Expression and characterization of protein kinase C-delta

A cDNA encoding protein kinase C-delta (PKC-delta) was isolated from a rat brain library. The coding region was subcloned into the expression vector pmt2 and transfected into COS-1 cells. Expression of the protein led to an 11-fold increase in activity as determined with a synthetic peptide based on the PKC-delta pseudosubstrate site. The Mr of PKC-delta as determined by SDS/PAGE and immunoblot analysis using anti-(PKC-delta C-terminal) antibodies was 77,000. The enzyme was purified to near homogeneity and showed total dependency on phospholipid and diacylglycerol (or phorbol esters) for activity. Like PKC-epsilon, PKC-delta displays no Ca2+ dependence for activation. The substrate specificity of PCK-delta is similar to that of PKC-epsilon but quite different from other PKCs.     

Mouse "protective protein". cDNA cloning, sequence comparison, and expression

The "protective protein" is the glycoprotein that forms a complex with the lysosomal enzymes beta-galactosidase and neuraminidase. Its deficiency in man leads to the metabolic storage disorder galactosialidosis. The primary structure of human protective protein, deduced from its cloned cDNA, shows homology to yeast serine carboxypeptidases. We have isolated a full-length cDNA encoding murine protective protein. The nucleotide sequences as well as the predicted amino acid sequences are highly conserved between man and mouse. Domains important for the protease function are completely identical in the two proteins. Both human and mouse mature protective proteins covalently bind radiolabeled diisopropyl fluorophosphate. Transient expression of the murine cDNA in COS-1 cells yields a protective protein precursor of 54 kDa, a size characteristic of the glycosylated form. This cDNA-encoded precursor, endocytosed by human galactosialidosis fibroblasts, is processed into a 32- and a 20-kDa heterodimer and corrects beta-galactosidase and neuraminidase activities. A tissue-specific expression of protective protein mRNA is observed when total RNA from different mouse organs is analyzed on Northern blots.     

Sequencing, expression, and characterization of cDNA expressed flavin-containing monooxygenase 2 from mouse

The cDNA clone of mouse flavin-containing monooxygenase 2 (FMO2) was obtained as an expressed sequence tag (EST) isolated from a female mouse kidney cDNA library from the I.M.A.G.E. consortium (I.M.A.G.E. CloneID 1432164). Complete sequencing of the EST derived a nucleotide sequence for mouse FMO2, which contains 112 bases of 5' flanking region, 1607 bases of coding region, and 309 bases of 3' flanking region. This FMO2 sequence encodes a protein of 535 amino acids including two putative pyrophosphate binding sequences (GxGxxG/A) beginning at positions 9 and 191. Additionally, this mouse FMO protein sequence shows 87 and 86% homology to rabbit and human FMO2 respectively. The mouse FMO2 sequence was subcloned into the expression vector pJL-2, a derivative of pKK233-2 and used to transform XL1-Blue Escherichia coli. FMO activity in particulate fractions isolated from isopropyl-beta-D-thiogalactopyanoside (IPTG) induced cells was heat stable (45 degrees C for 5 min) and demonstrated optimal activity at a relatively high pH of 10.5. The expressed FMO2 enzyme showed catalytic activity towards the FMO substrate methimazole and further analysis of E. coli fractions utilizing NADPH oxidation demonstrated that the mouse FMO2 enzyme also exhibits catalytic activity towards thiourea, trimethylamine, and the insecticide phorate.     

Rat RI beta isoform of type I regulatory subunit of cyclic adenosine monophosphate-dependent protein kinase: cDNA sequence analysis, mRNA tissue specificity, and rat/mouse difference in expression in testis

A rat complementary DNA (cDNA) for the RI beta isoform of type I cyclic adenosine monophosphate (cAMP)-dependent protein kinase regulatory subunit was cloned and sequenced and was found to contain the entire protein coding and 3'-untranslated regions, with a single (ATTAAA) poly-adenylation site. The largest open reading frame was preceded by a short out-of-phase open reading frame, which is not seen in the corresponding mouse RI beta cDNA due to a single base substitution. The rat RI beta cDNA clone was 2,374 bases long and detected a rat mRNA of approximately 2.8 kilobases. Rat RI beta mRNA was abundant in adult rat brain and testis but was undetectable in other rat tissues. The rat RI beta cDNA also detected RI beta mRNA in mouse brain, but not mouse testis, from 10-week-old BALB/c or 10- and 6-week-old Swiss Webster mice. Thus, despite a 96% nucleotide identity in the coding region of RI beta in rat vs. mouse, there are at least two differences in these closely related species. First, there is a short open reading frame, which precedes the coding region in the rat but not the mouse. Second, unlike the mouse testis, the rat testis contains abundant levels of RI beta mRNA.     

