The Polo-like Kinase Plx1 Is Required for Activation of the Phosphatase Cdc25C and Cyclin B-Cdc2 in Xenopus Oocytes

In the Xenopus oocyte system mitogen treatment triggers the G2/M transition by transiently inhibiting the cAMP-dependent protein kinase (PKA); subsequently, other signal transduction pathways are activated, including the mitogen-activated protein kinase (MAPK) and polo-like kinase pathways. To study the interactions between these pathways, we have utilized a cell-free oocyte extract that carries out the signaling events of oocyte maturation after addition of the heat-stable inhibitor of PKA, PKI. PKI stimulated the synthesis of Mos and activation of both the MAPK pathway and the Plx1/Cdc25C/cyclin B-Cdc2 pathway. Activation of the MAPK pathway alone by glutathione S-transferase (GST)-Mos did not lead to activation of Plx1 or cyclin B-Cdc2. Inhibition of the MAPK pathway in the extract by the MEK1 inhibitor U0126 delayed, but did not prevent, activation of the Plx1 pathway, and inhibition of Mos synthesis by cycloheximide had a similar effect, suggesting that MAPK activation is the only relevant function of Mos. Immunodepletion of Plx1 completely inhibited activation of Cdc25C and cyclin B-Cdc2 by PKI, indicating that Plx1 is necessary for Cdc25C activation. In extracts containing fully activated Plx1 and Cdc25C, inhibition of cyclin B-Cdc2 by p21Cip1 had no significant effect on either the phosphorylation of Cdc25C or the activity of Plx1. These results demonstrate that maintenance of Plx1 and Cdc25C activity during mitosis does not require cyclin B-Cdc2 activity.

BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} acts as a phosphate analog in proteins phosphorylated on aspartate: Structure of a BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} complex with phosphoserine phosphatase

Protein phosphoaspartate bonds play a variety of roles. In response regulator proteins of two-component signal transduction systems, phosphorylation of an aspartate residue is coupled to a change from an inactive to an active conformation. In phosphatases and mutases of the haloacid dehalogenase (HAD) superfamily, phosphoaspartate serves as an intermediate in phosphotransfer reactions, and in P-type ATPases, also members of the HAD family, it serves in the conversion of chemical energy to ion gradients. In each case, lability of the phosphoaspartate linkage has hampered a detailed study of the phosphorylated form. For response regulators, this difficulty was recently overcome with a phosphate analog, BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}, which yields persistent complexes with the active site aspartate of their receiver domains. We now extend the application of this analog to a HAD superfamily member by solving at 1.5-Å resolution the x-ray crystal structure of the complex of BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} with phosphoserine phosphatase (PSP) from Methanococcus jannaschii. The structure is comparable to that of a phosphoenzyme intermediate: BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} is bound to Asp-11 with the tetrahedral geometry of a phosphoryl group, is coordinated to Mg2+, and is bound to residues surrounding the active site that are conserved in the HAD superfamily. Comparison of the active sites of BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}⋅PSP and BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}⋅CeY, a receiver domain/response regulator, reveals striking similarities that provide insights into the function not only of PSP but also of P-type ATPases. Our results indicate that use of BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} for structural studies of proteins that form phosphoaspartate linkages will extend well beyond response regulators.

Induction of pheromone production in a moth by topical application of a pseudopeptide mimic of a pheromonotropic neuropeptide.

An amphiphilic analog of Locusta myotropin II (Lom-MT-II), Glu-Gly-Asp-Phe-Thr-Pro-Arg-Leu-amide, was synthesized by addition of 6-phenylhexanoic acid (6-Pha) linked through alanine to the amino terminus. This pseudopeptide, [6-Pha-Ala0]Lom-MT-II, was found to have pheromonotropic activity equivalent to pheromone biosynthesis activating neuropeptide when injected into females of Heliothis virescens. Topical application of [6-Pha-Ala0]Lom-MT-II or Helicoverpa zea-pheromone biosynthesis activating neuropeptide (PBAN), dissolved in dimethyl sulfoxide, to the descaled abdomen of females induced production of pheromone, although more Hez-PBAN than [6-Pha-Ala0]Lom-MT-II was required to obtain significant production of pheromone. Application of [6-Pha-Ala0]Lom-MT-II, dissolved in water, to the abdomen induced production of pheromone, but neither Hez-PBAN nor Lom-MT-II dissolved in water stimulated production of significant amounts of pheromone. Dose- and time-response studies indicated that application of the amphiphilic mimetic in water induced pheromone production in as little as 15 min after application and that the effects were maintained for prolonged periods. These findings show that amphiphilic pseudopeptide mimics of insect neuropeptides will penetrate the insect cuticle when applied topically in water and induce an endogenous response.

