Complexes Formation between Insulin Receptor and Extracellular Signal-Regulated Kinases ERKs Yea-Lih Lin,* ,1 Cle ´ment Mettling,* and Chen-Kung Chou† *Institut de Ge ´ne ´tique Humaine, Centre National de la Recherche Scientifique, 141 rue de la Cardonille, 34396 Montpellier Cedex 5, France; and †Department of Medical Research, Veterans General Hospital, Shih-Pai, Taipei, Taiwan Received March 30, 2001; published online May 24, 2001 A property of signal transduction pathways that might explain their efficiency and specificity is the formation of signaling complexes. The recent demon- stration that adaptor proteins can interact with many components of the extracellular signal-regulated ki- nases (ERKs) signaling cascade leads us to investigate whether such complexes may include the transmem- brane receptor. The present work shows that in hu- man hepatoma Hep3B cells, insulin receptor (IR) can be coimmunoprecipitated with other components of the ERKs cascade: insulin receptor substrate (IRS), Raf-1, and ERKs. Furthermore, these complexes formed near the cytoplasmic membrane even prior to insulin stimulation. © 2001 Academic Press The sequential activation of the components of the signaling cascade induced by insulin has been heavily described. Binding of the ligand to the insulin receptor tyrosine kinase results in the tyrosine phosphorylation of two immediate substrates, insulin receptor substrate-1 and 2 (IRS-1 and IRS-2) (1, 2). The IRS proteins have an NH 2 -terminal pleckstrin homology (PH) domain followed by a phosphotyrosine-binding (PTB) domain and 18 potential tyrosine phosphoryla- tion sites (3) from a recent review. Under physiological condition, both IRS-1 PH and PTB domains are re- quired for insulin-induced tyrosine phosphorylation of IRS-1 and mitogenic signaling (4, 5). The phosphory- lated IRS proteins can act as docking proteins to bind the p85 regulatory subunit of phosphatidylinositol-3 kinase (PI-3 K) and growth factor receptor binding protein 2 (Grb2) through their Src homology-2 (SH2) domain (6 – 8). Grb2 physically interacts with Ras- GEF, SOS. The formation of IRS-Grb2-SOS ternary complexes is thought to induce the conversion of the inactive GDP-bound Ras to the active GTP-bound state. Once in the active GTP-bound state, Ras directly associates and activates members of Raf family serine/ threonine kinases (9, 10). In turn, activated Raf phos- phorylates the dual specificity kinases MEKs (MAP/ ERK kinases) which phosphorylate and stimulate ERKs (extracellular signal-regulated kinase) activity providing an important bifurcation point for the regu- lation of metabolic, transcriptional and mitogenic events (8, 10). The view of a sequential cascade has recently been refined by the discovery of complexes where the whole signaling components interact (11, 12). An example showing that protein complexes may ensure the specificity and efficiency of signal transduc- tion is the MAP kinase complexes coordinated by the scaffold proteins Ste5p and Pbs2p in the yeast Saccha- romyces cerevisiae. Ste5p binds components of the pheromone mating response pathway and Pbs2p can coordinate components of the osmoregulatory pathway (13). Two scaffold-like components have been reported recently for MAP kinase pathways in higher eukary- otic systems. A protein called MP1 (MEK partner 1) was identified that binds specifically to MEK1 and ERK1 and facilitates their activation (14). The cyto- plasmic protein JIP-1 (JNK interacting protein-1) was demonstrated to bind selectively to JNK (c-Jun NH 2 - terminal kinase), MKK7 (MAP kinase kinase 7) and MLK (mixed-lineage kinase, MAPKKK) (15). These scaffold proteins appear to play a dual regulatory role. First, they ensure precise regulation of the MAP kinase pathway by colocalizing and facilitating phosphoryla- tion of successive members of the cascade. Second, they insulate the MAP kinase pathway to prevent cross talk with functionally unrelated members of other MAPK signaling modules. We have previously shown that insulin suppresses hepatitis B surface antigen (HBsAg) and stimulates cell proliferation and proto-oncogene expression in hu- man hepatoma Hep3B cells (16). In these cells, insulin induces the phosphorylation of its own receptors and 180/200 kDa IRS proteins, furthermore, it also induces MEK/ERKs activation within minutes (unpublished 1 To whom correspondence and reprint requests should be ad- dressed. Fax: (33) 4 99 61 99 01. E-mail: ylin@igh.cnrs.fr. Molecular Cell Biology Research Communications 4, 234 –238 (2000) doi:10.1006/mcbr.2001.0286, available online at http://www.idealibrary.com on 234 1522-4724/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.