Mouse preproacrosin: cDNA sequence, primary structure and postmeiotic expression in spermatogenesis

The primary structure of mouse preproacrosin was deduced by nucleotide sequencing of cDNA clones isolated from a mouse testis cDNA library. The largest cDNA, with 1373 bp, consists of a 11-bp 5'untranslated sequence, a 1254-bp open reading frame terminated by a TGA triplet and a 105-bp 3' untranslated end, including one potential polyadenylation signal. The NH2-terminus of the polypeptide contains a hydrophobic 15-amino acid signal peptide. This cleavable signal sequence is followed by 403 amino acids, representing the acrosin light and the heavy chain of 23 and 380 amino acid residues, respectively. The proteolytic active site segments His, Asp and Ser are part of the heavy chain, as well as a proline-rich COOH-terminus, which is not present in any other serine proteinase studied so far. Furthermore the postmeiotic expression of the preproacrosin gene during mouse spermatogenesis was studied.     

Protein kinase C isoform expression and activity in the mouse heart

The expression of protein kinase C (PKC) isoforms in the developing murine ventricle was studied using Western blotting, assays of PKC activity, and immunoprecipitations. The abundance of two Ca2+-dependent isoforms, PKCalpha and PKCbetaII, as well as two Ca2+-independent isoforms, PKCdelta and PKCepsilon, decreased during postnatal development to <15% of the levels detected at embryonic day 18. The analysis of the subcellular distribution of the four isoforms showed that PKCdelta and PKCepsilon were associated preferentially with the particulate fraction in fetal ventricles, indicating a high intrinsic activation state of these isoforms at this developmental time point. The expression of PKCalpha in cardiomyocytes underwent a developmental change. Although preferentially expressed in neonatal cardiomyocytes, this isoform was downregulated in adult cardiomyocytes. In fast-performance liquid chromatography-purified ventricular extracts, the majority of PKC activity was Ca2+-independent in both fetal and adult ventricles. Immunoprecipitation assays indicated that PKCdelta and PKCepsilon were responsible for the majority of the Ca2+-independent activity. These studies indicate a prominent role for Ca2+-independent PKC isoforms in the mouse heart.     

Mouse in red: red fluorescent protein expression in mouse ES cells, embryos, and adult animals

Spectral variants of green fluorescent protein are widely used in live samples for a broad range of applications: from visualization of protein interactions, through following gene expression, to marking particular cells in complex tissues. Higher wavelength emissions (such as red) are preferred due to the lower background-autofluorescence in tissues (Miyawaka et al., Nat Cell Biol Suppl S1-7, 2003). Until now, however, red fluorescent proteins (RFP) have displayed toxicity in murine embryos, which has hampered its application in this model. Here we report strong expression of a recently developed RFP variant, DsRed.T3, in mouse ES cells, embryos, and adult mice. Our results show that the red fluorescent wavelength has a superior tissue penetrance compared with spectral variants of lower wavelength. Furthermore, we have generated an ES cell line and a corresponding transgenic mouse line in which red fluorescence is activated upon Cre excision. Finally, we introduced cell type-specifically expressed Cre transgenes into this Cre recombinase reporter cell line, and by using the tetraploid embryo complementation assay, we could directly verify the Cre recombinase specificity on ES cell-derived embryos/animals.     

