Mutations of the 22- and 27-kD zein promoters affect transactivation by the Opaque-2 protein.
The Plant cell. 4:701-9. Abstract
By utilizing a homologous transient expression system, we have demonstrated that the Opaque-2 (O2) gene product O2 confers positive trans-regulation on a 22-kD zein promoter. This trans-acting function of the O2 protein is mediated by its sequence-specific binding to a cis element (the O2 target site) present in the 22-kD zein promoter. A multimer of a 32-bp promoter fragment containing this O2 target site confers transactivation by O2. A single nucleotide substitution in the O2 target sequence not only abolishes O2 binding in vitro, but also its response to transactivation by O2 in vivo. We have also demonstrated that an amino acid domain including the contiguous basic region and the heptameric leucine repeat is essential for the trans-acting function of the O2 protein. Similar but not identical O2 target sequence motifs can be found in the promoters of zein genes of different molecular weight classes. Conversion of such a motif in the 27-kD zein promoter to an exact O2 target sequence by site-directed mutagenesis was sufficient to increase the binding affinity of the O2 protein in vitro and to confer transactivation by O2 in vivo.
The role of regulatory domain interactions in UNC-43 CaMKII localization and trafficking.
J Cell Sci. 118:3327-38. Abstract
Calcium and calmodulin-dependent protein kinase II (CaMKII) plays a fundamental role in the synaptic plasticity events that underlie learning and memory. Regulation of CaMKII kinase activity occurs through an autoinhibitory mechanism in which a regulatory domain of the kinase occupies the catalytic site and calcium/calmodulin activates the kinase by binding to and displacing this regulatory domain. A single putative ortholog of CaMKII, encoded by unc-43, is present in the Caenorhabditis elegans nervous system. Here we examined UNC-43 subcellular localization in the neurons of intact animals and show that UNC-43 is localized to clusters in ventral cord neurites, as well as to an unlocalized pool within these neurites. A mutation that mimics autophosphorylation within the regulatory domain results in an increase in the levels of UNC-43 in the unlocalized neurite pool. Multiple residues of CaMKII facilitate the interaction between the catalytic domain and the regulatory domain, thereby keeping the kinase inactive. Whereas most mutations in these residues result in an increased neurite pool of UNC-43, we have identified two residues that result in the opposite effect when mutated: a decreased neurite pool of UNC-43. The activity of UNC-2, a voltage-dependent calcium channel, is also required for UNC-43 to accumulate in the neurites, suggesting that neural activity regulates the localization of UNC-43. Our results suggest that the activation of UNC-43 by calcium/calmodulin displaces the autoinhibitory domain, thereby exposing key residues of the catalytic domain that allow for protein translocation to the neurites.