Dominant negative forms of Akt (protein kinase B) and atypical protein kinase Cλ do not prevent insulin inhibition of phosphoenolpyruvate carboxykinase gene transcription

Ko Kotani, Wataru Ogawa, Yasuhisa Hino, Tadahiro Kitamura, Hikaru Ueno, Wataru Sano, Calum Sutherland, Daryl K. Granner, Masato Kasuga

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    Abstract

    Transcriptional regulation of phosphoenolpyruvate carboxykinase (PEPCK), the rate limiting enzyme in hepatic gluconeogenesis, by insulin was investigated with the use of adenovirus vectors encoding various mutant signaling proteins. Insulin inhibited transcription induced by dexamethasone and cAMP of a chloramphenicol acetyltransferase (CAT) reporter gene fused with the PEPCK promoter sequence in HL1C cells stably transfected with this construct. A dominant negative mutant of phosphoinositide (PI) 3-kinase blocked insulin inhibition of transcription of the PEPCK-CAT fusion gene, whereas a constitutively active mutant of PI 3-kinase mimicked the effect of insulin. Although a constitutively active mutant of Akt (protein kinase B) inhibited PEPCK-CAT gene transcription induced by dexamethasone and cAMP, a mutant Akt (Akt-AA) in which the phosphorylation sites targeted by insulin are replaced by alanine did not affect the ability of insulin to inhibit transcription of the fusion gene. Akt-AA almost completely inhibited insulin-induced activation of both endogenous and recombinant Abt in HL1C cells. Furthermore, neither a kinase-defective mutant protein kinase C lambda (PKC lambda), which blocked insulin-induced activation of endogenous PKC lambda, nor a dominant negative mutant of the small GTPase Pac prevented inhibition of PEPCK-CAT gene transcription by insulin. These data suggest that phosphoinositide 3-kinase is important for insulin-induced inhibition of PEPCK gene transcription and that a downstream effector of phosphoinositide 3-kinase distinct from Akt, PKC lambda, and Rac may exist for mediating the effect of insulin.

    Original languageEnglish
    Pages (from-to)21305-21312
    Number of pages8
    JournalJournal of Biological Chemistry
    Volume274
    Issue number30
    DOIs
    Publication statusPublished - 1999

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