Masaharu Mizutani

CYTOCHROME P450 SUPERFAMILY IN ARABIDOPSIS THALIANA : ISOLATION OF CDNAS, DIFFERENTIAL EXPRESSION, AND RFLP MAPPING OF MULTIPLE CYTOCHROMES P450

We have isolated multiple cDNAs encoding cytochromes P450 (P450s) from Arabidopsis thaliana employing a PCR strategy. Degenerate oligonucleotide primers were designed from amino acid sequences conserved between two plant P450s, CYP71A1 and CYP73A2, including the heme-binding site and the proline-rich motif found in the N-terminal region, and 11 putative P450 fragments were amplified from first-strand cDNA from 7-day-old Arabidopsis as a template. With these PCR fragments as hybridization probes, 13 full-length and 3 partial cDNAs encoding different P450s have been isolated from an Arabidopsis cDNA library. These P450s have been assigned to either one of the established subfamilies: CYP71B, CYP73A, and CYP83A; or novel subfamilies: CYP76C, CYP83B, and CYP91A. The primary protein structures predicted from the cDNA sequences revealed that the regions around both the heme-binding site and the proline-rich motif were highly conserved among all these P450s. The N-terminal structures of the predicted P450 proteins suggested that these Arabidopsis P450s were located at the endoplasmic reticulum membrane. The loci of four P450 genes were determined by RFLP mapping. One of the clones, CYP71B2, was located at a position very close to the ga4 and gai mutations. RNA blot analysis showed expression patterns unique to each of the P450s in terms of tissue specificity and responsiveness to wounding and light/dark cycle, implicating involvement of these P450s in diverse metabolic processes.

Brain-derived neurotrophic factor promotes interaction of the Nck2 adaptor protein with the TrkB tyrosine kinase receptor

Brain-derived neurotrophic factor (BDNF) binds to and activates the TrkB tyrosine kinase receptor to regulate cell differentiation, survival, and neural plasticity in the nervous system. However, the identities of the downstream signaling proteins involved in this process remain unclear. Using a yeast two-hybrid screen with the intracellular domain (ICD-TrkB) of the TrkB BDNF receptor, we identified the Nck2 adaptor protein as a novel interaction partner of the active form of TrkB. Additionally, we identified three tyrosines in ICD-TrkB (Y694, Y695, and Y771) that are crucial for this interaction. Similar results were obtained for Nck1, an Nck2 homolog. We also found that TrkB could be co-precipitated with GST-Nck2 recombinant protein or anti-Nck antibody in BDNF-activated cortical neurons. These results suggest that BDNF stimulation promotes interaction of Ncks with TrkB in cortical neurons.