title: Akt and Mammalian target of rapamycin regulate separate systems of proteolysis in renal tubular cells.
abstract: EGF suppresses proteolysis via class 1 phosphatidylinositol 3-kinase (PI3K) in renal tubular cells. EGF also increases the abundance of glycolytic enzymes (e.g., glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) and transcription factors (e.g., pax2) that are degraded by the lysosomal pathway of chaperone-mediated autophagy. To determine if EGF regulates chaperone-mediated autophagy through PI3K signaling, this study examined the effect of inhibiting PI3K and its downstream mediators Akt and the mammalian target of rapamycin (mTOR). Inhibition of PI3K with LY294002 prevented EGF-induced increases in GAPDH and pax2 abundance in NRK-52E renal tubular cells. Similar results were seen with an adenovirus encoding a dominant negative Akt (DN Akt). Expression of a constitutively active Akt increased GAPDH and pax2 abundance. An mTOR inhibitor, rapamycin, did not prevent EGF-induced increases in these proteins. Neither DN Akt nor rapamycin alone had an effect on total cell protein degradation, but both partially reversed EGF-induced suppression of proteolysis. DN Akt no longer affected proteolysis after treatment with a lysosomal inhibitor, methylamine. In contrast, methylamine or the inhibitor of macroautophagy, 3-methyladenine, did not prevent rapamycin from partially reversing the effect of EGF on proteolysis. Notably, rapamycin did not increase autophagasomes detected by monodansylcadaverine staining. Blocking the proteasomal pathway with either MG132 or lactacystin prevented rapamycin from partially reversing the effect of EGF on proteolysis. It is concluded that EGF regulates pax2 and GAPDH abundance and proteolysis through a PI3K/Akt-sensitive pathway that does not involve mTOR. Rapamycin has a novel effect of regulating proteasomal proteolysis in cells that are stimulated with EGF.
authors.affiliation: Address correspondence to: Dr. Harold A. Franch, Renal Division, Emory University School of Medicine, W.M.B., Room #338, 1639 Pierce Drive, N.E., Atlanta, GA 30322, USA.
authors.forename: Wen
authors.initials: W
authors.lastname: Shen
authors.forename: Nikia S
authors.initials: NS
authors.lastname: Brown
authors.forename: Patrick F
authors.initials: PF
authors.lastname: Finn
authors.forename: J Fred
authors.initials: JF
authors.lastname: Dice
authors.forename: Harold A
authors.initials: HA
authors.lastname: Franch
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001343:Autophagy / Q000187:drug effects; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000502:physiology; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D007684:Kidney Tubules / Q000166:cytology / Q000502:physiology*; D007976:Leupeptins / Q000494:pharmacology; D008247:Lysosomes / Q000187:drug effects; D008744:Methylamines / Q000494:pharmacology; D009025:Morpholines / Q000494:pharmacology; D051762:PAX2 Transcription Factor / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects / Q000378:metabolism; D011494:Protein Kinases / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt / Q000502:physiology*; D051381:Rats; D015398:Signal Transduction; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases
chemical_list: D000111:Acetylcysteine; D000225:Adenine; D002867:Chromones; D004815:Epidermal Growth Factor; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D007976:Leupeptins; D008744:Methylamines; D009025:Morpholines; D051762:PAX2 Transcription Factor; D010447:Peptide Hydrolases; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D046988:Proteasome Endopeptidase Complex; D011494:Protein Kinases; D051057:Proto-Oncogene Proteins c-akt; D020123:Sirolimus; D058570:TOR Serine-Threonine Kinases; C067713:lactacystin; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C025946:3-methyladenine; C027451:methylamine; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde