Pseudopodium-enriched atypical kinase 1 mediates angiogenesis by modulating GATA2-dependent VEGFR2 transcription

PEAK1 is a newly described tyrosine kinase and scaffold protein that transmits integrin-mediated extracellular matrix (ECM) signals to facilitate cell movement and growth. While aberrant expression of PEAK1 has been linked to cancer progression, its normal physiological role in vertebrate biology is not known. Here we provide evidence that PEAK1 plays a central role in orchestrating new vessel formation in vertebrates. Deletion of the PEAK1 gene in zebrafish, mice, and human endothelial cells (ECs) induced severe defects in new blood vessel formation due to deficiencies in EC proliferation, survival, and migration. Gene transcriptional and proteomic analyses of PEAK1-deficient ECs revealed a significant loss of vascular endothelial growth factor receptor 2 (VEGFR2) mRNA and protein expression, as well as downstream signaling to its effectors, ERK, Akt, and Src kinase. PEAK1 regulates VEGFR2 expression by binding to and increasing the protein stability of the transcription factor GATA-binding protein 2 (GATA2), which controls VEGFR2 transcription. Importantly, PEAK1-GATA2-dependent VEGFR2 expression is mediated by EC adhesion to the ECM and is required for breast cancer-induced new vessel formation in mice. Also, elevated expression of PEAK1 and VEGFR2 mRNA are highly correlated in many human cancers including breast cancer. Together, our findings reveal a novel PEAK1-GATA2-VEGFR2 signaling axis that integrates cell adhesion and growth factor cues from the extracellular environment necessary for new vessel formation during vertebrate development and cancer.NIHNCIAHANIGMS/NIHRay Thomas Edwards FoundationUniv Calif San Diego, Dept Pathol, La Jolla, CA 92093 USAUniv Calif San Diego, Moores Canc Ctr, La Jolla, CA 92093 USAUniv Calif San Diego, Dept Pharmacol, La Jolla, CA 92093 USAUniv Calif San Diego, Skaggs Sch Pharm & Pharmaceut Sci, La Jolla, CA 92093 USAUniv Calif San Diego, Dept Med, La Jolla, CA 92093 USAUniv Fed Sao Paulo, Dept Biochem, Sao Paulo, SP, BrazilUniv Calif San Diego, Sanford Consortium Regenerat Med, La Jolla, CA 92093 USAComenius Univ, Jessenius Fac Med Martin, Dept Mol Med, Biomed Ctr Martin, Martin 03601, SlovakiaUniv Fed Sao Paulo, Dept Biochem, Sao Paulo, SP, BrazilNIH: CA182495NIH: CA184594NIH: CA097022NIH: HL135737NIH: CA050286NCI: CA180374AHA: 16POST27250126NIGMS/NIH: K12GM068524Web of Scienc

Mitogen- and Stress-Activated Kinase 1 (MSK1) Regulates Cigarette Smoke-Induced Histone Modifications on NF-κB-dependent Genes

Cigarette smoke (CS) causes sustained lung inflammation, which is an important event in the pathogenesis of chronic obstructive pulmonary disease (COPD). We have previously reported that IKKα (I kappaB kinase alpha) plays a key role in CS-induced pro-inflammatory gene transcription by chromatin modifications; however, the underlying role of downstream signaling kinase is not known. Mitogen- and stress-activated kinase 1 (MSK1) serves as a specific downstream NF-κB RelA/p65 kinase, mediating transcriptional activation of NF-κB-dependent pro-inflammatory genes. The role of MSK1 in nuclear signaling and chromatin modifications is not known, particularly in response to environmental stimuli. We hypothesized that MSK1 regulates chromatin modifications of pro-inflammatory gene promoters in response to CS. Here, we report that CS extract activates MSK1 in human lung epithelial (H292 and BEAS-2B) cell lines, human primary small airway epithelial cells (SAEC), and in mouse lung, resulting in phosphorylation of nuclear MSK1 (Thr581), phospho-acetylation of RelA/p65 at Ser276 and Lys310 respectively. This event was associated with phospho-acetylation of histone H3 (Ser10/Lys9) and acetylation of histone H4 (Lys12). MSK1 N- and C-terminal kinase-dead mutants, MSK1 siRNA-mediated knock-down in transiently transfected H292 cells, and MSK1 stable knock-down mouse embryonic fibroblasts significantly reduced CS extract-induced MSK1, NF-κB RelA/p65 activation, and posttranslational modifications of histones. CS extract/CS promotes the direct interaction of MSK1 with RelA/p65 and p300 in epithelial cells and in mouse lung. Furthermore, CS-mediated recruitment of MSK1 and its substrates to the promoters of NF-κB-dependent pro-inflammatory genes leads to transcriptional activation, as determined by chromatin immunoprecipitation. Thus, MSK1 is an important downstream kinase involved in CS-induced NF-κB activation and chromatin modifications, which have implications in pathogenesis of COPD

Epigenetic Variability Confounds Transcriptome but not Proteome Profiling for Coexpression-based Gene Function Prediction

