Abstract
Hippocalcin is a member of the neuronal calcium sensors (NCS) family predominantly expressed in the hippocampal pyramidal cells. Here we show the interaction of hippocalcin with mixed- lineage kinase (MLK) 3, which has been identified as a hippocalcin-binding protein by using yeast two-hybrid screening of mouse brain cDNA library, and indicate the possible involvement of hippocalcin in MLK3 activation process. Immunoprecipitation study using antigen-purified anti-hippocalcin antibody displayed the co-precipitation of MLK3 in 1% TritonX-100 soluble hippocampal lysate. In vitro co-precipitation analysis using domain specific constructs of MLK3 revealed that hippocalcin bound with C-terminal Pro/Ser/Thr-rich region of MLK3. Recombinant fragments for each EF-hand domain of hippocalcin failed to bind with MLK3, indicating that whole hippocalcin molecule involved in the interaction with MLK3. Immunocomplex kinase assay displayed that resting kinase activity of MLK3 in hippocalcin deficient mice was higher than that in wild type. Then we detected the phosphorylation state of MLK3 because phosphorylation events in MLK3 play crucial roles in its activation process. Immunoblot analysis using anti-phospho-Ser/Thr antibodies revealed that immunoprecipitated MLK3 in hippocalcin deficient mice was higher phosphorylated than that in wild type. These observations suggest that hippocalcin inhibits the MLK3 activation via regulating MLK3 phosphorylation state. [Jpn J Physiol 54 Suppl:S215 (2004)]
