Protein kinase A modulates transforming growth factor-β signaling through a direct interaction with Smad4 protein.
Transforming growth factor β (TGFβ) signaling normally functions to regulate embryonic development and cellular homeostasis. It is increasingly recognized that TGFβ signaling is regulated by cross-talk with other signaling pathways. We previously reported that TGFβ activates protein kinase A (PKA) independent of cAMP through an interaction of an activated Smad3-Smad4 complex and the regulatory subunit of the PKA holoenzyme (PKA-R). Here we define the interaction domains of Smad4 and PKA-R and the functional consequences of this interaction. Using a series of Smad4 and PKA-R truncation mutants, we identified amino acids 290-300 of the Smad4 linker region as critical for the specific interaction of Smad4 and PKA-R. Co-immunoprecipitation assays showed that the B cAMP binding domain of PKA-R was sufficient for interaction with Smad4. Targeting of B domain regions conserved among all PKA-R isoforms and exposed on the molecular surface demonstrated that amino acids 281-285 and 320-329 were required for complex formation with Smad4. Interactions of these specific regions of Smad4 and PKA-R were necessary for TGFβ-mediated increases in PKA activity, CREB (cAMP-response element-binding protein) phosphorylation, induction of p21, and growth inhibition. Moreover, this Smad4-PKA interaction was required for TGFβ-induced epithelial mesenchymal transition, invasion of pancreatic tumor cells, and regulation of tumor growth in vivo.
Department of Surgery, University of Michigan, Ann Arbor, Michigan 48109.
Huibin
H
Yang
Department of Surgery, University of Michigan, Ann Arbor, Michigan 48109.
Gangyong
G
Li
Department of Surgery, University of Michigan, Ann Arbor, Michigan 48109.
Jing-Jiang
JJ
Wu
Department of Surgery, University of Michigan, Ann Arbor, Michigan 48109.
Lidong
L
Wang
Department of Biochemistry, University of Michigan, Ann Arbor, Michigan 48109.
Michael
M
Uhler
Department of Surgery, University of Michigan, Ann Arbor, Michigan 48109; Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan 48109; Translational Oncology Program, University of Michigan, Ann Arbor, Michigan 48109. Electronic address: simeone@umich.edu.
Diane M
DM
Simeone
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002465:Cell Movement; D000242:Cyclic AMP / Q000737:chemistry; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit / Q000378:metabolism* / Q000502:physiology; D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit / Q000737:chemistry; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism / Q000502:physiology; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D004789:Enzyme Activation; D058750:Epithelial-Mesenchymal Transition; D006801:Humans; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D008907:Mink; D009368:Neoplasm Transplantation; D010766:Phosphorylation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011499:Protein Processing, Post-Translational; D017384:Sequence Deletion; D015398:Signal Transduction; D051901:Smad4 Protein / Q000235:genetics / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000378:metabolism* / Q000502:physiology
C115163:CREB1 protein, human; D017362:Cyclic AMP Response Element-Binding Protein; D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit; D050759:Cyclin-Dependent Kinase Inhibitor p21; C517819:PRKAR2A protein, human; C497796:SMAD4 protein, human; D051901:Smad4 Protein; D016212:Transforming Growth Factor beta; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases