Identification of βIIΣ1-Spectrin as a Binding Partner of the GH-regulated Human Obesity Scaffold Protein SH2B1

SH2B1β is a multifunctional scaffold protein that modulates cytoskeletal processes such as cellular motility and neurite outgrowth. To identify novel SH2B1β-interacting proteins involved in these processes, a yeast 2-hybrid assay was performed. The C-terminal 159 residues of the cytoskeleton structural protein, βIIΣ1-spectrin, interacted with the N-terminal 260 residues of SH2B1β, a region implicated in SH2B1β enhancement of cell motility and localization at the plasma membrane. The interaction between SH2B1β and βIIΣ1-spectrin (2205-2363) requires residues 1 through 150 in SH2B1β, with residues 105 through 120 playing a key role. While βIIΣ1-spectrin (2205-2363) was expressed throughout the cell, it colocalized with SH2B1β when coexpressed with SH2B1β mutants with varied intracellular localizations. The SH2B1β-βIIΣ1-spectrin (2205-2363) interaction impaired the ability of SH2B1β to enter the nucleus. A slightly larger βIIΣ1-spectrin fragment (2170-2363) with an intact pleckstrin homology domain localized primarily to the plasma membrane and cytoplasm, similar to SH2B1β. Similarly, full-length βIIΣ1-spectrin colocalized at the plasma membrane and cytoplasm with SH2B1β as well as the SH2B1β-regulated tyrosine kinase, JAK2. Phosphorylation of spectrins has been shown to regulate their localization and function. Coexpression of βIIΣ1-spectrin, JAK2, and SH2B1β resulted in SH2B1β-dependent tyrosyl phosphorylation of βIIΣ1-spectrin. Finally, stimulation with GH induced formation of an endogenous complex containing βII-spectrin, SH2B1, and JAK2 in 3T3-F442A cells and increased tyrosyl phosphorylation of βII-spectrin. Our results identify a novel interaction between SH2B1β, βIIΣ1-spectrin, and JAK2 resulting in JAK2- and SHB1-dependent tyrosyl phosphorylation of βII-spectrin. This raises the possibility that the many other ligand-activated tyrosine kinases that signal through SH2B1 form similar complexes with βIIΣ1-spectrin.