Hippocalcin acts as a possible suppressor of neuronal apoptosis via NAIP-caspase and MLK3-JNK cascades

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Hippocalcin (HIP) is a member of neuronal calcium sensors family predominantly expressed in the hippocampal pyramidal cells. Primary cultured hippocampal neuron from HIP deficient (-/-) mice showed a low survivability compared to that from wild type (+/+) mice. Contrary, in the survivability of cultured glia, there is no difference between -/- and +/+ mice. Calcium influx into primary cultured neurons was induced by glutamate, high potassium and inomycin, and the influx and efflux processes were monitored using Fra2AM. Both processes were impaired in -/- mice. Contrary, both were accelerated in HIP over expressed COS7 cells. These indicate that HIP acts not only as a calcium buffer but also as calcium channel and pump activators. Glutamate- and high potassium- induced JNK activations in -/- hippocampal slices were much higher than those in +/+ slices, indicating that HIP acts as a possible calcium-dependent suppressor of JNK cascade. Then, we screened to search for HIP target proteins by using yeast two-hybrid system composed of mouse brain cDNA library. Among 3x10^6 transformants, 76 positive clones were obtained. Β-galactosidase expression analysis revealed that 12 clones interact with HIP. These clones were classified into mixed lineage kinase (MLK) 3 (7 clones), protein 1B; a transcription factor having f-box/WD40 domain (3 clones), NAIP (neuronal apoptosis inhibitory protein) 3 (3 clones) and complexin 2 by sequence analysis. MLK3 is a JNK activator, displayed the strongest interaction. HIP was found to interact with C-terminal Pro/Ser/Thr-rich region of MLK3. Whole HIP conformation involved in the interaction. Neural visinin-like protein (NVP) 1, 2 and 3, structurally related proteins to HIP, did not show the binding activity to MLK3, NAIP is known to be an inhibitor of caspase cascade that suppress the neuronal apoptosis. These indicate that HIP acts as a possible suppressor of neuronal apoptosis via NAIP-caspase and MLK3-JNK cascades.