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PubMed1995

A catalytic mechanism for the dual-specific phosphatases.

Dual-specific protein-tyrosine phosphatases have the common active-site sequence motif HCXXGXXRS(T). The role of the conserved hydroxyl was investigated by changing serine-131 to an alanine (S131A) in the dual-specific protein-tyrosine phosphatase VHR. The pH profile of the kcat/Km value for the S131A mutant is indistinguishable from that of the native enzyme. In contrast, the kcat value for S131A mutant is 100-fold lower than that for the native enzyme, and the shape of the pH profile was perturbed from bell-shaped in the native enzyme to a pH-independent curve over the pH range 4.5-9.0. This evidence, along with results from a previous study, suggests that the S131A mutation alters the rate-limiting step in the catalytic mechanism. Formation of a phosphoenzyme intermediate appears to be rate-limiting with the native enzyme, whereas in the S131A mutant breakdown of the intermediate is rate-limiting. This was confirmed by the appearance of a burst of p-nitrophenol formation when p-nitrophenyl phosphate rapidly reacted with the S131A enzyme in a stopped-flow spectrophotometer. Loss of this hydroxyl group at the active site dramatically diminished the ability of the enzyme to hydrolyze the thiol-phosphate intermediate without exerting any significant change in the steps leading to and including the formation of the intermediate. Consistent with rate-limiting intermediate formation in the native enzyme, the rate of burst in the S131A mutant was 1.5 s-1, which agrees well with the kcat value of 5 s-1 observed for native enzyme. The amplitude of the burst was stoichiometric with final enzyme concentration, and the slow linear rate (0.06 s-1) of p-nitrophenol formation after the burst was in agreement with the steady-state determined value of kcat (0.055 s-1).

Alanine↗

PubMed1995

A single mutation converts a novel phosphotyrosine binding domain into a dual-specificity phosphatase.

Dual-specificity protein-tyrosine phosphatases (dsPTPases) have been implicated in the inactivation of mitogen-activated protein kinases (MAPKs). We have identified a novel phosphoserine/threonine/tyrosine-binding protein (STYX) that is related in amino acid sequence to dsPTPases, except for the substitution of Gly for Cys in the conserved dsPTPase catalytic loop (HCXXGXXR(S/T)). cDNA subcloning and Northern blot analysis in mouse shows poly(A+) hybridization bands of 4.6, 2.4, 1.5, and 1.2 kilobases, with highest abundance in skeletal muscle, testis, and heart. Polymerase chain reaction amplification of reverse-transcribed poly(A+) RNA revealed an alternatively spliced form of STYX containing a unique carboxyl terminus. Bacterially expressed STYX is incapable of hydrolyzing Tyr(P)-containing substrates; however, mutation of Gly120 to Cys (G120C), which structurally mimics the active site of dsPTPases, confers phosphatase activity to this molecule. STYX-G120C mutant hydrolyzes p-nitrophenyl phosphate and dephosphorylates both Tyr(P) and Thr(P) residues of peptide sequences of MAPK homologues. The kinetic parameters of dephosphorylation are similar to human dsPTPase, Vaccinia H1-related, including inhibition by vanadate. We believe this is the first example of a naturally occurring "dominant negative" phosphotyrosine/serine/threonine-binding protein which is structurally related to dsPTPases.

Alternative Splicing↗

PubMed2025

Dual-specific phosphatases-8: a new target for clinical disease intervention.

Dual-specific phosphatase-8 (DUSP8), identified as the first gene in a genome-wide association study (GWAS), is implicated in cellular oxidative stress, proliferation, apoptosis, and drug resistance through its negative regulation of the dephosphorylation activities of JNK, ERK, and p38 within the MAPK pathway. Recent studies have shown that DUSP8 plays a pivotal role in the progression of several human diseases, notably colorectal cancer, diabetic kidney disease, and breast cancer. This suggests that DUSP8 may represent a novel target for clinical intervention in these diseases. This review first introduces the biological structure and function of DUSP8, with a focus on its relationship with a series of diseases and the regulatory mechanisms involved. Furthermore, we concentrate on unresolved scientific questions in the current research, aiming to establish a new theoretical foundation for the diagnosis and treatment of related diseases.

Humans↗

PubMed1995

The dual-specificity phosphatase encoded by vaccinia virus, VH1, is essential for viral transcription in vivo and in vitro.

