Cdc14 Phosphatases Preferentially Dephosphorylate a Subset of Cyclin-dependent kinase (Cdk) Sites Containing Phosphoserine

Bremmer, Steven C.; Hall, Hana; Martinez, Juan S.; Eissler, Christie L.; Hinrichsen, Thomas H.; Rossie, Sandra; Parker, Laurie L.; Hall, Mark; Charbonneau, Harry

doi:10.1074/jbc.m111.281105

Cited by 82 publications

(118 citation statements)

References 29 publications

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“…Of the 68 hypo-phosphorylated peptides, 65 (95.6%) contained phosphoserine (P-serine) residues and only 3 (4.4%) phospho-threonine (P-threonine), whereas P-serine represented 80.6% and P-threonine 18.6% of the global phospho-proteome of these cells. Thus, in line with in vitro data, in vivo hCDC14A also appears to preferentially target P-serine residues (24,25).…”

supporting

confidence: 67%

Human phosphatase CDC14A regulates actin organization through dephosphorylation of epithelial protein lost in neoplasm

Chen

Uddin

Hardt

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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CDC14 is an essential dual-specificity phosphatase that counteracts CDK1 activity during anaphase to promote mitotic exit in Saccharomyces cerevisiae. Surprisingly, human CDC14A is not essential for cell cycle progression. Instead, it regulates cell migration and cell adhesion. Little is known about the substrates of hCDC14A and the counteracting kinases. Here, we combine phospho-proteome profiling and proximity-dependent biotin identification to identify hCDC14A substrates. Among these targets were actin regulators, including the tumor suppressor eplin. hCDC14A counteracts EGF-induced rearrangements of actin cytoskeleton by dephosphorylating eplin at two known extracellular signal-regulated kinase sites, serine 362 and 604. hCDC14A(PD) and eplin knockout cell lines exhibited down-regulation of E-cadherin and a reduction in alpha/beta-catenin at cell-cell adhesions. Reduction in the levels of hCDC14A and eplin mRNA is frequently associated with colorectal carcinoma and is correlated with poor prognosis. We therefore propose that eplin dephosphorylation by hCDC14A reduces actin dynamics to restrict tumor malignancy

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supporting

confidence: 67%

Human phosphatase CDC14A regulates actin organization through dephosphorylation of epithelial protein lost in neoplasm

Chen

Uddin

Hardt

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…These substrates are derived from a known Cdc14 target Cdh1-pS239 (46). This assay enabled us to directly compare the phosphatase activity of the phosphomimetic S429EE mutant with wild type.…”

Section: (Ms) +mentioning

confidence: 99%

Method for identifying phosphorylated substrates of specific cyclin/cyclin-dependent kinase complexes

Cross

Chait

2014

Proc. Natl. Acad. Sci. U.S.A.

