however, measuring the phosphorylating activity of kinases, and potentially monitoring the functionality of the entire human phospho-reactome at once, remains largely unexplored. We developed a semi-high throughput assay to monitor the phospho-catalytic activity of kinase enzymes, using their biological targets as phospho-sensors. We successfully used this assay to identify the active, oncogenic phospho-signature of breast cancer cells. Methods: We first built a library of peptide sensors established from confirmed kinase substrates' phosphorylation sites. Precisely, we used computational methods to create a unique phospho-repertoire cataloguing 3,408 peptide sequences established from validated human proteins' phosphorylation sites, curated from 38 public databases. Second, we experimentally used these biologically relevant probes in multiplex assays to measure the catalytic state of kinases. Specifically, 228 peptides were developed into an ATP-consumption screen to identify the activity signatures of AKT, EGFR, MAPK, ABL and SRC family kinases. We developed analysis methods to derive phospho-signatures from semi-high throughput measurements. Finally, we validated the bio-applicability of the assay using isogenic culture model of basal-like breast cancer (HMT-3522 S1 and T4-2), and cell lines harboring EGFR/HER2-oncogenic alterations (MDA-MB-231, AU565, MCF7, T47D). Results: The differential phosphorylation activity of 25 recombinant, active kinase enzymes was successfully captured on 228 phospho-sensing peptides. In cancer cell extracts, hyper-activated AKT, SRC, EGFR, or MAPK kinases originally identified by immuno-detection were reliably and specifically detectable using the peptide-based kinase-activity assay. The phospho-sensing assay revealed the heterogeneity of kinase activity networks in breast cancer cells, and possible new drug-targeting opportunities. Conclusion: This unique strategy and resources allows to comprehensively measure the catalytic activity of a multitude of kinases at once, ultimately representing a new molecular dimension to characterize biological samples. Such new phospho-reactome profiling system suggests how chemotherapeutic interventions affect the activity of kinases and lead to reprogramming of phospho-circuits. Our efforts now concentrate on expanding this approach into a high-content, functional kinome-screening platform to map phosphorylation signatures that cause breast malignancies, therapeutic resistance, or recurrence. Citation Format: Jean-Philippe F. Coppé, Miki Mori, Evelyn Lee, Bo Pan, Aaron Boudreau, Zhongzhong Chen, Laura van 't Veer. Phospho-reactome profiling reveals the heterogenic, targetable kinase signature of breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5309. doi:10.1158/1538-7445.AM2014-5309

The late endosomal transporter CD222 directs the spatial distribution and activity of Lck.

The spatial and temporal organization of T cell signaling molecules is increasingly accepted as a crucial step in controlling T cell activation. CD222, also known as the cation-independent mannose 6-phosphate/insulin-like growth factor 2 receptor, is the central component of endosomal transport pathways. In this study, we show that CD222 is a key regulator of the early T cell signaling cascade. Knockdown of CD222 hampers the effective progression of TCR-induced signaling and subsequent effector functions, which can be rescued via reconstitution of CD222 expression. We decipher that Lck is retained in the cytosol of CD222-deficient cells, which obstructs the recruitment of Lck to CD45 at the cell surface, resulting in an abundant inhibitory phosphorylation signature on Lck at the steady state. Hence, CD222 specifically controls the balance between active and inactive Lck in resting T cells, which guarantees operative T cell effector functions.

Profiling the tyrosine phosphoproteome of different mouse mammary tumour models reveals distinct, model-specific signalling networks and conserved oncogenic pathways.

