Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

B-Raf, a member of the Raf family of serine/threonine kinases, is expressed primarily in the brain and in the nervous system. In this study, the biochemical properties of the B-Raf protein were investigated in nerve growth factor (NGF)-responsive cell lines and in brain tissues. B-Raf was identified by using phosphopeptide mapping analysis and cDNA analysis as a 95-kDa protein which is primarily localized in the cytosol. NGF rapidly stimulated both serine and threonine phosphorylation in vivo and autophosphorylation activity in vitro of the B-Raf protein. In PC12 cells, B-Raf autokinase activity was induced by both differentiation factors and mitogens, with maximal activity observed after 5 min of factor addition. B-Raf kinase activity was also observed following NGF treatment of SH-SY5Y neuroblastoma cells and in adult mouse brain and hippocampus. Induction of B-Raf kinase activity in NGF-treated PC12 cells required expression of kinase-active trk receptors. Exogenous substrates or a peptide containing the autophosphorylation site became phosphorylated when added to immune complex kinase assays and reduced the in vitro autophosphorylation activity of B-Raf, suggesting that in vitro autophosphorylation sites and exogenous substrates compete for active sites of the B-Raf kinase. Finally, the major in vitro autophosphorylation site of B-Raf was identified as threonine 372 in the conserved region 2 domain. A threonine residue is present at similar positions in all three mammalian Raf family members and may represent a regulatory site for these proteins.
Mol Cell Biol 1992 Sep
PMID:95-kilodalton B-Raf serine/threonine kinase: identification of the protein and its major autophosphorylation site. 150 79

The compartmentalization of glycolytic enzymes into specialized organelles, the glycosomes, allows the bloodstream form of Trypanosoma brucei to rely solely on glycolysis for its energy production. The biogenesis of glycosomes in these parasites has been studied intensively as a potential target for chemotherapy. We have adapted the recently developed methods for stable transformation of T. brucei to the in vivo analysis of glycosomal protein import. Firefly luciferase, a peroxisomal protein in the lantern of the insect, was expressed in stable transformants of the procyclic form of T. brucei, where it was found to accumulate inside the glycosomes. Mutational analysis of the peroxisomal targeting signal serine-lysine-leucine (SKL) located at the C-terminus of luciferase showed that replacement of the serine residue (Serine548) with a small neutral amino acid (A, C, G, H, N, P, T) still resulted in an import efficiency of 50-100% of the wild-type luciferase. Lysine549 could be substituted with an amino acid capable of hydrogen bonding (H, M, N, Q, R, S), whereas the C-terminal leucine550 could be replaced with a subset of hydrophobic amino acids (I, M, Y). Thus, a peroxisome-like C-terminal SKL-dependent targeting mechanism may function in T. brucei to import luciferase into the glycosomes. However, a few significant differences exist between the glycosomal targeting signals identified here and the tripeptide sequences that direct proteins to mammalian or yeast peroxisomes.
Mol Biol Cell 1992 Jul
PMID:In vivo import of firefly luciferase into the glycosomes of Trypanosoma brucei and mutational analysis of the C-terminal targeting signal. 151 76

