Abstract
Csk homologous kinase (CHK), a member of the Csk regulatory tyrosine kinase family, is expressed primarily in brain and hematopoietic cells. The role of CHK in the nervous system is as yet unknown. Using PC12 cells as a model system of neuronal cells, we show that CHK participates in signaling mediated by TrkA receptors. CHK was found to be associated with tyrosine-phosphorylated TrkA receptors in PC12 cells upon stimulation with NGF. Binding assays and far Western blotting analysis, using glutathione S-transferase fusion proteins containing the Src homology 2 (SH2) and SH3 domains of CHK, demonstrate that the SH2 domain of CHK binds directly to the tyrosine-phosphorylated TrkA receptors. Site-directed mutagenesis of TrkA cDNA, as well as phosphopeptide inhibition of the in vitro interaction of the CHK-SH2 domain or native CHK with TrkA receptors, indicated that the residue Tyr-785 on TrkA is required for its binding to the CHK-SH2 domain upon NGF stimulation. In addition, overexpression of CHK resulted in enhanced activation of the mitogen-activated protein kinase pathway upon NGF stimulation, and microinjection of anti-CHK antibodies, but not anti-Csk antibodies, inhibited neurite outgrowth of PC12 cells in response to NGF. Thus, CHK is a novel signaling molecule that participates in TrkA signaling, associates directly with TrkA receptors upon NGF stimulation, and is involved in neurite outgrowth of PC12 cells in response to NGF.
Highlights
Nerve growth factor (NGF)1 regulates the survival, development, and differentiation of the sympathetic and sensory neurons in the peripheral nervous system and the differentiation of certain cholinergic neurons in the central nervous system [1, 2]
Csk homologous kinase (CHK) Is Associated with Activated TrkA Receptors upon Stimulation with NGF—Because CHK is a second member of the Csk family and both kinases are expressed in brain, we investigated whether CHK and/or Csk might be involved in one of the main signaling pathways in neuronal cells that is mediated by the neurotrophin NGF
Microinjection of CHK-specific Antibodies Inhibited Neuronal Differentiation of PC12 Cells—To determine the requirement for the binding of the CHK-Src homology 2 (SH2) domain to TrkA receptors during neurite outgrowth, we examined the effects of decreasing the intracellular levels of CHK by microinjecting purified anti-CHK antibodies, anti-Csk antibodies, control preimmune rabbit antiserum, or control monoclonal antibody into living PC12 cells
Summary
Nerve growth factor (NGF) regulates the survival, development, and differentiation of the sympathetic and sensory neurons in the peripheral nervous system and the differentiation of certain cholinergic neurons in the central nervous system [1, 2]. With NGF, these cells acquire the phenotype of sympathetic neurons as characterized by neurite outgrowth and persistent activation of the ERK family of mitogen-activated protein (MAP) kinase [5]. The phosphotyrosines on activated TrkA serve as docking sites for signaling substrates such as phospholipase C-␥1 (PLC-␥1), phosphatidylinositol 3-kinase (PI-3 kinase), and the Shc adaptor protein [6, 12]. These molecules trigger kinase cascades resulting in the phosphorylation and activation of transcription factors that direct gene expression. We identified and characterized the association of CHK in TrkA signaling and investigated CHK involvement in neuronal differentiation of PC12 cells
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