GARNL1, a major RalGAP α subunit in skeletal muscle, regulates insulin-stimulated RalA activation and GLUT4 trafficking via interaction with 14-3-3 proteins.
Insulin and muscle contraction each stimulate translocation of the glucose transporter GLUT4 to the plasma membrane in skeletal muscle, an important process regulating whole-body glucose homeostasis. RalA mediates insulin-stimulated GLUT4 translocation; however, it is unclear how this small GTPase is regulated in skeletal muscle in response to insulin. Here, we identified GARNL1/RalGAPα1, a major α subunit of the Ral-GTPase activating protein in skeletal muscle, as a protein whose phosphorylation and binding to the regulatory 14-3-3 proteins is stimulated by insulin and also by muscle contraction. The insulin-stimulated interaction with 14-3-3 involved PKB/Akt-mediated phosphorylation of Thr(735) on GARNL1/RalGAPα1. Knockdown of GARNL1/RalGAPα1 increased, while overexpression of GARNL1/RalGAPα1(Thr735Ala) mutant protein decreased, the RalA activation and the RalA-dependent GLUT4 translocation in response to insulin in muscle cells. These findings show that GARNL1/RalGAPα1 is the missing link that connects the insulin-PKB/Akt signaling pathway with the activation of the RalA small GTPase in muscle cells. GARNL1/RalGAPα1 and its phosphorylation and/or binding to 14-3-3s are critical for GLUT4 trafficking through RalA in muscle cells.
MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Nanjing Biomedical Research Institute, Nanjing University, Nanjing, 210061, China.
Qiaoli
Q
Chen
MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Nanjing Biomedical Research Institute, Nanjing University, Nanjing, 210061, China.
Chao
C
Quan
MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Nanjing Biomedical Research Institute, Nanjing University, Nanjing, 210061, China.
Bingxian
B
Xie
MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Nanjing Biomedical Research Institute, Nanjing University, Nanjing, 210061, China.
Liang
L
Chen
MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Nanjing Biomedical Research Institute, Nanjing University, Nanjing, 210061, China.
Shuilian
S
Zhou
MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.
Rachel
R
Toth
MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.
David G
DG
Campbell
MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Nanjing Biomedical Research Institute, Nanjing University, Nanjing, 210061, China.
Shuangshuang
S
Lu
Department of Molecular and Cellular Biology, Institute of Development, Aging and Cancer, Tohoku University, Sendai, 980-8575, Japan.
Ryutaro
R
Shirakawa
Department of Molecular and Cellular Biology, Institute of Development, Aging and Cancer, Tohoku University, Sendai, 980-8575, Japan.
Hisanori
H
Horiuchi
MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Nanjing Biomedical Research Institute, Nanjing University, Nanjing, 210061, China.
Chaojun
C
Li
MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Nanjing Biomedical Research Institute, Nanjing University, Nanjing, 210061, China.
Zhongzhou
Z
Yang
Division of Cell and Developmental Biology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.
Carol
C
MacKintosh
MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Nanjing Biomedical Research Institute, Nanjing University, Nanjing, 210061, China. Electronic address: wanghy@nicemice.cn.
Hong Yu
HY
Wang
MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Nanjing Biomedical Research Institute, Nanjing University, Nanjing, 210061, China. Electronic address: schen6@163.com.
Shuai
S
Chen
D048948:14-3-3 Proteins / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D020690:GTPase-Activating Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051275:Glucose Transporter Type 4 / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007328:Insulin / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D051379:Mice; D009119:Muscle Contraction; D018482:Muscle, Skeletal / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D020725:ral GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D048948:14-3-3 Proteins; D020690:GTPase-Activating Proteins; D051275:Glucose Transporter Type 4; D007328:Insulin; D009419:Nerve Tissue Proteins; C495326:RALGAPA1 protein, human; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D051057:Proto-Oncogene Proteins c-akt; C492198:RALA protein, human; D020725:ral GTP-Binding Proteins
14-3-3 interaction; GARNL1/RalGAPα1; GLUT4 trafficking; Insulin signaling; Protein phosphorylation