It was shown that in LA-N-2 cells prelabeled with [3H-methyl]choline for 24 hr (Singh et al.: Mol Chem Neuropathol 14:53-66, 1991) the major intracellular and extracellular hydrophilic compound was phosphorylcholine. LA-N-2 cells were labeled with [14C-methyl]choline for 24 hr, harvested, and incubated in Hepes/BSA/saline buffer for varying periods of time. The radioactive compound present in the cytosol and released into Hepes/BSA/saline buffer medium in the presence or absence of TPA was phosphorylcholine. There was a gradual increase in the appearance of radioactivity in the medium and this corresponded to a gradual decline in the radioactivity present in the cytosolic compartment with a statistically significant P value of less than .005. Identical results were obtained with prelabeled cells subsequently incubated with TPA. There was no significant change in the amount of radioactivity associated with lipid suggesting that the phosphorylcholine may be released directly from the cytosolic compartment into the medium rather than originating through a phospholipase-C catalyzed hydrolysis of phosphatidylcholine. This possibility received support from experiments in which cells were electropermeabilized in the presence of radioactive phosphorylcholine. It was found that the introduced [14C]phosphorylcholine was released intact into the incubation medium from the cytosolic compartment. The incorporation of [14C]choline, [14C]ethanolamine, and [14C]serine by LA-N-2 cells into their corresponding phospholipids was investigated in the presence or absence of TPA. The presence of TPA increased the amount of radioactivity incorporated into the phospholipids with a corresponding decrease in the amount of radioactivity in the cytosolic compartment compared to control cultures. There were no detectable differences between TPA exposed and control cells in the distribution of radioactivity in free choline, phosphorylcholine, or CDP-choline of [14C] choline labeled cells. This indicates that the increased lipid labeling was not accompanied by enhanced labeling of the intermediates of the de novo pathway. This effect of TPA in altering the distribution of labeling of the cytosolic and lipid components was not demonstrable with cells grown in the presence of 10(-5) M retinoic acid.
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PMID:Phorbol esters modulate phospholipid metabolism in a human cholinergic cell line, LA-N-2: a possible role for the base exchange enzymes. 152 4

Serine hydroxymethyltransferase (SHMT) occupies a central position in one-carbon (C1) metabolism, catalyzing the reaction of serine and tetrahydrofolate to yield glycine and 5,10-methylenetetrahydrofolate. Methylenetetrahydrofolate serves as a donor of C1 units for the synthesis of numerous compounds, including purines, thymidylate, lipids, and methionine. We provide evidence that the formate (for) locus of Neurospora crassa encodes cytosolic SHMT. The for+ gene was localized to a 2.8-kb BglII fragment by complementation (restoration to formate-independent growth) of a strain carrying a recessive for allele, which confers a growth requirement for formate. The for+ gene encodes a polypeptide of 479 amino acids which shows significant similarity to amino acid sequences of SHMT from bacterial and mammalian sources (47 and 60% amino acid identity, respectively). The for+ mRNA has several different start and stop sites. The abundance of for+ mRNA increased in response to amino acid imbalance induced by glycine supplementation, suggesting regulation by the N. crassa cross-pathway control system, which is analogous to general amino acid control in Saccharomyces cerevisiae. This was confirmed by documenting that for+ expression increased in response to histidine limitation (induced by 3-amino-1,2,4-triazole) and that this response was dependent on the presence of a functional cross-pathway control-1 (cpc-1) gene, which encodes CPC1, a positively acting transcription factor. There are at least five potential CPC1 binding sites upstream of the for+ transcriptional start, as well as one that exactly matches the consensus CPC1 binding site in the first intron of the for+ gene.
Mol Cell Biol 1992 Apr
PMID:Characterization of the formate (for) locus, which encodes the cytosolic serine hydroxymethyltransferase of Neurospora crassa. 153 27

The proto-oncogene Wnt-1 encodes a cysteine-rich, secretory glycoprotein implicated in virus-induced mouse mammary cancer and intercellular signaling during vertebrate neural development. To attempt to correlate structural motifs of Wnt-1 protein with its function, 12 mutations were introduced singly and in several combinations into the coding sequence of Wnt-1 cDNA by site-directed mutagenesis. Mutant alleles in a retroviral vector were tested for their ability to transform the mouse mammary epithelial cell line C57MG in two ways: by direct infection of C57MG cells and by infection of NIH3T3 cells that serve as donors of Wnt-1 protein to adjacent C57MG cells in a secretion-dependent (paracrine) assay. In addition, the synthesis and secretion of mutant proteins were monitored in multiple cell types by immunological assays. Deletion of the signal peptide demonstrated that transformation in both direct and paracrine assays depends upon entry of Wnt-1 protein into the endoplasmic reticulum. Changes in potential proteolytic processing sites (two basic dipeptides and a probable signal peptidase cleavage site) did not adversely impair biological activity or protein processing and uncovered a second site for cleavage by signal peptidase. Replacement of each of the four asparagine-linked glycosylation sites did not affect transforming activity at normal temperatures, but one glycosylation site mutant was found to be temperature-sensitive for transformation. An allele encoding a protein that lacks all four glycosylation sites was also transformation competent. In two of four cases, substitution of serine for a cysteine residue impaired transforming activity at the usual temperature, and transformation was temperature sensitive in a third case, implying that at least some of the highly conserved cysteine residues are important for Wnt-1 function.
Mol Biol Cell 1992 May
PMID:Mutational analysis of mouse Wnt-1 identifies two temperature-sensitive alleles and attributes of Wnt-1 protein essential for transformation of a mammary cell line. 153 41

