RET(MEN 2B) is active in the endoplasmic reticulum before reaching the cell surface.

MEN 2B (multiple endocrine neoplasia type 2B) is an autosomal dominant cancer syndrome caused by an oncogenic form of the receptor tyrosine kinase REarranged during transfection (RET). The MEN 2B syndrome is associated with an abnormal autophosphorylation of the mutated receptor even without ligand-stimulation. Here, we characterize the activation of a RET(MEN 2B) variant carrying the point mutation Met918Thr, and show that the 150 kDa precursor of RET(MEN 2B) becomes phosphorylated already during synthesis in the endoplasmic reticulum (ER). At least three different tyrosine residues (Tyr905, Tyr1062, Tyr1096) of the RET(MEN 2B) precursor are phosphorylated before the oncogenic receptor reaches the cell surface. We also demonstrate that the precursor of RET(MEN 2B) interacts with both growth factor receptor-bound protein and Src homology 2 domain-containing already in the ER, and that this interaction is dependent on the kinase activity of RET. With the aid of two RET mutants (RET(MEN 2B/S32L) and RET(MEN 2B/F393L)), which accumulate in the ER, we show that the oncogenic precursor of the receptor has the capacity to activate AKT, extracellular signal-regulated kinase and signal transducer and activator of transcription 3 from the ER. Taken together, our data demonstrate that the oncogenic precursor of RET(MEN 2B) is phosphorylated, interacts with adapter proteins and induces downstream signalling from the ER.

The mouse soluble GFRalpha4 receptor activates RET independently of its ligand persephin.

Glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) all signal through the transmembrane receptor tyrosine kinase RET. The signalling complex consists of GFLs, GPI-anchored ligand binding GDNF family receptor alphas (GFRalphas) and RET. Signalling via RET is required for the development of the nervous system and the kidney, as well as for spermatogenesis. However, constitutive activation of RET is implicated as a cause in several diseases. Mutations of the RET proto-oncogene cause the inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN 2). Recently, it has been suggested that mutations in the persephin binding GFRalpha4 receptor may have a potentially modifying role in MEN 2. Several naturally occurring, different splice variants of the mammalian GFRalpha4 have been reported. A 7 bp insertion-mutation in the human GFRalpha4 gene causes a shift of reading frame and thereby changes the balance between the transcripts encoding GPI-anchored and soluble GFRalpha4 receptors. We report here that the mammalian soluble GFRalpha4 can activate RET independently of its preferential ligand, persephin. Our data show that soluble GFRalpha4 can associate with, and induce, phosphorylation of RET. In addition, our data show that this isoform of GFRalpha4 can induce downstream signalling, as well as neuronal survival and differentiation, in the absence of persephin. These results suggest that, in line with the previous report, GFRalpha4 may be a candidate gene for, or modifier of, the MEN 2 diseases.

Glycosylation of phytepsin and expression of dad1, dad2 and ost1 during onset of cell death in germinating barley scutella.

Dysfunction and downregulation of dad (defending against death) has been linked to programmed cell death (PCD) in animals and plants. As DAD is an essential subunit of the oligosaccharyltransferase that is located in the ER membrane, the results have raised the possibility that downregulation of N-linked glycosylation could be involved in the regulation of PCD. Here we show that the 16 kDa subunit of phytepsin, a vacuolar proteinase, is normally processed and glycosylated at the onset of DNA fragmentation in germinating barley scutella. Two cDNA clones encoding dad (dad1, dad2), and one cDNA encoding another subunit of the same oligosaccharyltransferase complex (ost1) were isolated from barley. Northern analysis of germinating scutella show that the expression of only dad1 is declining before onset of DNA fragmentation. In contrast to this, the expression of both dad2 and ost1 increase before onset of DNA fragmentation.

Phytepsin, a barley vacuolar aspartic proteinase, is highly expressed during autolysis of developing tracheary elements and sieve cells.

Vacuolarisation, formation of autophagocytotic vacuoles and tonoplast disruption have been reported in plant cells undergoing developmentally regulated programmed cell death (PCD), but little is known about the vacuolar proteins involved. In HeLa cells, cathepsin D, a lysosomal aspartic proteinase has been shown to mediate PCD. Based on immunohistochemical staining of barley roots, we show here that the previously well characterised barley vacuolar aspartic proteinase (phytepsin), a plant homologue to cathepsin D, is highly expressed both during formation of tracheary elements and during partial autolysis of sieve cells. In serial transverse sections of the vascular cylinder, starting from the root tip, phytepsin is expressed in root cap cells, in the tracheary elements of early and late metaxylem, and in the sieve cells of the protophloem and metaphloem. Aleurain, a barley vacuolar cysteine proteinase, is expressed similarly in root cap cells but differently in the tracheary elements of protoxylem and early metaxylem. This is the first evidence that a vacuolar aspartic proteinase, in analogy to cathepsin D in animals, may play a role in the active autolysis of plant cells.

The aspartic proteinase of barley is a vacuolar enzyme that processes probarley lectin in vitro.

