Dual-specificity Phosphatase Family Research Articles

Pseudo-phosphatase Sbf1 contains an N-terminal GEF homology domain that modulates its growth regulatory properties.

Sbf1 (SET binding factor 1) is a pseudo-phosphatase related to the myotubularin family of dual specificity phosphatases, some of which have been implicated in cellular growth and differentiation by virtue of their mutation in human genetic disorders. Sbf1 contains germline-encoded alterations of its myotubularin homology domain that render it non-functional as a phosphatase. We report here the complete structure of Sbfl and further characterization of its growth regulatory properties. In addition to its similarity to myotubularin, the predicted full-length Sbf1 protein contains pleckstrin (PH) and GEF homology domains that are conserved in several proteins implicated in signaling and growth control. Forced expression of wild-type Sbfl in NIH 3T3 cells inhibited their proliferation and altered their morphology. These effects required intact PH, GEF and myotubularin homology domains, implying that growth inhibition may be an intrinsic property of wild-type Sbf1. Conversely, deletion of its conserved N-terminal 44 amino acids alone was sufficient to convert Sbf1 from an inhibitor of cellular growth to a transforming protein in NIH 3T3 cells. Oncogenic forms of Sbf1 partially localized to the nucleus, in contrast to the exclusively cytoplasmic subcellular localization of endogenous Sbf1 in all cell lines and mammalian tissues tested. These data show that the N-terminal GEF homology domain serves to inhibit the transforming effects of Sbf1, possibly sequestering the protein to the cytoplasm, and suggest that this region may be a modulatory domain that relays growth control signals.

A growing family of dual specificity phosphatases with low molecular masses.

Five putative dual specificity protein phosphatases (DSPs), designated LMW-DSP1, -DSP4, -DSP6, -DSP10, and -DSP11, were cloned with a combination of RT-PCR and cDNA library screening strategies. Sequencing analysis revealed that all lacked the cdc25 homology domain that is conserved in most known DSPs/MAP kinase phosphatases (MKPs). LMW-DSP1 exhibited the highest similarity to plant DSPs. LMW-DSP4 exhibited the highest similarity to human YVH1 and rat GKAP, but its C-terminal region was much shorter than that of the human and rat clones. LMW-DSP6 was found to be identical to recently cloned TMDP, and LMW-DSP11 seemed to be a mouse ortholog of human VHR. LMW-DSP10 was found to have a DSP catalytic-like domain, but the critical cysteine residue for catalytic activity was missing. Recombinant LMW-DSP1, -DSP6, and -DSP11 exhibited obvious and strong activity against an artificial low molecular substrate, para-nitrophenyl phosphate (pNPP). Recombinant LMW-DSP4 exhibited slight but significant activity, whereas no activity was detected for LMW-DSP10. The phosphatase activity of the recombinant LMW-DSPs was inhibited by orthovanadate but not sodium fluoride. However, none of the DSPs could dephosphorylate MAP kinases such as ERK1, p38, and SAPK/JNK in transiently transfected COS7 cells under the conditions used. Northern blot analysis revealed that LMW-DSP1, -DSP6, -DSP10, and -DSP11 were specifically expressed in testis, while LMW-DSP4 was broadly expressed. The testis-specific expression and apparent absence of dephosphorylation action on MAP kinases suggest that LMW-DSP1, -DSP6, -DSP10, and -DSP11 play specific roles in testis. Taken together, it is conceivable that a distinct class of low molecular mass DSPs is present and plays a role in dephosphorylating unknown molecules other than MAP kinases.

The dual-specificity phosphatase MKP-1 limits the cardiac hypertrophic response in vitro and in vivo.

Mitogen-activated protein kinase (MAPK) signaling pathways are important regulators of cell growth, proliferation, and stress responsiveness. A family of dual-specificity MAP kinase phosphatases (MKPs) act as critical counteracting factors that directly regulate the magnitude and duration of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) activation. Here we show that constitutive expression of MKP-1 in cultured primary cardiomyocytes using adenovirus-mediated gene transfer blocked the activation of p38, JNK1/2, and ERK1/2 and prevented agonist-induced hypertrophy. Transgenic mice expressing physiological levels of MKP-1 in the heart showed (1) no activation of p38, JNK1/2, or ERK1/2; (2) diminished developmental myocardial growth; and (3) attenuated hypertrophy in response to aortic banding and catecholamine infusion. These results provide further evidence implicating MAPK signaling factors as obligate regulators of cardiac growth and hypertrophy and demonstrate the importance of dual-specificity phosphatases as counterbalancing regulatory factors in the heart.

