Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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The p34cdc2 protein kinase is a component of maturation-promoting factor, the master regulator of the cell cycle in all eukaryotes. The activity of p34cdc2 is itself tightly regulated by phosphorylation and dephosphorylation. Predicted regulatory phosphorylation sites of Xenopus p34cdc2 were mutated in vitro, and in vitro-transcribed RNAs were injected into Xenopus oocytes. The cdc2 single mutants Thr-14----Ala and Tyr-15----Phe did not induce germinal vesicle breakdown (BVBD) upon microinjection into oocytes. In contrast, the cdc2 double mutant Ala-14/Phe-15 did induce GVBD. Both the Ala-14 and Ala-14/Phe-15p34cdc2 mutants were shown to coimmunoprecipitate cyclin B1 and to phosphorylate histone H1 in immune complex kinase assays. Microinjection of antisense oligonucleotides to c-mosXe was used to demonstrate the role of mos protein synthesis in the induction of GVBD by the Ala-14/Phe-15 cdc2 mutant. Thr-161 was also mutated. p34cdc2 single mutants Ala-161 and Glu-161 and triple mutants Ala-14/Phe-15/Ala-161 and Ala-14/Phe-15/Glu-161 failed to induce GVBD in oocytes and showed a decreased binding to cyclin B1 in coimmunoprecipitations. Each of the cdc2 mutants was also assayed by coinjection with cyclin B1 or c-mosXe RNA into oocytes. Several of the cdc2 mutants were found to affect the kinetics of cyclin B1 and/or mos-induced GVBD upon coinjection, although none affected the rate of progesterone-induced maturation. We demonstrate here the significance of Thr-14, Tyr-15, and Thr-161 of p34cdc2 in Xenopus oocyte maturation. In addition, these results suggest a regulatory role for mosXe in induction of oocyte maturation by the cdc2 mutant Ala-14/Phe-15.
Mol Cell Biol 1992 Jul
PMID:Requirement of mosXe protein kinase for meiotic maturation of Xenopus oocytes induced by a cdc2 mutant lacking regulatory phosphorylation sites. 137 75

The protein kinase C (PKC) family of phospholipid-dependent serine-threonine kinases has been implicated in keratinocyte differentiation and neoplastic transformation. To determine if Ca(2+)-mediated keratinocyte differentiation is associated with changes in PKC isozyme gene expression, RNA was isolated from primary mouse keratinocytes grown in medium with 0.05, 0.12, or 1.4 mM Ca2+. Based on northern blot analysis, primary keratinocytes expressed mRNA encoding PKC-alpha, -delta, -epsilon, -zeta, and -eta, but not PKC-beta or -gamma. Relatively little change was detected in the level of these transcripts in cells induced to differentiate by exposure to elevated extracellular Ca2+. Interestingly, the PKC-zeta transcripts detected in RNA isolated from keratinocytes were approximately 200 nucleotides longer than those from mouse brain, suggesting the existence of an alternative form of this isozyme. An early change in benign neoplastic transformation of keratinocytes is the inability to differentiate in response to Ca2+ or the PKC activator 12-O-tetradecanoylphorbol-13-acetate, which is consistent with altered PKC function in these cells. The PKC isozyme mRNA profile was examined in two benign neoplastic keratinocyte cell lines, 308 and SP-1, which contain an activating mutation of the c-Ha-ras gene. Like normal keratinocytes. 308 and SP-1 cells expressed mRNA encoding PKC-alpha, -delta, -epsilon, -zeta, and -eta. However, the abundance of PKC-zeta transcripts in both cell lines was reduced by 74-89% when compared with normal keratinocytes at similar Ca2+ levels. In addition, SP-1 but not 308 cells exhibited a sevenfold increase in PKC-eta mRNA when cultured in medium with 1.4 mM Ca2+. To address whether these changes were related to the presence of an activated ras gene, RNA was isolated from primary keratinocytes transduced to a benign neoplastic phenotype with the v-Ha-ras oncogene. As with normal, 308, and SP-1 cells, v-Ha-ras keratinocytes expressed mRNA encoding PKC-alpha, -delta, -epsilon, -zeta and -eta. The level of PKC-zeta transcripts was similar in normal and v-Ha-ras keratinocytes, indicating that reduction of this mRNA in both 308 and SP-1 cells was not a direct result of ras activation. As in SP-1 cells, PKC-eta in v-Ha-ras keratinocytes was responsive to extracellular Ca2+, with a four-fold increase in transcript abundance in 0.12 mM Ca2+ medium relative to 0.05 mM Ca2+ medium.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Carcinog 1992
PMID:Transcripts encoding protein kinase C-alpha, -delta, -epsilon, -zeta, and -eta are expressed in basal and differentiating mouse keratinocytes in vitro and exhibit quantitative changes in neoplastic cells. 137 14

