Insulin activates a novel adipocyte mitogen-activated protein kinase kinase kinase that shows rapid phasic kinetics and is distinct from c-Raf.
Treatment of adipocytes with insulin or phorbol 12-myristate 13-acetate (PMA) results in transient activation of mitogen-activated protein kinase kinase (MEK) (Tmax = 90 s) and mitogen-activated protein kinase (MAPK) (Tmax = 300 s). We have identified a novel insulin-stimulated MEK kinase (I-MEKK) in the 100,000 x g infranatant that shows rapid phasic kinetics that temporally precede that of MEK. Maximal activation of I-MEKK occurs within 20 +/- 5 s (S.D., n = 3) followed by complete inactivation by 30 +/- 10 s (S.D., n = 3). I-MEKK was characterized by anion-exchange and gel filtration chromatography and separated into two distinct activities of approximately 56 kDa that phosphorylated and activated MEK. I-MEKKs did not co-elute on anion exchange with c-Raf or 73-kDa MEK kinase (MEKK), suggesting they are distinct enzymes. Protein phosphatase 2A inactivated both I-MEKKs in vitro and in the intact cell okadaic acid blocked inactivation in the presence of insulin. These results suggest activation of I-MEKK involves phosphorylation on serine or threonine residues. I-MEKK was not activated by PMA, suggesting that in adipocytes the enzyme represents a divergence point between signal transduction pathways mediated by insulin and those activating protein kinase C.
Haystead
C M
CM
Department of Pharmacology, University of Virginia, Charlottesville 22908.
Gregory
P
P
Shirazi
A
A
Fadden
P
P
Mosse
C
C
Dent
P
P
Haystead
T A
TA
D017667:Adipocytes / Q000201:enzymology*; D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004789:Enzyme Activation; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D020930:MAP Kinase Kinase Kinases; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D054648:Protein Phosphatase 2; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D051381:Rats; D017208:Rats, Wistar; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D007328:Insulin; D011518:Proto-Oncogene Proteins; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D019950:Mitogen-Activated Protein Kinase 1; D020930:MAP Kinase Kinase Kinases; C087095:insulin-stimulated MEK kinase; D020929:Mitogen-Activated Protein Kinase Kinases; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D013755:Tetradecanoylphorbol Acetate