Murine Protein Kinase Research Articles

The trkB tyrosine protein kinase is a receptor for neurotrophin-4

Neurotrophin-4 is a novel member of the nerve growth factor family of neurotrophins recently isolated from Xenopus and viper DNA. We now report that the Xenopus NT-4 protein (XNT-4) can mediate some of its biological properties through gp 145trkB a murine tyrosine protein kinase previously identified as a primary receptor for the related brain-derived neurotrophic factor (BDNF). XNT-4 displaces 125I-labeled BDNF from binding to cells expressing gp 145trkB receptors, induces their rapid phosphorylation on tyrosine residues, and causes the morphologic transformation of NIH 3T3 cells when coexpressed with gp 145trkB. Moreover, XNT-4 induces the differentiation of PC12 cells into sympathetic-like neurons only if they ectopically express gp 145trkB receptors. None of these biochemical or biological effects could be observed when XNT-4 was added to cells expressing the related receptors. Replacement of one of the extracellular cysteines (Cys-345) of gp 145trkB by a serine residue prevents its activation by XNT-4 but not by BDNF. Therefore, XNT-4 and BDNF may interact with at least partially distinct domains within the gp 145trkB receptor.

Transfection-mediated expression of a dominant cAMP-resistant phenotype in the opossum kidney (OK) cell line prevents parathyroid hormone-induced inhibition of Na-phosphate cotransport. A protein kinase-A-mediated event.

Sodium-phosphate cotransport in the PTH-responsive opossum kidney (OK) cell line is inhibited by PTH, cAMP, and activators of protein kinase C. In order to probe the role of cAMP, we stably transfected OK cells with an expression vector for a cAMP-binding mutation of the murine protein kinase A regulatory subunit. Two-dimensional electrophoresis of cAMP-binding proteins from transfected cells indicated a 20-fold overexpression of the mutant regulatory unit. Protein kinase A from these cells had a 20-fold increase in the concentration of cAMP required for half-maximal activation, 2.8 microM vs. 0.15 microM for wild type cells. In the transfected cells, Na-phosphate cotransport was insensitive to up to 1 mM 8-Br-cAMP and 1 microM PTH, while these same agonists caused a significant inhibition of transport in the wild type cells. The effects on Na-phosphate cotransport of the protein kinase C activators oleoyl-acetyl glycerol and tetradecanoyl-phorbol acetate, which were marked in the wild type cells, were still present, although attenuated, in the transfected mutants. With prolonged passage, the cAMP-insensitive phenotype reverted to wild type cAMP sensitivity despite continued selection for the cotransfected neo marker. The revertant cells had a normal cAMP requirement for half-maximal activation of protein kinase A, 0.13 microM, and the PTH and cAMP-sensitive inhibition of Na-phosphate cotransport was restored. We suggest that an intact and normally cAMP-sensitive protein kinase A pathway is an absolute requirement for PTH inhibition of Na-phosphate cotransport in the OK cell.

Casein kinase II from Caenorhabditis elegans. Properties and developmental regulation of the enzyme; cloning and sequence analyses of cDNA and the gene for the catalytic subunit.

The nematode Caenorhabditis elegans provides a model system for investigating the structure, function, and regulation of casein kinase II. Cytosols from C. elegans embryos and gravid adults, which contain fertilized eggs and embryos, are enriched in casein kinase II activity; cytosols from newly hatched larva, four subsequent larval stages, and immature adults exhibit casein kinase II levels that are 3-10-fold lower than those observed in embryo cytosol. C. elegans casein kinase II contains alpha (Mr = 42,000) and beta (Mr = 29,000) subunits and has a Stokes radius of 50 nm. The enzyme utilizes ATP and GTP as substrates, is potently inhibited by heparin and undergoes autophosphorylation. Sequence analyses of cloned cDNAs corresponding to the 1.7-kilobase mRNA encoding the alpha (catalytic) subunit of casein kinase II indicate that the alpha polypeptide contains 359 amino acid residues. Variations in the abundance of casein kinase II alpha mRNA are coordinated with changes in enzyme activity during C. elegans development, indicating that alpha subunit expression is controlled at a pretranslational level. However, the magnitude of the developmentally controlled changes in phosphotransferase activity exceeded the corresponding increments in alpha subunit mRNA content. This suggests that translational and/or post-translational mechanisms also play an important role in the developmental regulation of C. elegans casein kinase II activity. The 2.9-kilobase casein kinase II alpha gene is divided into eight exons by intervening sequences ranging from 48 to 457 base pairs in length. The alpha gene promoter contains a TATA box, and a unique transcription start site has been identified. The intron/exon organization of the casein kinase II alpha gene differs markedly from the gene structure of the catalytic subunit of murine cAMP-dependent protein kinase (Chrivia, J. C., Uhler, M. D., and McKnight, G. S. (1988) J. Biol. Chem. 263, 5739-5744).

