Identification of epidermal growth factor receptor as a target of Cdc25A protein phosphatase

Cdc25A, a dual-specificity protein phosphatase, plays a critical role in cell cycle progression. Although cyclin-dependent kinases are established substrates, Cdc25A may also affect other proteins. We have shown here that Cdc25A interacts with epidermal growth factor receptor (EGFR) both physically and functionally in Hep3B human hepatoma cells. Cdc25A inhibitor Cpd 5, a vitamin K analog, inhibited Cdc25A activity in the Cdc25A-EGFR immunocomplex and consequently caused prolonged EGFR tyrosine phosphorylation. Both purified GST-Cdc25A protein and endogenous Hep3B cellular Cdc25A dephosphorylated tyrosine-phosphorylated EGFR, and Cpd 5 antagonized the phosphatase activity of Cdc25A. A functional Cdc25A-EGFR interaction was seen in NR-6 fibroblasts expressing ectopic EGFR but not with a receptor lacking the C terminus or a mutated kinase domain. These data link the cell cycle control Cdc25A phosphatase to an EGFR-linked mitogenic signaling pathway specifically involving EGFR dephosphorylation.     

Arylstibonic acids are potent and isoform-selective inhibitors of Cdc25a and Cdc25b phosphatases

Arylstibonates structurally resemble phosphotyrosine side chains in proteins and here we addressed the ability of such compounds to act as inhibitors of a panel of mammalian tyrosine and dual-specificity phosphatases. Two arylstibonates both possessing a carboxylate side chain were identified as potent inhibitors of the protein tyrosine phosphatase PTP-ß. In addition, they inhibited the dual-specificity, cell cycle regulatory phosphatases Cdc25a and Cdc25b with sub-micromolar potency. However, the Cdc25c phosphatase was not affected demonstrating that arylstibonates may be viable leads from which to develop isoform specific Cdc25 inhibitors.     

Normal cell cycle and checkpoint responses in mice and cells lacking Cdc25B and Cdc25C protein phosphatases

The Cdc25 family of protein phosphatases positively regulates cell division by activating cyclin-dependent protein kinases (CDKs). In humans and rodents, there are three Cdc25 family members--denoted Cdc25A, Cdc25B, and Cdc25C--that can be distinguished based on their subcellular compartmentalizations, their abundances and/or activities throughout the cell cycle, the CDKs that they target for activation, and whether they are overexpressed in human cancers. In addition, murine forms of Cdc25 exhibit distinct patterns of expression throughout development and in adult tissues. These properties suggest that individual Cdc25 family members contribute distinct biological functions in embryonic and adult cell cycles of mammals. Interestingly, mice with Cdc25C disrupted are healthy, and cells derived from these mice exhibit normal cell cycles and checkpoint responses. Cdc25B-/- mice are also generally normal (although females are sterile), and cells derived from Cdc25B-/- mice have normal cell cycles. Here we report that mice lacking both Cdc25B and Cdc25C are obtained at the expected Mendelian ratios, indicating that Cdc25B and Cdc25C are not required for mouse development or mitotic entry. Furthermore, cell cycles, DNA damage responses, and Cdc25A regulation are normal in cells lacking Cdc25B and Cdc25C. These findings indicate that Cdc25A, or possibly other phosphatases, is able to functionally compensate for the loss of Cdc25B and Cdc25C in mice.     

Identification of the phosphatase deinhibitor protein phosphatases in rabbit skeletal muscle.

In rabbit skeletal muscle the polycation-stimulated (PCS) protein phosphatases [Merlevede (1985) Adv. Protein Phosphatases 1, 1-18] are the only phosphatases displaying significant activity toward the deinhibitor protein. Among them, the PCSH protein phosphatase represents more than 80% of the measurable deinhibitor phosphatase activity associated with the PCS phosphatases. The deinhibitor phosphatase activity co-purifies with the PCSH phosphatase to apparent homogeneity. In the last purification step two forms of PCSH phosphatase were separated (PCSH1, containing 62, 55 and 34 kDa subunits, and PCSH2, containing 62 and 35 kDa subunits), both showing the same deinhibitor/phosphorylase phosphatase activity ratio. The activity of the PCSH phosphatase toward the deinhibitor is not stimulated by polycations such as protamine, histone H1 or polylysine, unlike the stimulation observed with phosphorylase as the substrate. The phosphorylase phosphatase activity of PCSH phosphatase is inhibited by ATP, PPi and Pi, whereas the deinhibitor phosphatase activity of the enzyme is much less sensitive to these agents.     

