Roles of Active Site Residues and the NH2-terminal Domain in the Catalysis and Substrate Binding of Human Cdc25

Xu, Xu; Burke, Shannon Plisinski

doi:10.1074/jbc.271.9.5118

Cited by 72 publications

(14 citation statements)

References 56 publications

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“…Table 1 shows that the K m values of each of these proteins are in the millimolar range. These values are in reasonable agreement with those measured previously for either the GST-fused or untagged Cdc25-cd constructs (24, 34–36). The k cat and k cat / K m values of pNPP for Cdc25B-cd were 0.3 s −1 and 33 M −1 s −1 , respectively, at pH 8.5 and 30 °C.…”

Section: Resultssupporting

confidence: 92%

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Adventitious Arsenate Reductase Activity of the Catalytic Domain of the Human Cdc25B and Cdc25C Phosphatases

Bhattacharjee¹,

Sheng

Ajees³

et al. 2010

Biochemistry

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A number of eukaryotic enzymes that function as arsenate reductases are homologues of the catalytic domain of the human Cdc25 phosphatase. For example, the Leishmania major enzyme LmACR2 is both a phosphatase and an arsenate reductase, and its structure bears similarity to the structure of the catalytic domain of human Cdc25 phosphatase. These reductases contain an active site C-X5-R signature motif, where C is the catalytic cysteine, the five X residues form a phosphate binding loop, and R is a highly conserved arginine, which is also present in human Cdc25 phosphatases. We therefore investigated the possibility that the three human Cdc25 isoforms might have adventitious arsenate reductase activity. The sequences for the catalytic domains of Cdc25A, -B, and -C were cloned individually into a prokaryotic expression vector, and their gene products were purified from a bacterial host using nickel affinity chromatography. While each of the three Cdc25 catalytic domains exhibited phosphatase activity, arsenate reductase activity was observed only with Cdc25B and -C. These two enzymes reduced inorganic arsenate but not methylated pentavalent arsenicals. Alteration of either the cysteine and arginine residues of the Cys-X5-Arg motif led to the loss of both reductase and phosphatase activities. Our observations suggest that Cdc25B and -C may adventitiously reduce arsenate to the more toxic arsenite and may also provide a framework for identifying other human protein tyrosine phosphatases containing the active site Cys-X5-Arg loop that might moonlight as arsenate reductases.

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Section: Resultssupporting

confidence: 92%

“…It should be noted that most of the studies with human Cdc25 enzymes, including purification, characterization, and crystallization, have been performed with the catalytic domain and not the full-length enzyme (28, 29, 34, 35, 37). Whether the full-length Cdc25 phosphatase can reduce As(V) was examined.…”

Section: Discussionmentioning

confidence: 99%

Adventitious Arsenate Reductase Activity of the Catalytic Domain of the Human Cdc25B and Cdc25C Phosphatases

Bhattacharjee¹,

Sheng

Ajees³

et al. 2010

Biochemistry

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“…The reaction is proposed to proceed via a phospho-cysteine intermediate (Figure 9). This mechanism is supported by mutagenesis studies that show replacement of the catalytic Cys (Cys473 in Cdc25B) with Ser yields a protein with no phosphatase activity (25). It is also supported by pH-dependent kinetics that show the apparent pH dependence of the cysteine using two different substrates in both steady-state kinetics and rapid quench kinetics (20), by the pH-dependent inactivation of Cdc25B by iodoacetic acid (Figure 1), and by the crystal structures of the catalytic domain (18,19).…”

Section: Discussionmentioning

confidence: 93%

Catalytic Mechanism of Cdc25

Rudolph

2002

Biochemistry

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Cdc25 is a dual-specificity phosphatase that catalyzes the activation of the cyclin-dependent kinases, thus causing initiation and progression of successive phases of the cell cycle. Although it is not significantly homologous in sequence or structure to other dual-specificity phosphatases, Cdc25 belongs to the class of well-studied cysteine phosphatases as it contains their active site signature motif. Like other dual-specificity phosphatases, Cdc25 contains an active site cysteine whose pK(a) of 5.9 can be measured in pH-dependent kinetics using both small molecule and protein substrates such as Cdk2-pTpY/CycA. We have previously shown that the catalytic acid expected in phosphatases of this family and apparent in kinetics with the natural protein substrate does not appear to lie within the known structure of Cdc25 [Chen, W., et al. (2000) Biochemistry 39, 10781]. Here we provide experimental evidence for a novel mechanism wherein Cdc25 uses as its substrate a monoprotonated phosphate in contrast to the more typical bisanionic phosphate. Our pH-dependent studies, including one-turnover kinetics, solvent kinetic isotope effects, equilibrium perturbation, substrate depletion, and viscosity measurements, show that the monoprotonated phosphate of the protein substrate Cdk2-pTpY/CycA provides the critical proton to the leaving group. Additionally, we provide evidence that Glu474 on the Cdc25 enzyme serves an important role as a base in the transfer of the proton from the phosphate to the leaving group. Because of its greater intrinsic reactivity, the use of a monoprotonated phosphate as a phosphatase substrate is a chemically attractive solution and suggests the possibility of designing inhibitors specific for the Cdc25 dual-specificity phosphatase, an important anticancer target.

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“…CDC25A phosphatase activity is required for dephosphorylation, as well as activation of CDK [38] and the interaction with CDK/cyclin complexes [39]. We investigated if the interaction of FOXM1 and CDC25A is dependent on CDC25A phosphatase activity.…”

Section: Resultsmentioning

confidence: 99%

Novel Interactions between FOXM1 and CDC25A Regulate the Cell Cycle

Sullivan

Liu

Shen³

et al. 2012

PLoS ONE

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FOXM1 is a critical regulator of the G1/S and G2/M cell cycle transitions, as well as of the mitotic spindle assembly. Previous studies have suggested that FOXM1 regulates CDC25A gene transcription, but the mechanism remains unknown. Here, we provide evidence that FOXM1 directly regulates CDC25A gene transcription via direct promoter binding and indirect activation of E2F-dependent pathways. Prior literature reported that CDC25B and CDC25C activate CDK1/cyclinB complexes in order to enable phosphorylation of FOXM1. It was unknown if CDC25A functions in a similar manner. We report that FOXM1 transcriptional activity is synergistically enhanced when co-expressed with CDC25A. The increase is dependent upon CDK1 phosphorylation of FOXM1 at T600, T611 and T620 residues. We also report a novel protein interaction between FOXM1 and CDC25A via the C-terminus of FOXM1. We demonstrate that the phosphorylation of Thr 600 and Thr 611 residues of FOXM1 enhanced this interaction, and that the interaction is dependent upon CDC25A phosphatase activity. Our work provides novel insight into the underlying mechanisms by which FOXM1 controls the cell cycle through its association with CDC25A.

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Roles of Active Site Residues and the NH2-terminal Domain in the Catalysis and Substrate Binding of Human Cdc25

Cited by 72 publications

References 56 publications

Adventitious Arsenate Reductase Activity of the Catalytic Domain of the Human Cdc25B and Cdc25C Phosphatases

Adventitious Arsenate Reductase Activity of the Catalytic Domain of the Human Cdc25B and Cdc25C Phosphatases

Catalytic Mechanism of Cdc25

Novel Interactions between FOXM1 and CDC25A Regulate the Cell Cycle

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