title: Different Raf protein kinases mediate different signaling pathways to stimulate E3 ligase RFFL gene expression in cell migration regulation.
abstract: We previously characterized a Gα12-specific signaling pathway that stimulates the transcription of the E3 ligase RFFL via the protein kinase ARAF and ERK. This pathway leads to persistent PKC activation and is important for sustaining fibroblast migration. However, questions remain regarding how Gα12 specifically activates ARAF, which transcription factor is involved in Gα12-mediated RFFL expression, and whether RFFL is important for cell migration stimulated by other signaling mechanisms that can activate ERK. In this study, we show that replacement of the Gα12 residue Arg-264 with Gln, which is the corresponding Gα13 residue, abrogates the ability of Gα12 to interact with or activate ARAF. We also show that Gα12 can no longer interact with and activate an ARAF mutant with its C-terminal sequence downstream of the kinase domain being replaced with the corresponding CRAF sequence. These results explain why Gα12, but not Gα13, specifically activates ARAF but not CRAF. Together with our finding that recombinant Gα12 is sufficient for stimulating the kinase activity of ARAF, this study reveals an ARAF activation mechanism that is different from that of CRAF. In addition, we show that this Gα12-ARAF-ERK pathway stimulates RFFL transcription through the transcription factor c-Myc. We further demonstrate that EGF, which signals through CRAF, and an activated BRAF mutant also activate PKC and stimulate cell migration through up-regulating RFFL expression. Thus, RFFL-mediated PKC activation has a broad significance in cell migration regulation.
authors.affiliation: Department of Pharmacology and Program in Vascular Biology and Therapeutics, Yale School of Medicine, New Haven, Connecticut 06520.
authors.forename: Xiaoqing
authors.initials: X
authors.lastname: Gan
authors.affiliation: Department of Pharmacology and Program in Vascular Biology and Therapeutics, Yale School of Medicine, New Haven, Connecticut 06520.
authors.forename: Chen
authors.initials: C
authors.lastname: Wang
authors.affiliation: Department of Pharmacology, University of Illinois at Chicago, Chicago, Illinois 60612.
authors.forename: Maulik
authors.initials: M
authors.lastname: Patel
authors.affiliation: Department of Pharmacology, University of Illinois at Chicago, Chicago, Illinois 60612.
authors.forename: Barry
authors.initials: B
authors.lastname: Kreutz
authors.affiliation: Department of Pharmacology and Program in Vascular Biology and Therapeutics, Yale School of Medicine, New Haven, Connecticut 06520.
authors.forename: Maggie
authors.initials: M
authors.lastname: Zhou
authors.affiliation: Department of Pharmacology, University of Illinois at Chicago, Chicago, Illinois 60612; Research Center for Advanced Science and Technology, University of Tokyo, Tokyo 153, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Kozasa
authors.affiliation: Department of Pharmacology and Program in Vascular Biology and Therapeutics, Yale School of Medicine, New Haven, Connecticut 06520. Electronic address: dan.wu@yale.edu.
authors.forename: Dianqing
authors.initials: D
authors.lastname: Wu
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D002465:Cell Movement / Q000502:physiology*; D004789:Enzyme Activation / Q000502:physiology; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology*; D051379:Mice; D020125:Mutation, Missense; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000096:biosynthesis* / Q000235:genetics; D048490:raf Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D051017:Apoptosis Regulatory Proteins; C489427:MYC protein, human; D016271:Proto-Oncogene Proteins c-myc; D004815:Epidermal Growth Factor; C502761:Rffl protein, mouse; D044767:Ubiquitin-Protein Ligases; D048490:raf Kinases; D011493:Protein Kinase C; D044365:GTP-Binding Protein alpha Subunits, G12-G13
keywords: Cell Migration; Cell Signaling; G Proteins; Raf; Signal Transduction; Signaling