Global and Site-Specific Effect of Phosphorylation on Protein Turnover.
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ABSTRACT:
SUBMITTER: Wu C
PROVIDER: S-EPMC7855865 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The effects of specific modification types and sites on protein lifetime have not been illustrated in large scale. We describe a proteomic method, DeltaSILAC, to quantify the impact of site-specific phosphorylation on the turnover of thousands of proteins in live cells. Being configured on the accurate and reproducible mass spectrometry, the pulse labeling approach using stable isotope-labeled amino acids in cells (pSILAC), phosphoproteomics, as well as a novel peptide-level matching strategy, our DeltaSILAC profiling revealed a global, unexpected delaying effect of many phosphosites on protein turnover. We further found that phosphorylated sites accelerating protein turnover are functionally selected for cell fitness, enriched in Cyclin-dependent kinase substrates, and evolutionarily conserved, whereas the Glutamic acids surrounding phosphosites significantly delay protein turnover. Our investigation provides a generalizable approach and a rich resource for prioritizing the effects of phosphorylation sites on protein lifetime in the context of cell signaling and disease biology.
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Project description:Protein S-acylation (palmitoylation) is a reversible lipid modification that is an important regulator of dynamic membrane-protein interactions. Proteomic approaches have uncovered many putative palmitoylated proteins however, methods for comprehensive palmitoylation site characterization are lacking. We demonstrate a quantitative site-specific-Acyl-Biotin-Exchange (ssABE) method that allowed the identification of 906 putative palmitoylation sites on 641 proteins from mouse forebrain. 62% of sites map to known palmitoylated proteins and 102 individual palmitoylation sites are known from the literature. 54% of palmitoylation sites map to synaptic proteins including many GPCRs, receptors/ion channels and peripheral membrane proteins. Phosphorylation sites were also identified on a subset of peptides that were palmitoylated, demonstrating for the first time co-identification of these modifications by mass spectrometry. Palmitoylation sites were identified on over half of the family of palmitoyl-acyltransferases (PATs) that mediate protein palmitoylation, including active site thioester-linked palmitoyl intermediates. Distinct palmitoylation motifs and site topology were identified for integral membrane and soluble proteins, indicating potential differences in associated PAT specificity and palmitoylation function. ssABE allows the global identification of palmitoylation sites as well as measurement of the active site modification state of PATs, enabling palmitoylation to be studied at a systems level.
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Project description:Purpose: CREB (cAMP response element binding protein) is a transcription factor that is critical for learning and memory. The activity of CREB is mediated through its post-translational modifications (PTMs); specifically, phosphorylation at serine 133 and glycosylation at serine 40. In this study, we used RNA-Seq and weighted gene network coexpression analysis (WGCNA) to determine the CREB-mediated transcriptional programmes that are regulated by phosphorylation at serine 133 and glycosylation at serine 40 through the use of various PTM-deficient CREB mutants. Methods: The mRNA profiles of E16.5 Creb1-/- mouse cortical neurons expressing GFP, WT CREB, S40A-CREB, S133A-CREB, and S40A-S133A-CREB were generated by deep sequencing, in triplicate, using Illumina HiSeq 2500. The sequence reads that passed quality filters were analyzed using Bowtie, aligned using TopHat, and quantified using Cufflinks in Galaxy. Results: Through differential expression analysis with glycosylation-deficient (S40A) and phosphorylation-deficient (S133A) CREB mutants, we show that CREB O-GlcNAcylation is important for neuronal activity and excitability, while phosphorylation at serine 133 regulates the expression of genes involved in neuronal differentiation. Furthermore, many of the S40A and S133A differentially-expressed genes were directly bound by (1) CREB and its co-activators, CREB-binding protein and p300, (2) activating histone modifications, (3) OGT and O-GlcNAc, and (4) Tet1, an critical regulator of neuronal activity and differentiation. Finally, we observed a positive correlation between S40A and activity- and excitotoxicity-related gene networks and a negative correlation between S133A and neuronal differentiation and amino and fatty acid metabolism-related gene networks. This study demonstrates that CREB O-GlcNAcylation at serine 40 and phosphorylation mediate mutually exclusive gene networks. Together, O-GlcNAc and phosphorylation impart a TF code, which CREB must integrate and decode to modulate neuronal activity, differentiation, and metabolism.