IntroductionAlthough aberrant tyrosine kinase signalling characterises particular breast cancer subtypes, a global analysis of tyrosine phosphorylation in mouse models of breast cancer has not been undertaken to date. This may identify conserved oncogenic pathways and potential therapeutic targets.MethodsWe applied an immunoaffinity/mass spectrometry workflow to three mouse models: murine stem cell virus-Neu, expressing truncated Neu, the rat orthologue of human epidermal growth factor receptor 2, Her2 (HER2); mouse mammary tumour virus-polyoma virus middle T antigen (PyMT); and the p53−/− transplant model (p53). Pathways and protein–protein interaction networks were identified by bioinformatics analysis. Molecular mechanisms underpinning differences in tyrosine phosphorylation were characterised by Western blot analysis and array comparative genomic hybridisation. The functional role of mesenchymal–epithelial transition factor (Met) in a subset of p53-null tumours was interrogated using a selective tyrosine kinase inhibitor (TKI), small interfering RNA (siRNA)–mediated knockdown and cell proliferation assays.ResultsThe three models could be distinguished on the basis of tyrosine phosphorylation signatures and signalling networks. HER2 tumours exhibited a protein–protein interaction network centred on avian erythroblastic leukaemia viral oncogene homologue 2 (Erbb2), epidermal growth factor receptor and platelet-derived growth factor receptor α, and they displayed enhanced tyrosine phosphorylation of ERBB receptor feedback inhibitor 1. In contrast, the PyMT network displayed significant enrichment for components of the phosphatidylinositol 3-kinase signalling pathway, whereas p53 tumours exhibited increased tyrosine phosphorylation of Met and components or regulators of the cytoskeleton and shared signalling network characteristics with basal and claudin-low breast cancer cells. A subset of p53 tumours displayed markedly elevated cellular tyrosine phosphorylation and Met expression, as well as Met gene amplification. Treatment of cultured p53-null cells exhibiting Met amplification with a selective Met TKI abrogated aberrant tyrosine phosphorylation and blocked cell proliferation. The effects on proliferation were recapitulated when Met was knocked down using siRNA. Additional subtypes of p53 tumours exhibited increased tyrosine phosphorylation of other oncogenes, including Peak1/SgK269 and Prex2.ConclusionThis study provides network-level insights into signalling in the breast cancer models utilised and demonstrates that comparative phosphoproteomics can identify conserved oncogenic signalling pathways. The Met-amplified, p53-null tumours provide a new preclinical model for a subset of triple-negative breast cancers.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0437-3) contains supplementary material, which is available to authorized users.

Glycogen Synthase Kinase-3β Stabilizes the Interleukin (IL)-22 Receptor from Proteasomal Degradation in Murine Lung Epithelia

Signaling through the interleukin (IL)-22 cytokine axis provides essential immune protection in the setting of extracellular infection as part of type 17 immunity. Molecular regulation of IL-22 receptor (IL-22R) protein levels is unknown. In murine lung epithelia, IL-22R is a relatively short-lived protein (t½ ∼1.5 h) degraded by the ubiquitin proteasome under normal unstimulated conditions, but its degradation is accelerated by IL-22 treatment. Lys(449) within the intracellular C-terminal domain of the IL-22R serves as a ubiquitin acceptor site as disruption of this site by deletion or site-directed mutagenesis creates an IL-22R variant that, when expressed in cells, is degradation-resistant and not ubiquitinated. Glycogen synthase kinase (GSK)-3β phosphorylates the IL-22R within a consensus phosphorylation signature at Ser(410) and Ser(414), and IL-22 treatment of cells triggers GSK-3β inactivation. GSK-3β overexpression results in accumulation of IL-22R protein, whereas GSK-3β depletion in cells reduces levels of the receptor. Mutagenesis of IL-22R at Ser(410) and Ser(414) results in receptor variants that display reduced phosphorylation levels and are more labile as compared with wild-type IL-22R when expressed in cells. Further, the cytoskeletal protein cortactin, which is important for epithelial spreading and barrier formation, is phosphorylated and activated at the epithelial cell leading edge after treatment with IL-22, but this effect is reduced after GSK-3β knockdown. These findings reveal the ability of GSK-3β to modulate IL-22R protein stability that might have significant implications for cytoprotective functions and therapeutic targeting of the IL-22 signaling axis.

