Regulated expression of a cell division control-related protein kinase during development

Protein kinases are important signaling molecules that are known constituents of cellular pathways critical for normal cellular growth and development. We have recently identified a new protein kinase, p58, which contains a large domain that is highly homologous to the cell division control p34cdc2 protein kinase. This new cell division control-related protein kinase was originally identified as a component of semipurified galactosyltransferase; thus, it has been denoted galactosyltransferase-associated protein kinase. In vitro, this protein kinase has been shown to phosphorylate a number of substrates, including histone H1, casein, and galactosyltransferase. In vivo, we have found that this protein kinase affects galactosyltransferase enzyme activity and that it is apparently involved in some aspect of normal cell cycle regulation. In this report, we find that the p58 gene is evolutionarily well conserved and expressed ubiquitously, but to varying extents, in adult tissues. In developmentally staged embryos, p58 expression was elevated early in embryogenesis and then decreased dramatically. In the murine submandibular gland, p58 expression was elevated between day 14 and day 16 post coitus. Expression in the submandibular gland appeared to parallel the proliferation and differentiation of specific cell types as judged by in situ hybridization. These studies indicate that the p58 protein kinase may have a critical function during normal embryonic development and that this protein kinase continues to be expressed in differentiated adult tissues.     

Human p68 kinase exhibits growth suppression in yeast and homology to the translational regulator GCN2.

The human p68 kinase is an interferon-regulated enzyme that inhibits protein synthesis when activated by double-stranded RNA. We show here that when expressed in Saccharomyces cerevisiae, the p68 kinase produced a growth suppressing phenotype resulting from an inhibition of polypeptide chain initiation consistent with functional protein kinase activity. This slow growth phenotype was reverted in yeast by two different mechanisms: expression of the p68 kinase N-terminus, shown to bind double-stranded RNA in vitro and expression of a mutant form of the alpha-subunit of yeast initiation factor 2, altered at a single phosphorylatable site. These results provide the first direct in vivo evidence that the p68 kinase interacts with the alpha-subunit of eukaryotic initiation factor 2. Sequence similarity with a yeast translational regulator, GCN2, further suggests that this enzyme may be a functional homolog in higher eukaryotes, where its normal function is to regulate protein synthesis through initiation factor 2 phosphorylation.     

The inner nuclear membrane protein p58 associates in vivo with a p58 kinase and the nuclear lamins.

p58, also referred to as the lamin B receptor, is an intrinsic protein of the inner nuclear membrane that binds in vitro to lamin B. Previous studies have demonstrated that p58 is phosphorylated in vivo and removal of its phosphate moieties affects lamin B binding. Using affinity-purified antipeptide antibodies, we have now immunoisolated p58 from bird erythrocyte lysates under isotonic, non-denaturing conditions. Analysis of the immunopurified material shows that five distinct proteins are tightly and specifically associated with p58. Two of these polypeptides can be identified as nuclear lamins A and B. The immunoisolate also contains a kinase activity that phosphorylates p58 in vivo and in vitro, exclusively at serine residues, as indicated by phosphoamino acid analysis and two-dimensional phosphopeptide mapping. Cell fractionation experiments and in vitro phosphorylation assays demonstrate that the p58 kinase resides in the nuclear envelope and is distinct from protein kinase A and cdc2 kinase, for both of which p58 is an in vitro substrate. These data suggest that p58 is interacting in vivo with a p58 kinase and the nuclear lamins.     

Characterization of the cytoplasmic proline-directed protein kinase in proliferative cells and tissues as a heterodimer comprised of p34cdc2 and p58cyclin A.