Covalent HLA-B27/peptide complex induced by specific recognition of an aziridine mimic of arginine.

The class I major histocompatibility complex (MHC) glycoprotein HLA-B27 binds short peptides containing arginine at peptide position 2 (P2). The HLA-B27/peptide complex is recognized by T cells both as part of the development of the repertoire of T cells in the cellular immune system and during activation of cytotoxic T cells. Based on the three-dimensional structure of HLA-B27, we have synthesized a ligand with an aziridine-containing side chain designed to mimic arginine and to bind covalently in the arginine-specific P2 pocket of HLA-B27. Using tryptic digestion followed by mass spectrometry and amino acid sequencing, the aziridine-containing ligand is shown to alkylate specifically cysteine 67 of HLA-B27. Neither free cysteine in solution nor an exposed cysteine on a class II MHC molecule can be alkylated, showing that specific recognition between the anchor side-chain pocket of an MHC class I protein and the designed ligand (propinquity) is necessary to induce the selective covalent reaction with the MHC class I molecule.

IA-2, a transmembrane protein of the protein tyrosine phosphatase family, is a major autoantigen in insulin-dependent diabetes mellitus.

IA-2 is a 105,847 Da transmembrane protein that belongs to the protein tyrosine phosphatase family. Immunoperoxidase staining with antibody raised against IA-2 showed that this protein is expressed in human pancreatic islet cells. In this study, we expressed the full-length cDNA clone of IA-2 in a rabbit reticulocyte transcription/translation system and used the recombinant radiolabeled IA-2 protein to detect autoantibodies by immunoprecipitation. Coded sera (100) were tested: 50 from patients with newly diagnosed insulin-dependent diabetes mellitus (IDDM) and 50 from age-matched normal controls. Sixty-six percent of the sera from patients, but none of the sera from controls, reacted with IA-2. The same diabetic sera tested for autoantibodies to islet cells (ICA) by indirect immunofluorescence and glutamic acid decarboxylase (GAD65Ab) by depletion ELISA showed 68% and 52% positivity, respectively. Up to 86% of the IDDM patients had autoantibodies to IA-2 and/or GAD65. Moreover, greater than 90% (14 of 15) of the ICA-positive but GAD65Ab-negative sera had autoantibodies to IA-2. Absorption experiments showed that the immunofluorescence reactivity of ICA-positive sera was greatly reduced by prior incubation with recombinant IA-2 or GAD65 when the respective antibody was present. A little over one-half (9 of 16) of the IDDM sera that were negative for ICA were found to be positive for autoantibodies to IA-2 and/or GAD65, arguing that the immunofluorescence test for ICA is less sensitive than the recombinant tests for autoantibodies to IA-2 and GAD65. It is concluded that IA-2 is a major islet cell autoantigen in IDDM, and, together with GAD65, is responsible for much of the reactivity of ICA with pancreatic islets. Tests for the detection of autoantibodies to recombinant IA-2 and GAD65 may eventually replace ICA immunofluorescence for IDDM population screening.

Activation and phosphorylation of a pleckstrin homology domain containing protein kinase (RAC-PK/PKB) promoted by serum and protein phosphatase inhibitors.

