title: Requirement for protein kinase A in the phosphorylation of the TGFβ receptor-interacting protein km23-1 as a component of TGFβ downstream effects.
abstract: km23-1 was previously identified as a TGFβ-receptor interacting protein that was phosphorylated on serines after TGFβ stimulation. In the current report, we examined the role of km23-1 phosphorylation in the downstream effects of TGFβ/protein kinase A (PKA) signaling. Using phosphorylation site prediction software, we found that km23-1 has two potential PKA consensus phosphorylation sites. In vitro kinase assays further demonstrated that PKA directly phosphorylates km23-1 on serine 73 (S73). Moreover, our results show that the PKA-specific inhibitor H89 diminishes phosphorylation of km23-1 on S73 after TGFβ stimulation. Taken together, our results demonstrate that TGFβ induction of PKA activity results in phosphorylation of km23-1 on S73. In order to assess the mechanisms underlying PKA phosphorylation of km23-1 on S73 (S73-km23-1) after TGFβ stimulation, immunoprecipitation (IP)/blot analyses were performed, which demonstrate that TGFβ regulates complex formation between the PKA regulatory subunit RIβ and km23-1 in vivo. In addition, an S73A mutant of km23-1 (S73A-km23-1), which could not be phosphorylated by PKA, inhibited TGFβ induction of the km23-1-dynein complex and transcriptional activation of the activin-responsive element (ARE). Furthermore, our results show that km23-1 is required for cAMP-responsive element (CRE) transcriptional activation by TGFβ, with S73-km23-1 being required for the CRE-dependent TGFβ stimulation of fibronectin (FN) transcription. Collectively, our results demonstrate for the first time that TGFβ/PKA phosphorylation of km23-1 on S73 is required for ARE- and CRE-mediated downstream events that include FN induction.
authors: Department of Biochemistry and Molecular Biology, Penn State Hershey, College of Medicine, PA 17033, USA.
authors: Qunyan
authors: Q
authors: Jin
authors: Yan
authors: Y
authors: Zhong
authors: Kathleen M
authors: KM
authors: Mulder
mesh_terms: D000242:Cyclic AMP / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D056445:Cytoplasmic Dyneins / Q000235:genetics / Q000378:metabolism*; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D010766:Phosphorylation; D025941:Protein Interaction Mapping; D020239:Receptor Cross-Talk; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D051899:Smad2 Protein / Q000235:genetics / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D015533:Transcriptional Activation; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism* / Q000494:pharmacology
chemical_list: C115163:CREB1 protein, human; D017362:Cyclic AMP Response Element-Binding Protein; C504797:DYNLRB1 protein, human; D005353:Fibronectins; D007546:Isoquinolines; D018125:Receptors, Transforming Growth Factor beta; C497782:SMAD2 protein, human; D051899:Smad2 Protein; D013449:Sulfonamides; D016212:Transforming Growth Factor beta; D012694:Serine; D000242:Cyclic AMP; D008156:Luciferases; D017868:Cyclic AMP-Dependent Protein Kinases; D056445:Cytoplasmic Dyneins; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide