Phosphatase specificity principles uncovered by MRBLE:Dephos and global substrate identification
Abstract Phosphoprotein phosphatases (PPPs) regulate major signaling pathways, but the determinants of phosphatase specificity are poorly understood. This is because methods to investigate this at scale are lacking. Here, we develop a novel in vitro assay, MRBLE:Dephos, that allows multiplexing of d...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Springer Nature
2023-12-01
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| Series: | Molecular Systems Biology |
| Subjects: | |
| Online Access: | https://doi.org/10.15252/msb.202311782 |
| _version_ | 1797279662272937984 |
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| author | Jamin B Hein Hieu T Nguyen Dimitriya H Garvanska Isha Nasa Thomas Kruse Yinnian Feng Blanca Lopez Mendez Norman Davey Arminja N Kettenbach Polly M Fordyce Jakob Nilsson |
| author_facet | Jamin B Hein Hieu T Nguyen Dimitriya H Garvanska Isha Nasa Thomas Kruse Yinnian Feng Blanca Lopez Mendez Norman Davey Arminja N Kettenbach Polly M Fordyce Jakob Nilsson |
| author_sort | Jamin B Hein |
| collection | DOAJ |
| description | Abstract Phosphoprotein phosphatases (PPPs) regulate major signaling pathways, but the determinants of phosphatase specificity are poorly understood. This is because methods to investigate this at scale are lacking. Here, we develop a novel in vitro assay, MRBLE:Dephos, that allows multiplexing of dephosphorylation reactions to determine phosphatase preferences. Using MRBLE:Dephos, we establish amino acid preferences of the residues surrounding the dephosphorylation site for PP1 and PP2A‐B55, which reveals common and unique preferences. To compare the MRBLE:Dephos results to cellular substrates, we focused on mitotic exit that requires extensive dephosphorylation by PP1 and PP2A‐B55. We use specific inhibition of PP1 and PP2A‐B55 in mitotic exit lysates coupled with phosphoproteomics to identify more than 2,000 regulated sites. Importantly, the sites dephosphorylated during mitotic exit reveal key signatures that are consistent with MRBLE:Dephos. Furthermore, integration of our phosphoproteomic data with mitotic interactomes of PP1 and PP2A‐B55 provides insight into how binding of phosphatases to substrates shapes dephosphorylation. Collectively, we develop novel approaches to investigate protein phosphatases that provide insight into mitotic exit regulation. |
| first_indexed | 2024-03-07T16:29:16Z |
| format | Article |
| id | doaj.art-2b7f9cdcf70344a5bb703577c2b23118 |
| institution | Directory Open Access Journal |
| issn | 1744-4292 |
| language | English |
| last_indexed | 2024-03-07T16:29:16Z |
| publishDate | 2023-12-01 |
| publisher | Springer Nature |
| record_format | Article |
| series | Molecular Systems Biology |
| spelling | doaj.art-2b7f9cdcf70344a5bb703577c2b231182024-03-03T11:22:53ZengSpringer NatureMolecular Systems Biology1744-42922023-12-011912n/an/a10.15252/msb.202311782Phosphatase specificity principles uncovered by MRBLE:Dephos and global substrate identificationJamin B Hein0Hieu T Nguyen1Dimitriya H Garvanska2Isha Nasa3Thomas Kruse4Yinnian Feng5Blanca Lopez Mendez6Norman Davey7Arminja N Kettenbach8Polly M Fordyce9Jakob Nilsson10Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences University of Copenhagen Copenhagen DenmarkBiochemistry and Cell Biology Geisel School of Medicine at Dartmouth College Hanover NH USANovo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences University of Copenhagen Copenhagen DenmarkDepartment of Bioengineering Stanford University Stanford CA USANovo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences University of Copenhagen Copenhagen DenmarkDepartment of Genetics Stanford University Stanford CA USANovo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences University of Copenhagen Copenhagen DenmarkDivision of Cancer Biology The Institute of Cancer Research London UKBiochemistry and Cell Biology Geisel School of Medicine at Dartmouth College Hanover NH USADepartment of Bioengineering Stanford University Stanford CA USANovo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences University of Copenhagen Copenhagen DenmarkAbstract Phosphoprotein phosphatases (PPPs) regulate major signaling pathways, but the determinants of phosphatase specificity are poorly understood. This is because methods to investigate this at scale are lacking. Here, we develop a novel in vitro assay, MRBLE:Dephos, that allows multiplexing of dephosphorylation reactions to determine phosphatase preferences. Using MRBLE:Dephos, we establish amino acid preferences of the residues surrounding the dephosphorylation site for PP1 and PP2A‐B55, which reveals common and unique preferences. To compare the MRBLE:Dephos results to cellular substrates, we focused on mitotic exit that requires extensive dephosphorylation by PP1 and PP2A‐B55. We use specific inhibition of PP1 and PP2A‐B55 in mitotic exit lysates coupled with phosphoproteomics to identify more than 2,000 regulated sites. Importantly, the sites dephosphorylated during mitotic exit reveal key signatures that are consistent with MRBLE:Dephos. Furthermore, integration of our phosphoproteomic data with mitotic interactomes of PP1 and PP2A‐B55 provides insight into how binding of phosphatases to substrates shapes dephosphorylation. Collectively, we develop novel approaches to investigate protein phosphatases that provide insight into mitotic exit regulation.https://doi.org/10.15252/msb.202311782mitotic exitMRBLE:DephosMRBLE‐Pepprotein phosphatasesubstrates |
| spellingShingle | Jamin B Hein Hieu T Nguyen Dimitriya H Garvanska Isha Nasa Thomas Kruse Yinnian Feng Blanca Lopez Mendez Norman Davey Arminja N Kettenbach Polly M Fordyce Jakob Nilsson Phosphatase specificity principles uncovered by MRBLE:Dephos and global substrate identification Molecular Systems Biology mitotic exit MRBLE:Dephos MRBLE‐Pep protein phosphatase substrates |
| title | Phosphatase specificity principles uncovered by MRBLE:Dephos and global substrate identification |
| title_full | Phosphatase specificity principles uncovered by MRBLE:Dephos and global substrate identification |
| title_fullStr | Phosphatase specificity principles uncovered by MRBLE:Dephos and global substrate identification |
| title_full_unstemmed | Phosphatase specificity principles uncovered by MRBLE:Dephos and global substrate identification |
| title_short | Phosphatase specificity principles uncovered by MRBLE:Dephos and global substrate identification |
| title_sort | phosphatase specificity principles uncovered by mrble dephos and global substrate identification |
| topic | mitotic exit MRBLE:Dephos MRBLE‐Pep protein phosphatase substrates |
| url | https://doi.org/10.15252/msb.202311782 |
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