Site-specific analysis of tyrosine hydroxylase phosphorylation in rat pheochromocytoma led previously to the identification of a novel growth factor-sensitive serine/threonine protein kinase, designated proline-directed protein kinase (PDPK). In this article we describe further the activation, purification, subunit configuration, and biochemical characteristics of this cytoplasmic enzyme system. In human A431 epidermoid carcinoma cells PDPK activity was found to be stimulated by epidermal growth factor in a dose-dependent, time-dependent manner. The PDPK purified from the cytosol of mouse FM3A mammary carcinoma cells exhibited the same chromatographic behavior and biochemical properties as the tyrosine hydroxylase-associated enzyme purified originally from rat pheochromocytoma. The presence of p34cdc2 was ultimately detected in all active fractions of highly purified PDPK by Western blotting and immunoprecipitation; however, it was determined that this catalytic subunit is complexed with a 58-kDa regulatory subunit that is clearly distinct from that of the "growth-associated" M phase-specific histone H1 kinase (i.e. cyclin B). The 58 kDa regulatory subunit of PDPK was identified by direct immunoblotting as a mammalian A-type cyclin. Furthermore, the p58cyclin A subunit of PDPK was found to be phosphorylated on tyrosine residues in vivo and in vitro, the latter of which resulted in a significant increase in PDPK activity. Additional distinctions between this growth factor-sensitive PDPK (p34cdc2-p58cyclin A) and the M phase-specific histone H1 kinase (p34cdc2-p62cyclin B-p13suc1) are identified on the basis of chromatographic behavior, enzyme kinetics, and physicochemical properties. Based on these findings, it is proposed that PDPK represents a unique complex of the p34cdc2 protein kinase which is active in the cytoplasm of proliferative cells, is regulated differently from the M phase-specific histone H1 kinase by phosphorylation reactions, and is modulated selectively by growth factors.     

Activity of dsRNA-dependent protein kinase in rat lymphoid cells under the effect of X-ray irradiation

The activity of dsRNA-dependent protein kinase, which is the key enzyme of the interferon signal system, was studied in the rat spleen and thymus lymphocytes under the influence of X-ray irradiation at 0.5 and 1 Gy doses and interferon inducers administration. An increase of the enzyme activity was established in the presence of FGA, concanavaline A, poly(I).poly(C) in vitro. The effect is intensified under the irradiation by 0.5 Gy dose. The protein kinase activity in lymphocytes is amplified in proportion to poly(I).poly(C) concentration, that was most pronounced in the irradiated animals. The comparative analysis of the action of interferon inducers on the dsRNA-dependent protein kinase activity was carried out. Two biological systems were used: in vivo (when the preparations were injected to the experimental animals) and in vivo (under the preincubation of isolated lymphocytes with the inducers). It was shown that the combined action of radiation and interferon inducers causes the stimulation of dsRNA-dependent protein kinase activity.     

Differences in thermal sensitivities of the regulatory-catalytic and catalytic forms of cyclic AMP-dependent protein kinases from various tissues.

The cAMP-dependent protein kinase from various tissues was more thermally sensitive when activated by cAMP than the non-activated enzyme. For example, when the activity ratio (the activity of protein kinase assayed -cAMP/+cAMP) was 0.40, 80% and 76% of total hepatic cAMP dependent protein kinase activity was recoverable after incubations at 45 degrees C for 15 and 30 minutes, respectively. However, when the activity ratio was elevated to about 0.80 - 0.90 by increasing cAMP levels in vivo or adding exogenous cAMP to soluble liver extracts, the total protein kinase activity recoverable after incubations at 45 degrees C for 15 minutes was 34-44% and 19-22%, respectively. This observation was used to estimate the degree of activation of the enzyme in vivo and in vitro, since the loss of enzyme activity at 45 degrees C was directly related to the degree of activation of the enzyme in tissue extracts. The regulatory-catalytic form of cAMP-dependent protein kinase was thermally resistant at 45 degrees C unless activated by incubation with exogenous cAMP, histones or NaCl, while the catalytic form of the enzyme was highly thermally sensitive at this same temperature. These data describe a new property of the cAMP-dependent protein kinase and suggest an alternative method which measure the degree of activation of the enzyme.     