Rat major acute-phase protein: biosynthesis and characterization of cDNA clone

The major acute-phase protein (alpha 1-MAP) of rat serum is induced in response to inflammation. This induction may be attributed to a corresponding increase in the level of translatable mRNA for the protein. Using in vitro and in vivo systems, various biosynthetic processing intermediates of this glycoprotein have been isolated. alpha 1-MAP is translated in a rabbit reticulocyte system as a preprotein with an amino-terminal signal peptide and an apparent molecular weight of 51,000. Translation of rough microsomes yields a product with a mass of 57,000 Da, representing the core glycosylated form of alpha 1-MAP. Cotranslational glycosylation appears to occur in a stepwise fashion, since three glycosylated forms of alpha 1-MAP (51,000, 54,000, and 57,000 Da) were detected in polysome translations; these products were digested by endoglycosidase H to a 48,000-Da protein. Two intracellular forms of alpha 1-MAP were observed in vivo, a 57,000-Da (core carbohydrate sidechains) and a 66,000-Da protein (mature complex carbohydrate side-chains); the latter was the only component secreted into the culture medium. To extend our studies on this protein, a cDNA clone specific for alpha 1-MAP was isolated. The recombinant was positively identified by hybrid selection procedures and contains a 1.55-kb insert. Partial radiosequence analysis of the primary translation product indicated the distribution of Leu, Ile, Cys, and Met in the amino-terminal region of this protein. To relate the location of these amino acids with the nucleotide sequence, cDNA was analyzed by the method of Maxam and Gilbert. These results indicate that the cDNA insert contains the 3' poly(A) tail, and alignment of the 5' end of the cDNA with the available amino acid sequence of the primary translation product corroborated that the insert encodes the entire alpha 1-MAP protein except for the first four amino acids of the signal peptide.     

Characterization of protein kinase C-delta in mouse oocytes throughout meiotic maturation and following egg activation

Changes in protein kinase C (PKC) activity influence the progression of meiosis; however, the specific function of the various PKC isoforms in female gametes is not known. In the current study, the protein expression and subcellular distribution profile of PKC-delta (PKC-delta), a novel isoform of the PKC family, was determined in mouse oocytes undergoing meiotic maturation and following egg activation. The full-length protein was observed as a doublet (76 and 78 kDa) on Western blot analysis. A smaller (47 kDa) carboxyl-terminal fragment, presumably the truncated catalytic domain of PKC-delta, was also strongly expressed. Both the full-length protein and the catalytic fragment became phosphorylated coincident with the resumption of meiosis and remained phosphorylated throughout metaphase II (MII) arrest. Immunofluorescence staining showed PKC-delta distributed diffusely throughout the cytoplasm of oocytes during maturation and associated with the spindle apparatus during the first meiotic division. Discrete foci of the protein also localized with the chromosomes in some mature eggs. Following the completion of meiosis, PKC-delta became dephosphorylated within 2 h of in vitro fertilization or parthenogenetic activation. The protein also accumulated in the nuclei of early embryos and was phosphorylated during M-phase of the initial mitotic cleavage division. By the two-cell stage, expression of the truncated catalytic fragment was minimal. These data demonstrate that the subcellular distribution and posttranslational modification of PKC-delta is cell cycle dependent, suggesting that its activity and/or function likely vary with the progression of meiosis and egg activation.     

Cloning and expression of a novel MAPKK-like protein kinase, lymphokine-activated killer T-cell-originated protein kinase, specifically expressed in the testis and activated lymphoid cells

A novel protein kinase, TOPK (T-LAK cell-originated protein kinase), was isolated from a lymphokine-activated killer T (T-LAK) cell subtraction cDNA fragment library. The open reading frame of the TOPK gene encodes a protein of 322 amino acids, possessing a protein kinase domain profile. The cap site analysis of the 5'-end of TOPK mRNA revealed two forms, a major full-length form and a minor spliced form at the 5'-site, both encoding the same protein. A BLAST homology search and phylogenetic analysis indicated that TOPK is related to dual specific mitogen-activated protein kinase kinase (MAPKK). The transfection of the TOPK gene to COS-7 cells up-regulated a phosphorylation of p38 MAPK but not ERK1/2 or SAPK/JNK. Gel precipitation study indicated that TOPK protein can be associated with p38 in vitro. Tissue distribution of TOPK mRNA expression was specific for the testis, T-LAK cells, activated lymphoid cells, and lymphoid tumors. On the other hand, deactivated T-LAK cells did not show TOPK mRNA expression. These data suggest that TOPK is a newly identified member of a novel MEK3/6-related MAPKK that may be enrolled in the activation of lymphoid cells and support testicular functions.