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Phosphoproteome analysis

ABSTRACT: The phosphatase activirty and dephosphorylation site are determined by using the recombinant protein. After treatment, the level of specific phosphorylation site is decreased. Also, the biological importance is evaluated through a serial of experiments including immunoprecipitation, western blotting, etc,. Based on this workflow, it is possible to determine the tyrosine phosphorylation since the sample complexity is reduced. This finding suggested this phosphatase play a key role in phosphoregulation.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

SUBMITTER: Miao-Hsia Lin

LAB HEAD: miao-hsia lin

PROVIDER: PXD002894 | Pride | 2022-04-13

REPOSITORIES: Pride

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			Action	DRS
	control-1.raw	Raw
	control-2.raw	Raw
	shp2-1.raw	Raw
	shp2-2.raw	Raw
	txt.zip	Other

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Publications

The Shp2-induced epithelial disorganization defect is reversed by HDAC6 inhibition independent of Cdc42.

Tien Sui-Chih SC Lee Hsiao-Hui HH Yang Ya-Chi YC Lin Miao-Hsia MH Chen Yu-Ju YJ Chang Zee-Fen ZF

Nature communications 20160119

Regulation of Shp2, a tyrosine phosphatase, critically influences the development of various diseases. Its role in epithelial lumenogenesis is not clear. Here we show that oncogenic Shp2 dephosphorylates Tuba to decrease Cdc42 activation, leading to the abnormal multi-lumen formation of epithelial cells. HDAC6 suppression reverses oncogenic Shp2-induced multiple apical domains and spindle mis-orientation during division in cysts to acquire normal lumenogenesis. Intriguingly, Cdc42 activity is no ...[more]

PMID: 26783207

Publication: 1/2

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Chemical phosphoproteomic approaches reveal substrate specificity of phosphoprotein phosphatases-1 and -2

Project description:The phosphatases PP1 and PP2A are responsible for the majority of dephosphorylation reactions on phosphoserine (pSer) and â€“threonine (pThr), and are involved in basically all cellular processes and many related diseases. They are thought to have no appreciable intrinsic substrate specificity, but to gain specificity only in their holoenzyme forms. Through the development of a peptide library approach and application of a complementary phosphoproteomics assay, we uncover that PP1 and PP2A show intrinsic specificity towards pThr over pSer, as well as toward the sequence context surrounding the phospho-site. Our data reveal that PP1 is a key phosphatase of the 14-3-3 protein binding motif. This result enabled us to establish a previously unknown role for PP1 as regulator of the GRB-associated-binding-protein 2 (Gab2)/14-3-3 complex, exemplifying predictive potential of the data. Thus, our work should serve as a rich resource for (de)phosphorylation studies covering multiple cellular processes and phosphoproteins.

2020-07-14 | PXD013775 | Pride

RNA polymerase II termination involves CTD tyrosine dephosphorylation by CPF subunit Glc7

Project description:At the 3'-ends of genes, RNA polymerase (Pol) II is dephosphorylated at tyrosine 1 residues of its C-terminal domain (CTD), resulting in recruitment of transcription termination factors. We show that the multisubunit cleavage and polyadenylation factor (CPF) is a Pol II CTD phosphatase and its Glc7 subunit is required for Tyr1 dephosphorylation at the poly-adenylation site and Pol II termination in vivo. ChIP-chip was performed to examine the effect of Glc7 nuclear depletion on genome-wide Pol II occupancy [using ?-Rpb3 (1Y26, cat. no. W0012, neoclone) antibody] and CTD tyrosine 1 phosphorylation levels [using ?-TyrY1P (3D12, D. Eick) antibody].

2013-12-16 | E-MTAB-1528 | biostudies-arrayexpress

Systematic Identification of SHP2 Phosphatase Substrates

Project description:Phosphatases play essential roles in normal cell physiology and diseases like cancer. The challenges of studying phosphatases have limited our understanding of their substrates, molecular mechanisms, and unique functions within highly complicate networks. Here we introduce a novel strategy using substrate trapping mutant coupled with quantitative mass spectrometry to identify physiological substrates of protein tyrosine phosphatase Src homology 2 containing protein tyrosine phosphatase 2 (SHP2) in a high-throughput manner. SHP2 plays a critical role in numerous cellular processes through the regulation of various signaling pathways. The method integrates three separate MS-based experiments; in vitro dephosphorylation assay, in vivo global phosphoproteomics, and pull down of substrate trapping mutant complex using HEK293SHP2KO cells. PTPN11-C459S/D425A is an optimized substrate trap mutant of SHP2. We identified eleven direct substrates, including both known and novel SHP2 substrates in EGFR signaling pathways, among which docking protein 1 (DOK1) was further validated as a new SHP2 substrate. This advanced workflow significantly improves the systemic identification of direct substrates of phosphatase, facilitating the comprehension of equally important roles of phosphatase signaling.