Interaction of p58(PITSLRE), a G2/M-specific protein kinase,with cyclin D3

The p58(PITSLRE) is a p34(cdc2)-related protein kinase that plays an important role in normal cell cycle progression. Elevated expression of p58(PITSLRE) in eukaryotic cells prevents them from undergoing normal cytokinesis and appears to delay them in late telophase. To investigate the molecular mechanism of p58(PITSLRE) action, we used the yeast two-hybrid system, screened a human fetal liver cDNA library, and identified cyclin D3 as an interacting partner of p58(PITSLRE). In vitro binding assay, in vivo coimmunoprecipitation, and immunofluorescence cell staining further confirmed the association of p58(PITSLRE) with cyclin D3. This binding was observed only in the G(2)/M phase but not in the G(1)/S phase of the cell cycle; meanwhile, no interaction between p110(PITSLRE) and cyclin D3 was observed in all the cell cycle. The overexpression of cyclin D3 in 7721 cells leads to an exclusively accumulation of p58(PITSLRE) in the nuclear region, affecting its cellular distribution. Histone H1 kinase activity of p58(PITSLRE) was greatly enhanced upon interaction with cyclin D3. Furthermore, kinase activity of p58(PITSLRE) was found to increase greatly in the presence of cyclin D3 using a specific substrate, beta-1,4-galactosyltransferase 1. These data provide a new clue to our understanding of the cellular function of p58(PITSLRE) and cyclin D3.     

Differential regulation of lipid and protein kinase activities of phosphoinositide 3-kinase gamma in vitro

G protein sensitive phosphoinositide 3-kinase gamma (PI3Kgamma) has been characterised as a pleiotropic signalling protein expressing lipid kinase and protein kinase activities. Whereas the regulation of the lipid kinase activity has been investigated in detail, the regulatory features of PI3Kgamma protein kinase activity are unknown. Here we report that Gbetagamma subunits of heterotrimeric G proteins induce a biphasic response of PI3Kgamma autophosphorylation in vitro, which contrasts the regulatory effects of the G proteins on PI3Kgamma lipid kinase activity. In addition to autophosphorylation PI3Kgamma is able to catalyse transphosphorylation of the adapter protein p101 and the protein kinase MEK-1. In the presence of the p101, Gbetagamma affects PI3Kgamma protein kinase activities in a complex manner. In summary, the differential regulatory effects of heterotrimeric G proteins on PI3Kgamma lipid and protein kinase activities in vitro reflect the functional diversity of the enzyme observed in vivo.     

Phosphorylation pattern of the p90rsk and mitogen-activated protein kinase (MAPK) molecule: comparison of in vitro and in vivo matured porcine oocytes

The overall objective was to elucidate the phosphorylation pattern and activity of the kinase p90rsk, a substrate of mitogen-activated protein kinase (MAPK), during in vitro and in vivo maturation of pig oocytes. Cumulus-oocyte complexes were collected from slaughtered pigs and matured in vitro (0, 22, 26, 30, 34, 46 h) with and without the MEK inhibitor U0126. For in vivo maturation, gilts were stimulated with equine chorionic gonadotrophin (eCG) (600-800 IU). Maturation was induced 72 h later with hCG (500 IU). Oocytes were obtained surgically (0, 22, 30 h). The samples were submitted to electrophoresis and protein blotting analysis. Enhanced chemiluminescence was used for visualization. In vitro matured oocytes were further submitted to a commercially available radioactive kinase assay to determine kinase activity. It was shown that oocytes, as well as cumulus cells, already possess a partially phosphorylated p90rsk at the time of removal from follicles, with a further phosphorylation of the molecule occurring between 22-24 h after the initiation of culture, and in vivo maturation. The phosphorylation of p90rsk coincides with the phosphorylation of MAPK and can be prevented by U0126, indicating a MAPK-dependent phosphorylation of p90rsk. Phosphorylation of the in vivo matured oocytes occurred shown as a band of less than 200 kDa. This is presumably a molecule complex, with MAPK not being a component. Therefore, the p90rsk molecule in vivo exists as a dimer. Determination of kinase activity demonstrated decreasing enzyme activities. This led to the conclusion that the assay is not specific for p90rsk, instead measuring p70S6 kinase activities.     