Mutation spectrum and predicted function of laforin in Lafora's progressive myoclonus epilepsy.

Lafora's disease is a progressive myoclonus epilepsy with pathognomonic inclusions (polyglucosan bodies) caused by mutations in the EPM2A gene. EPM2A codes for laforin, a protein with unknown function. Mutations have been reported in the last three of the gene's exons. To date, the first exon has not been determined conclusively. It has been predicted based on genomic DNA sequence analysis including comparison with the mouse homologue. 1) To detect new mutations in exon 1 and establish the role of this exon in Lafora's disease. 2) To generate hypotheses about the biological function of laforin based on bioinformatic analyses. 1) PCR conditions and components were refined to allow amplification and sequencing of the first exon of EPM2A. 2) Extensive bioinformatic analyses of the primary structure of laforin were completed. 1) Seven new mutations were identified in the putative exon 1. 2) Laforin is predicted not to localize to the cell membrane or any of the organelles. It contains all components of the catalytic active site of the family of dual-specificity phosphatases. It contains a sequence predicted to encode a carbohydrate binding domain (coded by exon 1) and two putative glucohydrolase catalytic sites. The identification of mutations in exon 1 of EPM2A establishes its role in the pathogenesis of Lafora's disease. The presence of potential carbohydrate binding and cleaving domains suggest a role for laforin in the prevention of accumulation of polyglucosans in healthy neurons.

Dual G1 and G2/M phase inhibition by SC-alpha alpha delta 9, a combinatorially derived Cdc25 phosphatase inhibitor.

The Cdc25 dual specificity phosphatase family has a central role in controlling cell cycle progression and has been implicated in the etiology of cancer. One compound, 4-(benzyl-(2-[(2, 5-diphenyl-oxazole-4-carbonyl)-amino]-ethyl)-carbamoyl)-2-decanoylami no butyric acid (SC-alpha alpha delta 9), was previously identified as the most potent reported synthetic inhibitor of Cdc25 phosphatases in vitro. In the present study, we demonstrate that SC-alpha alpha delta 9 inhibited Cdc25-dependent cell cycle progression at both G1 and G2/M phase using tsFT210 cells, which express a temperature-sensitive Cdc2 mutant. SC-alpha alpha delta 9 blocked both G2/M transition and dephosphorylation of Cdc2 in a concentration-dependent manner. SC-alpha alpha delta 9 also enhanced tyrosine phosphorylation of both Cdk2 and Cdk4, and decreased Cdk4 kinase activity. Both of the kinases are potent regulators of G1 transition. Furthermore, closely related chemical analogs that lacked Cdc25 inhibitory activity failed to block cell cycle progression at both G1 and G2/M, and did not affect Cdc2 phosphorylation or Cdk4 kinase activity. SC-alpha alpha delta 9 did not alter p53, p21 or p16 levels. Our results support the hypothesis that the disruption in cell cycle transition caused by SC-alpha alpha delta 9 was due to intracellular Cdc25 inhibition. We propose that the SC-alpha alpha delta 9 pharmacophore could be useful in further clarifying the role of Cdc25 phosphatase-dependent pathways in checkpoint control, oncogenesis, and apoptosis.

