NORMAL GENOMIC

• Jeong DG et al.
• Structure of human DSP18, a member of the dual-specificity proteintyrosine phosphatase family.
• Acta Crystallogr D Biol Crystallogr. 2006; 62: 582-8
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• The human dual-specificity protein phosphatase 18 (DSP18) gene and itsprotein product have recently been characterized. Like most DSPs, DSP18displays dephosphorylating activity towards both phosphotyrosine andphosphothreonine residues. However, DSP18 is distinct from other knownDSPs in terms of the existence of approximately 30 residues at theC-terminus of the catalytic domain and an unusual optimum activity profileat 328 K. The crystal structure of human DSP18 has been determined at 2.0A resolution. The catalytic domain of DSP18 adopts a fold similar to thatknown for other DSP structures. Although good alignments are found withother DSPs, substantial differences are also found in the regionssurrounding the active site, suggesting that DSP18 constitutes a uniquestructure with a distinct substrate specificity. Furthermore, the residuesat the C-terminus fold into two antiparallel beta-strands and participatein extensive interactions with the catalytic domain, explaining thethermostability of DSP18.

• Uto K, Inoue D, Shimuta K, Nakajo N, Sagata N
• Chk1, but not Chk2, inhibits Cdc25 phosphatases by a novel commonmechanism.
• EMBO J. 2004; 23: 3386-96
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• Cdc25 phosphatases activate cyclin-dependent kinases (Cdks) and therebypromote cell cycle progression. In vertebrates, Chk1 and Chk2phosphorylate Cdc25A at multiple N-terminal sites and target it for rapiddegradation in response to genotoxic stress. Here we show that Chk1, butnot Chk2, phosphorylates Xenopus Cdc25A at a novel C-terminal site(Thr504) and inhibits it from C-terminally interacting with variousCdk-cyclin complexes, including Cdk1-cyclin A, Cdk1-cyclin B, andCdk2-cyclin E. Strikingly, this inhibition, rather than degradationitself, of Cdc25A is essential for the Chk1-induced cell cycle arrest andthe DNA replication checkpoint in early embryos. 14-3-3 proteins bind toChk1-phosphorylated Thr504, but this binding is not required for theinhibitory effect of Thr504 phosphorylation. A C-terminal site presumablyequivalent to Thr504 exists in all known Cdc25 family members from yeastto humans, and its phosphorylation by Chk1 (but not Chk2) can also inhibitall examined Cdc25 family members from C-terminally interacting with theirCdk-cyclin substrates. Thus, Chk1 but not Chk2 seems to inhibit virtuallyall Cdc25 phosphatases by a novel common mechanism.

• Ceulemans H, Vulsteke V, De Maeyer M, Tatchell K, Stalmans W, Bollen M
• Binding of the concave surface of the Sds22 superhelix to the alpha4/alpha 5/alpha 6-triangle of protein phosphatase-1.
• J Biol Chem. 2002; 277: 47331-7
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• Functional studies of the protein phosphatase-1 (PP1) regulator Sds22suggest that it is indirectly and/or directly involved in one of the mostancient functions of PP1, i.e. reversing phosphorylation by theAurora-related protein kinases. We predict that the conserved portion ofSds22 folds into a curved superhelix and demonstrate that mutation toalanine of any of eight residues (Asp(148), Phe(170), Glu(192), Phe(214),Asp(280), Glu(300), Trp(302), or Tyr(327)) at the concave surface of thissuperhelix thwarts the interaction with PP1. Furthermore, we show that allmammalian isoforms of PP1 have the potential to bind Sds22. Interactionstudies with truncated versions of PP1 and with chimeric proteinscomprising fragments of PP1 and the yeast PP1-like protein phosphatasePpz1 suggest that the site(s) required for the binding of Sds22 residebetween residues 43 and 173 of PP1gamma(1). Within this region, a majorinteraction site was mapped to a triangular region delineated by thealpha4-, alpha5-, and alpha6-helices. Our data also show that well knownregulatory binding sites of PP1, such as the RVXF-binding channel, thebeta12/beta13-loop, and the acidic groove, are not essential for theinteraction with Sds22.

