A method to identify serine kinase substrates. Akt phosphorylates a novel adipocyte protein with a Rab GTPase-activating protein (GAP) domain.
This study describes a method for the identification of the substrates of specific serine kinases. An antibody specific for the phosphomotif generated by the kinase is used to isolate phosphorylated substrates by immunoprecipitation, and the isolated proteins are identified by tandem mass spectrometry of peptides. This method was applied to the identification of substrates for the protein kinase Akt, which specifically phosphorylates the RXRXXS/T motif. 3T3-L1 adipocytes were treated with insulin to activate Akt, and the putative Akt substrate proteins were isolated by immunoprecipitation with an antibody against the phospho form of this motif. This led to the identification of a novel 160-kDa substrate for Akt. The 160-kDa substrate for Akt, which was designated AS160, has a Rab GAP domain. Recombinant AS160 was shown to be a substrate for Akt, and two sites of phosphorylation, both in RXRXXS/T motifs, were identified by mass spectrometry and mutation. Insulin treatment of adipocytes caused AS160 to redistribute from the low density microsomes to the cytosol.
Kane
Susan
S
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Sano
Hiroyuki
H
Liu
Simon C H
SC
Asara
John M
JM
Lane
William S
WS
Garner
Charles C
CC
Lienhard
Gustav E
GE
D016475:3T3 Cells; D017667:Adipocytes / Q000378:metabolism*; D020816:Amino Acid Motifs; D000818:Animals; D001671:Biochemistry / Q000379:methods*; D019556:COS Cells; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007328:Insulin / Q000494:pharmacology; D013058:Mass Spectrometry; D051379:Mice; D008861:Microsomes / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D012694:Serine / Q000737:chemistry; D013912:Threonine / Q000737:chemistry; D020691:rab GTP-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*
D007328:Insulin; D011518:Proto-Oncogene Proteins; D013912:Threonine; D012694:Serine; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D020691:rab GTP-Binding Proteins