A Maternal Form of the Phosphatase Cdc25A Regulates Early Embryonic Cell Cycles in Xenopus laevis

In mammalian cells the Cdc25 family of dual-specificity phosphatases has three distinct isoforms, termed A, B, and C, which are thought to play discrete roles in cell-cycle control. In this paper we report the cloning of Xenopus Cdc25A and demonstrate its developmental regulation and key role in embryonic cell-cycle control. Northern and Western blot analyses show that Cdc25A is absent in oocytes, and synthesis begins within 30 min after fertilization. The protein product is localized in the nucleus in interphase and accumulates continuously until the midblastula transition (MBT), after which it is degraded. Upon injection into newly fertilized eggs, wild-type Cdc25A shortened the cell cycle and accelerated the timing of cleavage, whereas embryos injected with phosphatase-dead Cdc25A displayed a dose-dependent increase in the length of the cell cycle and a slower rate of cleavage. In contrast, injection of the phosphatase-dead Cdc25C isoform had no effect. Western blotting with an antibody specific for phosphorylated tyr15 in Cdc2/Cdk2 revealed a cycle of phosphorylation/dephosphorylation in each cell cycle in control embryos, and in embryos injected with phosphatase-dead Cdc25A there was a twofold increase in the level of p-tyr in Cdc2/Cdk2. Consistent with this, the levels of cyclin B/Cdc2 and cyclin E/Cdk2 histone H1 kinase activity were both reduced by approximately 50% after phosphatase-dead Cdc25A injection. The phosphatase-dead Cdc25A could be recovered in a complex with both Cdks, suggesting that it acts in a dominant-negative fashion. These results indicate that periodic phosphorylation of Cdc2/Cdk2 on tyr15 occurs in each pre-MBT cell cycle, and dephosphorylation of Cdc2/Cdk2 by Cdc25A controls at least in part the length of the cell cycle and the timing of cleavage in pre-MBT embryos. The disappearance of Cdc25A after the MBT may underlie in part the lengthening of the cell cycle at that time.

RNA-Mediated Interference of acdc25Homolog inCaenorhabditis elegansResults in Defects in the Embryonic Cortical Membrane, Meiosis, and Mitosis

The CDC25 dual-specificity phosphatase family has been shown to play a key role in cell cycle regulation. The phosphatase activity of CDC25 drives the cell cycle by removing inhibitory phosphates from cyclin-dependent kinase/cyclin complexes. Although the regulation of CDC25 phosphatase activity has been elucidated both biochemically and genetically in other systems, the role of this enzyme during development is not well understood. To examine the expression pattern and function of CDC25 inCaenorhabditis elegans, we characterized acdc25homolog,cdc-25.1, during early embryonic development. The CDC-25.1 protein localizes to oocytes, embryonic nuclei, and embryonic cortical membranes. When the expression of CDC-25.1 was disrupted by RNA-mediated interference, the anterior cortical membrane of fertilized eggs became very fluid during meiosis and subsequent mitotic cell cycles. Mispositioning of the meiotic spindle, defects in polar body extrusion and chromosome segregation, and abnormal cleavage furrows were also observed. We conclude that CDC-25.1 is required for a very early developmental process—the proper completion of meiosis prior to embryogenesis.

The role of protein phosphatases in the regulation of mitogen and stress-activated protein kinases

It is now established that a family of dual-specificity protein phosphatases are able to interact with mitogen and stress-activated protein kinases in a highly specific manner to differentially regulate these enzymes in mammalian cells. A role for these proteins in negative feedback regulation of MAP kinase activity is also supported by genetic and biochemical studies in yeasts and Drosophila. More recently it has become clear that other classes of protein phosphatase also play key roles in the regulated dephosphorylation of MAP kinases, including tyrosine-specific protein phosphatases and serine/threonine protein phosphatases. It is likely that a complex balance between upstream activators and these different classes of MAP kinase specific phosphatase are responsible for determining, at least in part, the magnitude and duration of MAP kinase activation and hence the physiological outcome of signalling.

PTP-SL and STEP protein tyrosine phosphatases regulate the activation of the extracellular signal-regulated kinases ERK1 and ERK2 by association through a kinase interaction motif.

