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

Transcriptional regulation of phosphoenolpyruvate carboxykinase (PEPCK), th e rate limiting enzyme in hepatic gluconeogenesis, by insulin was investiga ted with the use of adenovirus vectors encoding various mutant signaling pr oteins. Insulin inhibited transcription induced by dexamethasone and cAMP o f a chloramphenicol acetyltransferase (CAT) reporter gene fused with the PE PCK promoter sequence in HL1C cells stably transfected with this construct. A dominant negative mutant of phosphoinositide (PI) 3-kinase blocked insul in inhibition of transcription of the PEPCK-CAT fusion gene, whereas a cons titutively active mutant of PI 3-kinase mimicked the effect of insulin. Alt hough a constitutively active mutant of Akt (protein kinase B) inhibited PE PCK-CAT gene transcription induced by dexamethasone and cAMP, a mutant Akt (Akt-AA) in which the phosphorylation sites targeted by insulin are replace d by alanine did not affect the ability of insulin to inhibit transcription of the fusion gene. Akt-AA almost completely inhibited insulin-induced act ivation of both endogenous and recombinant Abt in HL1C cells. Furthermore, neither a kinase-defective mutant protein kinase C lambda (PKC lambda), whi ch blocked insulin-induced activation of endogenous PKC lambda, nor a domin ant negative mutant of the small GTPase Pac prevented inhibition of PEPCK-C AT gene transcription by insulin. These data suggest that phosphoinositide 3-kinase is important for insulin-induced inhibition of PEPCK gene transcri ption and that a downstream effector of phosphoinositide 3-kinase distinct from Akt, PKC lambda, and Rac may exist for mediating the effect of insulin .