Stimulation of neuropeptide Y gene expression by brain-derived neurotrophic factor requires both the phospholipase Cgamma and Shc binding sites on its receptor, TrkB.
In PC12 cells, it has been previously reported that nerve growth factor stimulates neuropeptide Y (NPY) gene expression. In the current study we examined the signalling pathways involved in this effect by transiently expressing in PC12 cells the receptor (TrkB) for the related neurotrophin, brain-derived neurotrophic factor (BDNF). BDNF caused a 3-fold induction of luciferase expression from a transiently co-transfected plasmid possessing the firefly luciferase gene under the control of the NPY promoter. This effect of BDNF was completely blocked by either a Y484F mutation in TrkB (which blocks high-affinity Shc binding to TrkB) or by a Y785F substitution [which blocks the binding, phosphorylation and activation of phospholipase Cgamma (PLCgamma)]. Activation of the NPY promoter by neurotrophin-3 in PC12 cells overexpressing TrkC was also completely blocked by a naturally occurring kinase insert which prevents the high-affinity binding of Shc and PLCgamma. NPY promoter activation by BDNF was blocked by PD98059, suggesting a role for mitogen-activated protein kinase (MAP kinase). Stimulation of NPY gene expression by PMA, but not by BDNF, was blocked by Ro-31-8220, a protein kinase C inhibitor, excluding a role for this serine/threonine protein kinase in the effect of BDNF. In addition, BDNF did not cause an elevation in cytosolic Ca2+ concentration. Taken together, our results suggest that stimulation of the NPY promoter by BDNF requires the simultaneous activation of two distinct pathways; one involves Shc and MAP kinase, and the other appears to be PLCgamma-independent but requires an intact tyrosine-785 on TrkB and so may involve an effector of TrkB signalling that remains to be identified.
Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK.
A G
AG
Williams
A C
AC
Hargreaves
F J
FJ
Gunn-Moore
J M
JM
Tavaré
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D019208:Brain-Derived Neurotrophic Factor / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation; D015870:Gene Expression / Q000187:drug effects; D007527:Isoenzymes / Q000378:metabolism*; D009154:Mutation; D009414:Nerve Growth Factors / Q000494:pharmacology*; D009478:Neuropeptide Y / Q000096:biosynthesis*; D016716:PC12 Cells; D051966:Phospholipase C gamma; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D020813:Receptor, trkB; D020812:Receptor, trkC; D017475:Receptors, Nerve Growth Factor / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013268:Stimulation, Chemical; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D019208:Brain-Derived Neurotrophic Factor; D007527:Isoenzymes; D009414:Nerve Growth Factors; D009478:Neuropeptide Y; D011506:Proteins; D017475:Receptors, Nerve Growth Factor; D055765:Shc Signaling Adaptor Proteins; C528035:Shc1 protein, rat; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D020794:Receptor Protein-Tyrosine Kinases; D020813:Receptor, trkB; D020812:Receptor, trkC; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D002118:Calcium