Glycogen synthase kinase-3 is the predominant insulin-regulated eukaryotic initiation factor 2B kinase in skeletal muscle.
Eukaryotic initiation factor eIF2B is a guanine nucleotide exchange protein involved in regulation of translation initiation. Phosphorylation of the epsilon-subunit is thought to be important in insulin-mediated changes in eIF2B activity. However, elucidation of insulin's action has proven elusive, primarily because eIF2B epsilon is a substrate in vitro for at least three different protein kinases. In the present study, we observed changes in eIF2B epsilon kinase activity only in those muscles previously shown to exhibit alterations in protein synthesis in response to insulin. Specifically, eIF2B epsilon kinase activity was increased in psoas muscle from diabetic rats compared to controls. Treating diabetic rats with insulin rapidly reduced eIF2B epsilon kinase activity below control values. Changes were not observed in heart. To identify the kinase(s) in psoas responsible for phosphorylating eIF2B epsilon, the wildtype and two variant forms of the epsilon-subunit were expressed in and purified from Sf9 insect cells, and were used as substrates in protein kinase assays. The first variant contained a point mutation in the eIF2B epsilon cDNA that converted the glycogen synthase kinase-3 (GSK-3) phosphorylation site, Ser535, to a nonphosphorylatable Ala residue. In the second variant, the putative GSK-3 'priming' site, Ser539, was converted to Asp. Based on the pattern of phosphorylation of the wildtype and two variant forms of eIF2B epsilon using casein kinase (CK)-I, CK-II, or GSK-3 as well as that observed with skeletal muscle extracts, we conclude that the predominant eIF2B epsilon kinase in psoas muscle is GSK-3. Thus, insulin-mediated changes in eIF2B activity are likely to involve GSK-3.
Jefferson
L S
LS
Department of Cellular and Molecular Physiology, Pennsylvania State University, College of Medicine, Hershey 17033, USA. jjefferson@psu.edu
Fabian
J R
JR
Kimball
S R
SR
D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D003921:Diabetes Mellitus, Experimental / Q000188:drug therapy / Q000378:metabolism; D020717:Eukaryotic Initiation Factor-2B; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D020662:Guanine Nucleotide Exchange Factors; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D008297:Male; D008969:Molecular Sequence Data; D018656:Muscle Fibers, Fast-Twitch / Q000378:metabolism; D018482:Muscle, Skeletal / Q000201:enzymology*; D009154:Mutation; D009206:Myocardium / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D016658:Psoas Muscles / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D020717:Eukaryotic Initiation Factor-2B; D020662:Guanine Nucleotide Exchange Factors; D007328:Insulin; D010761:Phosphorus Radioisotopes; D011506:Proteins; D011994:Recombinant Proteins; D038341:Glycogen Synthase Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3