Interleukin 22 receptor abundance and signaling is regulated by the ubiquitin proteasome and glycogen synthase kinase 3β. (INM2P.422)

Abstract Signaling through the interleukin (IL)-22 cytokine axis provides essential immune protection in the setting of extracellular infection at mucosal and other epithelial surfaces. Molecular regulation of IL-22 receptor (IL-22R) protein has not been characterized. In mouse lung epithelia, IL-22R is expressed as a relatively short-lived protein and is degraded by the ubiquitin proteasome. Its degradation is accelerated when cells are stimulated by IL-22. Lys 449 within the intracellular C-terminal domain of the IL-22R serves as a ubiquitin acceptor site for proteasomal degradation as deletion or mutation of K449 creates a degradation resistant IL-22R mutant protein that is not ubiquitinated in cells. IL-22 treatment of cells triggers glycogen synthase kinase (GSK)-3β inactivation, and GSK-3β phosphorylates the IL-22R within a consensus phosphorylation signature at Ser 410. GSK-3β overexpression results in accumulation of IL-22R protein while GSK-3β depletion in cells reduces levels of the receptor, and this effect is abolished on mutation of the 410-414 phosphorylation domain. Signal transduction of IL-22 appears increased when GSK-3β is overexpressed. This study shows GSK-3β as a modulator of IL-22R protein stability, which may be important for host defense and could impact therapeutic targeting of the IL-22 signaling axis.

Acute exercise and physiological insulin induce distinct phosphorylation signatures on TBC1D1 and TBC1D4 proteins in human skeletal muscle

We investigated the phosphorylation signatures of two Rab-GTPase activating proteins TBC1D1 and TBC1D4 in human skeletal muscle in response to physical exercise and physiological insulin levels induced by a carbohydrate rich meal using a paired experimental design. Eight healthy male volunteers exercised in the fasted or fed state and muscle biopsies were taken before and immediately after exercise. We identified TBC1D1/4 phospho-sites that (1) did not respond to exercise or postprandial increase in insulin (TBC1D4: S666), (2) responded to insulin only (TBC1D4: S318), (3) responded to exercise only (TBC1D1: S237, S660, S700; TBC1D4: S588, S751), and (4) responded to both insulin and exercise (TBC1D1: T596; TBC1D4: S341, T642, S704). In the insulin-stimulated leg, Akt phosphorylation of both T308 and S473 correlated significantly with multiple sites on both TBC1D1 (T596) and TBC1D4 (S318, S341, S704). Interestingly, in the exercised leg in the fasted state TBC1D1 phosphorylation (S237, T596) correlated significantly with the activity of the α2/β2/γ3 AMPK trimer, whereas TBC1D4 phosphorylation (S341, S704) correlated with the activity of the α2/β2/γ1 AMPK trimer. Our data show differential phosphorylation of TBC1D1 and TBC1D4 in response to physiological stimuli in human skeletal muscle and support the idea that Akt and AMPK are upstream kinases. TBC1D1 phosphorylation signatures were comparable between in vitro contracted mouse skeletal muscle and exercised human muscle, and we show that AMPK regulated phosphorylation of these sites in mouse muscle. Contraction and exercise elicited a different phosphorylation pattern of TBC1D4 in mouse compared with human muscle, and although different circumstances in our experimental setup may contribute to this difference, the observation exemplifies that transferring findings between species is problematic.

Identification of Potential Plk1 Targets in a Cell-Cycle Specific Proteome through Structural Dynamics of Kinase and Polo Box-Mediated Interactions

Polo like kinase 1 (Plk1) is a key player in orchestrating the wide variety of cell-cycle events ranging from centrosome maturation, mitotic entry, checkpoint recovery, transcriptional control, spindle assembly, mitotic progression, cytokinesis and DNA damage checkpoints recovery. Due to its versatile nature, Plk1 is considered an imperative regulator to tightly control the diverse aspects of the cell cycle network. Interactions among Plk1 polo box domain (PBD) and its putative binding proteins are crucial for the activation of Plk1 kinase domain (KD). To date, only a few substrate candidates have been characterized through the inclusion of both polo box and kinase domain-mediated interactions. Thus it became compelling to explore precise and specific Plk1 substrates through reassessment and extension of the structure-function paradigm. To narrow this apparently wide gap in knowledge, here we employed a thorough sequence search of Plk1 phosphorylation signature containing proteins and explored their structure-based features like conceptual PBD-binding capabilities and subsequent recruitment of KD directed phosphorylation to dissect novel targets of Plk1. Collectively, we identified 4,521 phosphodependent proteins sharing similarity to the consensus phosphorylation and PBD recognition motifs. Subsequent application of filters including similarity index, Gene Ontology enrichment and protein localization resulted in stringent pre-filtering of irrelevant candidates and isolated unique targets with well-defined roles in cell-cycle machinery and carcinogenesis. These candidates were further refined structurally using molecular docking and dynamic simulation assays. Overall, our screening approach enables the identification of several undefined cell-cycle associated functions of Plk1 by uncovering novel phosphorylation targets.