2022-09-20 | PXD028404 | Pride

Tyrosine 601 of Bacillus subtilis DnaK undergoes phosphorylation and is crucial for chaperone activity and heat shock survival

Project description:In order to screen for cellular substrates of the Bacillus subtilis BY-kinase PtkA, and its cognate phosphotyrosine-protein phosphatase PtpZ, we performed a triple SILAC-based quantitative phosphoproteome analysis. Detected tyrosine phosphorylation sites for which the phosphorylation level decreased in the ΔptkA strain and increased in the ΔptpZ strain, compared to the wild type, were considered as potential substrates of PtkA/PtpZ. One of those sites was the residue tyrosine 601 of the molecular chaperone DnaK. We confirmed that DnaK is a substrate of PtkA and PtpZ by in vitro phosphorylation and dephosphorylation assays. In vitro, the non-phosphorylatable mutant DnaK Y601F exhibited impaired interaction with its co-chaperones DnaJ and GrpE, along with diminished capacity to hydrolyze ATP and assist the re-folding of denatured proteins. In vivo, loss of DnaK phosphorylation in the mutant strain dnaK Y601F, or in the strain overexpressing the phosphatase PtpZ, led to diminished survival upon heat shock, consistent with the in vitro results. The decreased survival of the mutant dnaK Y601F at an elevated temperature could be rescued by complementing with the wild type dnaK allele expressed ectopically. We concluded that the residue tyrosine 601 of DnaK can be phosphorylated and dephosphorylated by PtkA and PtpZ, respectively. Furthermore, Y601 is important for DnaK chaperone activity and heat shock survival of B. subtilis.

2016-05-09 | PXD003764 | Pride

Control of RNA pol II speed by PNUTS-PP1 and Spt5 dephosphorylation facilitates termination by a “sitting duck torpedo” mechanism

Project description:Control of transcription speed, which influences many co-transcriptional processes, is poorly understood. We report that PNUTS-PP1 phosphatase is a negative regulator of RNA pol II elongation rate. The PNUTS W401A mutation, which disrupts PP1 binding, causes genome-wide acceleration of transcription associated with hyper-phosphorylation of the Spt5 elongation factor. Immediately downstream of poly(A) sites, pol II decelerates from >2kb/min to <1 kb/min, which correlates with Spt5 dephosphorylation. Pol II deceleration and Spt5 dephosphorylation require poly(A) site recognition and the PNUTS-PP1 complex, which is in turn necessary for transcription termination. These results lead to a new model for termination, the “sitting duck torpedo” mechanism, where poly(A) site-dependent deceleration caused by PNUTS-PP1 and Spt5 dephosphorylation is required to convert pol II into a viable target for the Xrn2 terminator exonuclease. Spt5 and its bacterial homologue NusG therefore have related functions controlling kinetic competition between RNA polymerases and the termination factors that pursue them.

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Integrator-PP2A complex dephosphorylates the C-terminal repeat domain of RNA Polymerase II

Project description:The phosphorylation and dephosphorylation of transcription machinery are essential for the precise control of gene expression. A non-canonical protein phosphatase 2A (PP2A) holoenzyme (denoted INTAC), in which the 14-subunit Integrator recruits RNA polymerase II (Pol II) and the PP2A core enzyme dephosphorylates the C-terminal repeat domain (CTD) of Pol II at Serine-5 and Serine-2.

2020-12-02 | GSE154093 | GEO

Distinct Cdk9-phosphatase switches act on elongation factor Spt5 at the beginning and end of the RNA polymerase II transcription cycle

Project description:The Pol II transcription cycle is ordered by CDKs and phosphatases. In fission yeast, Cdk9 phosphorylates carboxy-terminal repeats (CTRs) of Spt5 while inhibiting PP1 during elongation. Transcription past the cleavage and polyadenylation signal (CPS) coincides with PP1-dependent Spt5 dephosphorylation and leads to Pol II pausing with phosphorylated CTD-Ser2 (pSer2). Here we show this switch is conserved in humans: Cdk9 inhibition decreases phosphorylation of both PP1g and Spt5-Thr806 (pThr806), and induces pSer2 upstream of the CPS, whereas PP1 depletion increases pThr806. Moreover, in unperturbed cells, pThr806 is diminished in 3’-paused complexes where pSer2 is maximal. Cdk9 also phosphorylates Spt5 on Ser666, a PP1-refractory site between conserved KOW4 and KOW5 motifs; pSer666 increases upon promoter-proximal pause release and, in contrast to pThr806, persists beyond the CPS. We identify PP4—another target of inhibitory phosphorylation by Cdk9—as a pSer666 phosphatase. PP4 and PP1g are enriched at 5’ and 3’ ends of genes, respectively, consistent with pSer666 and pThr806 distributions. Therefore, distinct Cdk9-phosphatase switches operate on Spt5 at different steps in transcription.

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Distinct dephosphorylation site signatures of PP1 and PP2A-B55 coordinate mitotic exit.

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