title: Identification of a novel phosphorylation site on TBC1D4 regulated by AMP-activated protein kinase in skeletal muscle.
abstract: TBC1D4 (also known as AS160) regulates glucose transporter 4 (GLUT4) translocation and glucose uptake in adipocytes and skeletal muscle. Its mode of action involves phosphorylation of serine (S)/threonine (T) residues by upstream kinases resulting in inactivation of Rab-GTPase-activating protein (Rab-GAP) activity leading to GLUT4 mobilization. The majority of known phosphorylation sites on TBC1D4 lie within the Akt consensus motif and are phosphorylated by insulin stimulation. However, the 5'-AMP-activated protein kinase (AMPK) and other kinases may also phosphorylate TBC1D4, and therefore we hypothesized the presence of additional phosphorylation sites. Mouse skeletal muscles were contracted or stimulated with 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), and muscle lysates were subjected to mass spectrometry analyses resulting in identification of novel putative phosphorylation sites on TBC1D4. The surrounding amino acid sequence predicted that S711 would be recognized by AMPK. Using a phosphospecific antibody against S711, we found that AICAR and contraction increased S711 phosphorylation in mouse skeletal muscle, and this increase was abolished in muscle-specific AMPKalpha2 kinase-dead transgenic mice. Exercise in human vastus lateralis muscle also increased TBC1D4 S711 phosphorylation. Recombinant AMPK, but not Akt1, Akt2, or PKCzeta, phosphorylated purified muscle TBC1D4 on S711 in vitro. Interestingly, S711 was also phosphorylated in response to insulin in an Akt2- and rapamycin-independent, but a wortmannin-sensitive, manner, suggesting this site is regulated by one or more additional upstream kinases. Despite increased S711 phosphorylation with AICAR, contraction, and insulin, mutation of S711 to alanine did not alter glucose uptake in response to these stimuli. S711 is a novel TBC1D4 phosphorylation site regulated by AMPK in skeletal muscle.
authors.affiliation: Joslin Diabetes Center, Section on Metabolism, Harvard Medical School, Boston, MA 02215, USA.
authors.forename: Jonas T
authors.initials: JT
authors.lastname: Treebak
authors.forename: Eric B
authors.initials: EB
authors.lastname: Taylor
authors.forename: Carol A
authors.initials: CA
authors.lastname: Witczak
authors.forename: Ding
authors.initials: D
authors.lastname: An
authors.forename: Taro
authors.initials: T
authors.lastname: Toyoda
authors.forename: Ho-Jin
authors.initials: HJ
authors.lastname: Koh
authors.forename: Jianxin
authors.initials: J
authors.lastname: Xie
authors.forename: Edward P
authors.initials: EP
authors.lastname: Feener
authors.forename: Jørgen F P
authors.initials: JF
authors.lastname: Wojtaszewski
authors.forename: Michael F
authors.initials: MF
authors.lastname: Hirshman
authors.forename: Laurie J
authors.initials: LJ
authors.lastname: Goodyear
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D000328:Adult; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D004558:Electric Stimulation; D018274:Electroporation; D005260:Female; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D018014:Gene Transfer Techniques; D005947:Glucose / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D018345:Mice, Knockout; D009119:Muscle Contraction; D018482:Muscle, Skeletal / Q000187:drug effects / Q000201:enzymology*; D009154:Mutation; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D052097:Quadriceps Muscle / Q000201:enzymology; D011994:Recombinant Proteins / Q000378:metabolism; D012265:Ribonucleotides / Q000494:pharmacology; D012694:Serine; D020123:Sirolimus / Q000494:pharmacology; D053719:Tandem Mass Spectrometry; D013997:Time Factors; D000077191:Wortmannin; D055815:Young Adult; D000099221:Protein Kinase C zeta
chemical_list: D055372:AMP-Activated Protein Kinases; D000620:Aminoimidazole Carboxamide; D000730:Androstadienes; D020690:GTPase-Activating Proteins; D005947:Glucose; D007328:Insulin; D011493:Protein Kinase C; D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins; D012265:Ribonucleotides; D012694:Serine; D020123:Sirolimus; D000077191:Wortmannin; D000099221:Protein Kinase C zeta; C518767:TBC1D4 protein, human; C505647:Tbc1d4 protein, mouse; C534978:Prkaa2 protein, mouse; C494922:Akt2 protein, mouse; C031143:AICA ribonucleotide