Adapter Protein SH2B1β Cross-Links Actin Filaments and Regulates Actin Cytoskeleton

The Src homology 2 (SH2) domain-containing adapter protein SH2B1 beta plays a role in severe obesity, leptin and insulin resistance, and infertility. SH2B1 beta was initially identified as a Janus tyrosine kinase 2 (JAK2) substrate, and it has been implicated in cell motility and regulation of the actin rearrangement in response to GH and platelet-derived growth factor. SH2B1 beta is also required for maximal actin-based motility of Listeria. Here we have used a low-speed pelleting assay and electron microscopy to demonstrate that SH2B1 beta has two actin-binding sites and that it cross-links actin filaments in vitro. Wild-type SH2B1 beta localized to cell ruffles and along filopodia, but deletion of amino acids 150-200 (the first actin-binding site) led to mislocalization of the protein to filopodia tip complexes where it colocalized with vasodilator-stimulated phosphoprotein (VASP). Based on studies performed in VASP-deficient MVD7(-/-) cells, with or without green fluorescent protein-VASP reconstitution, we concluded that the proper intracellular localization of native SH2B1 beta required the presence of the first SH2B1 beta actin-binding site and VASP. Finally, we found that both SH2B1 beta actin-binding domains were required for maximal GH- and prolactin-induced cell ruffling. Together, these results suggest that SH2B1 beta functions as an adapter protein that cross-links actin filaments, leading to modulation of cellular responses in response to JAK2 activation. (Molecular Endocrinology 23: 1065-1076, 2009)