2020-12-17 | GSE109349 | GEO
Project description:Reactivation of androgen receptor (AR) may drive recurrent prostate cancer in castrate patients. Ack1 tyrosine kinase is overexpressed in prostate cancer and promotes castrate resistant xenograft tumor growth and enhances androgen target gene expression and AR recruitment to enhancers. Ack1 phosphorylates AR at Tyr-267 in the N-terminal transactivation domain. In this study, the role of this phosphorylation site was investigated by characterizing the phosphorylation site mutant in the context of full length and truncated AR lacking the ligand-binding domain. The Y267F mutant showed decreased transactivation of reporters. Expression of wild type full length and truncated AR in LNCaP cells increased cell proliferation in androgen-depleted conditions and increased colony formation. However, the Y267F mutant of full length and truncated AR was defective in stimulating cell proliferation. The full length AR Y267F mutant was defective in nuclear translocation induced by androgen or Ack1 kinase. The truncated AR was constitutively localized to the nucleus. Chromatin immunoprecipitation analysis showed that it was recruited to the target enhancers without androgen. The truncated Y267F AR mutant did not exhibit constitutive nuclear localization and androgen enhancer binding activity. Expression of AR responsive genes in cells expressing truncated AR wt and AR-Y267F mutant under androgen deprived conditions was assessed by microarray gene expression analysis. The AR pathway score was increased in cells expressing truncated AR wt and decreased in cells expressing truncated AR-Y267F. These results support the concept that phosphorylation of Tyr-267 is required for AR nuclear translocation and recruitment and DNA binding and transcription of AR responsive genes. Gene expression profiling was performed using RNA samples from LNCaP cells expressing truncated AR-wt and truncated AR-Y267F growing in androgen deprived conditions. The RNA sample from vector control cells were used as reference sample. Two biological replicates were used per each cell line.
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Project description:In all organisms, chromosome replication is regulated mainly at the initiation step. Most of the knowledge about the mechanisms that regulate replication initiation in bacteria has come from studies on rod-shaped bacteria, such as Escherichia coli and Bacillus subtilis Streptomyces is a bacterial genus that is characterized by distinctive features and a complex life cycle that shares some properties with the developmental cycle of filamentous fungi. The unusual lifestyle of streptomycetes suggests that these bacteria use various mechanisms to control key cellular processes. Here, we provide the first insights into the phosphorylation of the bacterial replication initiator protein, DnaA, from Streptomyces coelicolor We suggest that phosphorylation of DnaA triggers a conformational change that increases its ATPase activity and decreases its affinity for the replication origin, thereby blocking the formation of a functional orisome. We suggest that the phosphorylation of DnaA is catalyzed by Ser/Thr kinase AfsK, which was shown to regulate the polar growth of S. coelicolor Together, our results reveal that phosphorylation of the DnaA initiator protein functions as a negative regulatory mechanism to control the initiation of chromosome replication in a manner that presumably depends on the cellular localization of the protein.IMPORTANCE This work provides insights into the phosphorylation of the DnaA initiator protein in Streptomyces coelicolor and suggests a novel bacterial regulatory mechanism for initiation of chromosome replication. Although phosphorylation of DnaA has been reported earlier, its biological role was unknown. This work shows that upon phosphorylation, the cooperative binding of the replication origin by DnaA may be disturbed. We found that AfsK kinase is responsible for phosphorylation of DnaA. Upon upregulation of AfsK, chromosome replication occurred further from the hyphal tip. Orthologs of AfsK are exclusively found in mycelial actinomycetes that are related to Streptomyces and exhibit a complex life cycle. We propose that the AfsK-mediated regulatory pathway serves as a nonessential, energy-saving mechanism in S. coelicolor.
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