Cdc25A and ERK interaction: EGFR-independent ERK activation by a protein phosphatase Cdc25A inhibitor, compound 5

Extracellular signal-regulated kinase (ERK) plays a central role in regulating cell growth, differentiation, and apoptosis. We previously found that 2-(2-mercaptoethanol)-3-methyl-1,4-napthoquinone or Compound 5 (Cpd 5), is a Cdc25A protein phosphatase inhibitor and causes prolonged, strong ERK phosphorylation which is triggered by epidermal growth factor receptor (EGFR) activation. We now report that Cpd 5 can directly cause ERK phosphorylation by inhibiting Cdc25A activity independently of the EGFR pathway. We found that Cdc25A physically interacted with and de-phosphorylated phospho-ERK both in vitro and in cell culture. Inhibition of Cdc25A activity by Cpd 5 resulted in ERK hyper-phosphorylation. Transfection of Hep3B human hepatoma cells with inactive Cdc25A mutant enhanced Cpd 5 action on ERK phosphorylation, whereas over-expression of Cdc25A attenuated this Cpd 5 action. Furthermore, endogenous Cdc25A knock-down by Cdc25A siRNA resulted in a constitutive-like ERK phosphorylation and Cpd 5 treatment further enhanced it. In EGFR-devoid NR6 fibroblasts and MEK (ERK kinase) mutated MCF7 cells, Cpd 5 treatment also resulted in ERK phosphorylation, providing support for the idea that Cpd 5 can directly act on ERK phosphorylation by inhibiting Cdc25A activity. These data suggest that phospho-ERK is likely another Cdc25A substrate, and Cpd 5-caused ERK phosphorylation is probably regulated by both EGFR-dependent and EGFR-independent pathways.     

Identification of new Cdc25 dual specificity phosphatase inhibitors in a targeted small molecule array

Dual specificity protein phosphatases (DSPases) are key regulators of signal transduction, oncogenesis and the cell cycle. Few potent or specific inhibitors of DSPases, however, are readily available for these pharmacological targets. We have used a combinatorial/parallel synthetic approach to rigidify the variable core region and modify the side chains of 4-(benzyl-(2-[2,5-diphenyl-oxazole-4-carbonyl)-amino]-ethyl)-carbamoyl)- 2-decanoylamino butyric acid (or SC-alphaalphadelta9), which is the most active element in a previously described library of phosphatase inhibitors (Rice, R. L.; Rusnak, J. M.; Yokokawa, F.; Yokokawa, S.; Messner, D. J.; Boynton, A. L.; Wipf, P.; Lazo, J. S. Biochemistry 1997, 36, 15965). Several analogues were identified as effective inhibitors of the protein tyrosine phosphatase (PTPase) PTP1B and the DSPases VHR and Cdc25B2. Two compounds, FY3-alphaalpha09 and FY21-alphaalpha09, were partial competitive inhibitors of Cdc25B2 with Ki values of 7.6+/-0.5 and 1.6+/-0.2 microM, respectively. FY21-alphaalpha09 possessed only moderate activity against PTP1B. Consistent with its in vitro anti-phosphatase activity, FY21-alphaalpha09 inhibited growth in MDA-MB-231 and MCF-7 human breast cancer cell lines. FY21-alphaalpha09 also inhibited the G2/M transition in tsFT210 cells, consistent with Cdc25B inhibition. Several architectural requirements for DSPase inhibition were revealed through modification of the side chain moieties or variable core region of the pharmacophore, which resulted in decreased compound potency. The structure of FY21-alphaalpha09 provides a useful platform from which additional potent and more highly selective phosphatase inhibitors might be generated.     

Mechanistic studies of protein tyrosine phosphatases YopH and Cdc25A with m-nitrobenzyl phosphate

