Phosphorylation of the Unique C-Terminal Tail of the Alpha Isoform of the Scaffold Protein SH2B1 Controls the Ability of SH2B1α To Enhance Nerve Growth Factor Function

ABSTRACT

The scaffold protein SH2B1, a major regulator of body weight, is recruited to the receptors of multiple cytokines and growth factors, including nerve growth factor (NGF). The β isoform but not the α isoform of SH2B1 greatly enhances NGF-dependent neurite outgrowth of PC12 cells. Here, we asked how the unique C-terminal tails of the α and β isoforms modulate SH2B1 function. We compared the actions of SH2B1α and SH2B1β to those of the N-terminal 631 amino acids shared by both isoforms. In contrast to the β tail, the α tail inhibited the ability of SH2B1 to both cycle through the nucleus and enhance NGF-mediated neurite outgrowth, gene expression, phosphorylation of Akt and phospholipase C-gamma (PLC-γ), and autophosphorylation of the NGF receptor TrkA. These functions were restored when Tyr753 in the α tail was mutated to phenylalanine. We provide evidence that TrkA phosphorylates Tyr753 in SH2B1α, as well as tyrosines 439 and 55 in both SH2B1α and SH2B1β. Finally, coexpression of SH2B1α but not SH2B1α with a mutation of Y to F at position 753 (Y753F) inhibited the ability of SH2B1β to enhance neurite outgrowth. These results suggest that the C-terminal tails of SH2B1 isoforms are key determinants of the cellular role of SH2B1. Furthermore, the function of SH2B1α is regulated by phosphorylation of the α tail.

KEYWORDS:

• SH2B1
• phosphorylation
• nerve growth factor signaling
• scaffold protein
• TrkA
• isoform

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ACKNOWLEDGMENTS

We thank Jessica Cote and Martin Myers, Donna Martin, Ben Margolis, Ken Inoki, Lei Yin, Xin (Tony) Tong, Deqiang Zhang, Jessica Schwartz, and Ram Menon for their helpful comments, Steven Lentz for help with the confocal microscopy, and Sarah Cain for administrative support. We thank Chengbiao Wu (University of California at San Diego) and David Ginty (Harvard University) for their kind gifts of cDNA encoding rat mCherry-TrkA and untagged TrkA, respectively. Confocal microscopy was performed using the Morphology and Image Analysis Core of the Michigan Diabetes Research Center (NIH grant no. P60-DK20572). Flow cytometry and cDNA sequencing were supported by the University of Michigan Comprehensive Cancer Center (NIH grant no. P30-CA46592).

We declare no conflict of interest.

R.M.J., A.F., C.C.-S., and M.E.D. designed experiments; R.M.J., A.F., J.M.C., and M.E.D. performed experiments; H.R. contributed new reagents and helped edit the manuscript; R.M.J., A.F., L.S.A., E.S.C., P.B.V., and C.C.-S. analyzed the data; and R.M.J., C.C.-S., and L.S.A. wrote the manuscript.

This study was supported by grants from the National Institutes of Health (NIH) (grant no. F31-DK100217 to R.M.J., grant no. R01-DK054222, grant no. R01-DK107730, and research supplement no. R01DK054222 to promote diversity in health-related research to C.C.-S., and predoctoral fellowships from the Systems and Integrative Biology training grant no. T32-GM008322 to A.F. and M.E.D.).