Treatment of quiescent Swiss 3T3 fibroblasts with serum, or with the phosphatase inhibitors okadaic acid and vanadate, induced a 2- to 11-fold activation of the serine/ threonine RAC protein kinase (RAC-PK). Kinase activation was accompanied by decreased mobility of RAC-PK on SDS/PAGE such that three electrophoretic species (a to c) of the kinase were detected by immunoblot analysis, indicative of differentially phosphorylated forms. Addition of vanadate to arrested cells increased the RAC-PK phosphorylation level 3-to 4-fold. Unstimulated RAC-PK was phosphorylated predominantly on serine, whereas the activated kinase was phosphorylated on both serine and threonine residues. Treatment of RAC-PK in vitro with protein phosphatase 2A led to kinase inactivation and an increase in electrophoretic mobility. Deletion of the N-terminal region containing the pleckstrin homology domain did not affect RAC-PK activation by okadaic acid, but it reduced vanadate-stimulated activity and also blocked the serum-induced activation. Deletion of the serine/threonine rich C-terminal region impaired both RAC-PKalpha basal and vanadate-stimulated activity. Studies using a kinase-deficient mutant indicated that autophosphorylation is not involved in RAC-PKalpha activation. Stimulation of RAC-PK activity and electrophoretic mobility changes induced by serum were sensitive to wortmannin. Taken together the results suggest that RAC-PK is a component of a signaling pathway regulated by phosphatidylinositol (PI) 3-kinase, whose action is required for RAC-PK activation by phosphorylation.

A specific protein carboxyl methylesterase that demethylates phosphoprotein phosphatase 2A in bovine brain.

Phosphoprotein phosphatase 2A (PP2A) is one of the four major protein serine/threonine phosphatases found in all eukaryotic cells. We have shown that the 36-kDa catalytic subunit of PP2A is carboxyl methylated in eukaryotic cells, and we have previously identified and purified a novel methyltransferase (MTase) that is responsible for this modification. Here, we describe a novel protein carboxyl methyl-esterase (MEase) from bovine brain that demethylates PP2A. The enzyme has been purified to homogeneity as a monomeric 46-kDa soluble protein. The MEase is highly specific for PP2A. It does not catalyze the demethylation of other protein or peptide methylesters. Moreover, MEase activity is dramatically inhibited by nanomolar concentrations of okadaic acid, a specific inhibitor of PP2A. From these results, we conclude that PP2A methylation is controlled by two specific enzymes, a MTase and a MEase. Since PP2A methylation is highly conserved in eukaryotes ranging from human to yeast, it is likely that this system plays an important role in phosphatase regulation.

The multidomain protein Trio binds the LAR transmembrane tyrosine phosphatase, contains a protein kinase domain, and has separate rac-specific and rho-specific guanine nucleotide exchange factor domains.

rho-like GTP binding proteins play an essential role in regulating cell growth and actin polymerization. These molecular switches are positively regulated by guanine nucleotide exchange factors (GEFs) that promote the exchange of GDP for GTP. Using the interaction-trap assay to identify candidate proteins that bind the cytoplasmic region of the LAR transmembrane protein tyrosine phosphatase (PT-Pase), we isolated a cDNA encoding a 2861-amino acid protein termed Trio that contains three enzyme domains: two functional GEF domains and a protein serine/threonine kinase (PSK) domain. One of the Trio GEF domains (Trio GEF-D1) has rac-specific GEF activity, while the other Trio GEF domain (Trio GEF-D2) has rho-specific activity. The C-terminal PSK domain is adjacent to an Ig-like domain and is most similar to calcium/calmodulin-dependent kinases, such as smooth muscle myosin light chain kinase which similarly contains associated Ig-like domains. Near the N terminus, Trio has four spectrin-like repeats that may play a role in intracellular targeting. Northern blot analysis indicates that Trio has a broad tissue distribution. Trio appears to be phosphorylated only on serine residues, suggesting that Trio is not a LAR substrate, but rather that it forms a complex with LAR. As the LAR PTPase localizes to the ends of focal adhesions, we propose that LAR and the Trio GEF/PSK may orchestrate cell-matrix and cytoskeletal rearrangements necessary for cell migration.

Role of the regulatory subunit of bovine pyruvate dehydrogenase phosphatase.

Bovine pyruvate dehydrogenase phosphatase (PDP) is a Mg2+-dependent and Ca2+-stimulated heterodimer that is a member of the protein phosphatase 2C family and is localized to mitochondria. Insight into the function of the regulatory subunit of PDP (PDPr) has been gained. It decreases the sensitivity of the catalytic subunit of PDP (PDPc) to Mg2+. The apparent Km of PDPc for Mg2+ is increased about 5-fold, from about 0.35 mM to 1.6 mM. The polyamine spermine increases the sensitivity of PDP but not PDPc to Mg2+, apparently by interacting with PDPr. PDPc but not PDP can use the phosphopeptide RRAT(P)VA as a substrate. These observations are interpreted to indicate that PDPr blocks or distorts the active site of PDPc and that spermine produces a conformational change in PDPr that reverses its inhibitory effect. These findings suggest that PDPr may be involved in the insulin-induced activation of the mitochondrial PDP in adipose tissue, which is characterized by a decrease in its apparent Km for Mg2+.