Phosphorylation of numatrin and other nuclear proteins by cdc2 containing CTD kinase cdc2/p58

Numatrin is a nuclear matrix phosphoprotein whose synthesis and abundance were shown to be regulated during the cell cycle in mitogen-stimulated lymphocytes (Feuerstein, N., and Mond, J. (1987) J. Biol. Chem. 262, 11389-11397). We examined the effect of (a) CTD-kinase, which contains the cdc2 catalytic component (p34) in a complex with a p58 subunit (cdc2/p58) and (b) the M phase-specific histone H1 kinase, which contains the cdc2 kinase in association with a p62 subunit (cdc2/p62), on phosphorylation of numatrin. We show that both cdc2 kinase complexes can phosphorylate numatrin. However, cdc2/p58 at conditions that caused a similar effect to cdc2/p62 on phosphorylation of histone H1 (dpm/micrograms of substrate/micrograms of enzyme) was found to have a 5-25-fold higher catalytic activity in the phosphorylation of numatrin. Analysis of the tryptic phosphopeptide map of numatrin phosphorylated by these cdc2 kinase complexes showed that both kinase complexes phosphorylated two major identical peptides, but minor additional peptides were differentially phosphorylated by each of these kinases. This indicates that under certain experimental conditions cdc2/p58 and cdc2/p62 may express some differences in their catalytic activity. In vitro phosphorylation by CTD kinase of a whole nuclear protein extract from murine fibroblasts showed that numatrin is the most prominent substrate for CTD kinase in this nuclear extract. CTD kinase cdc2/p58 was found to induce significantly the phosphorylation of five other discrete nuclear substrates. Particularly, two nuclear proteins at 75 kDa/pI approximately 6.5 and 85 kDa/pI approximately 5.3, which were not Coomassie Blue stainable, were found to be markedly phosphorylated by CTD kinase. The results of this study call for further study of the role of CTD kinase cdc2/p58 in the phosphorylation of numatrin under physiological conditions and to further characterization of the other nuclear substrates for CTD kinase.     

Localization of an alpha 1,2 galactosyltransferase activity to the Golgi apparatus of Schizosaccharomyces pombe.

We have cloned a gene encoding an alpha 1,2 galactosyltransferase activity from Schizosaccharomyces pombe. The open reading frame of the gene (gma12 for galactomannan, alpha 1,2), combined with the previous protein purification (Chappell and Warren, 1989), predicts an O-linked glycoprotein with type II transmembrane topology. By homologous gene disruption, we have demonstrated that the gma12 gene product (gma12p) is nonessential. The deletion strain (gma12-D10::ura4) has a significantly reduced level of galactosyltransferase activity relative to the parental strain, but both in situ lectin binding and in vitro biochemical assays demonstrate the presence of further galactosyltransferase activity in addition to gma12p. Although gma12p is not the only galactosyltransferase in S. pombe, it produces a unique carbohydrate structure on the surface of the yeast cells. We have generated a polyclonal antiserum against this carbohydrate epitope and shown that gma12p is capable of synthesizing the epitope both in vitro and in vivo. Electron microscopic localization of the gma12+ specific epitope in gma12+ cells revealed that gma12p synthesizes the carbohydrate structure in the Golgi apparatus, and subsequent intracellular transport distributes the epitope to later stages of the secretory pathway. The immunolocalization studies confirm the presence of one or more galactosyltransferase activities in the Golgi apparatus in fission yeast.     

Identification of Ser424 as the protein kinase A phosphorylation site in CTP synthetase from Saccharomyces cerevisiae.

