Phosphoproteomic Studies Research Articles

Advances in quantitative high-throughput phosphoproteomics with sample multiplexing.

Eukaryotic protein phosphorylation modulates nearly every major biological process. Phosphorylation regulates protein activity, mediates cellular signal transduction, and manipulates cellular structure. Consequently, the dysregulation of kinase and phosphatase pathways has been linked to a multitude of diseases. Mass spectrometry-based proteomic techniques are increasingly used for the global interrogation of perturbations in phosphorylation-based cellular signaling. Strategies for studying phosphoproteomes require high-specificity enrichment, sensitive detection, and accurate localization of phosphorylation sites with advanced LC-MS/MS techniques and downstream informatics. Sample multiplexing with isobaric tags has also been integral to recent advancements in throughput and sensitivity for phosphoproteomic studies. Each of these facets of phosphoproteomics analysis present distinct challenges and thus opportunities for improvement and innovation. Here, we review current methodologies, explore persistent challenges, and discuss the outlook for isobaric tag-based quantitative phosphoproteomic analysis.

Comparative phosphoproteomic analysis unravels MAPK1 regulated phosphoproteins in Leishmania donovani

Mitogen Activated Protein Kinase1 (MAPK1) of Leishmania donovani functions as key regulators of various cellular activities, which seem to be imperative for parasite survival, infectivity, drug resistance and post-translational modification of chaperones/co-chaperones. However, very less is known about LdMAPK1 target proteins. With recent advancements in proteomics, we aimed to identify phosphoproteins which were differentially expressed in LdMAPK1 overexpressing (Dd8++/++) and single replacement mutants (Dd8+/) as compared to wild type (Dd8+/+) parasites, utilizing LC-MS/MS approach. An in-depth label-free phospoproteomic analysis revealed that modulation of LdMAPK1 expression significantly modulates expression levels of miscellaneous phosphoproteins which may act as its targets/substrates. Out of 1974 quantified phosphoproteins in parasite, 140 were significantly differentially expressed in MAPK1 overexpressing and single replacement mutants. These differentially expressed phosphoproteins are majorly associated with metabolism, signal transduction, replication, transcription, translation, transporters and cytoskeleton/motor proteins, hence suggested that MAPK1 may act in concert to modulate global biological processes. The study further implicated possible role of LdMAPK1 in regulation and management of stress machinery in parasite through post translational modifications. Precisely, comparative phosphoproteomics study has elucidated significant role of LdMAPK1 in regulating various pathways contributing in parasite biology with relevance to future drug development. SignificanceMAPKinase1, the downstream kinase of MAPK signal transduction pathway, has drawn much attention as potential therapeutic drug target due to their indispensable role in survival and infectivity of Leishmania donovani. However, limited information is available about its downstream effector proteins/signaling networks. Utilizing label free LC-MS/MS analysis, phosphoproteome of LdMAPK1 over-expressing (Dd8++/++) and LdMAPK1 single replacement mutants (Dd8+/−) with wild type (Dd8+/+) parasites was compared and identified 140 LdMAPK1 modulated phosphoproteins, mainly involved in pathways like signal transduction, metabolism, transcriptional, translational, post-translational modification and regulation of heat shock proteins. Interestingly, LdMAPK1 interacts directly with only six phosphoproteins i.e. casein kinase, casein kinase II, HSP83/HSP90, LACK, protein kinase and serine/threonine protein kinase. Thus, the study elucidates significant role of LdMAPK1 in Leishmania biology which may drive drug-discovery efforts against visceral leishmaniasis.

Extracellular signal-regulated kinase regulates microglial immune responses in Alzheimer's disease.

