title: Interaction of phosphorylated Rab11-FIP2 with Eps15 regulates apical junction composition.
abstract: MARK2 regulates the establishment of polarity in Madin-Darby canine kidney (MDCK) cells in part through phosphorylation of serine 227 of Rab11-FIP2. We identified Eps15 as an interacting partner of phospho-S227-Rab11-FIP2 (pS227-FIP2). During recovery from low calcium, Eps15 localized to the lateral membrane before pS227-FIP2 arrival. Later in recovery, Eps15 and pS227-FIP2 colocalized at the lateral membrane. In MDCK cells expressing the pseudophosphorylated FIP2 mutant FIP2(S227E), during recovery from low calcium, Eps15 was trapped and never localized to the lateral membrane. Mutation of any of the three NPF domains within GFP-FIP2(S227E) rescued Eps15 localization at the lateral membrane and reestablished single-lumen cyst formation in GFP-FIP2(S227E)-expressing cells in three-dimensional (3D) culture. Whereas expression of GFP-FIP2(S227E) induced the loss of E-cadherin and occludin, mutation of any of the NPF domains of GFP-FIP2(S227E) reestablished both proteins at the apical junctions. Knockdown of Eps15 altered the spatial and temporal localization of pS227-FIP2 and also elicited formation of multiple lumens in MDCK 3D cysts. Thus an interaction of Eps15 and pS227-FIP2 at the appropriate time and location in polarizing cells is necessary for proper establishment of epithelial polarity.
authors.affiliation: Section of Surgical Sciences, Vanderbilt University School of Medicine, Nashville, TN 37232.|Epithelial Biology Center, Vanderbilt University School of Medicine, Nashville, TN 37232.|Nashville VA Medical Center, Nashville, TN 37212.
authors.forename: Lynne A
authors.initials: LA
authors.lastname: Lapierre
authors.affiliation: Section of Surgical Sciences, Vanderbilt University School of Medicine, Nashville, TN 37232.|Epithelial Biology Center, Vanderbilt University School of Medicine, Nashville, TN 37232.|Nashville VA Medical Center, Nashville, TN 37212.
authors.forename: Elizabeth H
authors.initials: EH
authors.lastname: Manning
authors.affiliation: Section of Surgical Sciences, Vanderbilt University School of Medicine, Nashville, TN 37232.|Epithelial Biology Center, Vanderbilt University School of Medicine, Nashville, TN 37232.|Nashville VA Medical Center, Nashville, TN 37212.
authors.forename: Kenya M
authors.initials: KM
authors.lastname: Mitchell
authors.affiliation: Section of Surgical Sciences, Vanderbilt University School of Medicine, Nashville, TN 37232.|Epithelial Biology Center, Vanderbilt University School of Medicine, Nashville, TN 37232.|Nashville VA Medical Center, Nashville, TN 37212.
authors.forename: Cathy M
authors.initials: CM
authors.lastname: Caldwell
authors.affiliation: Section of Surgical Sciences, Vanderbilt University School of Medicine, Nashville, TN 37232 jim.goldenring@vanderbilt.edu.|Epithelial Biology Center, Vanderbilt University School of Medicine, Nashville, TN 37232.|Nashville VA Medical Center, Nashville, TN 37212.|Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232.|Vanderbilt Ingram Cancer Center, Nashville, TN 37232.
authors.forename: James R
authors.initials: JR
authors.lastname: Goldenring
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D015820:Cadherins / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D016764:Cell Polarity / Q000502:physiology; D004285:Dogs; D011992:Endosomes / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D055786:Gene Knockout Techniques; D057809:HEK293 Cells; D006801:Humans; D007365:Intercellular Junctions / Q000378:metabolism*; D061985:Madin Darby Canine Kidney Cells; D008565:Membrane Proteins / Q000378:metabolism*; D062793:Occludin / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D021381:Protein Transport; D020691:rab GTP-Binding Proteins / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D015820:Cadherins; D002352:Carrier Proteins; C486206:EPS15 protein, human; D008565:Membrane Proteins; D062793:Occludin; C463963:RAB11FIP2 protein, human; D020691:rab GTP-Binding Proteins