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-acetat e (PMA) results in transient activation of mitogen-activated protein k inase kinase (MEK) (T-max = 90 s) and mitogen-activated protein kinase (MAPK) (T-max = 300 s). We have identified a novel insulin-stimulated MEK kinase (I-MEKK) in the 100,000 x g infranatant that shows rapid p hasic 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 b y anion-exchange and gel filtration chromatography and separated into two distinct activities of similar to 56 kDa that phosphorylated and a ctivated MEK. I-MEKKs did not co-elute on anion exchange with c-Raf or 73-kDa MEK kinase (MEKK), suggesting they are distinct enzymes. Prote in phosphatase 2A inactivated both I-MEKKs in vitro and in the intact cell okadaic acid blocked inactivation in the presence of insulin. The se results suggest activation of I-MEKK involves phosphorylation on se rine or threonine residues. I-MEKK was not activated by PMA, suggestin g that in adipocytes the enzyme represents a divergence point between signal transduction pathways mediated by insulin and those activating protein kinase C.