Novel Insights into Biased Agonism at G Protein-Coupled Receptors and their Potential for Drug Design

G-protein coupled receptors (GPCRs) are the most important class of current pharmacological targets. However, it is now widely acknowledged that their regulation is more complex than previously thought: the evidence that GPCRs can couple to several effector pathways, and the existence of biased agonists able to activate them differentially, has introduced a new level of complexity in GPCR drug research. Considering bias represents a challenge for the research of new GPCR modulators, because it demands a detailed characterization of compound properties for several effector pathways. Still, biased ligands could provide an opportunity to modulate GPCR function in a finer way and to separate therapeutic from side effects. Nowadays, a variety of agonists for GPCRs have been described, which differ in their ability to promote receptor coupling to different Gprotein families or even subunits, recruit signal transducers such as arrestins, activate a variety of downstream molecular pathways and induce certain phosphorylation signatures or gene expression patterns. In this review, we will cover some of the experimental techniques currently used to understand and characterize biased agonism and discuss their strengths and limitations. Additionally, we will comment on the computational efforts that are being devoted to study ligand-induced bias and on the potential they hold for rationalizing its structural determinants. Finally, we will discuss which of these strategies could be used for the rational design of biased ligands and give some examples of the potential therapeutic value of this class of compounds.

A Separate Pool of Cardiac Phospholemman That Does Not Regulate or Associate with the Sodium Pump: MULTIMERS OF PHOSPHOLEMMAN IN VENTRICULAR MUSCLE

Phospholemman regulates the plasmalemmal sodium pump in excitable tissues. In cardiac muscle, a subpopulation of phospholemman with a unique phosphorylation signature associates with other phospholemman molecules but not with the pump. Phospholemman oligomers exist in cardiac muscle. Much like phospholamban regulation of SERCA, phospholemman exists as both a sodium pump inhibiting monomer and an unassociated oligomer. Phospholemman (PLM), the principal quantitative sarcolemmal substrate for protein kinases A and C in the heart, regulates the cardiac sodium pump. Much like phospholamban, which regulates the related ATPase SERCA, PLM is reported to oligomerize. We investigated subpopulations of PLM in adult rat ventricular myocytes based on phosphorylation status. Co-immunoprecipitation identified two pools of PLM: one not associated with the sodium pump phosphorylated at Ser(63) and one associated with the pump, both phosphorylated at Ser(68) and unphosphorylated. Phosphorylation of PLM at Ser(63) following activation of PKC did not abrogate association of PLM with the pump, so its failure to associate with the pump was not due to phosphorylation at this site. All pools of PLM co-localized to cell surface caveolin-enriched microdomains with sodium pump α subunits, despite the lack of caveolin-binding motif in PLM. Mass spectrometry analysis of phosphospecific immunoprecipitation reactions revealed no unique protein interactions for Ser(63)-phosphorylated PLM, and cross-linking reagents also failed to identify any partner proteins for this pool. In lysates from hearts of heterozygous transgenic animals expressing wild type and unphosphorylatable PLM, Ser(63)-phosphorylated PLM co-immunoprecipitated unphosphorylatable PLM, confirming the existence of PLM multimers. Dephosphorylation of the PLM multimer does not change sodium pump activity. Hence like phospholamban, PLM exists as a pump-inhibiting monomer and an unassociated oligomer. The distribution of different PLM phosphorylation states to different pools may be explained by their differential proximity to protein phosphatases rather than a direct effect of phosphorylation on PLM association with the pump.

Abstract 2507: Mechanistic and functional characterization of the atypical kinase SgK269/PEAK1 in breast cancer.