• Wang B, Lemay S, Tsai S, Veillette A
• SH2 domain-mediated interaction of inhibitory protein tyrosine kinase Cskwith protein tyrosine phosphatase-HSCF.
• Mol Cell Biol. 2001; 21: 1077-88
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• The protein tyrosine kinase (PTK) Csk is a potent negative regulator ofseveral signal transduction processes, as a consequence of its exquisiteability to inactivate Src-related PTKs. This function requires not onlythe kinase domain of Csk, but also its Src homology 3 (SH3) and SH2regions. We showed previously that the Csk SH3 domain mediates highlyspecific associations with two members of the PEP family of nonreceptorprotein tyrosine phosphatases (PTPs), PEP and PTP-PEST. In comparison, theCsk SH2 domain interacts with several tyrosine phosphorylated molecules,presumed to allow targetting of Csk to sites of Src family kinaseactivation. Herein, we attempted to understand better the regulation ofCsk by identifying ligands for its SH2 domain. Using a modified yeasttwo-hybrid screen, we uncovered the fact that Csk associates withPTP-HSCF, the third member of the PEP family of PTPs. This association wasdocumented not only in yeast cells but also in a heterologous mammaliancell system and in cytokine-dependent hemopoietic cells. Surprisingly, theCsk-PTP-HSCF interaction was found to be mediated by the Csk SH2 domainand two putative sites of tyrosine phosphorylation in the noncatalyticportion of PTP-HSCF. Transfection experiments indicated that Csk andPTP-HSCF synergized to inhibit signal transduction by Src family kinasesand that this cooperativity was dependent on the domains mediating theirassociation. Finally, we obtained evidence that PTP-HSCF inactivatedSrc-related PTKs by selectively dephosphorylating the positive regulatorytyrosine in their kinase domain. Taken together, these results demonstratethat part of the function of the Csk SH2 domain is to mediate an inducibleassociation with a PTP, thereby engineering a more efficient inhibitorymechanism for Src-related PTKs. Coupled with previously publishedobservations, these data also establish that Csk forms complexes with allthree known members of the PEP family.

• Sandhu C, Donovan J, Bhattacharya N, Stampfer M, Worland P, Slingerland J
• Reduction of Cdc25A contributes to cyclin E1-Cdk2 inhibition at senescencein human mammary epithelial cells.
• Oncogene. 2000; 19: 5314-23
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• Replicative senescence may be an important tumor suppressive mechanism forhuman cells. We investigated the mechanism of cell cycle arrest atsenescence in human mammary epithelial cells (HMECs) that have undergone aperiod of 'self-selection', and as a consequence exhibit diminishedp16INK4A levels. As HMECs approached senescence, the proportion of cellswith a 2N DNA content increased and that in S phase decreasedprogressively. Cyclin D1-cdk4, cyclin E-cdk2 and cyclin A-cdk2 activitieswere not abruptly inhibited, but rather diminished steadily withincreasing population age. In contrast to observations in fibroblast,p21Cip1 was not increased at senescence in HMECs. There was no increase inp27Kip1 levels nor in KIP association with targets cdks. While p15INK4Band its binding to both cdk4 and cdk6 increased with increasing passage,some cyclin D1-bound cdk4 and cdk6 persisted in senescent cells, whoseinhibition could not be attributed to p15INK4B. The inhibition of cyclinE-cdk2 in senescent HMECs was accompanied by increased inhibitoryphosphorylation of cdk2, in association with a progressive loss of Cdc25A.Recombinant Cdc25A strongly reactivated cyclin E-cdk2 from senescent HMECssuggesting that reduction of Cdc25A contributes to cyclin E-cdk2inhibition and G1 arrest at senescence. Although ectopic expression ofCdc25A failed to extend the lifespan of HMECs, the exogenous Cdc25Aappeared to lack activity in these cells, since it neither shortened theG1-to-S phase interval nor activated cyclin E-cdk2. In contrast, in thebreast cancer-derived MCF-7 line, Cdc25A overexpression increased bothcyclin E-cdk2 activity and the S phase fraction. Thus, mechanisms leadingto HMEC immortalization may involve not only the re-induction of Cdc25Aexpression, but also activation of this phosphatase.

• Tanoue T, Moriguchi T, Nishida E
• Molecular cloning and characterization of a novel dual specificityphosphatase, MKP-5.
• J Biol Chem. 1999; 274: 19949-56
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• A group of dual specificity protein phosphatases negatively regulatesmembers of the mitogen-activated protein kinase (MAPK) superfamily, whichconsists of three major subfamilies, MAPK/extracellular signal-regulatedkinase (ERK), stress-activated protein kinase (SAPK)/c-Jun N-terminalkinase (JNK), and p38. Nine members of this group of dual specificityphosphatases have previously been cloned. They show distinct substratespecificities for MAPKs, different tissue distribution and subcellularlocalization, and different modes of inducibility of their expression byextracellular stimuli. Here we have cloned and characterized a novel dualspecificity phosphatase, which we have designated MKP-5. MKP-5 is aprotein of 482 amino acids with a calculated molecular mass of 52.6 kDaand consists of 150 N-terminal amino acids of unknown function, two Cdc25homology 2 regions in the middle, and a C-terminal catalytic domain. MKP-5binds to p38 and SAPK/JNK, but not to MAPK/ERK, and inactivates p38 andSAPK/JNK, but not MAPK/ERK. p38 is a preferred substrate. The subcellularlocalization of MKP-5 is unique; it is present evenly in both thecytoplasm and the nucleus. MKP-5 mRNA is widely expressed in varioustissues and organs, and its expression in cultured cells is elevated bystress stimuli. These results suggest that MKP-5 is a novel type of dualspecificity phosphatase specific for p38 and SAPK/JNK.

