SH2B1 and IRSp53 proteins promote the formation of dendrites and dendritic branches.

SH2B1 is an adaptor protein known to enhance neurite outgrowth. In this study, we provide evidence suggesting that the SH2B1 level is increased during in vitro culture of hippocampal neurons, and the ß isoform (SH2B1ß) is the predominant isoform. The fact that formation of filopodia is prerequisite for neurite initiation suggests that SH2B1 may regulate filopodium formation and thus neurite initiation. To investigate whether SH2B1 may regulate filopodium formation, the effect of SH2B1 and a membrane and actin regulator, IRSp53 (insulin receptor tyrosine kinase substrate p53), is investigated. Overexpressing both SH2B1ß and IRSp53 significantly enhances filopodium formation, neurite outgrowth, and branching. Both in vivo and in vitro data show that SH2B1 interacts with IRSp53 in hippocampal neurons. This interaction depends on the N-terminal proline-rich domains of SH2B1. In addition, SH2B1 and IRSp53 co-localize at the plasma membrane, and their levels increase in the Triton X-100-insoluble fraction of developing neurons. These findings suggest that SH2B1-IRSp53 complexes promote the formation of filopodia, neurite initiation, and neuronal branching.

SH2B1 increases the numbers of IRSp53-induced filopodia.

BACKGROUND: Filopodia are actin-rich membrane protrusions that play instrumental roles in development, cell migration, pathogen detection, and wound healing. During neurogenesis, filopodium formation precedes the formation of dendrites and spines. The insulin receptor substrate protein of 53kDa (IRSp53) has been implicated in regulating the formation of filopodia. Our previous results suggest that a signaling adaptor protein SH2B1ß is required for neurite outgrowth of hippocampal neurons and neurite initiation of PC12 cells. Thus, we hypothesize that IRSp53 and SH2B1ß may act together to regulate filopodium formation. METHODS: To determine the contribution of IRSp53 and SH2B1ß in the formation of filopodia, we transiently transfect IRSp53 and/or SH2B1ß to 293T cells. Cell morphology and protein distribution are assessed via confocal microscopy and subcellular fractionation. Total numbers of filopodia and filopodium numbers per perimeter are calculated to show the relative contribution of IRSp53 and SH2B1ß. RESULTS: In this study, we show that SH2B1ß interacts with IRSp53 and increases the number of IRSp53-induced filopodia. One mechanism for this enhancement is that IRSp53 recruits SH2B1ß to the plasma membrane to actively promote membrane protrusion. The increased numbers of filopodia likely result from SH2B1-mediated cytoplasmic extension and thus increased cell perimeter as well as IRSp53-mediated filopodium formation. CONCLUSIONS: Taken together, this study provides a novel finding that SH2B1ß interacts with IRSp53-containing complexes to increase the number of filopodia. GENERAL SIGNIFICANCE: A better understanding of how SH2B1ß and IRSp53 promote filopodium formation may have clinical implication in neurogenesis and regeneration.