Expression of the Catalytic Subunit of cAMP-Dependent Protein Kinase in Escherichia coli

A cDNA clone for the catalytic subunit of murine cAMP-dependent protein kinase was placed into two expression vectors, pLWS-3 and pLSW-4. For pLWS-3, the entire coding region of the catalytic subunit was inserted into the NdeI site of pT7-7 under the control of the T7 promoter. pLWS-4 contains a polycistronic transcript under control of the lac UV5 promoter encoding for the type I regulatory subunit followed by the catalytic subunit. Significant expression was achieved with pLWS-4 in Escherichia coli 222 and JM101; however, the catalytic subunit was produced in an insoluble form. In the case of the catalytic subunit produced in E. coli BL21(DE3) by pLWS-3, the catalytic subunit accounted for approximately 30% of the total bacterial protein. Up to 5 mg of this catalytic subunit per liter of culture was in the soluble extract. Solubility was improved substantially when induction was carried out at 30 degrees C instead of 37 degrees C. This recombinant catalytic subunit was purified by phosphocellulose chromatography, followed by ammonium sulfate precipitation and gel filtration. A Mr of 38,000 was estimated based on size exclusion chromatography and on polyacrylamide gel electrophoresis. The recombinant protein had a free alpha-amino-terminal Gly in contrast to the mammalian enzyme which is myristylated at the amino-terminal glycine. The lack of acylation did not significantly alter the activity of the enzyme. The specific activity of 19 mumol/min/mg is comparable to the mammalian enzyme. The Km values for Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) (43 microM) and MgATP (18.5 microM also were comparable. The absence of the acyl group also did not prevent holoenzyme formation. Holoenzyme activation by cAMP was indistinguishable for holoenzyme made with mammalian catalytic subunit and recombinant catalytic subunit. The recombinant enzyme was more sensitive than the mammalian enzyme to heat denaturation at 49 degrees C. The t1/2 for the recombinant catalytic subunit was 0.7 min in contrast to 3.9 min for the mammalian enzyme. This difference in stability may be attributable to the lack of the acyl group. The recombinant enzyme was particularly sensitive to heat denaturation in the presence of low concentrations (0.01%) of Triton X-100.

Age-dependent changes in murine protein kinase and protease enzymes

Hormone responsiveness is mediated by signal transduction mechanisms involving second messengers, such as cAMP and Ca 2+, which regulate reversible changes in the phosphorylation state of proteins. During senescence individuals frequently exhibit a diminished responsiveness to hormones. We examined changes in enzymes involved in protein phosphorylation reactions that might account for this decreased adaptiveness in old mice, and observed the following post-maturational changes: (1) cAMP-dependent protein kinase (Pk-A) specific activity decreased in spleen cytosol and in the particulate fractions of lung, spleen and liver of 24-month-old mice as compared to 2-month-old mice. Splenic cytosolic Pk-A activity decreased by 18 months of age, while particulate activity decreased by 6 months; (2) The amount of 8-N 3-[ 32P]cAMP, a photoaffinity analog of cAMP, incorporated into Pk-A regulatory (R)-subunits from spleen and liver particulate fractions decreased, while photolabeling of R-subunit degradative products with this analog in heart and spleen cytosol increased. (3) Age-dependent increases in membrane-associated protease activities were found in all organs, along with a decrease in cytosolic lung calpain activity. these preteolytic changes may account for the enhanced R-subunit degradation and decreased Pk-A activities observed during senescence. (4) Age-dependent alterations in Ca 2+/phospholipid-dependent protein kinase (Pk-C) are organ specific: lung, liver, brain, and heart demonstrate no change in Pk-C activity, while spleen exhibits decreased activity. We hypothesize that these age-dependent alterations in kinase and proteolytic activities may be in part responsible for changes in cellular response to hormonal stimulation, differentiation signals, and antigen responsiveness during senescence.