The genetic complexity of vaccinia virus is such that as well as encoding its own transcription and replication machinery, it encodes two protein kinases and a protein phosphatase. The latter enzyme, designated VH1, is a prototype for the dual-specificity class of phosphatases. Here we report that the H1 phosphatase is encapsidated within vaccinia virions and describe the construction of a viral recombinant in which expression of the H1 gene is regulated by the presence or absence of isopropylthiogalactopyranoside (IPTG) in the culture medium. When expression of H1 is repressed, the number of viral particles produced is not compromised but the fraction of these particles which is infectious is significantly reduced. The lack of infectivity of the H1-deficient particles is specifically correlated with their inability to direct the transcription of early genes either in vitro or in vivo. A proximal role for the viral phosphatase in regulating the onset of viral gene expression is implied. Prominent among the encapsidated proteins found to be hyperphosphorylated in H1-deficient virions is the 11-kDa product of the F18 gene; this protein is the major DNA-binding component of the viral nucleoprotein complex. The ability of recombinant H1 phosphatase to reverse this hyperphosphorylation in permeabilized virions strengthens the conclusion that the F18 protein is a bona fide substrate for the H1 phosphatase.

Alleles↗

PubMed1992

Expression cloning of a human dual-specificity phosphatase.

Using an expression cloning strategy, we isolated a cDNA encoding a human protein-tyrosine-phosphatase. Bacteria expressing the kinase domain of the keratinocyte growth factor receptor (bek/fibroblast growth factor receptor 2) were infected with a fibroblast cDNA library in a phagemid prokaryotic expression vector and screened with a monoclonal anti-phosphotyrosine antibody. Among several clones showing decreased anti-phosphotyrosine recognition, one displayed phosphatase activity toward the kinase in vitro. The 4.1-kilobase cDNA encoded a deduced protein of 185 amino acids with limited sequence similarity to the vaccinia virus phosphatase VH1. The purified recombinant protein dephosphorylated several activated growth factor receptors, as well as serine-phosphorylated casein, in vitro. Both serine and tyrosine phosphatase activities were completely abolished by mutagenesis of a single cysteine residue conserved in VH1 and the VH1-related (VHR) human protein. These properties suggest that VHR is capable of regulating intracellular events mediated by both tyrosine and serine phosphorylation.

Amino Acid Sequence↗

PubMed2024

Relaxin Modulates the Genomic Actions and Biological Effects of Estrogen in the Myometrium.

Estradiol (E2) and relaxin (Rln) are steroid and polypeptide hormones, respectively, with important roles in the female reproductive tract, including myometrium. Some actions of Rln, which are mediated by its membrane receptor RXFP1, require or are augmented by E2 signaling through its cognate nuclear steroid receptor, estrogen receptor alpha (ERα). In contrast, other actions of Rln act in opposition to the effects of E2. Here we explored the molecular and genomic mechanisms that underlie the functional interplay between E2 and Rln in the myometrium. We used both ovariectomized female mice and immortalized human myometrial cells expressing wild-type or mutant ERα (hTERT-HM-ERα cells). Our results indicate that Rln modulates the genomic actions and biological effects of estrogen in the myometrium and myometrial cells by reducing phosphorylation of ERα on serine 118 (S118), as well as by reducing the E2-dependent binding of ERα across the genome. These effects were associated with changes in the hormone-regulated transcriptome, including a decrease in the E2-dependent expression of some genes and enhanced expression of others. The inhibitory effects of Rln cotreatment on the E2-dependent phosphorylation of ERα required the nuclear dual-specificity phosphatases DUSP1 and DUSP5. Moreover, the inhibitory effects of Rln were reflected in a concomitant inhibition of the E2-dependent contraction of myometrial cells. Collectively, our results identify a pathway that integrates Rln/RXFP1 and E2/ERα signaling, resulting in a convergence of membrane and nuclear signaling pathways to control genomic and biological outcomes.

Female↗

PubMed1995

Phosphorylation and activation of the Xenopus Cdc25 phosphatase in the absence of Cdc2 and Cdk2 kinase activity.

The M-phase inducer, Cdc25C, is a dual-specificity phosphatase that directly phosphorylates and activates the cyclin B/Cdc2 kinase complex, leading to initiation of mitosis. Cdc25 itself is activated at the G2/M transition by phosphorylation on serine and threonine residues. Previously, it was demonstrated that Cdc2 kinase is capable of phosphorylating and activating Cdc25, suggesting the existence of a positive feedback loop. In the present study, kinases other than Cdc2 that can phosphorylate and activate Cdc25 were investigated. Cdc25 was found to be phosphorylated and activated by cyclin A/Cdk2 and cyclin E/Cdk2 in vitro. However, in interphase Xenopus egg extracts with no detectable Cdc2 and Cdk2, treatment with the phosphatase inhibitor microcystin activated a distinct kinase that could phosphorylate and activate Cdc25. Microcystin also induced other mitotic phenomena such as chromosome condensation and nuclear envelope breakdown in extracts containing less than 5% of the mitotic level of Cdc2 kinase activity. These findings implicate a kinase other than Cdc2 and Cdk2 that may initially activate Cdc25 in vivo and suggest that this kinase may also phosphorylate M-phase substrates even in the absence of Cdc2 kinase.