Protein tyrosine phosphatases (PTPs) constitute a large family of signaling enzymes that include both tyrosine specific and dual-specificity phosphatases that hydrolyze pSer/Thr in addition to pTyr. Previous mechanistic studies of PTPs have relied on the highly activated substrate p-nitrophenyl phosphate (pNPP), an aryl phosphate with a leaving group pK(a) of 7. In the study presented here, we employ m-nitrobenzyl phosphate (mNBP), an alkyl phosphate with a leaving group pK(a) of 14.9, which mimics the physiological substrates of the PTPs. We have carried out pH dependence and kinetic isotope effect measurements to characterize the mechanism of two important members of the PTP superfamily: Yersinia PTP (YopH) and Cdc25A. Both YopH and Cdc25A exhibit bell-shaped pH-rate profiles for the hydrolysis of mNBP, consistent with general acid catalysis. The slightly inverse (18)(V/K)(nonbridge) isotope effects (0.9999 for YopH and 0.9983 for Cdc25A) indicate a loose transition state with little nucleophilic participation for both enzymes. The smaller (18)(V/K)(bridge) primary isotope effects (0.9995 for YopH and 1.0012 for Cdc25A) relative to the corresponding isotope effects for pNPP hydrolysis suggest that protonation of the leaving group oxygen at the transition state by the general acid is ahead of P-O bond fission with the alkyl substrate, while general acid catalysis of pNPP by YopH is more synchronous with P-O bond fission. The isotope effect data also confirm findings from previous studies that Cdc25A utilizes general acid catalysis for substrates with a leaving group pK(a) of >8, but not for pNPP. Interestingly, the difference in the kinetic isotope effects for the reactions of aryl phosphate pNPP and alkyl phosphate mNBP by the PTPs parallels what is observed in the uncatalyzed reactions of their monoanions. In these reactions, the leaving group is protonated in the transition state, as is the case in PTP-catalyzed reactions. Also, the phosphoryl group in the transition states of the enzymatic reactions does not differ substantially from those of the uncatalyzed reactions. These results provide further evidence that these enzymes do not change the transition state but simply stabilize it.     

Sesterterpenoids and an alkaloid from a Thorectandra sp. as inhibitors of the phosphatase Cdc25B

Bioassay-directed separation of an extract of a Thorectandra sp. sponge led to the isolation of three new sesterterpenoids, 16-oxoluffariellolide (1), 16-hydroxyluffariellolide (2) and (2E,6E,10E)-3-formyl-7,11-dimethyl-13-(2,6,6-trimethylcyclohex-1-enyl)trideca-2,6,10-trienoic acid (3); two known sesterterpenoids, luffariellolide (4) and dehydroluffariellolide diacid (5); and one known alkaloid, fascaplysin (6). The structures of the new compounds 1-3 were established on the basis of extensive 1D and 2D NMR spectroscopic data interpretation. Compound 6 showed inhibitory activity in the Cdc25B assay, with an IC50 value of 1.0 microg/mL.     

Reactivity of Cdc25 phosphatase at low pH and with thiophosphorylated protein substrate

Cdc25s, dual-specificity phosphatases that dephosphorylate and activate cyclin-dependent kinases, are important regulators of the eukaryotic cell cycle. Herein, we probe the protonation state of the phosphate on the protein substrate of Cdc25 by pH-dependent studies and thiosubstitution. We have extended the useable range of pH for this enzyme substrate pair by using high concentrations of glycerol under acidic conditions. Using the protein substrate, we find a slope of 2 for the acidic side of the bell-shaped pH-rate profile, as found with other protein tyrosine phosphatases. Using thiophosphorylated protein substrate, we find no change in the basic side of the pH-rate profile, despite a large reduction in activity as measured by kcat/Km (0.18%) or kcat (0. 11%). In contrast, the acidic side of the profile changes shows a slope of 1, consistent with the 1.5 pH unit shift associated with thiosubstitution. Thus, Cdc25, like other protein phosphatases, uses a dianionic phosphorylated substrate.     

Differential roles for checkpoint kinases in DNA damage-dependent degradation of the Cdc25A protein phosphatase

In response to DNA damage, cells activate a signaling pathway that promotes cell cycle arrest and degradation of the cell cycle regulator Cdc25A. Cdc25A degradation occurs via the SCFbeta-TRCP pathway and phosphorylation of Ser-76. Previous work indicates that the checkpoint kinase Checkpoint kinase 1 (Chk1) is capable of phosphorylating Ser-76 in Cdc25A, thereby promoting its degradation. In contrast, other experiments involving overexpression of dominant Chk2 mutant proteins point to a role for Chk2 in Cdc25A degradation. However, loss-of-function studies that implicate Chk2 in Cdc25A turnover are lacking, and there is no evidence that Chk2 is capable of phosphorylating Ser-76 in Cdc25A despite the finding that Chk1 and Chk2 sometimes share overlapping primary specificity. We find that although Chk2 can phosphorylate many of the same sites in Cdc25A that Chk1 phosphorylates, albeit with reduced efficiency, Chk2 is unable to efficiently phosphorylate Ser-76. Consistent with this, Chk2, unlike Chk1, is unable to support SCFbeta-TRCP-mediated ubiquitination of Cdc25A in vitro. In CHK2(-/-) HCT116 cells, the kinetics of Cdc25A degradation in response to ionizing radiation is comparable with that seen in HCT116 cells containing Chk2, indicating that Chk2 is not generally required for timely DNA damage-dependent Cdc25A turnover. In contrast, depletion of Chk1 by RNA interference in CHK2(-/-) cells leads to Cdc25A stabilization in response to ionizing radiation. These data support the idea that Chk1 is the primary signal transducer linking activation of the ATM/ATR kinases to Cdc25A destruction in response to ionizing radiation.     