The phosphatase activity of carbonic anhydrase III is reversibly regulated by glutathiolation.

Carbonic anhydrase isozyme III (CAIII) is unique among the carbonic anhydrases because it demonstrates phosphatase activity. CAIII forms a disulfide link between glutathione and two of its five cysteine residues, a process termed S-glutathiolation. Glutathiolation of CAIII occurs in vivo and is increased during aging and under acute oxidative stress. We show that glutathiolation serves to reversibly regulate the phosphatase activity of CAIII. Glutathiolation of Cys-186 is required for phosphatase activity, while glutathiolation of Cys-181 blocks activity. Phosphotyrosine is the preferred substrate, although phosphoserine and phosphothreonine can also be cleaved. Thus, glutathiolation is a reversible covalent modification that can regulate CAIII, a phosphatase that may function in the cellular response to oxidative stress.

Protein-tyrosine phosphatase activity regulates osteoclast formation and function: inhibition by alendronate.

Alendronate (ALN), an aminobisphosphonate used in the treatment of osteoporosis, is a potent inhibitor of bone resorption. Its molecular target is still unknown. This study examines the effects of ALN on the activity of osteoclast protein-tyrosine phosphatase (PTP; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48), called PTPepsilon. Using osteoclast-like cells generated by coculturing mouse bone marrow cells with mouse calvaria osteoblasts, we found by molecular cloning and RNA blot hybridization that PTPepsilon is highly expressed in osteoclastic cells. A purified fusion protein of PTPepsilon expressed in bacteria was inhibited by ALN with an IC50 of 2 microM. Other PTP inhibitors--orthovanadate and phenylarsine oxide (PAO)-inhibited PTPepsilon with IC50 values of 0.3 microM and 18 microM, respectively. ALN and another bisphosphonate, etidronate, also inhibited the activities of other bacterially expressed PTPs such as PTPsigma and CD45 (also called leukocyte common antigen). The PTP inhibitors ALN, orthovanadate, and PAO suppressed in vitro formation of multinucleated osteoclasts from osteoclast precursors and in vitro bone resorption by isolated rat osteoclasts (pit formation) with estimated IC50 values of 10 microM, 3 microM, and 0.05 microM, respectively. These findings suggest that tyrosine phosphatase activity plays an important role in osteoclast formation and function and is a putative molecular target of bisphosphonate action.

Identification of a second transmembrane protein tyrosine phosphatase, IA-2beta, as an autoantigen in insulin-dependent diabetes mellitus: precursor of the 37-kDa tryptic fragment.

A novel cDNA, IA-2beta, was isolated from a mouse neonatal brain library. The predicted protein sequence revealed an extracellular domain, a transmembrane region, and an intracellular domain. The intracellular domain is 376 amino acids long and 74% identical to the intracellular domain of IA-2, a major autoantigen in insulin-dependent diabetes mellitus (IDDM). A partial sequence of the extracellular domain of IA-2beta indicates that it differs substantially (only 26% identical) from that of IA-2. Both molecules are expressed in islets and brain tissue. Forty-six percent (23 of 50) of the IDDM sera but none of the sera from normal controls (0 of 50) immunoprecipitated the intracellular domain of IA-2beta. Competitive inhibition experiments showed that IDDM sera have autoantibodies that recognize both common and distinct determinants on IA-2 and IA-2beta. Many IDDM sera are known to immunoprecipitate 37-kDa and 40-kDa tryptic fragments from islet cells, but the identity of the precursor protein(s) has remained elusive. The current study shows that treatment of recombinant IA-2beta and IA-2 with trypsin yields a 37-kDa fragment and a 40-kDa fragment, respectively, and that these fragments can be immunoprecipitated with diabetic sera. Absorption of diabetic sera with unlabeled recombinant IA-2 or IA-2beta, prior to incubation with radiolabeled 37-kDa and 40-kDa tryptic fragments derived from insulinoma or glucagonoma cells, blocks the immunoprecipitation of both of these radiolabeled tryptic fragments. We conclude that IA-2beta and IA-2 are the precursors of the 37-kDa and 40-kDa islet cell autoantigens, respectively, and that both IA-2 and IA-2beta are major autoantigens in IDDM.

