SH2-B, a membrane-associated adapter, is phosphorylated on multiple serines/threonines in response to nerve growth factor by kinases within the MEK/ERK cascade.
SH2-B has been shown to be required for nerve growth factor (NGF)-mediated neuronal differentiation and survival, associate with NGF receptor TrkA, and be tyrosyl-phosphorylated in response to NGF. In this work, we examined whether NGF stimulates phosphorylation of SH2-B on serines/threonines. NGF promotes a dramatic upward shift in mobility of SH2-B, resulting in multiple forms that cannot be attributed to tyrosyl phosphorylation. Treatment of SH2-B with protein phosphatase 2A, a serine/threonine phosphatase, reduces the many forms to two. PD98059, a MEK inhibitor, dramatically inhibits NGF-promoted phosphorylation of SH2-B on serines/threonines, whereas depletion of 4beta-phorbol 12-myristate 13-acetate-sensitive protein kinase Cs does not. ERKs 1 and 2 phosphorylate SH2-Bbeta primarily on Ser-96 in vitro. However, NGF still stimulates serine/threonine phosphorylation of SH2-Bbeta(S96A). SH2-Bbeta(S96A), like wild-type SH2-Bbeta, enhances NGF-induced neurite outgrowth. In contrast, SH2-Bbeta(R555E) containing a defective SH2 domain blocks NGF-induced neurite outgrowth and displays greatly reduced phosphorylation on serines/threonines in response to NGF. SH2-Bbeta(R555E), like wild-type SH2-Bbeta, associates with the plasma membrane, suggesting that the dominant negative effect of SH2-Bbeta(R555E) cannot be explained by an abnormal subcellular distribution. In summary, NGF stimulates phosphorylation of SH2-B on serines/threonines by kinases downstream of MEK, which may be important for NGF-mediated neuronal differentiation and survival.
Rui
L
L
Department of Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0622, USA.
Herrington
J
J
Carter-Su
C
C
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D016297:Mutagenesis, Site-Directed; D009414:Nerve Growth Factors / Q000378:metabolism*; D016716:PC12 Cells; D010766:Phosphorylation; D051381:Rats; D012694:Serine / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013912:Threonine / Q000378:metabolism*
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009414:Nerve Growth Factors; C492010:SH2B1 protein, rat; D013912:Threonine; D012694:Serine; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D013755:Tetradecanoylphorbol Acetate