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) with high affinity. A number of naturally occurring splice variants of TrkC exist, including one (TrkC.ki14) with a 14 amino acid insertion between subdomains VII and VIII of the tyrosine kinase domain. This kinase insert blocks the ability of NT-3 to stimulate neurite outgrowth in PC12 cells and proliferation in fibroblasts. The inserts also block the ability of TrkC to form a high-affinity complex with Shc and phospholipase Cγ(PLCγ) and the activation of PtdIns 3-kinase, and attenuates the sustained activation of mitogen-activated protein kinase (MAPK). In the current study we set out to determine whether the attenuation of the activation of MAPK by the insert was the result of the inability of TrkC to activate the ShcŐRas pathway, PtdIns 3-kinase activation, PLCγ activation, or a combination thereof. Experiments with the use of cell-permeant inhibitors argue against a major role for PLCγ and PtdIns 3-kinase in the activation of MAPK by TrkC. The introduction of the 14 amino acid kinase insert appeared to slow the kinetics of NT-3-stimulated Shc phosphorylation and ShcŐGrb2 association and reduce their magnitude; an effect which was associated with a delayed, and only transient, activation of MAPK. Taken together, our data suggest that the apparent defect in MAPK activation caused by the kinase insert may result predominantly from an inhibition of high-affinity Shc binding, although a role for PLCγ and PtdIns 3-kinase cannot be completely excluded.