Cdc25A is a novel phosphatase functioning early in the cell cycle.

The cdc25+ tyrosine phosphatase is a key mitotic inducer of the fission yeast Schizosaccharomyces pombe, controlling the timing of the initiation of mitosis. Mammals contain at least three cdc25+ homologues called cdc25A, cdc25B and cdc25C. In this study we investigate the biological function of cdc25A. Although very potent in rescuing the S.pombe cdc25 mutant, cdc25A is less structurally related to the S.pombe enzyme. Northern and Western blotting detection reveals that unlike cdc25B, cdc25C and cdc2, cdc25A is predominantly expressed in late G1. Moreover, immunodepletion of cdc25A in rat cells by microinjection of a specific antibody effectively blocks their cell cycle progression from G1 into the S phase, as determined by laser scanning single cell cytometry. These results indicate that cdc25A is not a mitotic regulator but a novel phosphatase that plays a crucial role in the start of the cell cycle. In view of its strong ability to activate cdc2 kinase and its specific expression in late G1, cdc2-related kinases functioning early in the cell cycle may be targets for this phosphatase.     

Temperature dependence of binding and catalysis for the Cdc25B phosphatase

Using a combination of steady-state and single-turnover kinetics, we probe the temperature dependence of substrate association and chemistry for the reaction of Cdc25B phosphatase with its Cdk2-pTpY/CycA protein substrate. The transition state for substrate association is dominated by an enthalpic barrier (DeltaH(++) of 13 kcal/mol) and has a favorable entropic contribution of 4 kcal/mol at 298 K. Phosphate transfer from Cdk2-pTpY/CycA to enzyme (DeltaH(++) of 12 kcal/mol) is enthalpically more favorable than for the small molecule substrate p-nitrophenyl phosphate (DeltaH(++) of 18 kcal/mol), yet entropically less favorable (TDeltaS(++) of 2 vs. -6 kcal/mol at 298 K, respectively). By measuring the temperature dependence of binding and catalysis for several hotspot mutants involved in binding of protein substrate, we determine the enthalpy-entropy compensations for changes in rates of association and phosphate transfer compared to the wild type system. We conclude that the transition state for enzyme-substrate association involves tight and specific contacts at the remote docking site and that phospho-transfer from Cdk2-pTpY/CycA to the pre-organized active site of the enzyme is accompanied by unfavorable entropic rearrangements that promote rapid product dissociation.     

The energetic network of hotspot residues between Cdc25B phosphatase and its protein substrate

We have investigated the functional network of hotspot residues at the remote docking site of two cell cycle regulators, namely Cdc25B phosphatase and its native protein substrate Cdk2-pTpY/CycA. Specifically, we have studied the roles of energetically important residues (Arg488, Arg492, Tyr497 on Cdc25B and Asp206 and Asp210 on Cdk2-pTpY/CycA) by generating a diverse set of substitutions and performing double and triple mutant cycle analyses. This transient protein-protein interaction is particularly well-suited for this mutagenic approach because various control experiments ensure that the effect of each mutation is limited to the interaction of interest. We find binary coupling energies for ion pairs and hydrogen bonds ranging from 0.7 kcal/mol to 3.9 kcal/mol and ternary coupling energies of 1.9 kcal/mol and 2.8 kcal/mol. Overall our biochemical analyses are in good agreement with the docked structure of the complex and suggest the following roles for the individual hotspot residues on Cdc25B. The most important contributor, Arg492, forms a specific and tight bidentate interaction with Asp206 and a weaker interaction with Asp210 that cannot be replaced by a Lys. Although Tyr497 does not directly participate in this ionic network, it is important for buttressing Arg492 using both its hydrophobic (aromatic ring) and hydrophilic characteristics (hydrogen bonding). Arg488 participates less specifically in the electrostatic network with Asp206 and Asp210 of the protein substrate as it can partially be replaced by Lys. Our data provide insight how a cluster of residues in a docking site remote from the site of the chemical reaction can bring about efficient and specific substrate recognition.     

Identification of Cdc45p,an essential factor required for DNA replication

CDC45 is an essential gene required for initiation of DNA replication in the budding yeast Saccharomyces cerevisiae. CDC45 interacts genetically with CDC46 and CDC47, both members of the MCM family of genes which have been implicated in the licensing of DNA replication. In this report, the isolation of CDC45 is described. The complementing gene is linked to an essential open reading frame on chromosome XII. CDC45 was found to be cell cycle regulated and steady-state mRNA levels are G1/S-specific. CDC45 encodes a protein structurally related to Tsd2p, a protein required for DNA replication in Ustilago maydis. CDC45 also interacts genetically with ORC2, the gene encoding the second subunit of the origin recognition complex, ORC, and MCM3, another member of the MCM family. The cdc45-1 mutant has a plasmid maintenance defect which is rescued by the addition of multiple potential origins to the plasmid.     

