Critical Role for Kalirin in Nerve Growth Factor Signaling through TrkA

Abstract

Kalirin is a multidomain guanine nucleotide exchange factor (GEF) that activates Rho proteins, inducing cytoskeletal rearrangement in neurons. Although much is known about the effects of Kalirin on Rho GTPases and neuronal morphology, little is known about the association of Kalirin with the receptor/signaling systems that affect neuronal morphology. Our experiments demonstrate that Kalirin binds to and colocalizes with the TrkA neurotrophin receptor in neurons. In PC12 cells, inhibition of Kalirin expression using antisense RNA decreased nerve growth factor (NGF)-induced TrkA autophosphorylation and process extension. Kalirin overexpression potentiated neurotrophin-stimulated TrkA autophosphorylation and neurite outgrowth in PC12 cells at a low concentration of NGF. Furthermore, elevated Kalirin expression resulted in catalytic activation of TrkA, as demonstrated by in vitro kinase assays and increased NGF-stimulated cellular activation of Rac, Mek, and CREB. Domain mapping demonstrated that the N-terminal Kalirin pleckstrin homology domain mediates the interaction with TrkA. The effects of Kalirin on TrkA provide a molecular basis for the requirement of Kalirin in process extension from PC12 cells and for previously observed effects on axonal extension and dendritic maintenance. The interaction of TrkA with the pleckstrin homology domain of Kalirin may be one example of a general mechanism whereby receptor/Rho GEF pairings play an important role in receptor tyrosine kinase activation and signal transduction.

ACKNOWLEDGMENTS

We thank David Ginty (Johns Hopkins University) and Phil Barker (McGill University) for the CMX.TrkA expression construct. We thank Francesco Ferraro and Jackie Sobota for assistance with deconvolution microscopy experiments, Brian Piccirillo for preliminary studies with Kal-PH1, and Sandrine Mouradian-Garci and Nicolas Charnay for technical assistance. We are grateful to Betty Eipper and Richard Mains (University of Connecticut Health Center) for the pEAK10.HisMyc.Kal5, pEGFP.PH-GFP, and pCMS.GFP/Kal-as expression constructs and for their support. We thank Eric Levine and Hugh Blumenfeld (University of Connecticut Health Center) for suggestions in the preparation of the manuscript.

Support for this research was provided by NIMH (MH65567), NIDDK (DK32948), the Association pour la Recherche contre le Cancer, the Centre National pour la Recherche Scientifique, the Ministère pour la Recherche et des Nouvelles Technologies, the Ligue Nationale contre le Cancer, and a Career Development Award to M.R.S. from the University of Connecticut Health Center.