Biochemie und Chemie
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The transcription factor p63 is expressed as at least six different isoforms, of which two have been assigned critical biological roles within ectodermal development and skin stem cell biology on the one hand and supervision of the genetic stability of oocytes on the other hand. These two isoforms contain a C-terminal inhibitory domain that negatively regulates their transcriptional activity. This inhibitory domain contains two individual components: one that uses an internal binding mechanism to interact with and mask the transactivation domain and one that is based on sumoylation. We have carried out an extensive alanine scanning study to identify critical regions within the inhibitory domain. These experiments show that a stretch of ~13 amino acids is crucial for the binding function. Further, investigation of transcriptional activity and the intracellular level of mutants that cannot be sumoylated suggests that sumoylation reduces the concentration of p63. We therefore propose that the inhibitory function of the C-terminal domain is in part due to direct inhibition of the transcriptional activity of the protein and in part due to indirect inhibition by controlling the concentration of p63. Keywords: p63, transcriptional regulation, auto-inhibition, sumoylation
Group III presynaptic metabotropic glutamate receptors (mGluRs) play a central role in regulating presynaptic activity through G-protein effects on ion channels and signal transducing enzymes. Like all Class C G-protein coupled receptors, mGluR8 has an extended intracellular C-terminal domain (CTD) presumed to allow for modulation of downstream signaling. To elucidate the function and modulation of mGluR8, yeast two-hybrid screens of an adult rat brain cDNA library were performed with the CTDs of mGluR8a and 8b (mGluR8-C) as baits. Different components of the sumoylation cascade (ube2a, sumo-1, Pias1, Pias gamma and Pias xbeta) and some other proteins were identified as mGluR8 interacting proteins. Binding assays using recombinant GST-fusion proteins confirmed that Pias1 interacts not only with mGluR8-C, but all group III mGluR CTDs. Pias1 binding to mGluR8-C required a region N-terminally to a consensus sumoylation motif and was not affected by arginine substitution of the conserved lysine K882 within this motif. Co-transfection of fluorescently tagged mGluR8a-C, sumo-1 and enzymes of the sumoylation cascade into HEK 293 cells showed that mGluR8a-C can be sumoylated in cells. Arginine substitution of lysine K882 within the consensus sumoylation motif, but not of other conserved lysines within the CTD, abolished in vivo sumoylation. The results are consistent with post-translational sumoylation providing a novel mechanism of group III mGluR regulation.