The palmitoylation state of PMP22 modulates epithelial cell morphology and migration
Susie J. Zoltewicz*, Sooyeon Lee*, Vinita G. Chittoor*, Steven M. Freeland*, Sunitha Rangaraju*, David A. Zacharias*,{ and Lucia Notterpek*1
*Department of Neuroscience, College of Medicine, McKnight Brain Institute, University of Florida, Gainesville, FL 32610, U.S.A.
{The Marine Whitney Laboratories, University of Florida, St. Augustine, FL 32080, U.S.A.
Abstract
Peripheral myelin protein 22 (PMP22), also known as growth arrest specific 3 (gas 3), is a disease-linked tetraspan glycoprotein of peripheral nerve myelin and constituent of intercellular junctions in epithelia. To date, our knowledge on the posttranslational modification of PMP22 is limited. Using the CSS-Palm 2.0 software we predicted that cysteine 85 (C85), a highly-conserved amino acid located between the second and third transmembrane domains, is a potential site for palmitoylation. To test this, we mutated C85 to serine (C85S) and established stable cells lines expressing the wild type (Wt) or the C85S-PMP22. In Schwann and Madin-Darby Canine Kidney (MDCK) cells mutating C85 blocked the palmitoylation of PMP22, which we monitored using 17-Octadecynoic acid (17-ODYA). While palmitoylation was not necessary for processing the newly-synthesized PMP22 through the secretory pathway, overexpression of C85S-PMP22 led to pronounced cell spreading and uneven monolayer thinning. To further investigate the functional significance of palmitoylated PMP22, we evaluated MDCK cell migration in a wound-healing assay. While Wt-PMP22 expressing cells were resistant to migration, C85S cells displayed lamellipodial protrusions and migrated at similar rate to vector control. These findings indicate that palmitoylation of PMP22 at C85 is critical for the role of the protein in modulating epithelial cell shape and motility.