The importance of mitogen-activated protein kinase (MAPK) pathway signaling in regulating microglia-mediated neuroinflammation in Alzheimer's disease (AD) remains unclear. We examined the role of MAPK signaling in microglia using a preclinical model of AD pathology and quantitative proteomics studies of postmortem human brains. In multiplex immunoassay analyses of MAPK phosphoproteins in acutely isolated microglia and brain tissue from 5xFAD mice, we found phosphorylated extracellular signal-regulated kinase (ERK) was the most strongly upregulated phosphoprotein within the MAPK pathway in acutely isolated microglia, but not whole-brain tissue from 5xFAD mice. The importance of ERK signaling in primary microglia cultures was next investigated using transcriptomic profiling and functional assays of amyloid-β and neuronal phagocytosis, which confirmed that ERK is a critical regulator of IFNγ-mediated pro-inflammatory activation of microglia, although it was also partly important for constitutive microglial functions. Phospho-ERK was an upstream regulator of disease-associated microglial gene expression (Trem2, Tyrobp), as well as several human AD risk genes (Bin1, Cd33, Trem2, Cnn2), indicative of the importance of microglial ERK signaling in AD pathology. Quantitative proteomic analyses of postmortem human brain showed that ERK1 and ERK2 were the only MAPK proteins with increased protein expression and positive associations with neuropathological grade. In a human brain phosphoproteomic study, we found evidence for increased flux through the ERK signaling pathway in AD. Overall, our analyses strongly suggest that ERK phosphorylation, particularly in microglia in mouse models, is a regulator of pro-inflammatory immune responses in AD pathogenesis.

Phosphoproteomics-Based Characterization of Prostaglandin E2 Signaling in T Cells.

Prostaglandin E2 (PGE2) is a key lipid mediator in health and disease and serves as a crucial link between the immune response and cancer. With the advent of cancer therapies targeting PGE2 signaling pathways at different levels, there has been increased interest in mapping and understanding the complex and interconnected signaling pathways arising from the four distinct PGE2 receptors. Here, we review phosphoproteomics studies that have investigated different aspects of PGE2 signaling in T cells. These studies have elucidated PGE2's regulatory effect on T cell receptor signaling and T cell function, the key role of protein kinase A in many PGE2 signaling pathways, the temporal regulation of PGE2 signaling, differences in PGE2 signaling between different T cell subtypes, and finally, the crosstalk between PGE2 signaling pathways elicited by the four distinct PGE2 receptors present in T cells. SIGNIFICANCE STATEMENT: Through the reviewed studies, we now have a much better understanding of PGE2's signaling mechanisms and functional roles in T cells, as well as a solid platform for targeted and functional studies of specific PGE2-triggered pathways in T cells.

Cell Type-specific Adaptive Signaling Responses to KRASG12C Inhibition.

Covalent inhibitors of KRASG12C specifically target tumors driven by this form of mutant KRAS, yet early studies show that bypass signaling drives adaptive resistance. Although several combination strategies have been shown to improve efficacy of KRASG12C inhibitors (KRASi), underlying mechanisms and predictive strategies for patient enrichment are less clear. We performed mass spectrometry-based phosphoproteomics analysis in KRASG12C cell lines after short-term treatment with ARS-1620. To understand signaling diversity and cell type-specific markers, we compared proteome and phosphoproteomes of KRASG12C cells. Gene expression patterns of KRASG12C cell lines and lung tumor tissues were examined. Our analysis suggests cell type-specific perturbation to ERBB2/3 signaling compensates for repressed ERK and AKT signaling following ARS-1620 treatment in epithelial cell type, and this subtype was also more responsive to coinhibition of SHP2 and SOS1. Conversely, both high basal and feedback activation of FGFR or AXL signaling were identified in mesenchymal cells. Inhibition of FGFR signaling suppressed feedback activation of ERK and mTOR, while AXL inhibition suppressed PI3K pathway. In both cell lines and human lung cancer tissues with KRASG12C, we observed high basal ERBB2/3 associated with epithelial gene signatures, while higher basal FGFR1 and AXL were observed in cells/tumors with mesenchymal gene signatures. Our phosphoproteomic study identified cell type-adaptive responses to KRASi. Markers and targets associated with ERBB2/3 signaling in epithelial subtype and with FGFR1/AXL signaling in mesenchymal subtype should be considered in patient enrichment schemes with KRASi.

Phosphoproteomics Reveals the AMPK Substrate Network in Response to DNA Damage and Histone Acetylation

AMP-activated protein kinase (AMPK) is a conserved energy sensor that plays roles in diverse biological processes via phosphorylating various substrates. Emerging studies have demonstrated the regulatory roles of AMPK in DNA repair, but the underlying mechanisms remain to be fully understood. Herein, using mass spectrometry-based proteomic technologies, we systematically investigate the regulatory network of AMPK in DNA damage response (DDR). Our system-wide phosphoproteome study uncovers a variety of newly-identified potential substrates involved in diverse biological processes, whereas our system-wide histone modification analysis reveals a link between AMPK and histone acetylation. Together with these findings, we discover that AMPK promotes apoptosis by phosphorylating apoptosis-stimulating of p53 protein 2 (ASPP2) in an irradiation (IR)-dependent manner and regulates histone acetylation by phosphorylating histone deacetylase 9 (HDAC9) in an IR-independent manner. Besides, we reveal that disrupting the histone acetylation by the bromodomain BRD4 inhibitor JQ-1 enhances the sensitivity of AMPK-deficient cells to IR. Therefore, our study has provided a resource to investigate the interplay between phosphorylation and histone acetylation underlying the regulatory network of AMPK, which could be beneficial to understand the exact role of AMPK in DDR.

Abstract PR009: The functional genomic landscape of recurrent glioblastoma

Abstract Glioblastoma (GBM) is a highly fatal brain cancer where mortality is predominantly caused by disease recurrence and lack of second-line therapies. Here, utilizing genome-wide CRISPR-Cas9 screening, we uncover treatment-based conditional genetic interactions modulating sensitivity to combined radiation therapy (RT) and temozolomide (TMZ), the standard of care treatment administered to over 70% of glioblastoma patients. To investigate therapy-induced changes in the functional landscape of glioblastoma stem cells (GSCs) without confounding patient-to-patient differences, we systematically mapped genetic dependencies in patient-matched pre-treatment primary and post-treatment recurrent GSCs. This comparison highlights a remodelling of genes and pathways regulating tumor cell survival at disease relapse, arming recurrent GSCs with newly acquired genetic drivers and further loss of tumor suppressors. Among these genes, we identify PTP4A2, encoding protein tyrosine phosphatase 4A2, as a major regulator of stemness and tumorigenicity in recurrent GSCs. Genetic perturbation or pharmacological inhibition of PTP4A2 influences the phosphorylation status and expression of interacting proteins belonging to axonal guidance pathways, as phosphoproteomic studies showed specific enrichment in axon guidance regulator Roundabout Guidance Receptor 1 (ROBO1) in recurrent GSCs. We therefore designed and developed efficacious Camelid biotinylated single-domain antibody (bio-sdAb) and tetrameric complexes targeting human ROBO1 (MKRo-20). Subsequently, we orthotopically engrafted recurrent GSCs into immunocompromised mice and administered local treatments of tetrameric MKRo-20. All mice treated with tetrameric MKRo-20 were rendered tumor free, suggesting that modulation of ROBO1 with bio-sdAbs presents a new immunotherapeutic strategy to target recurrent glioblastoma. Our work reveals a context-specific dependence on axonal guidance through a PTP4A2-ROBO axis in recurrent glioblastoma, highlighting new avenues of therapeutic intervention for second-line therapy in GBM. This abstract is also being presented as PO025. Citation Format: Chirayu Chokshi, David Tieu, Kevin Brown, Chitra Venugopal, Lina Liu, Laura Kuhlman, Katherine Chan, Amy Tong, Neil Savage, Dillon McKenna, Nikoo Aghaei, Minomi Subapanditha, Joseph M Salamoun, Martin Rossotti, Peter Wipf, Elizabeth Sharlow, John S. Lazo, Thomas Kislinger, Kevin Henry, Yu Lu, Jason Moffat, Sheila K. Singh. The functional genomic landscape of recurrent glioblastoma [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PR009.

Impact of three-month morphine withdrawal on rat brain cortex, hippocampus, striatum and cerebellum: proteomic and phosphoproteomic studies

Opioid addiction is characterized by compulsive drug seeking and taking behavior, which is thought to result from persistent neuroadaptations. However, there is a lack of information about the changes at both the cellular and molecular levels occurring after cessation of drug administration. The aim of our study was to determine alterations of both phosphoproteome and proteome in selected brain regions of the rats (brain cortex, hippocampus, striatum, and cerebellum) 3 months after cessation of 10-day morphine treatment. Phosphoproteome profiling was performed by Pro-Q® Diamond staining. The gel-based proteomic approach accompanied by label-free quantification (MaxLFQ) was used for characterization of proteome changes.The phosphoproteomic analysis revealed the largest change in the hippocampus (14); only few altered proteins were detected in the forebrain cortex (5), striatum (4), and cerebellum (3). The change of total protein composition, determined by 2D electrophoresis followed by LFQ analysis, identified 22 proteins with significantly altered expression levels in the forebrain cortex, 19 proteins in the hippocampus, 12 in the striatum and 10 in the cerebellum. The majority of altered proteins were functionally related to energy metabolism and cytoskeleton reorganization. As the most important change we regard down-regulation of 14-3-3 proteins in rat cortex and hippocampus.Our findings indicate that i) different parts of the brain respond in a distinct manner to the protracted morphine withdrawal, ii) characterize changes of protein composition in these brain parts, and iii) enlarge the scope of evidence for adaptability and distinct neuroplasticity proceeding in the brain of drug-addicted organism.

Charting protein dephosphorylation triggered by Toll-like receptor (TLR) signaling in macrophages and its role in health and disease.

Toll-like receptor (TLR) signaling induces substantial changes in the phosphoproteome of innate immune cells, mainly in the form of increased phosphorylation of signaling intermediaries. Loss of constitutive phosphorylation occurs simultaneously, but these transitions from a stable, phosphorylated state in resting cells to a sustained underphosphorylated state in activated cells have received far less attention. This review provides an overview of phosphorylation sites downregulated during TLR-mediated signaling, with a particular focus on TLR4 activation by lipopolysaccharide (LPS). Energy homeostasis, the cell cycle, mitochondrial fission, and gene regulation are among the biological events in macrophages that are regulated through the downregulation of phosphoproteins as part of intracellular signaling events. Phosphoproteomics studies on innate immune cells have identified hundreds of hitherto uncharacterized phosphorylation sites that are lost upon stimulation, indicating that protein hypophosphorylation is a significant, largely unexplored layer of complexity in the TLR4 pathway.

Global phosphoproteomic study of high/low specific productivity industrially relevant mAb producing recombinant CHO cell lines

Abstract Understanding the biology of CHO cells at a cellular level is extremely valuable to the pharmaceutical industry, as it presents the opportunity to rationally engineer clonally derived cell lines (CDCLs) with elevated recombinant specific productivity (Qp). To date, relatively little is understood about the intracellular pathways which influence desirable phenotypes in CHO cell lines. In this study we identified phosphoproteins and total proteins which are differentially expressed between high and low Qp IgG4 producing industry CHO cell lines, some of which upon further investigation, could potentially be used as targets to engineer high Qp CDCLs. We highlighted the importance of phosphoproteins and total proteins associated with amino acid sensing in high Qp cell lines. We also identified increased expression of proteins which promote progression through the cell cycle in low Qp cell lines, suggesting that lower Qp CDCLs are focusing their translational machinery on translating cell cycle associated mRNAs.

Plant Phosphopeptides Enrichment by Immobilized Metal Ion Affinity Chromatography.

Protein phosphorylation plays important roles in the regulation of plant growth and development as well as adaption to changing environments. Large-scale identification of the phosphorylated proteins could provide both a global view of and specific targets involved in the mechanism underlying these processes. The progress of phosphoproteomic study for higher plants has lagged behind that of animals due to technical challenges, particularly the difficulty in solubilizing proteins from plant tissues with a rigid cell wall and the interference of the secondary metabolites, polysaccharides, and pigments throughout the whole processes of sample preparation and LC-MS analysis. Thus, it is critical to improve the efficiency of protein extraction and to remove the interfering metabolites before phosphopeptides enrichment. Here we describe a protocol for plant protein extraction and phosphopeptides enrichment by Fe3+-immobilized metal ion affinity chromatography (Fe3+-IMAC). Strong detergents such as SDS were used to extract proteins from plant tissues, and the secondary metabolites were removed by protein precipitation and washing of the pellets. The protein samples were digested and the resulting peptides were prefractionated. Phosphopeptides enriched from each fraction were combined before analysis with LC-MS.

