Abstract
SH2B1 is an adapter protein that promotes neurite outgrowth of PC12 cells, cortical and hippocampal neurons. In this study, we provide evidence suggesting that overexpression of SH2B1β promotes filopodium formation and dendritic branching of hippocampal neurons. In neurite initiation, filopodium formation is required and microtubule extension into filopodia, suggesting that SH2B1β may regulate actin remodeling to enhance neurite initiation and outgrowth. To understand how SH2B1β may affect neurite formation, the collaborative efforts of SH2B1β with two actin-remodeling proteins, IRSp53 (Insulin receptor tyrosine kinase substrate p53) and Eps8 (Epidermal growth factor receptor kinase substrate 8) have been investigated. IRSp53 is a multi-domain protein that can regulate actin cytoskeleton-associated proteins and thus filopodium formation. Eps8 regulates actin dynamics through actin barbed-ends capping activity. The formation of IRSp53-Eps8 complex, regulated by active Cdc42, contributes to the formation of actin bundles, thus promoting filopodium protrusions. We found that the proline-rich domains of SH2B1 interact with IRSp53 and Eps8. Overexpressing SH2B1β and IRSp53 enhanced filopodium formation and neurite initiation. In contrast, shRNA for SH2B1 to silence endogenous SH2B1 or the deletion mutants of SH2B1β which lack the proline-rich domains that inhibit filopdium formation and neuronal branching. In addition, SH2B1, IRSp53 and Eps8 co-localize at the plasma membrane and their levels increase in the Triton X-100-insoluble fraction of developing neurons. These results suggest that the interaction among SH2B1, IRSp53 and Eps8 is required to promote actin polymerization and thus filopodium formation, neurite initiation and neuronal branching.