title: Phosphatidylinositol 3-kinase and Rab5 GTPase inversely regulate the Smad anchor for receptor activation (SARA) protein independently of transforming growth factor-β1.
abstract: SARA has been shown to be a regulator of epithelial cell phenotype, with reduced expression during TGF-β1-mediated epithelial-to-mesenchymal transition. Examination of the pathways that might play a role in regulating SARA expression identified phosphatidylinositol 3-kinase (PI3K) pathway inhibition as sufficient to reduce SARA expression. The mechanism of PI3K inhibition-mediated SARA down-regulation differs from that induced by TGF-β1 in that, unlike TGF-β1, PI3K-dependent depletion of SARA was apparent within 6 h and did not occur at the mRNA or promoter level but was blocked by inhibition of proteasome-mediated degradation. This effect was independent of Akt activity because neither reducing nor enhancing Akt activity modulated the expression of SARA. Therefore, this is likely a direct effect of p85α action, and co-immunoprecipitation of SARA and p85α confirmed that these proteins interact. Both SARA and PI3K have been shown to be associated with endosomes, and either LY294002 or p85α knockdown enlarged SARA-containing endocytic vesicles. Inhibition of clathrin-mediated endocytosis blocked SARA down-regulation, and a localization-deficient mutant SARA was protected against down-regulation. As inhibiting PI3K can activate the endosomal fusion-regulatory small GTPase Rab5, we expressed GTPase-deficient Rab5 and observed endosomal enlargement and reduced SARA protein expression, similar to that seen with PI3K inhibition. Importantly, either interference with PI3K via LY294002 or p85α knockdown, or constitutive activity of the Rab5 pathway, enhanced the expression of smooth muscle α-actin. Together, these data suggest that although TGF-β1 can induce epithelial-to-mesenchymal transition through reduction in SARA expression, SARA is also basally regulated by its interaction with PI3K.
authors.affiliation: Department of Pediatrics, Northwestern University, Chicago, Illinois 60611, USA. c-runyan@northwestern.edu
authors.forename: Constance E
authors.initials: CE
authors.lastname: Runyan
authors.forename: Zongyi
authors.initials: Z
authors.lastname: Liu
authors.forename: H William
authors.initials: HW
authors.lastname: Schnaper
mesh_terms: D000199:Actins / Q000096:biosynthesis / Q000235:genetics; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D058543:Class Ia Phosphatidylinositol 3-Kinase / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects / Q000502:physiology*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D055785:Gene Knockdown Techniques; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D009025:Morpholines / Q000494:pharmacology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism; D059748:Proteolysis / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism*; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000199:Actins; D002867:Chromones; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D009025:Morpholines; D000081082:Phosphoinositide-3 Kinase Inhibitors; D053773:Transforming Growth Factor beta1; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D058543:Class Ia Phosphatidylinositol 3-Kinase; D051057:Proto-Oncogene Proteins c-akt; C116420:ZFYVE16 protein, human; D012697:Serine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D020696:rab5 GTP-Binding Proteins