Biological characteristics of aging in human acute myeloid leukemia cells: the possible importance of aldehyde dehydrogenase, the cytoskeleton and altered transcriptional regulation.

Patients with acute myeloid leukemia (AML) have a median age of 65-70 years at diagnosis. Elderly patients have more chemoresistant disease, and this is partly due to decreased frequencies of favorable and increased frequencies of adverse genetic abnormalities. However, aging-dependent differences may also contribute. We therefore compared AML cell proteomic and phosphoproteomic profiles for (i) elderly low-risk and younger low-risk patients with favorable genetic abnormalities; and (ii) high-risk patients with adverse genetic abnormalities and a higher median age against all low-risk patients with lower median age. Elderly low-risk and younger low-risk patients showed mainly phosphoproteomic differences especially involving transcriptional regulators and cytoskeleton. When comparing high-risk and low-risk patients both proteomic and phosphoproteomic studies showed differences involving cytoskeleton and immunoregulation but also transcriptional regulation and cell division. The age-associated prognostic impact of cyclin-dependent kinases was dependent on the cellular context. The protein level of the adverse prognostic biomarker mitochondrial aldehyde dehydrogenase (ALDH2) showed a similar significant upregulation both in elderly low-risk and elderly high-risk patients. Our results suggest that molecular mechanisms associated with cellular aging influence chemoresistance of AML cells, and especially the cytoskeleton function may then influence cellular hallmarks of aging, e.g. mitosis, polarity, intracellular transport and adhesion.

Proteome and phosphoproteome characterization of liver in the postprandial state from diet-induced obese and lean mice

A metabolic consequence of obesity is hepatosteatosis, which can develop into more serious diseases in the non-alcoholic fatty liver disease (NAFLD) spectrum. The goal of this study was to identify the protein signature of liver in the postprandial state in obesity compared to leanness. The postprandial state is of interest due to the central role of the liver in regulating macronutrient and energy homeostasis during the fed-fast cycle and lack of previously reported controlled studies in the postprandial state. Therefore, we assessed the proteome and phosphoproteome of liver in the postprandial state from diet-induced obese (DIO) and lean mice using untargeted LC-MS/MS analysis. We identified significant alterations in the levels of proteins involved in fatty acid oxidation, activation, and transport, as well as proteins involved in energy metabolism including ketogenesis, tricarboxylic acid cycle, and electron transport chain in liver of DIO compared to lean mice. Additionally, phosphorylated proteins in liver of DIO and lean mice reflect possible regulatory mechanisms controlling fatty acid metabolism and gene expression that may contribute to hepatic metabolic alterations in obesity. Our data indicates PPARα-mediated transcriptional regulation of lipid metabolism and adaptation to hepatic lipid overload. The results of this study expand our knowledge of the molecular changes that occur in liver in the postprandial state in obesity compared to leanness. SignificanceProteome and phosphoproteome studies of liver in a controlled postprandial state in obesity and leanness are lacking; however, this information is crucial to understanding how obesity-associated hepatosteatosis influences postprandial nutrient and energy metabolism. In this global shotgun proteome and phosphoproteome analysis, we identified unique protein signatures defining obesity and leanness in liver in the postprandial state and identified potential mechanisms contributing to hepatic metabolic alterations in obesity. The results of this study provide a foundation to focus future experiments on the contribution of altered protein and phosphorylation patterns to postprandial metabolism in obesity-associated hepatosteatosis.

CDK8 Fine-Tunes IL-6 Transcriptional Activities by Limiting STAT3 Resident Time at the Gene Loci

SummaryCytokines are highly pleiotropic ligands that regulate the immune response. Here, using interleukin-6 (IL-6) as a model system, we perform detailed phosphoproteomic and transcriptomic studies in human CD4+ T helper 1 (Th-1) cells to address the molecular bases defining cytokine functional pleiotropy. We identify CDK8 as a negative regulator of STAT3 transcriptional activities, which interacts with STAT3 upon IL-6 stimulation. Inhibition of CDK8 activity, using specific small molecule inhibitors, reduces the IL-6-induced phosphoproteome by 23% in Th-1 cells, including STAT3 S727 phosphorylation. STAT3 binding to target DNA sites in the genome is increased upon CDK8 inhibition, which results in a concomitant increase in STAT3-mediated transcriptional activity. Importantly, inhibition of CDK8 activity under Th-17 polarizing conditions results in an enhancement of Th-17 differentiation. Our results support a model where CDK8 regulates STAT3 transcriptional processivity by modulation of its gene loci resident time, critically contributing to diversification of IL-6 responses.

