Ras-activated endocytosis is mediated by the Rab5 guanine nucleotide exchange activity of RIN1.

RIN1 was originally identified by its ability to inhibit activated Ras and likely participates in multiple signaling pathways because it binds c-ABL and 14-3-3 proteins, in addition to Ras. RIN1 also contains a region homologous to the catalytic domain of Vps9p-like Rab guanine nucleotide exchange factors (GEFs). Here, we show that this region is necessary and sufficient for RIN1 interaction with the GDP-bound Rabs, Vps21p, and Rab5A. RIN1 is also shown to stimulate Rab5 guanine nucleotide exchange, Rab5A-dependent endosome fusion, and EGF receptor-mediated endocytosis. The stimulatory effect of RIN1 on all three of these processes is potentiated by activated Ras. We conclude that Ras-activated endocytosis is facilitated, in part, by the ability of Ras to directly regulate the Rab5 nucleotide exchange activity of RIN1.

[1]  H. Hama,et al.  Vps9p Is a Guanine Nucleotide Exchange Factor Involved in Vesicle-mediated Vacuolar Protein Transport* , 1999, The Journal of Biological Chemistry.

[2]  C. D’Souza-Schorey,et al.  Uncoupling of Membrane Ruffling and Pinocytosis during Ras Signal Transduction* , 1997, The Journal of Biological Chemistry.

[3]  S. Schmid,et al.  Regulation of receptor-mediated endocytosis by Rho and Rac , 1996, Nature.

[4]  M. White,et al.  Protein binding and signaling properties of RIN1 suggest a unique effector function. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[5]  A. Cherniack,et al.  Phosphorylation of the Ras nucleotide exchange factor son of sevenless by mitogen-activated protein kinase. , 1994, The Journal of biological chemistry.

[6]  F. McCormick,et al.  Regulation of Epidermal Growth Factor Receptor Signaling by Phosphorylation of the Ras Exchange Factor hSOS1 (*) , 1996, The Journal of Biological Chemistry.

[7]  D. Stern,et al.  Specificity within the EGF family/ErbB receptor family signaling network , 1998, BioEssays : news and reviews in molecular, cellular and developmental biology.

[8]  J. Bos,et al.  Shc associates with an unphosphorylated form of the p21ras guanine nucleotide exchange factor mSOS. , 1995, Oncogene.

[9]  S. Schmid,et al.  Control of EGF Receptor Signaling by Clathrin-Mediated Endocytosis , 1996, Science.

[10]  J. Welsh,et al.  Ligand-induced transformation by a noninternalizing epidermal growth factor receptor. , 1990, Science.

[11]  M. Zerial,et al.  rab5 controls early endosome fusion in vitro , 1991, Cell.

[12]  Kai Simons,et al.  The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway , 1992, Cell.

[13]  A. Klippel,et al.  Evidence for phosphatidylinositol 3-kinase as a regulator of endocytosis via activation of Rab5. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[14]  T. Pawson,et al.  MAP kinase phosphorylation of mSos1 promotes dissociation of mSos1-Shc and mSos1-EGF receptor complexes. , 1995, Oncogene.

[15]  Chris P. Ponting,et al.  A novel family ofras-binding domains , 1996 .

[16]  D. Bar-Sagi,et al.  Induction of membrane ruffling and fluid-phase pinocytosis in quiescent fibroblasts by ras proteins. , 1986, Science.

[17]  R. Roth,et al.  Protein Kinase B/akt and Rab5 Mediate Ras Activation of Endocytosis* , 1998, The Journal of Biological Chemistry.

[18]  P. Stahl,et al.  Endosome fusion in living cells overexpressing GFP-rab5. , 1999, Journal of cell science.

[19]  G. M. Walton,et al.  Functional independence of the epidermal growth factor receptor from a domain required for ligand-induced internalization and calcium regulation , 1989, Cell.

[20]  M. Colombo,et al.  GTP gamma S stimulation of endosome fusion suggests a role for a GTP-binding protein in the priming of vesicles before fusion. , 1989, Cell regulation.

[21]  A. Wells,et al.  Cell movement elicited by epidermal growth factor receptor requires kinase and autophosphorylation but is separable from mitogenesis , 1994, The Journal of cell biology.