Extensins comprise a family of structural cell wall hydroxyproline-rich glycoproteins in plants. Two tomato genomic clones, Tom J-10 and Tom L-4, were isolated from a tomato genomic DNA library by in situ plaque hybridization with extensin DNA probes. Tom J-10 encoded an extensin with 388 amino acid residues and a predicted molecular mass of 43 kDa. The Tom J-10 encoded extensin lacked a typical signal peptide sequence, but contained two distinct protein domains consisting of 19 tandem repeats of Ser-Pro4-Ser-Pro-Lys-Tyr-Val-Tyr-Lys at the amino terminus which were directly followed by 8 tandem repeats of the consensus sequence Ser-Pro4-Tyr3-Lys-Ser-Pro4-Ser-Pro at the carboxy terminus. RNA blot hybridization analysis with the Tom J-10 extensin probe demonstrated the presence of a 4.0 kb tomato stem mRNA which accumulated markedly in response to wounding. Tom L-4 encoded an extensin with 322 amino acid residues and a predicted molecular mass of 35 kDa. The Tom L-4 encoded extensin contained a typical signal peptide sequence at the amino terminus and was followed by at least 3 distinct domains. These domains consisted of an amino terminal domain containing several Lys-Pro and Ser-Pro4 repeat units, a central domain with repeats of the consensus sequence Ser-Pro2-5-Thr-Pro-Ser-Tyr-Glu-His-Pro-Lys-Thr-Pro, and a carboxy terminal domain containing repeats of the consensus sequence Ser-Ser-Pro4-Ser-Pro-Ser-Pro4-Thr-Tyr1-3. RNA blot hybridization analysis with the Tom L-4 extensin probe demonstrated the presence of a 2.6 kb tomato stem mRNA which accumulated in response to wounding.
Plant Mol Biol 1992 Oct
PMID:Isolation and characterization of two wound-regulated tomato extensin genes. 138 Dec 33

The ontogenesis of vasopressin receptors in the rat collecting duct was studied by measuring the binding of [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid),2-O-methyltyrosine,4-threonine,8-ornithine,9-125I-tyrosylamide+ ++]-vasotocin (125I-d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH(9)2]-OVT) to isolated cortical collecting ducts (CCD), outer medullary collecting ducts (OMCD) and inner medullary collecting ducts (IMCD) microdissected from collagenase-treated kidneys of 2- to 34-day-old rats and adult animals. The stereospecificity for recognition of a series of seven vasopressin structural analogues by CCD and OMCD receptors reveals that the labeled binding sites identified in 11- to 16-day-old and adult rats are homologous respectively and contain a major population of V2 type and a minor population of V1a type of vasopressin receptors. At all postnatal stages examined, the receptor density (expressed as 10(-18) mol radioligand bound per square millimeter tubular outer surface area) decreases gradually from the CCD to the IMCD. For the three segments, the numbers of receptors detected remained constant during the first 2 weeks after birth and increased sharply after 20 days to reach the corresponding adult levels during the fifth week.
Mol Cell Endocrinol 1992 Aug
PMID:Postnatal ontogenesis of vasopressin receptors in the rat collecting duct. 138 71

The importance of amino acid side-chains in helix stability has been investigated by making a series of mutations at the N-caps, C-caps and internal positions of the solvent-exposed faces of the two alpha-helices of barnase. There is a strong positional and context dependence of the effect of a particular amino acid on stability. Correlations have been found that provide insight into the physical basis of helix stabilization. The relative effects of Ala and Gly (or Ser) may be rationalized on the basis of solvent-accessible surface areas: burial of hydrophobic surface stabilizes the protein as does exposure to solvent of unpaired hydrogen bond donors or acceptors in the protein. There is a good correlation between the relative stabilizing effects of Ala and Gly at internal positions with the total change in solvent-accessible hydrophobic surface area of the folded protein on mutation of Ala----Gly. The relationship may be extended to the N and C-caps by including an extra term in hydrophilic surface area for the solvent exposure of the non-intramolecularly hydrogen-bonded main-chain CO, NH or protein side-chain hydrogen bonding groups. The requirement for solvent exposure of the C-cap main-chain CO groups may account for the strong preference for residues having positive phi and psi angles at this position, since this alpha L-conformation results in the largest solvent exposure of the C-terminal CO groups. Glycine in an alpha L-conformation results in the greatest exposure of these CO groups. Further, the side-chains of His, Asn, Arg and Lys may, with positive phi and psi-angles, form a hydrogen bond with the backbone CO of residue in position C -3 (residues are numbered relative to the C-cap). The preferences at the C-cap are Gly much greater than His greater than Asn greater than Arg greater than Lys greater than Ala approximately Ser approximately greater than Asp. The preferences at the N-cap are determined by hydrogen bonding of side-chains or solvent to the exposed backbone NH groups and are: Thr approximately Asp approximately Ser greater than Gly approximately Asn greater than Gln approximately Glu approximately His greater than Ala greater than Val much greater than Pro. These general trends may be obscured when mutation allows another side-chain to become a surrogate cap.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1992 Sep 20
PMID:Alpha-helix stability in proteins. I. Empirical correlations concerning substitution of side-chains at the N and C-caps and the replacement of alanine by glycine or serine at solvent-exposed surfaces. 140 68

