THE UNIQUE PHYLOGENETIC DISTRIBUTION OF VAULT PARTICLES REVEALS ITS FUNCTIONAL ROLES

Ph.DDOCTOR OF PHILOSOPH

Phosphoproteomics reveals network rewiring to a pro-adhesion state in annexin-1-deficient mammary epithelial cells

Abstract Background Annexin-1 (ANXA1) plays pivotal roles in regulating various physiological processes including inflammation, proliferation and apoptosis, and deregulation of ANXA1 functions has been associated with tumorigenesis and metastasis events in several types of cancer. Though ANXA1 levels correlate with breast cancer disease status and outcome, its distinct functional involvement in breast cancer initiation and progression remains unclear. We hypothesized that ANXA1-responsive kinase signaling alteration and associated phosphorylation signaling underlie early events in breast cancer initiation events and hence profiled ANXA1-dependent phosphorylation changes in mammary gland epithelial cells. Methods Quantitative phosphoproteomics analysis of mammary gland epithelial cells derived from ANXA1-heterozygous and ANXA1-deficient mice was carried out using stable isotope labeling with amino acids in cell culture (SILAC)-based mass spectrometry. Kinase and signaling changes underlying ANXA1 perturbations were derived by upstream kinase prediction and integrated network analysis of altered proteins and phosphoproteins. Results We identified a total of 8110 unique phosphorylation sites, of which 582 phosphorylation sites on 372 proteins had ANXA1-responsive changes. A majority of these phosphorylation changes occurred on proteins associated with cytoskeletal reorganization spanning the focal adhesion, stress fibers, and also the microtubule network proposing new roles for ANXA1 in regulating microtubule dynamics. Comparative analysis of regulated global proteome and phosphoproteome highlighted key differences in translational and post-translational effects of ANXA1, and suggested closely coordinated rewiring of the cell adhesion network. Kinase prediction analysis suggested activity modulation of calmodulin-dependent protein kinase II (CAMK2), P21-activated kinase (PAK), extracellular signal-regulated kinase (ERK), and IκB kinase (IKK) upon loss of ANXA1. Integrative analysis revealed regulation of the WNT and Hippo signaling pathways in ANXA1-deficient mammary epithelial cells, wherein there is downregulation of transcriptional effects of TEA domain family (TEAD) suggestive of ANXA1-responsive transcriptional rewiring. Conclusions The phosphoproteome landscape uncovered several novel perspectives for ANXA1 in mammary gland biology and highlighted its involvement in key signaling pathways modulating cell adhesion and migration that could contribute to breast cancer initiation

L'Auto-vélo : automobilisme, cyclisme, athlétisme, yachting, aérostation, escrime, hippisme / dir. Henri Desgranges

03 avril 19321932/04/03 (A33,N11432)

Additional file 13: Figure S5. of Phosphoproteomics reveals network rewiring to a pro-adhesion state in annexin-1-deficient mammary epithelial cells

Clusters enriched in ANXA1-regulated protein interaction network. Integrated protein-protein interaction network was constructed using those proteins with ANXA1-responsive phosphorylation changes along with transcription factors predicted from ANXA1-regulated proteome. Clusters were identified using GLay community structure detection and the top clusters identified along with their associated functions are shown. (PDF 5678 kb

Additional file 12: Table S8. of Phosphoproteomics reveals network rewiring to a pro-adhesion state in annexin-1-deficient mammary epithelial cells

Summary of associated pathways and localizations of ANXA1-responsive phosphoproteins. (XLSX 29 kb

Additional file 5: Figure S3. of Phosphoproteomics reveals network rewiring to a pro-adhesion state in annexin-1-deficient mammary epithelial cells

Comparison of quantified proteome and phosphoproteome in ANXA1-deficient mammary epithelial cells. A Number of class I phosphorylation sites with corresponding protein quantification. Except for 1550 sites on 765 proteins that had no corresponding protein measure, the rest of the sites mapped to 1765 proteins with abundance measures. B Intensity-based density plot comparing protein and phosphorylation abundance shows poor correlation. (PDF 1234 kb

Additional file 4: Table S2. of Phosphoproteomics reveals network rewiring to a pro-adhesion state in annexin-1-deficient mammary epithelial cells

List of ANXA1-responsive phosphorylation sites in mammary epithelial cells from ANXA1-heterozygous and ANXA1-deficient mice. (XLSX 1001 kb

Additional file 8: Table S5. of Phosphoproteomics reveals network rewiring to a pro-adhesion state in annexin-1-deficient mammary epithelial cells

Cellular component enrichment of ANXA1-modulated phosphoproteins. (XLSX 10 kb

Mass spectrometry based quantitative proteomics and integrative network analysis accentuates modulating roles of annexin-1 in mammary tumorigenesis

10.1002/pmic.201400175Proteomics1542065408-41

Additional file 2: Figure S1. of Phosphoproteomics reveals network rewiring to a pro-adhesion state in annexin-1-deficient mammary epithelial cells

Phosphoproteomic data quality and reproducibility. A Distribution of Andromeda scores of the identified phosphorylated peptides show that most peptides were identified with high scores with a median score of 130. B Distribution of localization probabilities of identified phosphorylation sites. The median probability was ~ 0.97. C Assessment of reproducibility between the four biological replicates carried out with SILAC label swapping shows optimal reproducibility among the forward and reverse experiments. The correlation scores are indicated in each scatter plot. (PDF 1803 kb