The URA7-encoded CTP synthetase [EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)] in the yeast Saccharomyces cerevisiae is phosphorylated on a serine residue and stimulated by cAMP-dependent protein kinase (protein kinase A) in vitro. In vivo, the phosphorylation of CTP synthetase is mediated by the RAS/cAMP pathway. In this work, we examined the hypothesis that amino acid residue Ser424 contained in a protein kinase A sequence motif in the URA7-encoded CTP synthetase is the target site for protein kinase A. A CTP synthetase synthetic peptide (SLGRKDSHSA) containing the protein kinase A motif was a substrate (Km = 30 microM) for protein kinase A. This peptide also inhibited (IC50 = 45 microM) the phosphorylation of purified wild-type CTP synthetase by protein kinase A. CTP synthetase with a Ser424 --> Ala (S424A) mutation was constructed by site-directed mutagenesis. The mutated enzyme was not phosphorylated in response to the activation of protein kinase A activity in vivo. Purified S424A mutant CTP synthetase was not phosphorylated and stimulated by protein kinase A. The S424A mutant CTP synthetase had reduced Vmax and elevated Km values for ATP and UTP when compared with the protein kinase A-phosphorylated wild-type enzyme. The specificity constants for ATP and UTP for the S424A mutant CTP synthetase were 4.2- and 2.9-fold lower, respectively, when compared with that of the phosphorylated enzyme. In addition, the S424A mutant enzyme was 2.7-fold more sensitive to CTP product inhibition when compared with the phosphorylated wild-type enzyme. These data indicated that the protein kinase A target site in CTP synthetase was Ser424 and that the phosphorylation of this site played a role in the regulation of CTP synthetase activity.     

Phosphorylation and regulation of choline kinase from Saccharomyces cerevisiae by protein kinase A

The CKI1-encoded choline kinase (ATP:choline phosphotransferase, EC 2.7.1.32) from Saccharomyces cerevisiae was phosphorylated in vivo on multiple serine residues. Activation of protein kinase A activity in vivo resulted in a transient increase in the phosphorylation of choline kinase. This phosphorylation was accompanied by a stimulation in choline kinase activity. In vitro, protein kinase A phosphorylated choline kinase on a serine residue with a stoichiometry (0.44 mol of phosphate/mol of choline kinase) consistent with one phosphorylation site/choline kinase subunit. The major phosphopeptide derived from the enzyme phosphorylated in vitro by protein kinase A was common to one of the major phosphopeptides derived from the enzyme phosphorylated in vivo. Protein kinase A activity was dose- and time-dependent and dependent on the concentrations of ATP (Km 2.1 microM) and choline kinase (Km 0.12 microM). Phosphorylation of choline kinase with protein kinase A resulted in a stimulation (1.9-fold) in choline kinase activity whereas alkaline phosphatase treatment of choline kinase resulted in a 60% decrease in choline kinase activity. The mechanism of the protein kinase A-mediated stimulation in choline kinase activity involved an increase in the apparent Vmax values with respect to ATP (2.6-fold) and choline (2.7-fold). Overall, the results reported here were consistent with the conclusion that choline kinase was regulated by protein kinase A phosphorylation.     

Evidence that 3-phosphoinositide-dependent protein kinase-1 mediates phosphorylation of p70 S6 kinase in vivo at Thr-412 as well as Thr-252