The crystal and molecular structure of a triacylglyceride lipase (EC 3.1.1.3) from the fungus Rhizomucor miehei was analyzed using X-ray single crystal diffraction data to 1.9 A resolution. The structure was refined to an R-factor of 0.169 for all available data. The details of the molecular architecture and the crystal structure of the enzyme are described. A single polypeptide chain of 269 residues is folded into a rather unusual singly wound beta-sheet domain with predominantly parallel strands, connected by a variety of hairpins, loops and helical segments. All the loops are right-handed, creating an uncommon situation in which the central sheet is asymmetric in that all the connecting fragments are located on one side of the sheet. A single N-terminal alpha-helix provides the support for the other, distal, side of the sheet. Three disulfide bonds (residues 29-268, 40-43, 235-244) stabilize the molecule. There are four cis peptide bonds, all of which precede proline residues. In all, 230 ordered water molecules have been identified; 12 of them have a distinct internal character. The catalytic center of the enzyme is made up of a constellation of three residues (His257, Asp203 and Ser144) similar in structure and function to the analogous (but not homologous) triad found in both of the known families of serine proteinases. The fourth residue in this system equivalent to Thr/Ser in proteinases), hydrogen bonded to Asp, is Tyr260. The catalytic site is concealed under a short amphipatic helix (residues 85 to 91), which acts as "lid", opening the active site when the enzyme is adsorbed at the oil-water interface. In the native enzyme the "lid" is held in place by hydrophobic interactions.
J Mol Biol 1992 Oct 05
PMID:The crystal and molecular structure of the Rhizomucor miehei triacylglyceride lipase at 1.9 A resolution. 140 90

A cDNA (xynA), encoding xylanase A (XYLA), was isolated from a cDNA library, derived from mRNA extracted from the rumen anaerobic fungus, Neocallimastix patriciarum. Recombinant XYLA, purified from Escherichia coli harbouring xynA, had a M(r) of 53,000 and hydrolysed oat-spelt xylan to xylobiose and xylose. The enzyme did not hydrolyse any cellulosic substrates. The nucleotide sequence of xynA revealed a single open reading frame of 1821 bp coding for a protein of M(r) 66,192. The predicted primary structure of XYLA comprised an N-terminal signal peptide followed by a 225-amino-acid repeated sequence, which was separated from a tandem 40-residue C-terminal repeat by a threonine/proline linker sequence. The large N-terminal reiterated regions consisted of distinct catalytic domains which displayed similar substrate specificities to the full-length enzyme. The reiterated structure of XYLA suggests that the enzyme was derived from an ancestral gene which underwent two discrete duplications. Sequence comparison analysis revealed significant homology between XYLA and bacterial xylanases belonging to cellulase/xylanase family G. One of these homologous enzymes is derived from the rumen bacterium Ruminococcus flavefaciens. The homology observed between XYLA and a rumen prokaryote xylanase could be a consequence of the horizontal transfer of genes between rumen prokaryotes and lower eukaryotes, either when the organisms were resident in the rumen, or prior to their colonization of the ruminant. It should also be noted that Neocallimastix XYLA is the first example of a xylanase which consists of reiterated sequences. It remains to be established whether this is a common phenomenon in other rumen fungal plant cell wall hydrolases.
Mol Microbiol 1992 Aug
PMID:Homologous catalytic domains in a rumen fungal xylanase: evidence for gene duplication and prokaryotic origin. 140 48

