RET PLCγ Phosphotyrosine Binding Domain Regulates Ca2+ Signaling and Neocortical Neuronal Migration

The receptor tyrosine kinase RET plays an essential role during embryogenesis in regulating cell proliferation, differentiation, and migration. Upon glial cell line-derived neurotrophic factor (GDNF) stimulation, RET can trigger multiple intracellular signaling pathways that in concert activate various downstream effectors. Here we report that the RET receptor induces calcium (Ca2+) signaling and regulates neocortical neuronal progenitor migration through the Phospholipase-C gamma (PLCγ) binding domain Tyr1015. This signaling cascade releases Ca2+ from the endoplasmic reticulum through the inositol 1,4,5-trisphosphate receptor and stimulates phosphorylation of ERK1/2 and CaMKII. A point mutation at Tyr1015 on RET or small interfering RNA gene silencing of PLCγ block the GDNF-induced signaling cascade. Delivery of the RET mutation to neuronal progenitors in the embryonic ventricular zone using in utero electroporation reveal that Tyr1015 is necessary for GDNF-stimulated migration of neurons to the cortical plate. These findings demonstrate a novel RET mediated signaling pathway that elevates cytosolic Ca2+ and modulates neuronal migration in the developing neocortex through the PLCγ binding domain Tyr1015

Positive Selection for New Disease Mutations in the Human Germline: Evidence from the Heritable Cancer Syndrome Multiple Endocrine Neoplasia Type 2B

Multiple endocrine neoplasia type 2B (MEN2B) is a highly aggressive thyroid cancer syndrome. Since almost all sporadic cases are caused by the same nucleotide substitution in the RET proto-oncogene, the calculated disease incidence is 100–200 times greater than would be expected based on the genome average mutation frequency. In order to determine whether this increased incidence is due to an elevated mutation rate at this position (true mutation hot spot) or a selective advantage conferred on mutated spermatogonial stem cells, we studied the spatial distribution of the mutation in 14 human testes. In donors aged 36–68, mutations were clustered with small regions of each testis having mutation frequencies several orders of magnitude greater than the rest of the testis. In donors aged 19–23 mutations were almost non-existent, demonstrating that clusters in middle-aged donors grew during adulthood. Computational analysis showed that germline selection is the only plausible explanation. Testes of men aged 75–80 were heterogeneous with some like middle-aged and others like younger testes. Incorporating data on age-dependent death of spermatogonial stem cells explains the results from all age groups. Germline selection also explains MEN2B's male mutation bias and paternal age effect. Our discovery focuses attention on MEN2B as a model for understanding the genetic and biochemical basis of germline selection. Since RET function in mouse spermatogonial stem cells has been extensively studied, we are able to suggest that the MEN2B mutation provides a selective advantage by altering the PI3K/AKT and SFK signaling pathways. Mutations that are preferred in the germline but reduce the fitness of offspring increase the population's mutational load. Our approach is useful for studying other disease mutations with similar characteristics and could uncover additional germline selection pathways or identify true mutation hot spots

Sorting of proteins to vacuoles in plant cells

for correspondence

The Glycosylation of the Aspartic Proteinases from Barley (Hordeum Vulgare L.) and Cardoon (Cynara Cardunculus L.)

Plant aspartic proteinases characterised at the molecular level contain one or more consensus N-glycosylation sites [Runeberg-Roos, P., Törmäkangas, K. & Östman, A. (1991) Eur. J. Biochem. 202, 102120131027; Asakura, T., Watanabe, H., Abe, K. & Arai, S. (1995) Eur. J. Biochem. 232, 77201383; Veríssimo, P., Faro, C., Moir, A. J. G., Lin, Y., Tang, J. & Pires, E. (1996) Eur. J. Biochem. 235, 76220137681. We found that the glycosylation sites are occupied for the barley (Hordeum vulgare L.) aspartic proteinase (Asn333) and the cardoon (Cynara cardunculus L.) aspartic proteinase, cardosin A (Asn70 and Asn363). The oligosaccharides from each site were released from peptide pools by enzymatic hydrolysis with peptide-N-glycanase A or by hydrazinolysis and their structures were determined by exoglycosidase sequencing combined with matrix-assisted laser desorption/ionization time of flight mass spectrometry. It was observed that 6% of the oligosaccharides from the first glycosylation site of cardosin A are of the oligomannose type. Modified type glycans with proximal Fuc and without Xyl account for about 82%, 14% and 3% of the total oligosaccharides from the first and the second glycosylation sites of cardosin A and from H. vulgare aspartic proteinase, respectively. Oligosaccharides with Xyl but without proximal Fuc were only detected in the latter proteinase (4%). Glycans with proximal Fuc and Xyl account for 6%, 86% and 92% of the total oligosaccharides from the first and second glycosylation sites of cardosin A and from H. vulgure aspartic proteinase, respectively

Purification, Characterization and Partial Amino Acid Sequencing of Two New Aspartic Proteinases from Fresh Flowers of Cynara cardunculus L.

Two new aspartic proteinases have been isolated from stigmas of the cardoon Cynara cardunculus L. by a two-step purification procedure including extraction at low pH, gel filtration on Superdex 200, and ion-exchange chromatography on Mono Q. To follow the conventional nomenclature for aspartic proteinases, we have named these proteinases cardosin A and cardosin B. On SDS/PAGE, cardosin A migrated as two bands with apparent molecular masses of 31 000 Da and 15000 Da where as the chains of cardosin B migrated as bands of 34000 Da and 14000 Da. The partial amino acid sequences of the two cardosins revealed that they are similar but not identical, and that they differ horn the previously reported cardoon proteinases named cynarases, which were assumed to be derived from a common precursor. Although the cardosins show some degree of similarity to each other, we could detect no immunological cross-reactivity between them. Both cardosins were active at low pH and were inhibited by pepstatin, with Ki values of 3 nM for cardosin A and 1 nM for cardosin B, indicating that they belong to the class of aspartic proteinases. Significant differences between the two enzymes were also found for the Kcat/Km values for the hydrolysis of two chromophoric synthetic peptides. The active-site ionization constants, pKe1 and pKe2, for cardosin A are 2.5±0.2 and 5.3±20.2, whereas for cardosin R they are 3.73±10.09 and 6.7±50.1. The results herein described on the structural and kinetic properties of the cardosins indicate that they are the products of distinct genes which have probably arisen by gene duplication. A scheme for the proteolytic processing of the two enzymes is also proposed