Genes are often coexpressed with their genomic neighbors, even if these are functionally unrelated. For small expression changes driven by genetic variation within the same cell type, non-functional mRNA coexpression is not propagated to the protein level. However, it is unclear if protein levels are also buffered against any non-functional mRNA coexpression accompanying large, regulated changes in the gene expression program, such as those occurring during cell differentiation. Here, we address this question by analyzing mRNA and protein expression changes for housekeeping genes across 20 mouse tissues. We find that a large proportion of mRNA coexpression is indeed non-functional and does not lead to coexpressed proteins. Chromosomal proximity of genes explains a proportion of this nonfunctional mRNA coexpression. However, the main driver of non-functional mRNA coexpression across mouse tissues is epigenetic similarity. Both factors together provide an explanation for why monitoring protein coexpression outperforms mRNA coexpression data in gene function prediction. Furthermore, this suggests that housekeeping genes translocating during evolution within genomic subcompartments might maintain their broad expression pattern

Co-regulation map of the human proteome enables identification of protein functions

This is the author accepted manuscript. The final version is available from Nature Research via the DOI in this recordData availability: All mass spectrometry raw files generated in-house have been deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository36 with the dataset identifier PXD008888. The co-regulation map is hosted on our website at www.proteomeHD.net, and pair-wise co-regulation scores are available through STRING (https://string-db.org). A network of the top 0.5% co-regulated protein pairs can be explored interactively on NDEx (https://doi.org/10.18119/N9N30Q).Code availability: Data analysis was performed in R 3.5.1. R scripts and input files required to reproduce the results of this manuscript are available in the following GitHub repository: https://github.com/Rappsilber-Laboratory/ProteomeHD. R scripts related specifically to the benchmarking of the treeClust algorithm using synthetic data are available in the following GitHub repository: https://github.com/Rappsilber-Laboratory/treeClust-benchmarking. The R package data.table was used for fast data processing. Figures were prepared using ggplot2, gridExtra, cowplot and viridis.Note that the title of the AAM is different from the published versionThe annotation of protein function is a longstanding challenge of cell biology that suffers from the sheer magnitude of the task. Here we present ProteomeHD, which documents the response of 10,323 human proteins to 294 biological perturbations, measured by isotope-labelling mass spectrometry. We reveal functional associations between human proteins using the treeClust machine learning algorithm, which we show to improve protein co-regulation analysis due to robust selectivity for close linear relationships. Our co-regulation map identifies a functional context for many uncharacterized proteins, including microproteins that are difficult to study with traditional methods. Co-regulation also captures relationships between proteins which do not physically interact or co-localize. For example, co-regulation of the peroxisomal membrane protein PEX11β with mitochondrial respiration factors led us to discover a novel organelle interface between peroxisomes and mitochondria in mammalian cells. The co-regulation map can be explored at www.proteomeHD.net .Biotechnology & Biological Sciences Research Council (BBSRC)European Commissio

Histidine and Aspartic Acid Phosphorylation in a Human Prostate Cancer Progression Model

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Environmental Medicine, Toxicology Training Program, 2013.Prostate cancer is the most common non-skin cancer in men, affecting 1 in 6 individuals at some point in their lifetime. Mortality rates are decreasing due to earlier detection through screening with the PSA test, yet PSA is not a specific marker for early detection. To facilitate the understanding of prostate cancer etiology and to provide an earlier and more specific diagnostic tool for prostate cancer detection, improved model systems for prostate cancer biomarker research are required. The PHEC model, currently the only human prostate cancer progression model, displays non-tumorigenic, tumorigenic and metastatic states in vivo, with the added advantage of being able to be maintained in vitro as independent cell lines. Our lab utilizes the PHEC model, combined with a differential proteomics approach, to better understand prostate cancer etiology and progression. Through this type of approach, we have identified NM23-H1 as a protein that is differentially expressed through prostate cancer progression, as well as a protein that is affected by drug treatment. NM23-H1 is currently the only well-characterized histidine kinase in eukaryotes. The histidine kinase activity of NM23-H1 association with metastasis has not been assessed, largely due to the acid labile nature of this protein modification. Our lab has developed a mass spectrometry approach to detect histidine and aspartic acid post-translational modifications (PTMs). By applying this technique to global proteome analysis, we have discovered that this modification is not uncommon in our PHEC model. Pathway analysis has been utilized to establish a signaling network involving proteins that display histidine phosphorylations and their relationship with NM23-H1. To characterize the role of the NM23-H1 histidine kinase activity and its effect on cellular mobility, point mutations to the catalytic domain of the enzyme have been generated and mobility assays used to assess their phenotypic changes in PHEC cells. This work has uncovered an extensive, previously unrealized phosphorylation network present in human cells, offering new targets and mechanisms of action for current and future prostate cancer treatment options

Mass Spectrometry Detection of Histidine Phosphorylation on NM23-H1

Phosphorylation is a ubiquitous protein post-translational modification that is intimately involved in most aspects of cellular regulation. Currently, most proteomic analyses are performed with phosphorylation searches for serine, threonine, and tyrosine modifications, as the phosphorylated residues of histidine and aspartic acid are acid labile and thus undetectable with most proteomic methodologies. Here, we present a novel buffer system to show histidine phosphorylation of NM23-H1, the product of the first identified putative human metastasis suppressor gene (NME1), which catalyzes the transfer of the γ-phosphate from nucleoside triphosphates to nucleoside diphosphates. On the basis of a pH titration of LC elution buffers and MS/MS identification, recombinant NM23-H1 subjected to autophosphorylation was shown to contain phosphorylated histidine at residue 118 at pH 5 and 6, with each level giving over 75% peptide coverage for identification. The solvent system presented permits the detection of all five possible phosphorylation moieties. Application of histidine and aspartic acid phosphorylation modifications to proteomic analyses will significantly advance the understanding of phosphorylation relay signaling in cellular regulation, including elucidation of the role of NM23-H1 in metastasis