RAPID PHOSPHORYLATION OF PHOSPHOLIPASE C-GAMMA-1 BY BRAIN-DERIVED NEUROTROPHIC FACTOR AND NEUROTROPHIN-3 IN CULTURES OF EMBRYONIC RAT CORTICAL-NEURONS

Phospholipase C-gamma1 (PLC-gamma1) is involved at an early step in si gnal transduction of many hormones and growth factors and catalyzes th e hydrolysis of phosphatidylinositol (PI) 4,5-bisphosphate to diacylgl ycerol and inositol trisphosphate, two potent intracellular second mes senger molecules. The transformation of PC12 cells into neuron-like ce lls induced by nerve growth factor is preceded by a rapid stimulation of PLC-gamma1 phosphorylation and PI hydrolysis. The present study ana lyzed the effects of brain-derived neurotrophic factor (BDNF) and neur otrophin-3 (NT-3) on phosphorylation of PLC-gamma1 in primary cultures of embryonic rat brain cells. BDNF and NT-3 stimulated the phosphoryl ation of PLC-gamma1, followed by hydrolysis of PI. The stimulation of PLC-gamma1 phosphorylation occurred within 20 s after addition of BDNF or NT-3 and lasted up to 30 min, with a peak after 4 min. ED50 values were similar for BDNF and NT-3, with approximately 25 ng/ml. Phosphor ylation of PLC-gamma1 by BDNF and NT-3 was found in cultures from all major brain areas. K-252b, a compound known to inhibit selectively neu rotrophin actions by interfering with the phosphorylation of trk-type neurotrophin receptors, prevented the BDNF- and NT-3-stimulated phosph orylation of PLC-gamma1. Receptors of the trk type were coprecipitated with anti-PLC-gamma1 antibodies. The presence of irk-B mRNA in the cu ltures was substantiated by northern blot analysis. The action of BDNF and NT-3 seems to be neuron specific because no phosphorylation of PL C-gamma1 was observed in cultures of nonneuronal brain cells. The resu lts provide evidence that developing neurons of the cerebral cortex an d other brain areas are responsive to BDNF and NT-3, and they indicate that the transduction mechanism of BDNF and NT-3 in the brain involve s rapid phosphorylation of PLC-gamma1 followed by PI hydrolysis.