Large-Scale Phosphoproteomic Study of Arabidopsis Membrane Proteins Reveals Early Signaling Events in Response to Cold.

Cold stress is one of the major factors limiting global crop production. For survival at low temperatures, plants need to sense temperature changes in the surrounding environment. How plants sense and respond to the earliest drop in temperature is still not clearly understood. The plasma membrane and its adjacent extracellular and cytoplasmic sites are the first checkpoints for sensing temperature changes and the subsequent events, such as signal generation and solute transport. To understand how plants respond to early cold exposure, we used a mass spectrometry-based phosphoproteomic method to study the temporal changes in protein phosphorylation events in Arabidopsis membranes during 5 to 60 min of cold exposure. The results revealed that brief cold exposures led to rapid phosphorylation changes in the proteins involved in cellular ion homeostasis, solute and protein transport, cytoskeleton organization, vesical trafficking, protein modification, and signal transduction processes. The phosphorylation motif and kinase–substrate network analysis also revealed that multiple protein kinases, including RLKs, MAPKs, CDPKs, and their substrates, could be involved in early cold signaling. Taken together, our results provide a first look at the cold-responsive phosphoproteome changes of Arabidopsis membrane proteins that can be a significant resource to understand how plants respond to an early temperature drop.

Recent Advances of Functional Proteomics in Gastrointestinal Cancers- a Path towards the Identification of Candidate Diagnostic, Prognostic, and Therapeutic Molecular Biomarkers.

Gastrointestinal (GI) cancer remains one of the common causes of morbidity and mortality. A high number of cases are diagnosed at an advanced stage, leading to a poor survival rate. This is primarily attributed to the lack of reliable diagnostic biomarkers and limited treatment options. Therefore, more sensitive, specific biomarkers and curative treatments are desirable. Functional proteomics as a research area in the proteomic field aims to elucidate the biological function of unknown proteins and unravel the cellular mechanisms at the molecular level. Phosphoproteomic and glycoproteomic studies have emerged as two efficient functional proteomics approaches used to identify diagnostic biomarkers, therapeutic targets, the molecular basis of disease and mechanisms underlying drug resistance in GI cancers. In this review, we present an overview on how functional proteomics may contribute to the understanding of GI cancers, namely colorectal, gastric, hepatocellular carcinoma and pancreatic cancers. Moreover, we have summarized recent methodological developments in phosphoproteomics and glycoproteomics for GI cancer studies.

The STRIPAK signaling complex regulates dephosphorylation of GUL1, an RNA-binding protein that shuttles on endosomes.

The striatin-interacting phosphatase and kinase (STRIPAK) multi-subunit signaling complex is highly conserved within eukaryotes. In fungi, STRIPAK controls multicellular development, morphogenesis, pathogenicity, and cell-cell recognition, while in humans, certain diseases are related to this signaling complex. To date, phosphorylation and dephosphorylation targets of STRIPAK are still widely unknown in microbial as well as animal systems. Here, we provide an extended global proteome and phosphoproteome study using the wild type as well as STRIPAK single and double deletion mutants (Δpro11, Δpro11Δpro22, Δpp2Ac1Δpro22) from the filamentous fungus Sordaria macrospora. Notably, in the deletion mutants, we identified the differential phosphorylation of 129 proteins, of which 70 phosphorylation sites were previously unknown. Included in the list of STRIPAK targets are eight proteins with RNA recognition motifs (RRMs) including GUL1. Knockout mutants and complemented transformants clearly show that GUL1 affects hyphal growth and sexual development. To assess the role of GUL1 phosphorylation on fungal development, we constructed phospho-mimetic and -deficient mutants of GUL1 residues. While S180 was dephosphorylated in a STRIPAK-dependent manner, S216, and S1343 served as non-regulated phosphorylation sites. While the S1343 mutants were indistinguishable from wild type, phospho-deficiency of S180 and S216 resulted in a drastic reduction in hyphal growth, and phospho-deficiency of S216 also affects sexual fertility. These results thus suggest that differential phosphorylation of GUL1 regulates developmental processes such as fruiting body maturation and hyphal morphogenesis. Moreover, genetic interaction studies provide strong evidence that GUL1 is not an integral subunit of STRIPAK. Finally, fluorescence microscopy revealed that GUL1 co-localizes with endosomal marker proteins and shuttles on endosomes. Here, we provide a new mechanistic model that explains how STRIPAK-dependent and -independent phosphorylation of GUL1 regulates sexual development and asexual growth.