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In eukaryotes, cell cycle progression is controlled by cyclin/cyclindependent kinase (CDK) pairs. To better understand the details of this process, it is necessary to dissect the CDK's substrate pool in a cyclin-and cell cycle stage-specific way. Here, we report a mass spectrometry-based method that couples rapid isolation of native kinase-substrate complexes to on-bead phosphorylation with heavy-labeled ATP O 4 ). This combined in vivo/in vitro method was developed for identifying cyclin/CDK substrates together with their sites of phosphorylation. We used the method to identify Clb5 (S-cyclin)/Cdc28 and Cln2 (G 1 /S-cyclin)/Cdc28 substrates during S phase in Saccharomyces cerevisiae (Cdc28 is the master CDK in budding yeast). During the work, we discovered that Clb5/Cdc28 specifically phosphorylates S429 in the disordered tail of Cdc14, an essential phosphatase antagonist of Cdc28. This phosphorylation severely decreases the activity of Cdc14, providing a means for modulating the balance of CDK and phosphatase activity.kinase substrate identification | NESKA D uring proliferation, a eukaryotic cell must faithfully replicate its genome and segregate the resulting genetic material equally into two progeny cells. A host of cellular events during this process must happen in a precisely ordered manner. Although it has been well established that cyclin-dependent kinases (CDKs) constitute the primary driving force that order and coordinate these cell cycle events (1-3), the detailed picture of how CDK orchestrates the myriad cellular events during cell cycle progression remains to be fully elucidated. At different phases of the cell cycle, the CDKs associate with specific cyclins. These particular cyclin/CDK pairs preferentially phosphorylate sets of largely distinct substrates (4-7). To investigate this intricate relationship among cell cycle phase, cyclin/CDKs, and their substrates, it is necessary to dissect the CDK phosphorylated substrates in a cyclin-specific and cell cycle phase-specific manner. We report here a mass spectrometry-based native enzymesubstrate complex kinase assay (NESKA) that was developed to address this challenge. NESKA combines rapid isolation of native kinase-substrate complexes with on-bead phosphorylation using heavy labeled ATP within these complexes, providing sitespecific in vivo and in vitro phosphorylation information. It allows us to screen for specific cyclin-CDK substrates in a hypothesis-free manner within a chosen time window during cell cycle progression.We applied NESKA to investigate phosphorylation substrates of Clb5/Cdc28 during S-phase in budding yeast (Cdc28 encodes the main CDK in budding yeast; Clb5 is the major S-phase cyclin that specializes in the regulation of DNA synthesis; refs. 8-10). It has been shown that CDK phosphorylation of two proteins, Sld2 and Sld3, initiates DNA replication (11)(12)(13)(14). Here, we find that NESKA correctly identified a crucial phosphorylation site on Sld2 with Clb5/Cdc28. In addition, we discovered that Cdc14 is specifically phosphor...

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“…Thus far, yeast Clp1 substrates have also been Cdk1 substrates, but it is unclear whether Cdc14 phosphatases exclusively dephosphorylate Cdk1 substrates. Furthermore, it is not known whether all Cdk1 phosphorylation events can be reversed by Cdc14 family phosphatases because Cdc14 exhibits target site preference both in vitro and in vivo (5,6). Tethering molecules involved in Cdc14 family sequestration and inhibition have also not been comprehensively identified.…”

mentioning

confidence: 99%

Comprehensive Proteomics Analysis Reveals New Substrates and Regulators of the Fission Yeast Clp1/Cdc14 Phosphatase

Chen

Broadus

McLean

et al. 2013

Molecular & Cellular Proteomics

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The conserved family of Cdc14 phosphatases targets cyclin-dependent kinase substrates in yeast, mediating late mitotic signaling events. To discover substrates and regulators of the Schizosaccharomyces pombe Cdc14 phosphatase Clp1, TAP-tagged Clp1, and a substrate trapping mutant (Clp1-C286S) were purified from asynchronous and mitotic (prometaphase and anaphase) cells and binding partners were identified by 2D-LC-MS/MS. Over 100 Clp1-interacting proteins were consistently identified, over 70 of these were enriched in Clp1-C286S-TAP (potential substrates) and we and others detected Cdk1 phosphorylation sites in over half (44/73) of these potential substrates. According to GO annotations, Clp1-interacting proteins are involved in many essential cellular processes including mitosis, cytokinesis, ribosome biogenesis, transcription, and trafficking among others. We confirmed association and dephosphorylation of multiple candidate substrates, including a key scaffolding component of the septation initiation network called Cdc11, an essential kinase of the conserved morphogenesis-related NDR kinase network named Shk1, and multiple Mlu1-binding factor transcriptional regulators. In addition, we identified Sal3, a nuclear ␤-importin, as the sole karyopherin required for Clp1 nucleoplasmic shuttling, a key mode of Cdc14 phosphatase regulation. Finally, a handful of proteins were more abundant in wild type Clp1-TAP

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Cdc14 Phosphatases Preferentially Dephosphorylate a Subset of Cyclin-dependent kinase (Cdk) Sites Containing Phosphoserine

Cited by 82 publications

References 29 publications

Human phosphatase CDC14A regulates actin organization through dephosphorylation of epithelial protein lost in neoplasm

Human phosphatase CDC14A regulates actin organization through dephosphorylation of epithelial protein lost in neoplasm

Method for identifying phosphorylated substrates of specific cyclin/cyclin-dependent kinase complexes

Comprehensive Proteomics Analysis Reveals New Substrates and Regulators of the Fission Yeast Clp1/Cdc14 Phosphatase

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