Amino Acid Sequence↗

PubMed1995

Cell-cycle control linked to extracellular environment by MAP kinase pathway in fission yeast.

In fission yeast the onset of mitosis is brought about by Cdc2/Cdc13 kinase, which is inhibited by the Wee1/Mik1 tyrosine kinases and activated by Cdc25 tyrosine phosphatase. This control network integrates many signals, including those that monitor DNA replication, DNA damage and cell size. We report here that a fission yeast MAP kinase pathway links the cell-cycle G2/M control with changes in the extracellular environment that affect cell physiology. Fission yeast spc1- mutants have a G2 delay that is greatly exacerbated by growth in high osmolarity media and nutrient limitation. A lethal interaction of spc1 and cdc25 mutations shows that Spc1 promotes the onset of mitosis. Spc1 is a MAP kinase homologue that is activated by Wis1 kinase in response to osmotic stress and nutrient limitation. Spc1 is inactivated by Pyp1, a phosphatase previously identified as a mitotic inhibitor. Pyp1 dephosphorylates only tyrosine-173 of Spc1, unlike the dual-specificity phosphatases that have been shown to regulate other MAP kinases.

Amino Acid Sequence↗

PubMed1995

Tyrosine phosphatase signalling in a lower plant: cell-cycle and oxidative stress-regulated expression of the Chlamydomonas eugametos VH-PTP13 gene.

The first evidence for tyrosine phosphatase signalling pathways in plants is presented by characterizing a putative protein tyrosine phosphatase gene from the unicellular green alga Chlamydomonas eugametos. This cDNA, referred to as VH-PTP13, contains an open reading frame specifying a protein with a molecular weight of 30.3 kDa, that has significant homology with a distinct group of dual-specificity phosphatases. The highest homology is found with CL-100, a human stress-response gene that regulates MAPkinase activity. The purified VH-PTP13 protein expressed in E. coli had phosphatase activity and inactivated MAPkinases from alfalfa and tobacco. Nondividing C. eugametos gametes did not express the VH-PTP13 gene whereas synchronously dividing vegetative cells only expressed VH-PTP13 in the early G1-phase of the cycle, implying a function there. When vegetative cells were subjected to oxidative stress, expression of the VH-PTP13 gene was strongly induced, analogous to the human CL-100 gene. Its potential role in plant signalling pathways is discussed.

Amino Acid Sequence↗

PubMed1995

Induction of neurite outgrowth by MAP kinase in PC12 cells.

Treatment of PC12 cells with nerve growth factor (NGF) results in neural differentiation of the cells, inducing neurite outgrowth. Ras protein has been shown to play an essential role in this process. To examine whether or not the MAP kinase (MAPK) cascade mediates the NGF- and Ras-induced neural differentiation process, we injected PC12 cells with constitutive active forms of each components of the MAPK cascade. When a moderately active mutant of Xenopus MAPK kinase (S222E-MAPKK) in which Ser 222 was changed into glutamic acid was injected, the neurite outgrowth of PC12 cells occurred to some extent. Injection of an N-terminal truncated STE11 protein (delta N-STE11), a constitutively active form of STE11 which is a yeast MAPKK kinase, induced neurite outgrowth in PC12 cells. Furthermore, injection of thiophosphorylated MAPK, but not purified active MAPK, into PC12 cells resulted in neurite outgrowth. Thiophosphorylated MAPK was resistant to protein phosphatase 2A treatment, while purified active MAPK was inactivated by this treatment. All these results have suggested that sustained activation of MAPK is sufficient for PC12 cell differentiation. In accord with this, the delta N-STE11- or S222E- MAPKK-induced neurite outgrowth was inhibited by coinjection of CL-100 protein, a dual-specificity phosphatase that is capable of inactivating MAPK.

Animals↗

PubMed1995

A novel mitogen-activated protein kinase phosphatase. Structure, expression, and regulation.

