SUMOylation regulates the intracellular fate of ZO-2.
The zonula occludens (ZO)-2 protein links tight junctional transmembrane proteins to the actin cytoskeleton and associates with splicing and transcription factors in the nucleus. Multiple posttranslational modifications control the intracellular distribution of ZO-2. Here, we report that ZO-2 is a target of the SUMOylation machinery and provide evidence on how this modification may affect its cellular distribution and function. We show that ZO-2 associates with the E2 SUMO-conjugating enzyme Ubc9 and with SUMO-deconjugating proteases SENP1 and SENP3. In line with this, modification of ZO-2 by endogenous SUMO1 was detectable. Ubc9 fusion-directed SUMOylation confirmed SUMOylation of ZO-2 and was inhibited in the presence of SENP1 but not by an enzymatic-dead SENP1 protein. Moreover, lysine 730 in human ZO-2 was identified as a potential modification site. Mutation of this site to arginine resulted in prolonged nuclear localization of ZO-2 in nuclear recruitment assays. In contrast, a construct mimicking constitutive SUMOylation of ZO-2 (SUMO1ΔGG-ZO-2) was preferentially localized in the cytoplasm. Based on previous findings the differential localization of these ZO-2 constructs may affect glycogen-synthase-kinase-3β (GSK3β) activity and β-catenin/TCF-4-mediated transcription. In this context we observed that ZO-2 directly binds to GSK3β and SUMO1ΔGG-ZO-2 modulates its kinase activity. Moreover, we show that ZO-2 forms a complex with β-catenin. Wild-type ZO-2 and ZO-2-K730R inhibited transcriptional activity in reporter gene assays, whereas the cytosolic SUMO1ΔGG-ZO-2 did not. From these data we conclude that SUMOylation affects the intracellular localization of ZO-2 and its regulatory role on GSK3β and β-catenin signaling activity.
Institute of Biochemistry II, Jena University Hospital, Friedrich-Schiller-University Jena, Nonnenplan 2-4, 07743, Jena, Germany.|Institut für Ernährungswissenschaften, Abt. Humanernährung, Dornburger Str. 29, 07743, Jena, Germany.
Franziska
F
Wetzel
Institute of Biochemistry II, Jena University Hospital, Friedrich-Schiller-University Jena, Nonnenplan 2-4, 07743, Jena, Germany.
Sonnhild
S
Mittag
Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studies (Cinvestav), Mexico City, 07360, Mexico.
Misael
M
Cano-Cortina
Institute of Biochemistry and Biophysics, Friedrich-Schiller-University Jena, CMB Center for Molecular Biomedicine, Hans-Knöll-Str. 2, 07745, Jena, Germany.
Tobias
T
Wagner
Department of Toxicology, University Medical Center Mainz, 55131, Mainz, Germany.
Oliver H
OH
Krämer
Institute of Physiological Chemistry/Biochemistry, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany.
Rainer
R
Niedenthal
Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studies (Cinvestav), Mexico City, 07360, Mexico.
Lorenza
L
Gonzalez-Mariscal
Institute of Biochemistry II, Jena University Hospital, Friedrich-Schiller-University Jena, Nonnenplan 2-4, 07743, Jena, Germany. otmar.huber@med.uni-jena.de.
Otmar
0000-0003-4359-1747
O
Huber
D000595:Amino Acid Sequence; D000818:Animals; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004285:Dogs; D000071679:Glycogen Synthase Kinase 3 beta / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism; D061985:Madin Darby Canine Kidney Cells; D051379:Mice; D010766:Phosphorylation; D011485:Protein Binding; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D058207:Sumoylation; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D062827:Zonula Occludens-2 Protein / Q000737:chemistry / Q000378:metabolism*; D051176:beta Catenin / Q000378:metabolism; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D062827:Zonula Occludens-2 Protein; D051176:beta Catenin; D044763:Ubiquitin-Conjugating Enzymes; D000071679:Glycogen Synthase Kinase 3 beta; D003546:Cysteine Endopeptidases; D000098843:Ubiquitin-Conjugating Enzyme UBC9; D008239:Lysine
Glycogen-synthase-kinase-3β; Occludin; Tight junction; Zonula occludens-2; β-Catenin