MARK2/EMK1/Par-1Balpha phosphorylation of Rab11-family interacting protein 2 is necessary for the timely establishment of polarity in Madin-Darby canine kidney cells.
Rab11a, myosin Vb, and the Rab11-family interacting protein 2 (FIP2) regulate plasma membrane recycling in epithelial cells. This study sought to characterize more fully Rab11-FIP2 function by identifying kinase activities modifying Rab11-FIP2. We have found that gastric microsomal membrane extracts phosphorylate Rab11-FIP2 on serine 227. We identified the kinase that phosphorylated Rab11-FIP2 as MARK2/EMK1/Par-1Balpha (MARK2), and recombinant MARK2 phosphorylated Rab11-FIP2 only on serine 227. We created stable Madin-Darby canine kidney (MDCK) cell lines expressing enhanced green fluorescent protein-Rab11-FIP2 wild type or a nonphosphorylatable mutant [Rab11-FIP2(S227A)]. Analysis of these cell lines demonstrates a new role for Rab11-FIP2 in addition to that in the plasma membrane recycling system. In calcium switch assays, cells expressing Rab11-FIP2(S227A) showed a defect in the timely reestablishment of p120-containing junctional complexes. However, Rab11-FIP2(S227A) did not affect localization with recycling system components or the normal function of apical recycling and transcytosis pathways. These results indicate that phosphorylation of Rab11-FIP2 on serine 227 by MARK2 regulates an alternative pathway modulating the establishment of epithelial polarity.
Ducharme
Nicole A
NA
Department of Surgery and Cell and Developmental Biology, Vanderbilt University School of Medicine, Vanderbilt-Ingram Cancer Center, and the Nashville VA Medical Center, Nashville, TN 37232, USA.
Hales
Chadwick M
CM
Lapierre
Lynne A
LA
Ham
Amy-Joan L
AJ
Oztan
Asli
A
Apodaca
Gerard
G
Goldenring
James R
JR
D022005:Adherens Junctions / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D020013:Calcium Signaling; D016764:Cell Polarity; D002478:Cells, Cultured; D004285:Dogs; D004847:Epithelial Cells / Q000166:cytology*; D015870:Gene Expression; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D024701:Myosin Type V / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D021381:Protein Transport; D011817:Rabbits; D044463:Receptor, PAR-1 / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000378:metabolism*; D000099193:rab11 GTP-Binding Proteins
D024701:Myosin Type V; D017346:Protein Serine-Threonine Kinases; D044463:Receptor, PAR-1; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C489440:EMK1 protein, Canis familiaris