Mitogen-activated protein (MAP) kinase lies at the convergence of various extracellular ligand-mediated signaling pathways. It is activated by the dual-specificity kinase, MAP kinase kinase or MEK. MAP kinase inactivation is mediated by dephosphorylation via specific MAP kinase phosphatases (MKPs). One MKP (MKP-1 (also known as 3CH134, Erp, or CL100)) has been reported to be expressed in a wide range of tissues and cells. We report the identification of a second widely expressed MKP, termed MKP-2, isolated from PC12 cells. MKP-2 showed significant homology with MKP-1 (58.8% at the amino acid level) and, like MKP-1, displayed vanadate-sensitive phosphatase activity against MAP kinase in vitro. Overexpression of MKP-2 in vivo inhibited MAP kinase-dependent gene transcription in PC12 cells. MKP-2 differed from MKP-1 in its tissue distribution and in its extent of induction by growth factors and agents that induce cellular stress, suggesting that these MKPs may have distinct physiological functions.

Amino Acid Sequence↗

PubMed1995

Genomic organization and chromosomal localization of the DUSP2 gene, encoding a MAP kinase phosphatase, to human 2p11.2-q11.

The mitogen-induced gene, DUSP2, encodes a nuclear protein, PAC1, that acts as a dual-specific protein phosphatase with stringent substrate specificity for MAP kinase. MAP kinase phosphorylation and consequent enzymatic activation is a central and often obligatory component in signal transduction initiated by growth factor stimulation or resulting from various types of oncogenic transformation. DUSP2 downregulates intracellular signal transduction through the dephosphorylation/inactivation of MAP kinases. To facilitate assessment of the possible role of DUSP2 in growth processes, the genomic structure and chromosomal location of the gene have been determined. DUSP2 has been localized to the pericentromeric region of human chromosome 2 (2p11.2-q11) by analysis of somatic cell hybrids, in situ chromosome hybridization, and genetic linkage analysis using a single-strand conformational polymorphism (SSCP) that has been identified in the 3' UTR of the gene. No consistent translocations or deletions at this chromosomal site have been reported in hematopoietic neoplasias or other tumors.

Base Sequence↗

PubMed1995

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.

3T3 Cells↗

PubMed1995

Dephosphorylation of Cdk2 Thr160 by the cyclin-dependent kinase-interacting phosphatase KAP in the absence of cyclin.

The activation of cyclin-dependent kinases (CDKs) requires the phosphorylation of a conserved threonine (Thr160 in Cdk2) by CDK-activating kinase (CAK). Human KAP (also called Cdi1), a CDK-associated phosphatase, was shown to dephosphorylate Thr160 in human Cdk2. KAP was unable to dephosphorylate Tyr15 and only dephosphorylated Thr160 in native monomeric Cdk2. The binding of cyclin A to Cdk2 inhibited the dephosphorylation of Thr160 by KAP but did not preclude the binding of KAP to the cyclin A-Cdk2 complex. Moreover, the dephosphorylation of Thr160 by KAP prevented Cdk2 kinase activity upon subsequent association with cyclin A. These results suggest that KAP binds to Cdk2 and dephosphorylates Thr160 when the associated cyclin subunit is degraded or dissociates.

Amino Acid Sequence↗

PubMed1995

hVH-5: a protein tyrosine phosphatase abundant in brain that inactivates mitogen-activated protein kinase.

A novel protein tyrosine phosphatase [homologue of vaccinia virus H1 phosphatase gene clone 5 (hVH-5)] was cloned; it shared sequence similarity with a subset of protein tyrosine phosphatases that regulate mitogen-activated protein kinase. The catalytic region of hVH-5 was expressed as a fusion protein and was shown to hydrolyze p-nitrophenylphosphate and inactivate mitogen-activated protein kinase, thus proving that hVH-5 possessed phosphatase activity. A unique proline-rich region distinguished hVH-5 from other closely related protein tyrosine phosphatases. Another feature that distinguished hVH-5 from related phosphatases was that hVH-5 was expressed predominantly in the adult brain, heart, and skeletal muscle. In addition, in situ hybridization histochemistry of mouse embryo revealed high levels of expression and a wide distribution in the central and peripheral nervous system. Some specific areas of abundant hVH-5 expression included the olfactory bulb, retina, layers of the cerebral cortex, and cranial and spinal ganglia. hVH-5 was induced in PC12 cells upon nerve growth factor and insulin treatment in a manner characteristic of an immediate-early gene, suggesting a possible role in the signal transduction cascade.

Amino Acid Sequence↗

PubMed1995

Yeast RLM1 encodes a serum response factor-like protein that may function downstream of the Mpk1 (Slt2) mitogen-activated protein kinase pathway.

