Ras subcellular localization defines extracellular signal-regulated kinase 1 and 2 substrate specificity through distinct utilization of scaffold proteins.

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 A(2) (cPLA(2)) 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 cPLA(2) 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.