Protein kinase B and p70 S6 kinase are members of the cyclic AMP-dependent/cyclic GMP-dependent/protein kinase C subfamily of protein kinases and are activated by a phosphatidylinositol 3-kinase-dependent pathway when cells are stimulated with insulin or growth factors. Both of these kinases are activated in cells by phosphorylation of a conserved residue in the kinase domain (Thr-308 of protein kinase B (PKB) and Thr-252 of p70 S6 kinase) and another conserved residue located C-terminal to the kinase domain (Ser-473 of PKB and Thr-412 of p70 S6 kinase). Thr-308 of PKBalpha and Thr-252 of p70 S6 kinase are phosphorylated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) in vitro. Recent work has shown that PDK1 interacts with a region of protein kinase C-related kinase-2, termed the PDK1 interacting fragment (PIF). Interaction with PIF converts PDK1 from a form that phosphorylates PKB at Thr-308 alone to a species capable of phosphorylating Ser-473 as well as Thr-308. This suggests that PDK1 may be the enzyme that phosphorylates both residues in vivo. Here we demonstrate that PDK1 is capable of phosphorylating p70 S6 kinase at Thr-412 in vitro. We study the effect of PIF on the ability of PDK1 to phosphorylate p70 S6 kinase. Surprisingly, we find that PDK1 bound to PIF is no longer able to interact with or phosphorylate p70 S6 kinase in vitro at either Thr-252 or Thr-412. The expression of PIF in cells prevents insulin-like growth factor 1 from inducing the activation of the p70 S6 kinase and its phosphorylation at Thr-412. Overexpression of PDK1 in cells induces the phosphorylation of p70 S6 kinase at Thr-412 in unstimulated cells, and a catalytically inactive mutant of PDK1 prevents the phosphorylation of p70 S6K at Thr-412 in insulin-like growth factor 1-stimulated cells. These observations indicate that PDK1 regulates the activation of p70 S6 kinase and provides evidence that PDK1 mediates the phosphorylation of p70 S6 kinase at Thr-412.     

Biochemical properties of p60v-src mutants that induce different cell transformation parameters.

PA101 and PA104 are Rous sarcoma virus variants that are differentially temperature sensitive in cell transformation parameters, including stimulation of cell proliferation, morphological alteration, and anchorage independence. To investigate the biochemical basis for the differential expression of these parameters, the tyrosine kinase activity and subcellular localization of the mutant p60v-src proteins encoded in the variants were examined. Analysis of chimeric src proteins derived from the mutant proteins revealed that lesions in the kinase domain inhibit in vitro kinase activity and confer temperature sensitivity on tyrosine phosphorylation of cellular protein p34 in vivo. The amino-terminal portions of the mutant src proteins also influence tyrosine phosphorylation in vivo and in vitro, which is consistent with an interaction between an amino-terminal region and the kinase domain. Large proportions of the mutant src proteins exist in soluble complexes with cellular proteins p50 and p90, even though the src proteins are myristylated. The formation of these soluble complexes segregates with lesions in the kinase domain and is independent of temperature. Our results demonstrate that the transformation parameters examined correlate to a limited extent with p34 phosphorylation but not with the levels of in vitro kinase activity or soluble complex formation.     

Spy1 interacts with p27Kip1 to allow G1/S progression

Progression through the G1/S transition commits cells to synthesize DNA. Cyclin dependent kinase 2 (CDK2) is the major kinase that allows progression through G1/S phase and subsequent replication events. p27 is a CDK inhibitor (CKI) that binds to CDK2 to prevent premature activation of this kinase. Speedy (Spy1), a novel cell cycle regulatory protein, has been found to prematurely activate CDK2 when microinjected into Xenopus oocytes and when expressed in mammalian cells. To determine the mechanism underlying Spy1-induced proliferation in mammalian cell cycle regulation, we used human Spy1 as bait in a yeast two-hybrid screen to identify interacting proteins. One of the proteins isolated was p27; this novel interaction was confirmed both in vitro, using bacterially expressed and in vitro translated proteins, and in vivo, through the examination of endogenous and transfected proteins in mammalian cells. We demonstrate that Spy1 expression can overcome a p27-induced cell cycle arrest to allow for DNA synthesis and CDK2 histone H1 kinase activity. In addition, we utilized p27-null cells to demonstrate that the proliferative effect of Spy1 depends on the presence of endogenous p27. Our data suggest that Spy1 associates with p27 to promote cell cycle progression through the G1/S transition.     

