A Role for Shc, Grb2, and Raf-1 in FcγRI Signal Relay

Rae Kil Park,Yenbou Liu,Donald L Durden

doi:10.1074/jbc.271.23.13342

Abstract

The activation of the serine/threonine kinase, Raf-1, serves to connect upstream protein tyrosine kinases to downstream signaling events. We previously reported that FcgammaRI stimulation of interferon gamma-differentiated U937 cells (termed U937IF cells) induces a mobility shift in Erk2. Herein, we report that cross-linking of FcgammaRI receptor in U937IF cells induces a marked tyrosine phosphorylation of Raf-1 (10-fold increase). Tyrosine phosphorylation of Raf-1 is induced by FcgammaRI activation and not by PMA (1 microg/ml), N-formyl-Met-Leu-Phe (1 microM), calcium ionophore (1 microM), thrombin (0.05 unit/ml), FcgammaRII, or FcgammaRIII stimulation. The kinetics of Raf-1 tyrosine phosphorylation is rapid, reaching peak levels 1-2 min after FcgammaRI activation, and the tyrosine phosphorylation of Raf-1 precedes the activation of the respiratory burst. FcgammaRI cross-linking induces the tyrosine phosphorylation of Shc; tyrosine-phosphorylated Shc binds to Grb2 forming a Shc-Grb2 complex. The data provide evidence that the FcgammaRI receptor signals via the upstream activation of nonreceptor protein tyrosine kinases, which leads to the subsequent activation of Ras family GTPases and serine/threonine kinases, Raf-1 and mitogen-activated protein kinase.

Highlights

Phosphorylation on tyrosine, serine, or threonine residues is a key regulatory mechanism in mammalian cells used to regulate the mitogenic or oncogenic potential of proteins by augmenting their enzymatic activity or modifying their association with other signal transducers [1]
Tyrosine Phosphorylation of Raf-1 upon Fc␥RI Activation—To evaluate the involvement of Raf-1 in Fc␥RI signaling, we examined whether Raf-1 is tyrosine-phosphorylated after Fc␥RI stimulation in U937IF cells
To determine whether Fc␥RI cross-linking can induce the tyrosine phosphorylation of Raf-1 in myeloid cells, 2 ϫ 107 U937IF cells were first incubated with mAb 197, followed by stimulation with rabbit anti-mouse F(abЈ)2 fragment (R␣M)

Summary

Introduction

Phosphorylation on tyrosine, serine, or threonine residues is a key regulatory mechanism in mammalian cells used to regulate the mitogenic or oncogenic potential of proteins by augmenting their enzymatic activity or modifying their association with other signal transducers [1]. We report that cross-linking of Fc␥RI receptor in U937IF cells induces a marked tyrosine phosphorylation of Raf-1 (10-fold increase). We previously reported that the stimulation of Fc␥RI receptor in U937IF cells results in tyrosine phosphorylation and activation of Syk [32].

Results

Conclusion