Phosphoproteome Dynamics in Onset and Maintenance of Oncogene-induced Senescence

Abstract

Expression of the BRAFV600E oncoprotein is known to cause benign lesions, such as melanocytic nevi (moles). Despite the oncogenic function of mutant BRAF, these lesions are arrested by a cell-autonomous mechanism called oncogene-induced senescence. Infrequently, nevi can progress to malignant melanoma, through mechanisms that are incompletely understood. To gain more insight into this vital tumor-suppression mechanism, we performed a mass-spectrometry-based screening of the proteome and phosphoproteome in cycling and senescent cells and in cells with abrogated senescence. Proteome analysis of senescent cells revealed the up-regulation of established senescence biomarkers, including specific cytokines, but also several proteins not previously associated with senescence, including extracellular matrix-interacting. Using both general and targeted phosphopeptide enrichment by Ti 4؉ -IMAC and phosphotyrosine antibody enrichment, we identified over 15,000 phosphorylation sites. Among the regulated phosphorylation sites we encountered components of the interleukin, BRAF/MAPK, and CDK-retinoblastoma pathways and several other factors. The extensive proteome and phosphoproteome dataset of BRAF V600E

Phosphoproteomics reveals ALK promote cell progress via RAS/JNK pathway in neuroblastoma

“…To enhance the depth of phosphoproteomics analysis, we employed high pH chromatography and pre-separated the full proteome digestion into 20 fractions followed by phosphopeptide enrichment [ 33 ]. This approach allowed identification of more than 19,500 phosphorylation sites from each cell line—of which approximately 10,000 could be quantitatively analyzed—representing the most in-depth phosphoproteome analysis to date [ 17 , 34 , 35 ]. Bioinformatic analysis of phosphoproteomic data revealed that either the GO categories or the KEGG signaling pathways overrepresented by the significantly regulated proteins were similar amongst all three cell lines examined.…”

Autophagy and senescence in fibrosing cholangiopathies

“…These cells do not respond to various external stimuli, but they remain metabolically active. Senescence can be triggered by multiple mechanisms, including derepression of the INK4a/ARF locus, telomere dysfunction, oxidative stress, and oncogene activation [25][26][27][28]38]. In contrast to apoptosis, senescence is typically a delayed stress response that involves multiple effector mechanisms, and it can serve a program that protects against cellular insults [39,40].…”

“…Lastly, we will discuss the inappropriate Cells respond to various stresses by means of adaptation, autophagy, and recovery, or they either commit to irreversible cell-cycle exit (senescence) or are eliminated through programmed cell death (apoptosis) [24]. While cellular senescence, autophagy and apoptosis are distinct cellular responses to stress, they are correlating with each other, and signaling pathways involved are often overlapping each other [25][26][27][28][29][30][31][32]. For example, autophagy is considered a survival mechanism when faced with cellular insults, however extensive autophagy can also lead to senescence or apoptosis ( Fig.…”

Proteomics Approaches to Define Senescence Heterogeneity and Chemotherapy Response

“…This approach is certainly complementary to transcriptomic analysis since the correlation between mRNA expression and the concentration of the corresponding protein is not always direct. We can expect that some SASP components will also be regulated at the level of protein synthesis and consequently, proteomic analysis will be a method of choice to characterize the different states of senescence …”