Nuclear phosphoproteome analysis of 3T3‐L1 preadipocyte differentiation reveals system‐wide phosphorylation of transcriptional regulators

摘要

Adipocytes (fat cells) are important endocrine and metabolic cells critical for systemic insulin sensitivity. Both adipose excess and insufficiency are associated with adverse metabolic function. Adipogenesis is the process whereby preadipocyte precursor cells differentiate into lipidladen mature adipocytes. This process is driven by a network of transcriptional regulators (TRs). We hypothesized that protein PTMs, in particular phosphorylation, play a major role in activating and propagating signals within TR networks upon induction of adipogenesis by extracellular stimulus. We applied MSbased quantitative proteomics and phosphoproteomics to monitor the alteration of nuclear proteins during the early stages (4 h) of preadipocyte differentiation. We identified a total of 4072 proteins including 2434 phosphorylated proteins, a majority of which were assigned as regulators of gene expression. Our results demonstrate that adipogenic stimuli increase the nuclear abundance and/or the phosphorylation levels of proteins involved in gene expression, cell organization, and oxidationreduction pathways. Furthermore, proteins acting as negative modulators involved in negative regulation of gene expression, insulin stimulated glucose uptake, and cytoskeletal organization showed a decrease in their nuclear abundance and/or phosphorylation levels during the first 4 h of adipogenesis. Among 288 identified TRs, 49 were regulated within 4 h of adipogenic stimulation including several known and many novel potential adipogenic regulators. We created a kinasesubstrate database for 3T3L1 preadipocytes by investigating the relationship between protein kinases and protein phosphorylation sites identified in our dataset. A majority of the putative protein kinases belong to the cyclindependent kinase family and the mitogenactivated protein kinase family including P38 and cJun Nterminal kinases, suggesting that these kinases act as orchestrators of early adipogenesis.