Abstract
Intersectin (ITSN) is a molecular scaffold involved in regulating endocytosis and mitogenic signaling. We previously demonstrated that ITSN transformed rodent fibroblasts, accelerated hormone-induced maturation of Xenopus oocytes, and activated the Elk-1 transcription factor through an MEK- and Erk-independent mechanism. We now demonstrate that ITSN complexes with the Ras guanine nucleotide exchange factor Sos1 leading to increased RasGTP levels. Using fluorescence resonant energy transfer analysis, we demonstrate that ITSN complexes with Ras in living cells leading to Ras activation on intracellular vesicles. These vesicles contain epidermal growth factor receptor but are distinct from transferrin-positive vesicles. However, Ras is not required for ITSN stimulation of transcription. Rather, we demonstrate that ITSN signals through JNK to activate Elk-1. Although ITSN activation of Elk-1 was Ras-independent, ITSN cooperates with Ras to synergistically activate JNK. These findings indicate that ITSN activates multiple intracellular signaling pathways and suggest that this adaptor protein may coordinately regulate the activity of these pathways in vivo.
Highlights
ITSN is a recently described endocytic adaptor protein consisting of multiple modular domains, including two aminoterminal Eps15 homology (EH)1 domains, a central coiled-coil domain, and five carboxyl-terminal Src homology 3 (SH3) domains [1,2,3,4,5]
ITSN synergized with EGF as well as other growth factors to stimulate Elk-1, we found that Elk-1 activation by ITSN was independent of EGFR kinase activity
These data indicate that ITSN signaling does not result merely from inhibition of EGFR endocytosis thereby prolonging signaling from the receptor at the cell surface
Summary
ITSN is a recently described endocytic adaptor protein consisting of multiple modular domains, including two aminoterminal Eps homology (EH) domains, a central coiled-coil domain, and five carboxyl-terminal Src homology 3 (SH3) domains [1,2,3,4,5]. Preliminary characterization of the pathway(s) involved in Elk-1 activation by ITSN revealed that this activity was independent of MEK1/2 and Erk MAPK [9]. A recent study demonstrated that Ras activates distinct signaling pathways at different endomembrane compartments [12]. ITSN stimulation of Elk-1 does not require Ras function. ITSN signaling does not require the kinase activity of the epidermal growth factor receptor EGFR and ITSN cooperate to synergistically activate Elk-1 and are co-localized on vesicles. ITSN stimulates Elk-1 through a JNK-dependent pathway. These data further define the biochemical pathways through which ITSN functions and support the model that ITSN is a pivotal component in integrating numerous cellular processes, including activation of GTPase cascades, endocytosis, and mitogenesis
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