The 145-kDa protein induced to associate with Shc by multiple cytokines is an inositol tetraphosphate and phosphatidylinositol 3,4,5-triphosphate 5-phosphatase.

A 145-kDa tyrosine-phosphorylated protein that becomes associated with Shc in response to multiple cytokines has been purified from the murine hemopoietic cell line B6SUtA1. Amino acid sequence data were used to clone the cDNA encoding this protein from a B6SUtA1 library. The predicted amino acid sequence encodes a unique protein containing an N-terminal src homology 2 domain, two consensus sequences that are targets for phosphotyrosine binding domains, a proline-rich region, and two motifs highly conserved among inositol polyphosphate 5-phosphatases. Cell lysates immunoprecipitated with antiserum to this protein exhibited both phosphatidylinositol 3,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate polyphosphate 5-phosphatase activity. This novel signal transduction intermediate may serve to modulate both Ras and inositol signaling pathways. Based on its properties, we suggest the 145-kDa protein be called SHIP for SH2-containing inositol phosphatase.

Ca(2+)-independent reduction of N-methyl-D-aspartate channel activity by protein tyrosine phosphatase.

Regulation of ion channel function by intracellular processes is fundamental for controlling synaptic signaling and integration in the nervous system. Currents mediated by N-methyl-D-aspartate (NMDA) receptors decline during whole-cell recordings and this may be prevented by ATP. We show here that phosphorylation is necessary to maintain NMDA currents and that the decline is not dependent upon Ca2+. A protein tyrosine phosphatase or a peptide inhibitor of protein tyrosine kinase applied intracellularly caused a decrease in NMDA currents even when ATP was included. On the other hand, pretreating the neurons with a membrane-permeant tyrosine kinase inhibitor occluded the decline in NMDA currents when ATP was omitted. In inside-out patches, applying a protein tyrosine phosphatase to the cytoplasmic face of the patch caused a decrease in probability of opening of NMDA channels. Conversely, open probability was increased by a protein tyrosine phosphatase inhibitor. These results indicate that NMDA channel activity is reduced by a protein tyrosine phosphatase associated with the channel complex.

Differential functions of the two Src homology 2 domains in protein tyrosine phosphatase SH-PTP1.

SH-PTP1 (also known as PTP1C, HCP, and SHP) is a non-transmembrane protein tyrosine phosphatase (PTPase) containing two tandem Src homology 2 (SH2) domains. We show here that the two SH2 (N-SH2 and C-SH2) domains in SH-PTP1 have different functions in regulation of the PTPase domain and thereby signal transduction. While the N-terminal SH2 domain is both necessary and sufficient for autoinhibition through an intramolecular association with the PTPase domain, truncation of the C-SH2 domain [SH-PTP1 (delta CSH2) construct] has little effect on SH-PTP1 activity. A synthetic phosphotyrosine residue (pY) peptide derived from the erythropoietin receptor (EpoR pY429) binds to the N-SH2 domain and activates both wild-type SH-PTP1 and SH-PTP1 (delta CSH2) 60- to 80-fold. Another pY peptide corresponding to a phosphorylation site on the IgG Fc receptor (Fc gamma RIIB1 pY309) associates with both the C-SH2 domain (Kd = 2.8 microM and the N-SH2 domain (Kd = 15.0 microM) and also activates SH-PTP1 12-fold. By analysis of the effect of the Fc gamma RIIB1 pY309 peptide on SH-PTP1 (delta CSH2), SH-PTP1 (R30K/R33E), SH-PTP1 (R30K/R136K), and SH-PTP1 (R136K) mutants in which the function of either the N- or C-SH2 domain has been impaired, we have determined that both synthetic pY peptides stimulate SH-PTP1 by binding to its N-SH2 domain; binding of pY ligand to the C-SH2 domain has no effect on SH-PTP1 activity. We propose that the N-terminal SH2 domain serves both as a regulatory domain and as a recruiting unit, whereas the C-terminal SH2 domain acts merely as a recruiting unit.