Ras Subcellular Localization Defines Extracellular Signal-Regulated Kinase 1 and 2 Substrate Specificity through Distinct Utilization of Scaffold Proteins

Abstract

Subcellular localization influences the nature of Ras/extracellular signal-regulated kinase (ERK) signals by unknown mechanisms. Herein, we demonstrate that the microenvironment from which Ras signals emanate determines which substrates will be preferentially phosphorylated by the activated ERK1/2. We show that the phosphorylation of epidermal growth factor receptor (EGFr) and cytosolic phospholipase A₂ (cPLA₂) is most prominent when ERK1/2 are activated from lipid rafts, whereas RSK1 is mainly activated by Ras signals from the disordered membrane. We present evidence indicating that the underlying mechanism of this substrate selectivity is governed by the participation of different scaffold proteins that distinctively couple ERK1/2, activated at defined microlocalizations, to specific substrates. As such, we show that for cPLA₂ activation, ERK1/2 activated at lipid rafts interact with KSR1, whereas ERK1/2 activated at the endoplasmic reticulum utilize Sef-1. To phosphorylate the EGFr, ERK1/2 activated at lipid rafts require the participation of IQGAP1. Furthermore, we demonstrate that scaffold usage markedly influences the biological outcome of Ras site-specific signals. These results disclose an unprecedented spatial regulation of ERK1/2 substrate specificity, dictated by the microlocalization from which Ras signals originate and by the selection of specific scaffold proteins.

ACKNOWLEDGMENTS

We are indebted to A. Pandiella, T. Iglesias, J. Lozano, and Z. Chang for providing reagents.

A.P. and L.A.-I. are Spanish Ministry of Education predoctoral fellows. P.C.'s lab is supported by grants BFU2005-00777 and GEN2003-20239-C06-03 from the Spanish Ministry of Education, the EU Sixth Framework Program under the GROWTHSTOP (LSHC CT-2006-037731) and SIMAP (IST-2004-027265) projects, the Red Temática de Investigación Cooperativa en Cáncer (RD06/0020/0105), the Fondo de Investigaciones Sanitarias, Carlos III Institute, and the Spanish Ministry of Health.