Activation and Decay Kinetics of the RET Receptor Tyrosine Kinase

Abstract

We use ZD6474, an inhibitor of the kinase domain of the RET receptor tyrosine kinase, as a chemical probe to obtain quantitative information on the molecular kinetics of RET activation, autophosphorylation, dephosphorylation and trafficking in mouse neuroblastoma cells. Stimulation with RET's cognate ligand artemin (ART) resulted in up to 65% of cellular RET becoming phosphorylated over several minutes, followed by a slow decay. Addition of ZD6474 6 min after stimulation resulted in loss of phosphoRET (pRET) with t1/2 蠄 1 min, indicating that overall pRET dynamics reflects an evolving steady state between rapid RET phosphorylation and its comparably rapid dephosphorylation. Reconciling the observed rate of pRET formation with the rapid rate of pRET dephosphorylation and the high level of pRET at the steady state required invoking a rate-limiting activation of RET by ART, prior to receptor phosphorylation which occurs rapidly (rate constant 1.2 min-1). Dephosphorylation of pRET regenerates activated but unphosphorylated RET, which autophosphorylates again rapidly rather than reverting to the resting state. We additionally show that this rapidly cycling pRET is rapidly internalized through clathrin-coated pits, where it continues to activate ERK and Akt signaling. A fraction of the internalized pRET slowly converts to a longer-lived form (t1/2 ~ 10 min) that resides in late endosomes where it is protected from phosphatases and loses its signaling ability. Our results kinetically distinguish ART-dependent assembly of the activated RET receptor complex from its subsequent autophosphorylation, and provide a quantitative picture of RET receptor activation and trafficking.