[22]  Jonathan A. Cooper,et al.  Mammalian Ras interacts directly with the serine/threonine kinase raf , 1993, Cell.

[23]  E. Saihan.  Regulation of Epidermal Growth , 1989 .

[24]  G. Hannon,et al.  Isolation of the Rb-related p130 through its interaction with CDK2 and cyclins. , 1993, Genes & development.

[25]  M. Wigler,et al.  Multiple ras functions can contribute to mammalian cell transformation , 1995, Cell.

[26]  I. Mellman.  Endocytosis and molecular sorting. , 1996, Annual review of cell and developmental biology.

[27]  C. Rice,et al.  Noncytopathic Sindbis virus RNA vectors for heterologous gene expression. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[28]  G. Tall,et al.  The phosphatidylinositol 3-phosphate binding protein Vac1p interacts with a Rab GTPase and a Sec1p homologue to facilitate vesicle-mediated vacuolar protein sorting. , 1999, Molecular biology of the cell.

[29]  Marino Zerial,et al.  A Novel Rab5 GDP/GTP Exchange Factor Complexed to Rabaptin-5 Links Nucleotide Exchange to Effector Recruitment and Function , 1997, Cell.

[30]  S. Schmid,et al.  Regulation of signal transduction by endocytosis. , 2000, Current opinion in cell biology.

[31]  M. Zerial,et al.  Co‐operative regulation of endocytosis by three RAB5 isoforms , 1995, FEBS letters.

[32]  G. M. Di Guglielmo,et al.  Compartmentalized signal transduction by receptor tyrosine kinases. , 1995, Trends in Cell Biology.

[33]  K A Overholser,et al.  Multiple autophosphorylation site mutations of the epidermal growth factor receptor. Analysis of kinase activity and endocytosis. , 1991, The Journal of biological chemistry.

[34]  S. Cohen,et al.  Direct visualization of the binding and internalization of a ferritin conjugate of epidermal growth factor in human carcinoma cells A-431 , 1979, The Journal of cell biology.

[35]  J. Schlessinger,et al.  Regulation of signal transduction and signal diversity by receptor oligomerization. , 1994, Trends in biochemical sciences.

[36]  J. Olefsky,et al.  Negative Feedback Regulation and Desensitization of Insulin- and Epidermal Growth Factor-stimulated p21ras Activation (*) , 1995, The Journal of Biological Chemistry.

[37]  P. Stahl,et al.  In vitro fusion of endosomes following receptor-mediated endocytosis. , 1988, The Journal of biological chemistry.

[38]  M. Wigler,et al.  Expression of three mammalian cDNAs that interfere with RAS function in Saccharomyces cerevisiae. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[39]  J. Colicelli,et al.  A human protein selected for interference with Ras function interacts directly with Ras and competes with Raf1 , 1995, Molecular and cellular biology.

[40]  P. Stahl,et al.  Evidence for a Symmetrical Requirement for Rab5-GTP in in Vitro Endosome-Endosome Fusion* , 1998, The Journal of Biological Chemistry.

[41]  G. Carpenter,et al.  Epidermal Growth Factor , 2002 .

[42]  A. Wells,et al.  Epidermal growth factor and membrane trafficking. EGF receptor activation of endocytosis requires Rab5a. , 2000 .

[43]  H. Mellor,et al.  Regulation of endocytic traffic by rho family GTPases. , 2000, Trends in cell biology.

[44]  M. Zerial,et al.  Inhibition of rab5 GTPase activity stimulates membrane fusion in endocytosis. , 1994, The EMBO journal.

[45]  A. Dhaka,et al.  Regulation of the oncogenic activity of BCR-ABL by a tightly bound substrate protein RIN1. , 1997, Immunity.

[46]  D. Lauffenburger,et al.  Internalized Epidermal Growth Factor Receptors Participate in the Activation of p21 ras in Fibroblasts* , 1999, The Journal of Biological Chemistry.

[47]  J. Pessin,et al.  SOS Phosphorylation and Disassociation of the Grb2-SOS Complex by the ERK and JNK Signaling Pathways (*) , 1996, The Journal of Biological Chemistry.