Fragment Mass Spectrum Prediction Facilitates Site Localization of Phosphorylation.

Liquid chromatography tandem mass spectrometry (LC-MS/MS) has been the most widely used technology for phosphoproteomics studies. As an alternative to database searching and probability-based phosphorylation site localization approaches, spectral library searching has been proved to be effective in the identification of phosphopeptides. However, incompletion of experimental spectral libraries limits the identification capability. Herein, we utilize MS/MS spectrum prediction coupled with spectral matching for site localization of phosphopeptides. In silico MS/MS spectra are generated from peptide sequences by deep learning/machine learning models trained with nonphosphopeptides. Then, mass shift according to phosphorylation sites, phosphoric acid neutral loss, and a "budding" strategy are adopted to adjust the in silico mass spectra. In silico MS/MS spectra can also be generated in one step for phosphopeptides using models trained with phosphopeptides. The method is benchmarked on data sets of synthetic phosphopeptides and is used to process real biological samples. It is demonstrated to be a method requiring only computational resources that supplements the probability-based approaches for phosphorylation site localization of singly and multiply phosphorylated peptides.

Abstract 1915: Intrinsic and acquired trastuzumab resistance pathways in breast cancer cell lines using DigiWest® phospho-protein profiling

Abstract Introduction The widespread clinical use of monoclonal antibodies such as trastuzumab in personalized medicine, targeting upstream proteins such as receptors with cascading effects on downstream signaling, elicits the importance of studying the phosphoproteome in the investigation of resistance to targeted therapy. This study sought to differentiate between the complex mechanisms of intrinsic and acquired trastuzumab resistance, by quantifying changes in expression and activity of proteins in key signal transduction pathways, in cellular models of resistance. Method DigiWest®, a Luminex® xMAP® bead-based high-throughput protein profiling method, was utilized to measure 194 proteins and phospho-proteins from 12µg of total protein, obtained from HER2-positive cell lines which were trastuzumab-sensitive (SKBR3), intrinsically resistant (JIMT-1, UACC-893) and with acquired resistance (T15, obtained by long-term culture of SKBR3 in 15µg/mL trastuzumab). The resulting median fluorescence intensities (MFI) were normalized to total protein content. Relative quantification was done against SKBR3 and a log2 fold-change of ±1 was considered to be significant. For each of the three trastuzumab-resistant cell lines, targets which were differentially expressed were organized into established signal transduction pathways, and specific phosphosite expression values were used to determine whether each protein was under- or over-activated. Results A total of 115 proteins were expressed in at least one of the HER2-positive cell lines at a normalized MFI above the set threshold. JIMT-1 showed the most concurrent resistance mechanisms, including PI3K/Akt and RAS/RAF/MEK/ERK activation, β-catenin stabilization, cell cycle progression by Rb suppression, and CREB-mediated cell survival. MAPK (ERK) pathway activation was common to both intrinsic and acquired resistance – it was over-activated in all the three trastuzumab-resistant cell lines. The overexpression of upstream RAS, however, was confined to the intrinsically resistant cell lines JIMT-1 and UACC-893; meanwhile, in T15, entry into the ERK pathway was mostly mediated by pan-PKC overexpression. PKCs, especially PKCα, were also highly activated in UACC-893. β-catenin stabilization was another mechanism observed only in the intrinsically trastuzumab-resistant cell lines. Conclusion Both distinct and overlapping mechanisms are involved in intrinsic and acquired trastuzumab resistance. Multiple pathways can also co-exist to promote cell survival, as was mostly exemplified by JIMT-1, derived from an aggressive, basal-like tumor. Furthermore, different models of intrinsic resistance (JIMT-1 vs UACC-893) showed significant differences in differentially activated signal transduction pathways. DigiWest® provided a simple and robust multiplexing solution for this phospho-proteomic study. Citation Format: Jeanesse Scerri, Felix Ruoff, Markus Templin, Godfrey Grech, Christian Scerri. Intrinsic and acquired trastuzumab resistance pathways in breast cancer cell lines using DigiWest® phospho-protein profiling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1915.