Gene technology-based antimetabolite design: the use of an in vitro protein expression system to facilitate antimetabolite design for virally-induced human diseases and malignant conditions

A precondition for the chemotherapeutic treatment of a variety of virally-induced human diseases and malignant conditions is a highly selective interaction of the drug molecule to be used with it's biological target. To ensure the development of novel, effective drugs, it is essential that the biological target is well characterised with regard to it's structure and activity. Such characterisation relies upon adequate amounts of pure target being available. One of the most important enzymatic importers for antimetabolites is the enzyme thymidine kinase. In this article an in vitro protein expression system is described which facilitates the production of milligram amounts of pure and biologically active thymidine kinase, from a number of important biological sources. Results have shown that the in vitro produced enzyme has the exact biochemical propeties of the in vivo enzyme. Thus the in vitro protein expression system is an ideal vechicle to facilitate an in depth investigation of the enzyme's biological properties.     

Identification of the human pim-1 gene product as a 33-kilodalton cytoplasmic protein with tyrosine kinase activity.

The human pim-1 gene was recently identified as a new putative oncogene located on chromosome 6p21, a region showing karyotypic abnormalities in particular leukemias. In the present work we characterized the pim protein product. In vitro translation of positively selected poly(A)+ mRNA indicates that this gene encodes a 33-kilodalton protein. Anti-pim antibodies were raised against a fused TrpE-pim protein induced in a bacterial expression vector. This antibody immunoprecipitated a 33-kilodalton protein from in vivo [35S]methionine-labeled K562 and KCl myelogenous origin cell lines. This protein was localized to the cytoplasm, and in vivo labeling as well as in vitro kinase assay suggests that it is a phosphoprotein with tyrosine kinase activity. This was further confirmed by performing autophosphorylation directly on a p33pim-containing gel band cut out after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results imply that the tyrosine kinase activity of pim can be recovered after boiling the pim-1 protein in sample buffer: a feature not described yet for this class of protein. These results suggest that pim-1 is a new member of the subgroup of oncogenes encoding tyrosine kinases.     

Phosphorylation of the kinase interaction motif in mitogen-activated protein (MAP) kinase phosphatase-4 mediates cross-talk between protein kinase A and MAP kinase signaling pathways

MAP kinase phosphatase 4 (DUSP9/MKP-4) plays an essential role during placental development and is one of a subfamily of three closely related cytoplasmic dual-specificity MAPK phosphatases, which includes the ERK-specific enzymes DUSP6/MKP-3 and DUSP7/MKP-X. However, unlike DUSP6/MKP-3, DUSP9/MKP-4 also inactivates the p38α MAP kinase both in vitro and in vivo. Here we demonstrate that inactivation of both ERK1/2 and p38α by DUSP9/MKP-4 is mediated by a conserved arginine-rich kinase interaction motif located within the amino-terminal non-catalytic domain of the protein. Furthermore, DUSP9/MKP-4 is unique among these cytoplasmic MKPs in containing a conserved PKA consensus phosphorylation site (55)RRXSer-58 immediately adjacent to the kinase interaction motif. DUSP9/MKP-4 is phosphorylated on Ser-58 by PKA in vitro, and phosphorylation abrogates the binding of DUSP9/MKP-4 to both ERK2 and p38α MAP kinases. In addition, although mutation of Ser-58 to either alanine or glutamic acid does not affect the intrinsic catalytic activity of DUSP9/MKP-4, phospho-mimetic (Ser-58 to Glu) substitution inhibits both the interaction of DUSP9/MKP-4 with ERK2 and p38α in vivo and its ability to dephosphorylate and inactivate these MAP kinases. Finally, the use of a phospho-specific antibody demonstrates that endogenous DUSP9/MKP-4 is phosphorylated on Ser-58 in response to the PKA agonist forskolin and is also modified in placental tissue. We conclude that DUSP9/MKP-4 is a bona fide target of PKA signaling and that attenuation of DUSP9/MKP-4 function can mediate cross-talk between the PKA pathway and MAPK signaling through both ERK1/2 and p38α in vivo.