• Kim SH, Li C, Maller JL
• A maternal form of the phosphatase Cdc25A regulates early embryonic cellcycles in Xenopus laevis.
• Dev Biol. 1999; 212: 381-91
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• In mammalian cells the Cdc25 family of dual-specificity phosphatases hasthree distinct isoforms, termed A, B, and C, which are thought to playdiscrete roles in cell-cycle control. In this paper we report the cloningof Xenopus Cdc25A and demonstrate its developmental regulation and keyrole in embryonic cell-cycle control. Northern and Western blot analysesshow that Cdc25A is absent in oocytes, and synthesis begins within 30 minafter fertilization. The protein product is localized in the nucleus ininterphase and accumulates continuously until the midblastula transition(MBT), after which it is degraded. Upon injection into newly fertilizedeggs, wild-type Cdc25A shortened the cell cycle and accelerated the timingof cleavage, whereas embryos injected with phosphatase-dead Cdc25Adisplayed a dose-dependent increase in the length of the cell cycle and aslower rate of cleavage. In contrast, injection of the phosphatase-deadCdc25C isoform had no effect. Western blotting with an antibody specificfor phosphorylated tyr15 in Cdc2/Cdk2 revealed a cycle ofphosphorylation/dephosphorylation in each cell cycle in control embryos,and in embryos injected with phosphatase-dead Cdc25A there was a twofoldincrease in the level of p-tyr in Cdc2/Cdk2. Consistent with this, thelevels of cyclin B/Cdc2 and cyclin E/Cdk2 histone H1 kinase activity wereboth reduced by approximately 50% after phosphatase-dead Cdc25A injection.The phosphatase-dead Cdc25A could be recovered in a complex with bothCdks, suggesting that it acts in a dominant-negative fashion. Theseresults indicate that periodic phosphorylation of Cdc2/Cdk2 on tyr15occurs in each pre-MBT cell cycle, and dephosphorylation of Cdc2/Cdk2 byCdc25A controls at least in part the length of the cell cycle and thetiming of cleavage in pre-MBT embryos. The disappearance of Cdc25A afterthe MBT may underlie in part the lengthening of the cell cycle at thattime.

• Zhao S, Lee EY
• A protein phosphatase-1-binding motif identified by the panning of arandom peptide display library.
• J Biol Chem. 1997; 272: 28368-72
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• An unusually large number of regulatory or targeting proteins that bind tothe catalytic subunit of protein phosphatase-1 have been recentlyreported. This can be explained by their possession of a common proteinmotif that interacts with a binding site on protein phosphatase-1. Theexistence of such a motif was established by the panning of a randompeptide library in which peptide sequences are displayed on theEscherichia coli bacterial flagellin protein for bacteria that bound toprotein phosphatase-1. There were 79 isolates containing 46 uniquesequences with the conserved motif VXF or VXW, where X was most frequentlyHis or Arg. In addition, this sequence was commonly preceded by 2-5 basicresidues and followed by 1 acidic residue. This study demonstrates thatbinding to protein phosphatase-1 can be conferred to a protein by thepresentation of a peptide motif on a surface loop. This binding motif isfound in a number of protein phosphatase-1-binding proteins.

• Dixon JE
• Protein tyrosine phosphatases: their roles in signal transduction.
• Recent Prog Horm Res. 1996; 51: 405-14
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• Protein tyrosine phosphatases play critical roles in a number of cellular signal transduction pathways. Receptor-like PTPases such as CD45 are essential for antigen-induced proliferative responses of T-cells. Intracellular PTPases have been shown to associate with specific growth factor receptors and this association has a dramatic effect on receptor signaling mechanisms. Other phosphatases (e.g., the product of the CDC25 gene) are essential for cell cycle progression. It appears that the cellular location of the intracellular PTPases plays an important role in defining the substrate specificity. Phosphatases are also present in both pathogenic bacteria and viruses. These PTPases most likely function to disrupt important signal transduction pathways present in the host. More than 30 different phosphatases have been cloned and characterized. A detailed understanding of their catalytic properties suggests that all PTPases use a common mechanism for removing phosphatase from various phosphoproteins. Two PTPase structures recently have been determined. The structural information along with biochemical and kinetic data provides a basis for understanding the catalytic properties of these enzymes.