In basophils, mast cells, and the RBL-2H3 tumor mast cell line, cross-linking the high-affinity immunoglobulin E receptor (Fc epsilon R1) stimulates a series of responses, particularly the activation of phospholipase C (PLC), that lead to allergic and other immediate hypersensitivity reactions. The mechanism of activation of PLC, however, is not clear. Here, we show that cross-linking Fc epsilon R1 on RBL-2H3 cells causes the tyrosine phosphorylation of at least 12 cellular proteins, including PLC gamma 1 (PLC gamma 1) and the receptor beta and gamma subunits. 32P-labeled PLC gamma 1 can be detected by anti-phosphotyrosine antibody as early as 10 s after the addition of antigen. The tyrosine-phosphorylated 33-kDa beta subunit and 9- to 11-kDa gamma subunit of the Fc epsilon R1 are additionally phosphorylated on serine and theonine residues, respectively, and are found as complexes with other phosphotyrosine-containing proteins in antigen-stimulated cells. Our results indicate a means by which the Fc epsilon R1 may control PLC activity in RBL-2H3 cells and raise the possibility that other receptor-mediated signalling events in mast cells may also be controlled through protein tyrosine phosphorylation.
Mol Cell Biol 1992 Jul
PMID:Fc epsilon R1-mediated tyrosine phosphorylation of multiple proteins, including phospholipase C gamma 1 and the receptor beta gamma 2 complex, in RBL-2H3 rat basophilic leukemia cells. 153 86

We have isolated and sequenced a cDNA clone encoding the apoprotein of a potato phytochrome. Based on the deduced amino acid sequence, which shows 78% amino acid identity to the Arabidopsis phyA and 50% identity to the Arabidopsis phyB open reading frame, we have classified this cDNA clone as potato phyA phytochrome. The amino acid immediately preceding cysteine 323, which is the homologue of oat cystein 321, to which the chromophore has been shown to be attached, is a tyrosine residue. This contrasts with six other type A phytochrome sequences from both monocots and dicots that encode serine in this position. As already observed in three other cDNAs isolated from dicot species, the potato phyA clone encodes a short open reading frame (13 amino acids) preceding the phyA open reading frame (1123 amino acids), supporting the idea that this type of leader sequence might be involved in the regulated expression of the phytochrome apoprotein. Southern blot analysis revealed a single phyA gene as well as other related phytochrome sequences in the potato genome. phyA mRNA levels varied in different organs and were modulated by white light; in seedlings and sprouts, highest levels of mRNA were detected in the etiolated stage. Upon illumination with white light, mRNA levels decreased to the amount found in leaves of re-etiolated plants. Lowest expression was observed in leaves of plants grown in the light, in tubers irrespective of light treatment, and in roots of plants grown in the dark. In roots of plants grown in the light, elevated levels of phyA mRNA were detected. Using a monoclonal antibody generated against pea phytochrome as an immunochemical probe, the protein was only detectable in protein extracts from etiolated seedlings and sprouts.
Plant Mol Biol 1992 Feb
PMID:Isolation and characterization of a cDNA-clone coding for potato type A phytochrome. 153 28

