Characterization of the Tyrosine Kinases RAFTK/Pyk2 and FAK in Nerve Growth Factor-induced Neuronal Differentiation

Shin-Young Park,Hava Avraham,Shalom Avraham

doi:10.1074/jbc.m909932199

Abstract

The related adhesion focal tyrosine kinase (RAFTK), a member of the focal adhesion kinase (FAK) family and highly expressed in brain, is a key mediator of various extracellular signals that elevate intracellular Ca(2+) concentration. We investigated RAFTK and FAK signaling upon nerve growth factor (NGF) stimulation of PC12 cells. NGF induced the tyrosine phosphorylation of RAFTK in a time- and dose-dependent manner, whereas no change in the tyrosine phosphorylation of FAK was observed. Chemical inhibition showed that RAFTK phosphorylation was inhibited by blocking phospholipase Cgamma activity or intracellular Ca(2+). Blocking of extracellular Ca(2+) or phosphatidylinositol 3-kinase activity partially reduced the phosphorylation of RAFTK. In addition, disruption of actin polymerization abolished RAFTK phosphorylation, indicating that an intact actin-based cytoskeletal organization is required for RAFTK phosphorylation. The focal adhesion molecule paxillin was co-immunoprecipitated with RAFTK, and its tyrosine phosphorylation was increased in a Ca(2+)-dependent manner upon NGF stimulation. Confocal microscopic analysis demonstrated that RAFTK translocated from the cytoplasm to potential neurite initiation sites at the cell periphery, where RAFTK co-localized with paxillin and bundled actin in the early phase (within 5 min) of NGF stimulation, whereas FAK co-localized with paxillin at "point contacts," which are the primary cell adhesion sites in neuronal cells. Significant distribution of RAFTK was observed in the neurites and growth cones of differentiated PC12 cells. Furthermore, potassium depolarization induced the tyrosine phosphorylation of both RAFTK and paxillin in an intracellular Ca(2+)-dependent manner in the differentiated PC12 cells. Taken together, these results demonstrate that RAFTK is involved in NGF-induced cytoskeletal organization and may play a role in neurite and growth cone function(s).

Highlights

This paper is dedicated in memory of Ronald Ansin for his friendship and support for our research program
Confocal microscopic analysis demonstrated that related adhesion focal tyrosine kinase (RAFTK) translocated from the cytoplasm to potential neurite initiation sites at the cell periphery, where RAFTK co-localized with paxillin and bundled actin in the early phase of nerve growth factor (NGF) stimulation, whereas focal adhesion kinase (FAK) co-localized with paxillin at “point contacts,” which are the primary cell adhesion sites in neuronal cells
The tyrosine phosphorylation of RAFTK was induced by NGF in a time-dependent manner, peaking at 5 min (Fig. 1A), whereas that of FAK was constitutively tyrosine-phosphorylated at a high level and showed no change in phosphorylation upon NGF stimulation

Summary

Introduction

This paper is dedicated in memory of Ronald Ansin for his friendship and support for our research program. Since RAFTK activation is induced by Ca2ϩ mobilization and is involved in cytoskeletal reorganization [22], the signaling of RAFTK and its homologous tyrosine kinase FAK and their role as mediators of intracellular Ca2ϩ mobilization during NGF signaling were investigated. RAFTK associates with paxillin inducing the tyrosine phosphorylation of paxillin in a Ca2ϩ-dependent manner and translocates from the cytoplasm to the cell periphery upon NGF stimulation, whereas FAK associates with paxillin at the plasma membrane and is involved in the regulation of cell adhesion.

Results

Conclusion