ANALYSIS OF MITOGEN-ACTIVATED PROTEIN-KINASE ACTIVATION BY NATURALLY-OCCURRING SPLICE VARIANTS OF TRKC, THE RECEPTOR FOR NEUROTROPHIN-3

TrkC is a receptor tyrosine kinase that binds neurotrophin-3 (NT-3) wi th high affinity. A number of naturally occurring splice variants of T rkC exist, including one (TrkC.kil4) with a 14 amino acid insertion be tween subdomains VII and VIII of the tyrosine kinase domain. This kina se insert blocks the ability of NT-3 to stimulate neurite outgrowth in PC12 cells and proliferation in fibroblasts. The inserts also block t he ability of TrkC to form a high-affinity complex with She and phosph olipase Cy (PLC gamma) and the activation of PtdIns 3-kinase, and atte nuates the sustained activation of mitogen-activated protein kinase (M APK). In the current study we set out to determine whether the attenua tion of the activation of MAPK by the insert was the result of the ina bility of TrkC to activate the Shc-Ras pathway, PtdIns 3-kinase activa tion, PLC gamma activation, or a combination thereof. Experiments with the use of cell-permeant inhibitors argue against a major role for PL C gamma and PtdIns 3-kinase in the activation of MAPK by TrkC. The int roduction of the 14 amino acid kinase insert appeared to slow the kine tics of NT-3-stimulated She phosphorylation and Shc-Grb2 association a nd reduce their magnitude; an effect which was associated with a delay ed, and only transient, activation of MAPK. Taken together, our data s uggest that the apparent defect in MAPK activation caused by the kinas e insert may result predominantly from an inhibition of high-affinity She binding, although a role for PLC gamma and PtdIns 3-kinase cannot be completely excluded.