Using a mutant of Saccharomyces cerevisiae defective in the NAT1 gene, that encodes one of the NH2-terminal acetyltransferases, we have identified 14 ribosomal proteins whose electrophoretic mobility at pH 5.0 suggests they carry an additional charge, presumably due to the lack of NH2-terminal acetylation. At least 30 other ribosomal proteins from the mutant are electrophoretically normal. Attempted NH2-terminal analysis of most of the presumed acetylated proteins from wild type cells indicated that all were blocked. NH2-terminal analysis of the same proteins from the nat1 mutant strain yielded unique sequences. Each one carries an NH2-terminal serine. We conclude that these are normally acetylated due to the presence of the NAT1 gene product. It seems surprising that cells whose ribosomes have been altered to this degree grow rather well and synthesize the same spectrum of proteins as do wild type cells (Mullen, J. R., Kayne, P. S., Moerschell, R. P., Tsunasawa, S. Gribskov, M., Sherman, F., and Sternglanz, R. (1989) EMBO J. 8, 2067-2075). Finally, this analysis has provided the first sequence information available for several of the acetylated ribosomal proteins and for one non-acetylated ribosomal protein, which is clearly the product of the MFT1 gene (Garrett, J. M., Singh, K. K., Vonder Haar, R. A., and Emr. S. D. (1991) Mol. Gen. Gen. 225, 483-491).
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PMID:NH2-terminal acetylation of ribosomal proteins of Saccharomyces cerevisiae. 154 21

Translation initiation factor eIF-4E, which binds to the 5' cap structure of eukaryotic mRNAs, is believed to play an important role in the control of cell growth. Consistent with this, overexpression of eIF-4E in fibroblasts results in their malignant transformation. The activity of eIF-4E is thought to be regulated by phosphorylation on a single serine residue (Ser-53). Treatment of rat pheochromocytoma (PC12) cells with nerve growth factor (NGF) strongly curtails their growth and causes their differentiation into cells that resemble sympathetic neurons. The present study shows that eIF-4E is rapidly phosphorylated in PC12 cells upon NGF treatment, resulting in a significant increase in the steady-state levels of the phosphorylated protein. In contrast, epidermal growth factor, a factor which elicits a weak mitogenic response in PC12 cells, did not significantly enhance eIF-4E phosphorylation. We also show that although the mitogen and tumor promoter, phorbol 12-myristate-13-acetate, is able to induce phosphorylation of eIF-4E in PC12 cells, the NGF-mediated increase is primarily a protein kinase C-independent response. The NGF-induced enhancement of eIF-4E phosphorylation is abrogated in PC12 cells expressing a dominant inhibitory ras mutant (Ser-17 replaced by Asn), indicating that eIF-4E phosphorylation is dependent on a ras signalling pathway. As phosphorylation of eIF-4E effects translation initiation, these results suggest that NGF-mediated and ras-dependent eIF-4E phosphorylation may play a role in switching the pattern of gene expression during the differentiation of PC12 cells.
Mol Cell Biol 1992 Mar
PMID:Phosphorylation of translation initiation factor eIF-4E is induced in a ras-dependent manner during nerve growth factor-mediated PC12 cell differentiation. 154 5

We demonstrate that members of the erk-encoded family of mitogen-activated protein (MAP) kinases (pp44/42mapk/erk) and members of the rsk-encoded protein kinases (RSKs or pp90rsk) are present in the cytoplasm and nucleus of HeLa cells. Addition of growth factors to serum-deprived cells results in increased tyrosine and threonine phosphorylation and in the activation of cytosolic and nuclear MAP kinases. Activated MAP kinases then phosphorylate (serine/threonine) and activate RSKs. Concurrently, a fraction of the activated MAP kinases and RSKs enter the nucleus. In addition, a distinct growth-regulated RSK-kinase activity (an enzyme[s] that phosphorylates recombinant RSK in vitro and that may be another member of the erk-encoded family of MAP kinases) was found associated with a postnuclear membrane fraction. Regulation of nuclear MAP kinase and RSK activities by growth factors and phorbol ester is coordinate with immediate-early gene expression. Indeed, in vitro, MAP kinase and/or RSK phosphorylates histone H3 and the recombinant c-Fos and c-Jun polypeptides, transcription factors phosphorylated in a variety of cells in response to growth stimuli. These in vitro studies raise the possibility that the MAP kinase/RSK signal transduction pathway represents a protein-Tyr/Ser/Thr phosphorylation cascade with the spatial distribution and temporal regulation that can account for the rapid transmission of growth-regulating information from the membrane, through the cytoplasm, and to the nucleus.
Mol Cell Biol 1992 Mar
PMID:Nuclear localization and regulation of erk- and rsk-encoded protein kinases. 154 23


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