Cell lines stably overexpressing protein kinase C (PKC)-alpha were previously described by us. These cell lines were generated by the introduction of the full length cDNA coding for PKC-alpha into Swiss/3T3 cells. Here we show that activation of PKC-alpha by phorbol-esters induced in these cells specific phosphorylation of two cellular proteins p90 and p52. Phosphorylation of p80 (MARCKS protein), previously identified as a substrate for PKC, was also enhanced. Phosphorylated p90 and p52 proteins were associated with particulate membrane-enriched fractions and were extractable with the use of nonionic detergents. Time course analysis of phorbol-ester induced phosphorylation of p90 and p52 revealed maximal stimulation of phosphorylation after 15-30 min. Phosphamino acid analysis showed that phosphorylation of p90 and p52 occurred mainly on serine residues. Phosphorylation of p52 was also on threonine residues. Whereas, phorbol ester activation induced phosphorylation of both p90 and p52, the mitogens platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) enhanced phosphorylation of p90, but not p52. Thus, our studies showed the involvement of PKC-alpha in the regulation of p90 and p52 phosphorylation and provided direct evidence for the role of PKC-alpha in cellular signaling by PDGF and FGF. Moreover, the fact that phosphorylation of p52 was specific to phorbol ester activation may suggest its involvement in tumor promotion. Characterization of p90 and p52 will enable us to reveal the phosphorylation cascade activated downstream to PKC-alpha and to determine their role in mitogenic signaling and tumor promotion.
Mol Biol Cell 1992 Sep
PMID:Phosphorylation of p90 and p52 in response to phorbol-esters in Swiss/3T3 cells overexpressing protein kinase C-alpha. 142 77

In order to obtain a better structural framework for understanding the catalytic mechanism of carbonic anhydrase, a number of inhibitor complexes of the enzyme were investigated crystallographically. The three-dimensional structure of free human carbonic anhydrase II was refined at pH 7.8 (1.54 A resolution) and at pH 6.0 (1.67 A resolution). The structure around the zinc ion was identical at both pH values. The structure of the zinc-free enzyme was virtually identical with that of the native enzyme, apart from a water molecule that had moved 0.9 A to fill the space that would be occupied by the zinc ion. The complexes with the anionic inhibitors bisulfite and formate were also studied at neutral pH. Bisulfite binds with one of its oxygen atoms, presumably protonized, to the zinc ion and replaces the zinc water. Formate, lacking a hydroxyl group, is bound with its oxygen atoms not far away from the position of the non-protonized oxygen atoms of the bisulfite complex, i.e. at hydrogen bond distance from Thr199 N and at a position between the zinc ion and the hydrophobic part of the active site. The result of these and other studies have implications for our view of the catalytic function of the enzyme, since virtually all inhibitors share some features with substrate, product or expected transition states. A reaction scheme where electrophilic activation of carbon dioxide plays an important role in the hydration reaction is presented. In the reverse direction, the protonized oxygen of the bicarbonate is forced upon the zinc ion, thereby facilitating cleavage of the carbon-oxygen bond. This is achieved by the combined action of the anionic binding site, which binds carboxyl groups, the side-chain of threonine 199, which discriminates between hydrogen bond donors and acceptors, and hydrophobic interaction between substrate and the active site cavity. The required proton transfer between the zinc water and His64 can take place through water molecules 292 and 318.
J Mol Biol 1992 Oct 20
PMID:Structure of native and apo carbonic anhydrase II and structure of some of its anion-ligand complexes. 143 93

Mucins are the structural components of the mucus gels that protect the respiratory, gastrointestinal, and reproductive tracts. These polydisperse glycoproteins (250,000 to 20,000,000 D) are approximately 80% carbohydrate on a mass basis and have a high intrinsic viscosity due to their large size and extreme hydrophilicity. Mucin oligosaccharides, the structures responsible for this hydrophilicity, are heterogeneous in size and structure but are chiefly O-linked, i.e., they initiate from N-acetylgalactosamine residues attached to threonine and serine residues of the polypeptide backbone. Our understanding of the structure of mucins has advanced rapidly in the last few years with the isolation and sequencing of cDNA clones that encode mucin polypeptide backbones. All currently well-characterized mucins have been found to contain extended arrays of tandemly repeated peptides rich in potential O-glycosylation sites. Less is known about the unique sequences that flank the tandem repeat arrays of secretory mucins, but currently available information indicates that these flanking regions contain cysteine-rich stretches that participate in mucin oligomer formation. Thus, secretory mucins appear to consist of oligomers containing heavily glycosylated domains flanked by unique sequences required for polymerization. Progress has also been made in characterizing the genes that encode mucins. At least four human mucin genes are known at present, although many others may remain to be discovered. Moreover, much work remains before we gain an understanding of the mechanisms involved in the expression of mucin genes and their tissue-specific regulation.
Am J Respir Cell Mol Biol 1992 Dec
PMID:Mucin genes and the proteins they encode: structure, diversity, and regulation. 144 3


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