Protein kinases and phosphatases regulate the activity of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by controlling the phosphorylation of specific residues. We report the physical and functional association of ERK1/2 with the PTP-SL and STEP protein tyrosine phosphatases (PTPs). Upon binding, the N-terminal domains of PTP-SL and STEP were phosphorylated by ERK1/2, whereas these PTPs dephosphorylated the regulatory phosphotyrosine residues of ERK1/2 and inactivated them. A sequence of 16 amino acids in PTP-SL was identified as being critical for ERK1/2 binding and termed kinase interaction motif (KIM) (residues 224-239); it was shown to be required for phosphorylation of PTP-SL by ERK1/2 at Thr253. Co-expression of ERK2 with catalytically active PTP-SL in COS-7 cells impaired the EGF-induced activation of ERK2, whereas a PTP-SL mutant, lacking PTP activity, increased the ERK2 response to EGF. This effect was dependent on the presence of the KIM on PTP-SL. Furthermore, ERK1/2 activity was downregulated in 3T3 cells stably expressing PTP-SL. Our findings demonstrate the existence of a conserved ERK1/2 interaction motif within the cytosolic non-catalytic domains of PTP-SL and STEP, which is required for the regulation of ERK1/2 activity and for phosphorylation of the PTPs by these kinases. Our findings suggest that PTP-SL and STEP act as physiological regulators of the ERK1/2 signaling pathway.

Characterization of the myotubularin dual specificity phosphatase gene family from yeast to human.

X-linked myotubular myopathy (XLMTM) is a severe congenital muscle disorder due to mutations in the MTM1 gene. The corresponding protein, myotubularin, contains the consensus active site of tyrosine phosphatases (PTP) but otherwise shows no homology to other phosphatases. Myotubularin is able to hydrolyze a synthetic analogue of tyrosine phosphate, in a reaction inhibited by orthovanadate, and was recently shown to act on both phosphotyrosine and phosphoserine. This gene is conserved down to yeast and strong homologies were found with human ESTs, thus defining a new dual specificity phosphatase (DSP) family. We report the presence of novel members of the MTM gene family in Schizosaccharomyces pombe, Caenorhabditis elegans, zebrafish, Drosophila, mouse and man. This represents the largest family of DSPs described to date. Eight MTM-related genes were found in the human genome and we determined the chromosomal localization and expression pattern for most of them. A subclass of the myotubularin homologues lacks a functional PTP active site. Missense mutations found in XLMTM patients affect residues conserved in a Drosophila homologue. Comparison of the various genes allowed construction of a phylogenetic tree and reveals conserved residues which may be essential for function. These genes may be good candidates for other genetic diseases.

Regulation of growth factor-induced signaling by protein-tyrosine-phosphatases.

The binding of a growth factor to its specific receptor catalyzes a complex cascade of intracellular signaling events, characterized by changes in the phosphorylation state of many key proteins. Among these phosphorylation events, tyrosine phosphorylation plays a prominent role in the transmission of postreceptor signals. The state of tyrosine phosphorylation is regulated by the actions of protein-tyrosine kinases (PTKs) and protein-tyrosine-phosphatases (PTPs). Dysregulation of either event can lead to abnormal cellular responses. PTPs generally act to regulate negatively-that is, to turn off-any signals generated by PTKs. However, this is not always the case, as seen by the phosphatase SHP-2, which can either be a positive or negative regulator of signal transduction depending on the particular cellular context. In addition, a novel family of dual specificity phosphatases has been recently discovered. These enzymes are capable of dephosphorylating phosphotyrosine and phosphothreonine/phosphoserine residues, and seem to play a significant role in attenuating the action of MAP kinases. Several themes appear throughout PTP regulation of growth factor signaling, including positive or negative regulation, importance of cell/ tissue type, identity of the receptor activated, and subcellular localization. Although only a handful of PTPs have been identified, the present work done in elucidating their function has revealed their significance in the maintenance of normal physiological responses to growth factors.

A dual-specificity phosphatase Cdc25B is an unstable protein and triggers p34(cdc2)/cyclin B activation in hamster BHK21 cells arrested with hydroxyurea.

