Calmyrin1 binds to SCG10 protein (stathmin2) to modulate neurite outgrowth |
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Authors: | Adam SobczakKatarzyna Debowska Magdalena BlazejczykMichael R. Kreutz Jacek Kuznicki Urszula Wojda |
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Affiliation: | a Laboratory of Neurodegeneration, International Institute of Molecular and Cell Biology, Warsaw, Polandb PG Neuroplasticity, Leibniz Institute for Neurobiology, Magdeburg, Germanyc Laboratory of Calcium Binding Proteins, Nencki Institute of Experimental Biology, Warsaw, Poland |
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Abstract: | Calmyrin1 (CaMy1) is an EF-hand Ca2+-binding protein expressed in several cell types, including brain neurons. Using a yeast two-hybrid screen of a human fetal brain cDNA library, we identified SCG10 protein (stathmin2) as a CaMy1 partner. SCG10 is a microtubule-destabilizing factor involved in neuronal growth during brain development. We found increased mRNA and protein levels of CaMy1 during neuronal development, which paralleled the changes in SCG10 levels. In developing primary rat hippocampal neurons in culture, CaMy1 and SCG10 colocalized in cell soma, neurites, and growth cones. Pull-down, coimmunoprecipitation, and proximity ligation assays demonstrated that the interaction between CaMy1 and SCG10 is direct and Ca2+-dependent in vivo and requires the C-terminal domain of CaMy1 (residues 99-192) and the N-terminal domain of SCG10 (residues 1-35). CaMy1 did not interact with stathmin1, a protein that is homologous with SCG10 but lacks the N-terminal domain characteristic of SCG10. CaMy1 interfered with SCG10 inhibitory activity in a microtubule polymerization assay. Moreover, CaMy1 overexpression inhibited SCG10-mediated neurite outgrowth in nerve growth factor (NGF)-stimulated PC12 cells. This CaMy1 activity did not occur when an N-terminally truncated SCG10 mutant unable to interact with CaMy1 was expressed. Altogether, these data suggest that CaMy1 via SCG10 couples Ca2+ signals with the dynamics of microtubules during neuronal outgrowth in the developing brain. This article is part of a Special Issue entitled: 11th European Symposium on Calcium. |
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Keywords: | 3-AT, 3-amino-1,2,4-triazole AD, activation domain ANOVA, analysis of variance BD, binding domain CaMy1, calmyrin1 CIB1, calcium and integrin binding protein 1 DIV, days in vitro DMEM, Dulbecco's Modified Eagle Medium FBS, fetal bovine serum GFP, green fluorescent protein GST, glutathione S-transferase IPTG, isopropyl-1-thio-β- smallcaps" >d-galactopyranoside MBP, maltose-binding protein NBR1, next to BRCA1 gene 1 protein NCS, neuronal calcium sensors NGF, nerve growth factor Ni-NTA, Ni2+-nitriloacetic acid agarose resin PAX3, paired box  3 protein PBS, phosphate-buffered saline PCR, polymerase chain reaction PFA, paraformaldehyde PLA, proximity ligation assay SCG10, superior cervical ganglia neural specific 10 SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis |
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