Identification of aspartate-184 as an essential residue in the catalytic subunit of cAMP-dependent protein kinase

The hydrophobic carbodiimide dicyclohexylcarbodiimide (DCCD) was previously shown to be an irreversible inhibitor of the catalytic subunit of cAMP-dependent protein kinase, and MgATP protected against inactivation [Toner-Webb, J., & Taylor, S. S. (1987) Biochemistry 26, 7371]. This inhibition by DCCD indicated that an essential carboxyl group was present at the active site of the enzyme even though identification of that carboxyl group was not possible. This presumably was because a nucleophile on the protein cross-linked to the electrophilic intermediate formed when the carbodiimide reacted with the carboxyl group. To circumvent this problem, the catalytic subunit first was treated with acetic anhydride to block accessible lysine residues, thus preventing intramolecular cross-linking. The DCCD reaction then was carried out in the presence of [14C]glycine ethyl ester in order to trap any electrophilic intermediates that were generated by DCCD. The modified protein was treated with trypsin, and the resulting peptides were separated by HPLC. Two major radioactive peptides were isolated as well as one minor peptide. MgATP protected all three peptides from covalent modification. The two major peaks contained the same modified carboxyl group, which corresponded to Asp-184. The minor peak contained a modified glutamic acid, Glu-91. Both of these acidic residues are conserved in all protein kinases, which is consistent with their playing essential roles. The positions of Asp-184 and Glu-91 have been correlated with the overall domain structure of the molecule. Asp-184 may participate as a general base catalyst at the active site. A third carboxyl group, Glu-230, also was identified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of the naphthalene-derived inhibitors against Cdc25A dual-specificity protein phosphatase and their biological activity

The novel naphthalene-type analogues 14 and 18 and the naphthoquinone-type analogues, 8, 9, 15, 16, 19, 21, 22, and 23-28 have been synthesized, and their in vitro Cdc25A phosphatase-inhibitory activity was examined. In assessment of the inhibitory activity, it was revealed that the naphthoquinone core is contributed to the activity, rather than the alkyl side chain.     

Identification of an essential glutamic acid residue in beta-lactamase II from Bacillus cereus.

Beta-Lactamase II from Bacillus cereus was readily inactivated by incubation at pH 4.75 with a water-soluble carbodiimide plus a suitable nucleophile. In the early stages of the reaction, 1 equivalent of nucleophile was incorporated/equivalent of enzyme, whereas during the later stages a second equivalent of nucleophile was also incorporated. This latter process correlated with the blocking of the enzyme's single thiol group. Enzyme inactivated in the presence of the coloured nucleophile N-(2,4-dinitrophenyl)ethylenediamine was fragmented by pepsin digestion, and coloured peptides were isolated by gel filtration and h.p.l.c. Two major peptides, representing 52% of the incorporated label, were isolated and sequenced. Both peptides contained the incorporated label on glutamic acid-37, and it is concluded that this latter residue represents a catalytically essential carboxylic residue in beta-lactamase II.     

Regulation of Mih1/Cdc25 by protein phosphatase 2A and casein kinase 1

The Cdc25 phosphatase promotes entry into mitosis by removing cyclin-dependent kinase 1 (Cdk1) inhibitory phosphorylation. Previous work suggested that Cdc25 is activated by Cdk1 in a positive feedback loop promoting entry into mitosis; however, it has remained unclear how the feedback loop is initiated. To learn more about the mechanisms that regulate entry into mitosis, we have characterized the function and regulation of Mih1, the budding yeast homologue of Cdc25. We found that Mih1 is hyperphosphorylated early in the cell cycle and is dephosphorylated as cells enter mitosis. Casein kinase 1 is responsible for most of the hyperphosphorylation of Mih1, whereas protein phosphatase 2A associated with Cdc55 dephosphorylates Mih1. Cdk1 appears to directly phosphorylate Mih1 and is required for initiation of Mih1 dephosphorylation as cells enter mitosis. Collectively, these observations suggest that Mih1 regulation is achieved by a balance of opposing kinase and phosphatase activities. Because casein kinase 1 is associated with sites of polar growth, it may regulate Mih1 as part of a signaling mechanism that links successful completion of growth-related events to cell cycle progression.