• Vinals C, De Bolle X, Depiereux E, Feytmans E
• Knowledge-based modeling of the D-lactate dehydrogenase three-dimensionalstructure.
• Proteins. 1995; 21: 307-18
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• A three-dimensional structure of the NAD-dependent D-lactate dehydrogenaseof Lactobacillus bulgaricus is modeled using the structure of the formatedehydrogenase of Pseudomonas sp. as template. Both sequences share only22% of identical residues. Regions for knowledge-based modeling aredefined from the structurally conserved regions predicted by multiplealignment of a set of related protein sequences with low homology. Themodel of the D-LDH subunit shows, as for the formate dehydrogenase, analpha/beta structure, with a catalytic domain and a coenzyme bindingdomain. It points out the catalytic histidine (His-296) and supports thehypothetical catalytic mechanism. It also suggests that the other residuesinvolved in the active site are Arg-235, possibly involved in the bindingof the carboxyl group of the pyruvate, and Phe-299, a candidate forstabilizing the methyl group of the substrate.

• Conklin DS, Galaktionov K, Beach D
• 14-3-3 proteins associate with cdc25 phosphatases.
• Proc Natl Acad Sci U S A. 1995; 92: 7892-6
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• The cdc25 phosphatases play key roles in cell cycle progression by activating cyclin-dependent kinases. Two members of the 14-3-3 protein family have been isolated in a yeast two-hybrid screen designed to identify proteins that interact with the human cdc25A and cdc25B phosphatases. Genes encoding the human homolog of the 14-3-3 epsilon protein and the previously described 14-3-3 beta protein have been isolated in this screening. 14-3-3 proteins constitute a family of well-conserved eukaryotic proteins that were originally isolated in mammalian brain preparations and that possess diverse biochemical activities related to signal transduction. We present evidence that indicates that cdc25 and 14-3-3 proteins physically interact both in vitro and in vivo. 14-3-3 protein does not, however, affect the phosphatase activity of cdc25A. Raf-1, which is known to bind 14-3-3 proteins, has recently been shown to associate with cdc25A and to stimulate its phosphatase activity. 14-3-3 protein, however, has no effect on the cdc25A-kinase activity of Raf-1. Instead, 14-3-3 may facilitate the association of cdc25 with Raf-1 in vivo, participating in the linkage between mitogenic signaling and the cell cycle machinery.

• Taylor SS, Knighton DR, Zheng J, Ten Eyck LF, Sowadski JM
• Structural framework for the protein kinase family.
• Annu Rev Cell Biol. 1992; 8: 429-62
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• In this review, we have summarized the general structural features of the catalytic subunit of cAMP-dependent protein kinase, emphasizing those features that will very likely be conserved in all members of the protein kinase family. The overall secondary structure of the catalytic core will probably be conserved throughout the catalytic core, as will the active site regions associated with MgATP binding and catalysis. The mechanisms for activation and the role of protein phosphorylation are unique for each kinase. The structure of the catalytic subunit now provides a general framework for modeling other protein kinases. Although this is no substitute for a crystal structure for each protein kinase, this one structure, nevertheless, does provide major insights to the molecular organization of each of these enzymes.

• Kumagai A, Dunphy WG
• Regulation of the cdc25 protein during the cell cycle in Xenopus extracts.
• Cell. 1992; 70: 139-51
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• The cdc25 protein is a highly specific tyrosine phosphatase that triggersmitosis by dephosphorylating the cdc2 protein kinase. Using Xenopusextracts, we have found that the cdc25 protein is active at a low levelthroughout interphase. Near the onset of mitosis, the cdc25 proteinundergoes a marked elevation in phosphatase activity that coincides withan extensive phosphorylation of the protein in its N-terminal region. Invitro dephosphorylation of this hyperphosphorylated form of cdc25 reducesits phosphatase activity back to the interphase level. Moreover, treatmentof interphase Xenopus extracts with okadaic acid, a phosphatase inhibitorthat accelerates the entry into mitosis, elicits both the prematurehyperphosphorylation of cdc25 and the stimulation of its cdc2-specifictyrosine phosphatase activity. These experiments demonstrate the existenceof a cdc25 regulatory system consisting of both a stimulatory kinase thatphosphorylates a putative regulatory domain of the cdc25 protein and aninhibitory serine/threonine phosphatase that counteracts this kinaseactivity.