Abstract The human genome encodes 518 protein kinases which can be mined for potential cancer drug targets and biomarkers. Previously, we performed SILAC labelling in combination with mass spectrometry-based phosphoproteomics to identify seven proteins exhibiting decreased tyrosine phosphorylation upon Lyn knockdown in the BT549 and MDA-MB-231 basal breast cancer cell lines. From these we selected the atypical kinase SgK269 (also known as PEAK1) for further study. SgK269 is classified as a pseudokinase since it contains critical substitutions in one of the three conserved motifs important for kinase activity. The primary objective of this study was to functionally characterize SgK269 and to identify its signaling role through proteomic and phosphoproteomic profiling in mammary epithelial cells. Initial pull-down experiments using SgK269 recombinant protein were performed to identify binding partners of SgK269 in MDA-MB-231 cells. Functional annotation of the identified binding partners subsequently indicated that SgK269 may be involved in MAPK signaling, regulation of the actin cytoskeleton and focal adhesions, and ubiquitin-mediated proteolysis. To further interrogate the role of SgK269 in breast cancer, SgK269 was overexpressed in the human mammary epithelial cell line MCF-10A. We also expressed a mutant form of SgK269 where the Lyn phosphorylation site, Y635, was altered to phenylalanine (F). Overexpression of SgK269 resulted in a change in cellular morphology. In monolayer, cells were elongated resembling a mesenchymal phenotype and exhibited a decreased expression of E-cadherin and an increase in N-cadherin, indicative of epithelial-to-mesenchymal transition (EMT). When grown in Matrigel, the SgK269-overexpressing cells formed large, aberrant acini, while the SgK269 Y635F mutant was unable to enhance acinar size. In terms of signaling, overexpression of SgK269 increased Erk phosphorylation in three-dimensional culture, an effect associated with SgK269/Grb2 binding and enhanced interaction between the EGFR and Grb2. Since SgK269 showed strong binding with Stat3 in pull-down studies, we also characterized Stat3 signalling in this model. SgK269 overexpression led to enhanced Stat3 tyrosine phosphorylation and transcriptional activity whilst the Y635F mutation abolished this effect. The global tyrosine phosphorylation signature associated with SgK269 overexpression in MCF-10A cells is currently under investigation. SgK269 is a poorly characterized kinase that may contribute to the progression of breast and other cancers. This study provides novel and highly significant insights into the signaling mechanism and function of SgK269 that highlights potential strategies for the improved treatment of basal breast cancers, as well as other cancers where SgK269 is overexpressed. Citation Format: Ling Liu, Howard Chan, David Croucher, Falco Hochgräfe, Luxi Zhang, Carole Tactacan, Roger Daly. Mechanistic and functional characterization of the atypical kinase SgK269/PEAK1 in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2507. doi:10.1158/1538-7445.AM2013-2507 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Phosphoproteome dynamics reveal novel ERK1/2 MAP kinase substrates with broad spectrum of functions

The ERK1/2 MAP kinase pathway is an evolutionarily conserved signaling module that controls many fundamental physiological processes. Deregulated activity of ERK1/2 MAP kinases is associated with developmental syndromes and several human diseases. Despite the importance of this pathway, a comprehensive picture of the natural substrate repertoire and biochemical mechanisms regulated by ERK1/2 is still lacking. In this study, we used large-scale quantitative phosphoproteomics and bioinformatics analyses to identify novel candidate ERK1/2 substrates based on their phosphorylation signature and kinetic profiles in epithelial cells. We identified a total of 7936 phosphorylation sites within 1861 proteins, of which 155 classify as candidate ERK1/2 substrates, including 128 new targets. Candidate ERK1/2 substrates are involved in diverse cellular processes including transcriptional regulation, chromatin remodeling, RNA splicing, cytoskeleton dynamics, cellular junctions and cell signaling. Detailed characterization of one newly identified substrate, the transcriptional regulator JunB, revealed that ERK1/2 phosphorylate JunB on a serine adjacent to the DNA-binding domain, resulting in increased DNA-binding affinity and transcriptional activity. Our study expands the spectrum of cellular functions controlled by ERK1/2 kinases.