By incubating at 30 degrees C in the presence of an energy source, p34(cdc2)/cyclin B was activated in the extract prepared from a temperature-sensitive mutant, tsBN2, which prematurely enters mitosis at 40 degrees C, the nonpermissive temperature (Nishimoto, T. , E. Eilen, and C. Basilico. 1978. Cell. 15:475-483), and wild-type cells of the hamster BHK21 cell line arrested in S phase, without protein synthesis. Such an in vitro activation of p34(cdc2)/cyclin B, however, did not occur in the extract prepared from cells pretreated with protein synthesis inhibitor cycloheximide, although this extract still retained the ability to inhibit p34(cdc2)/cyclin B activation. When tsBN2 cells arrested in S phase were incubated at 40 degrees C in the presence of cycloheximide, Cdc25B, but not Cdc25A and C, among a family of dual-specificity phosphatases, Cdc25, was lost coincidentally with the lack of the activation of p34(cdc2)/cyclin B. Consistently, the immunodepletion of Cdc25B from the extract inhibited the activation of p34(cdc2)/cyclin B. Cdc25B was found to be unstable (half-life < 30 min). Cdc25B, but not Cdc25C, immunoprecipitated from the extract directly activated the p34(cdc2)/cyclin B of cycloheximide-treated cells as well as that of nontreated cells, although Cdc25C immunoprecipitated from the extract of mitotic cells activated the p34(cdc2)/cyclin B within the extract of cycloheximide-treated cells. Our data suggest that Cdc25B made an initial activation of p34(cdc2)/cyclin B, which initiates mitosis through the activation of Cdc25C.

Chromosomal Localization of Three Human Dual Specificity Phosphatase Genes (DUSP4, DUSP6, and DUSP7)

Mitogen-activated protein (MAP) kinase phosphatases constitute a growing family of dual specificity phosphatases thought to play a role in the dephosphorylation and inactivation of MAP kinases and are therefore likely to be important in the regulation of diverse cellular processes such as proliferation, differentiation, and apoptosis. For this reason it has been suggested that MAP kinase phosphatases may be tumor suppressors. We have determined the chromosomal locations of three human dual specificity phosphatase genes by fluorescencein situhybridization and radiation hybrid mapping. The genes were localized to three different chromosomes,MKP2(DUSP4) to 8p11–p12,MKP3(DUSP6) to 12q22–q23, andMKPX(DUSP7) to 3p21. This will allow the potential roles of these genes in disease processes to be evaluated.

Mitogen-activated Protein Kinase Phosphatases Inactivate Stress-activated Protein Kinase Pathways in Vivo

The c-Jun N-terminal protein kinases (JNKs), also called stress-activated protein kinases, are members of the growing family of serine/threonine kinases in the mitogen-activated protein (MAP) kinase superfamily. Like other MAP kinases, JNKs are activated via phosphorylation on adjacent threonine and tyrosine residues and can be inactivated by a unique family of dual specificity phosphatases, called MAP kinase phosphatases (MKPs). MKPs are encoded by immediate early genes and induced in response to environmental stressors and growth factor stimulation. Two prevalent isoforms of MKP, MKP1 and MKP2, are co-expressed in a wide variety of cell types. In this study, we examined the actions of MKP1 and MKP2 on JNK1 and JNK2. JNK1 phosphorylation and activation was inhibited by expression of both MKP1 and MKP2, although MKP1 selectivity toward JNK1 appeared significantly higher than that of MKP2. In contrast, JNK2 activity was inhibited by either phosphatase to similar degrees. Both MKP1 and MKP2 were highly effective at blocking the activation of the physiological target of JNK activation, the transcription factor c-Jun. In PC12 cells, MKP1 and MKP2 are transcriptionally induced following stimulation by nerve growth factor. In these cells, UV light-evoked JNK activation was reduced by pretreatment with nerve growth factor. Therefore, JNKs may be selective targets of MKP action in certain cells.

The Dual Specificity Mitogen-activated Protein Kinase Phosphatase-1 and -2 Are Induced by the p42/p44MAPK Cascade

Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) and MKP-2 are two members of a recently described family of dual specificity phosphatases that are capable of dephosphorylating p42/p44MAPK. Overexpression of MKP-1 or MKP-2 inhibits MAP kinase-dependent intracellular signaling events and fibroblast proliferation. By using specific antibodies that recognize endogenous MKP-1 and MKP-2 in CCL39 cells, we show that MKP-1 and MKP-2 are not expressed in quiescent cells, but are rapidly induced following serum addition, with protein detectable as early as 30 min (MKP-1) or 60 min (MKP-2). Serum induction of MKP-1 and MKP-2 is sustained, with protein detectable up to 14 h after serum addition. Induction of MKP-1 and, to a lesser extent, MKP-2 temporally correlates with p42/p44MAPK inactivation. To analyze the contribution of the MAP kinase cascade to MKP-1 and MKP-2 induction, we examined CCL39 cells transformed with either v-ras or a constitutively active direct upstream activator of MAP kinase, mitogen-activated protein kinase kinase-1 (MKK-1; MKK-1(SD/SD) mutant). In both cell models, MKP-1 and MKP-2 are constitutively expressed, with MKP-2 being prevalent. In addition, in CCL39 cells expressing an estradiol-inducible deltaRaf-1::ER chimera, activation of Raf alone is sufficient to induce MKP-1 and MKP-2. The role of the MAP kinase cascade in MKP induction was highlighted by the MKK-1 inhibitor PD 098059, which blunted both the activation of p42/p44MAPK and the induction of MKP-1 and MKP-2. However, the MAP kinase cascade is not absolutely required for the induction of MKP-1, as this phosphatase, but not MKP-2, was induced to detectable levels by agents that stimulate protein kinases A and C. Thus, activation of the p42/p44MAPK cascade promotes the induction of MKP-1 and MKP-2, which may then attenuate p42/p44MAPK-dependent events in an inhibitory feedback loop.

Differential regulation of the MAP, SAP and RK/p38 kinases by Pyst1, a novel cytosolic dual-specificity phosphatase.

The Pyst1 and Pyst2 mRNAs encode closely related proteins, which are novel members of a family of dual-specificity MAP kinase phosphatases typified by CL100/MKP-1. Pyst1 is expressed constitutively in human skin fibroblasts and, in contrast to other members of this family of enzymes, its mRNA is not inducible by either stress or mitogens. Furthermore, unlike the nuclear CL100 protein, Pyst1 is localized in the cytoplasm of transfected Cos-1 cells. Like CL100/ MKP-1, Pyst1 dephosphorylates and inactivates MAP kinase in vitro and in vivo. In addition, Pyst1 is able to form a physical complex with endogenous MAP kinase in Cos-1 cells. However, unlike CL100, Pyst1 displays very low activity towards the stress-activated protein kinases (SAPKs) or RK/p38 in vitro, indicating that these kinases are not physiological substrates for Pyst1. This specificity is underlined by the inability of Pyst1 to block either the stress-mediated activation of the JNK-1 SAP kinase or RK/p38 in vivo, or to inhibit nuclear signalling events mediated by the SAP kinases in response to UV radiation. Our results provide the first evidence that the members of the MAP kinase family of enzymes are differentially regulated by dual-specificity phosphatases and also indicate that the MAP kinases may be regulated by different members of this family of enzymes depending on their subcellular location.

Isolation and Characterization of a Novel Dual Specific Phosphatase, HVH2, Which Selectively Dephosphorylates the Mitogen-activated Protein Kinase

The mitogen-activated protein kinase (MAPK) also known as extracellular signal-regulated kinase (ERK) plays a crucial role in various signal transduction pathways. ERK is activated by its upstream activator, MEK, via threonine and tyrosine phosphorylation. ERK activity in the cell is tightly regulated by phosphorylation and dephosphorylation. Here we report the cloning and characterization of a novel dual specific phosphatase, HVH2, which may function in vivo as a MAP kinase phosphatase. The deduced amino acid sequence of HVH2 shows significant identity to the VH1-related dual specific phosphatase family. In addition, the N-terminal region of HVH2 also displays sequence identity to the cell cycle regulator, Cdc25 phosphatase. Recombinant HVH2 phosphatase exhibited a high substrate specificity toward activated ERK and dephosphorylated both threonine and tyrosine residues of activated ERK1 and ERK2. Immunofluorescence studies with an epitope-tagged HVH2 showed that the enzyme was localized in cell nucleus. Transfection of HVH2 into NIH3T3 cells inhibited the v-src and MEK-induced transcriptional activation of serum-responsive element containing promoter, consistent with the notion that HVH2 promotes the inactivation of MAP kinase. HVH2 mRNA showed an expression pattern distinct from CL100 (human homologue of mouse MKP1) and PAC1, two previously identified MAP kinase phosphatases. Our data suggest a possible role of HVH2 in MAP kinase regulation.