Previous work suggested that the aspartic proteinase from Hordeum vulgare (HvAP) would be a vacuolar protein in plant cells. Based on N-terminal sequencing we show that the in vitro-translated protein was translocated into the lumen of microsomal membranes, causing a concomitant removal of 25 amino acid residues from the protein. Vacuoles were purified from barley leaf protoplasts and were shown to contain all of the aspartic proteinase activity found in the protoplasts. This vacuolar localization of HvAP was confirmed with immunocytochemical electron microscopy using antibodies to HvAP in both barley leaf and root cells. In an attempt to discern a function for this protease, we investigated the ability of HvAP to process the C-terminal proregion of barley lectin (BL) in vitro. Prolectin (proBL), expressed in bacteria, was processed rapidly when HvAP was added. Using several means, we were able to determine that 13 amino acid residues at the C terminus of proBL were cleaved off, whereas the N terminus stayed intact during this incubation. Immunohistochemical electron microscopy showed that HvAP and BL are co-localized in the root cells of developing embryos and germinating seedlings. Thus, we propose that the vacuolar HvAP participates in processing the C terminus of BL.

Primary structure of a barley-grain aspartic proteinase. A plant aspartic proteinase resembling mammalian cathepsin D.

Two enzymatically active heterodimeric forms of an aspartic proteinase, a putative 32 kDa + 16 kDa precursor form and a putative 29 kDa + 11 kDa mature form, are present in resting barley grains (Sarkkinen, P., Kalkkinen, N., Tilgmann, C., Siuro, J., Kervinen, J. & Mikola, L., 1990, in the press). The cDNA corresponding to this enzyme has been cloned and sequenced. The full-length 1863-bp cDNA sequence codes for an open reading frame of 508 amino acids. The open reading frame consists of a 66-amino acid preprosequence and a 442-amino acid mature protein. Comparison of the N-terminal amino acid sequences of the enzyme subunits with the sequence of the cDNA clone indicates that the heterodimeric enzyme is translated as a proenzyme which is processed into two subunits. The localisation of the experimentally determined N-terminal amino acid sequences of all four subunits (32 kDa + 16 kDa and 29 kDa + 11 kDa) in the same transcript, as well as the detection of only one 2.0-kb mRNA on Northern blots from resting seeds, clearly indicates that the larger (32 kDa + 16 kDa) enzyme is an intermediate precursor form of the smaller (29 kDa + 11 kDa) enzyme. The processing pattern of the barley enzyme, which is the first sequenced plant aspartic proteinase, differs from that of all other known aspartic proteinases. The barley enzyme is highly similar to mammalian and yeast aspartic proteinases, especially to human and porcine cathepsin D. This similarity is clearly dispersed over two regions, separated by a dissimilar, barley-specific region of 104 amino acids.

Homology in the region containing a tRNA(Trp) gene and a (complete or partial) tRNA(Pro) gene in wheat mitochondrial and chloroplast genomes.

We have used bean mitochondrial (mt) and chloroplast (cp) tRNA(Trp) as probes to locate the corresponding genes on the mt and cp genomes of wheat and we have determined the nucleotide sequences of the wheat mt and cp tRNA(Trp) genes and of the flanking regions. Sequence comparisons show that the wheat mt and cp tRNA(Trp) genes are 97% homologous. On the wheat cp DNA, a tRNA(UGGPro) gene was found 139 bp upstream of the cp tRNA(Trp) gene. On the wheat mt DNA, a sequence of 23 nucleotides completely homologous with the 3' end of this cp tRNA(Pro) gene was found 136 bp upstream of the mt tRNA(Trp) gene, but there is only 38% homology between cp and mt wheat genomes in the intergenic regions. The overall organization of this region in the chloroplast genome (a tRNA(Trp) gene separated by about 140 bp from a tRNA(Pro) gene) is also found in the mitochondrial genome, suggesting that this mitochondrial fragment might have originated from a chloroplast DNA insertion. A comparison of the genes and of the intergenic regions located between the tRNA(Trp) gene and the tRNA(Pro) (or partial tRNA(Pro)) gene shows that there is an almost complete conservation of these sequences in the mitochondrial DNA of wheat and maize, whereas wheat mt and cp intergenic regions show more sequence divergence. Wheat mt tRNA(Trp) gene is encoded by the main mt genome (accounted for by the master chromosome) but, in the case of maize mitochondria, this gene was found to be encoded by the 2.3 kb linear plasmid, indicating that this plasmid is not dispensable in maize mitochondria.

Localization, sequence and expression of the gene coding for tRNA(Pro) (UGG) in plant mitochondria.

The four Sal I fragments of wheat mitochondrial DNA containing the 18S and 5S ribosomal RNA genes were screened for the presence of tRNA genes. Upon sequencing, a tRNA(Pro) (UGG) gene was found in two of these four fragments. The localization of the corresponding gene on the maize mitochondrial genome was established. Transcriptional studies have shown that this gene is transcribed in wheat and maize mitochondria. The sequence of the corresponding tRNA(Pro) (UGG) of bean mitochondria was determined using in vitro post-labeling techniques.