The MPK1 (SLT2) gene of Saccharomyces cerevisiae encodes a mitogen-activated protein kinase that is regulated by a kinase cascade whose known elements are Pkc1 (a homolog of protein kinase C), Bck1 (Slk1) (a homolog of MEK kinase), and the functionally redundant Mpk1 activators Mkk1 and Mkk2 (homologs of MEK). An activated mutation of MKK1, MKK1P386, inhibits growth when overexpressed. This growth-inhibitory effect was suppressed by the mpk1 delta mutation, suggesting that hyperactivation of the Mpk1 pathway is toxic to cells. To search for genes that interact with the Mpk1 pathway, we isolated both chromosomal mutations and dosage suppressor genes that ameliorate the growth-inhibitory effect of overexpressed Mkk1P386. One of the genes identified by the analysis of chromosomal mutations is RLM1 (resistance to lethality of MKK1P386 overexpression), which encodes a protein homologous to a conserved domain of the MADS (Mcm1, Agamous, Deficiens, and serum response factor) box family of transcription factors. Although rlm1 delta cells grow normally at any temperature, they display a caffeine-sensitive phenotype similar to that observed in mutants defective in BCK1, MKK1/MKK2, or MPK1. A gene fusion that provides Rlm1 with a transcriptional activation domain of Gal4 suppresses bck1 delta and mpk1 delta. A screening for dosage suppressors yielded the MSG5 genes, which encode a dual-specificity protein phosphatase. Our results suggest that Rlm1 functions as a transcription factor downstream of Mpk1 that is subject to activation by the Mpk1 mitogen-activated protein kinase pathway.

Amino Acid Sequence↗

PubMed2026

RAS signaling in lung adenocarcinoma is defined by lineage context and DUSP4 loss.

BACKGROUNDThe molecular landscape of lung adenocarcinoma (LUAD) is often illustrated as a driver-oncogene pie chart, but identical mutations exhibit heterogeneous signaling shaped by comutations, transcriptional programs, and lineage context. We propose a lineage-integrated signaling framework using an EGFR mutation signature (mSig).METHODSWe defined EGFR mSig using differentially expressed genes in EGFR-mutant (EGFR-mt) LUADs. Semisupervised clustering and machine learning models were used to test reproducibility in different combinations of datasets. We analyzed molecular subtypes, lineage markers, co-occurring mutations, and EGFR copy number alterations in EGFR mSig-defined subtypes of LUAD.RESULTSEGFR mSig showed robust classification performance (area under receiver operating characteristic curve = 0.83-0.95; mean negative predictive value = 96.3%). Validated gene expression subtypes and lung lineage markers were closely aligned with EGFR mSig status. Most EGFR mSig+ tumors, including many without EGFR mutations, belonged to the bronchioid subtype. A subset of canonical RAS mutations were mSig+ and mirrored the EGFR mutation pattern. EGFR WT/mSig- tumors were enriched for nonbronchioid subtypes and had comutations in TP53 or RAS/RAF/RTKs. We highlight a parsimonious collection of coordinated mutations, including RAS, KEAP1, STK11, TP53, and CDKN2A, that taken together suggest coordination of tumor signaling previously suggested but now reproduced and expanded.CONCLUSIONA potentially novel EGFR mSig that captures the transcriptional footprint of EGFR activation revealed a subset of EGFR WT LUADs with mt-like features. mSig refines LUAD taxonomy beyond mutation-only pie-chart models by incorporating lineage and comutation context. Lineage-directed stratification with coalteration identifies clinically relevant groups across EGFR and RAS states and highlights treatment opportunities for patients currently considered oncogene-negative.FUNDINGNational Cancer Institute (NCI) U01CA272541, R01CA262296, U24CA264021, UG1CA233333, R01CA211939.

Humans↗

PubMed1995

[Expression of sdi1, a potent inhibitor of cdk2 kinase, cdk2 and G1 cyclins and mutation of sdi1 in human gastric carcinomas].

Sdi1, also known as Cip1/Waf1, is a potent inhibitor of G1 cyclin-dependent kinases, which is induced by wild type p53 but not by mutant p53. Expression of mRNAs for sdil, cdk2 and G1 cyclins was examined in gastric carcinomas. All the cell lines expressing very low or undetectable level of sdil mRNA contains p53 gene abnormalities, while the cell lines expressing high level of sdil shares wild type p53 gene. An exception was a cell line MKN-28 with mutated p53 gene which expressed mRNAs for sdi1, cdk2 and G1 cyclins at high levels, p21 point mutation was detected in one (MKN-28) of the eight cell lines. These result suggest that low level of sdil and subsequent overexpression of cdk2 and G1 cyclins might be involved in deregulated growth of gastric carcinomas. It is likely that gene alteration of sdil and subsequent loss of function may have implication for cdk2 and G1 cyclins expression.

CDC2-CDC28 Kinases↗