Abstract 3649: Alternative RNA splicing of U2AF2, induced by AKT3-phosphorylated IWS1, promotes tumor growth, by activating a CDCA5-pERK positive feedback loop

Abstract A phosphoproteomics study of isogenic cell lines expressing the 3 different Akt isoforms identified 606 proteins that are phosphorylated by at least one isoform. About 30 of these proteins were involved in various steps of RNA processing. One of them, IWS1, is a transcription elongation factor, which was originally identified in the yeast Saccharomyces cerevisiae, as a protein that interacts with the histone H3/H4 chaperone Spt6 or as a suppressor of TATA binding protein (TBP) mutations that impair post-recruitment transcriptional activation. The human IWS1 is an 819aa protein, which contains a C-terminal domain that is similar to domain I of the transcription elongation factor TFIIS, and to related domains in Elongin A and the Mediator Complex subunit 26 (Med26). IWS1 was shown to be phosphorylated, primarily by Akt3, at two neighboring sites (Ser720/Thr721). To address the role of phosphorylated IWS1 in RNA processing, we performed an RNA-seq study, using human lung adenocarcinoma cell lines in which IWS1 was knocked down or replaced by its phosphorylation site mutant. This identified the splicing factor U2AF2 as a target of IWS1 phosphorylation. Specifically, phosphorylated IWS1 regulated the alternative splicing of U2AF2 and its loss resulted in U2AF2 transcripts lacking exon 2. This exon encodes part of the U2AF2 Serine-Rich (SR) Domain, required for the binding of U2AF2 with Prp19. Exploring the mechanism of this alternative splicing event revealed that the loss of phosphorylated IWS1 interferes with the recruitment of the histone H3K36 trimethyltransferase SETD2 to an Spt6/IWS1/Aly complex, which assembles on the Ser-2-phosphorylated CTD of RNA-Pol II. The absence of SETD2 recruitment to this complex impairs histone H3K36 trimethylation and the assembly of LEDGF/SRSF1 splicing complexes inthe U2AF2 gene, resulting in the exclusion of exon 2 from the mature U2AF2 mRNA transcript. The loss of the U2AF2/Prp19 interaction results in the downregulation of CDCA5, a component of the cohesin complex, giving rise to genomic instability. Phosphorylation of CDCA5 by p-ERK at Ser79 and Ser209 has a major impact in the regulation of cell proliferation and cancer stem cell renewal, although does not affect its role in the cohesin complex. The effect of phosphorylated CDCA5 on cell proliferation appears to depend on the transcriptional regulation of a set of genes involved in the control of the G2/M phase of the cell cycle, including Cyclin B1 and CDK1. Overall, these data describe a novel pathway, which starts with the phosphorylation of IWS1 by AKT3 and results in the epigenetic modulation of RNA splicing and cell cycle regulation. The importance of this pathway to human cancer was confirmed by meta-analysis of pre-existing patient data, tumor xenograft models and prospective studies on human lung adenocarcinomas. Citation Format: Georgios I. Laliotis, Evangelia Chavdoula, Maria D. Paraskevopoulou, Vollter Anastas, Ioannis Vlachos, Vasiliki Tarasslia, Artemis Hatzigeorgiou, Dario Palmieri, Abdul Kaba, Marina Capece, Arturo Orlacchio, Lalit Sehgal, Vincenzo Coppola, Philip N. Tsichlis. Alternative RNA splicing of U2AF2, induced by AKT3-phosphorylated IWS1, promotes tumor growth, by activating a CDCA5-pERK positive feedback loop [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3649.