Proteomics Screen Identifies Class I Rab11 Family Interacting Proteins as Key Regulators of Cytokinesis

ABSTRACT

The 14-3-3 protein family orchestrates a complex network of molecular interactions that regulates various biological processes. Owing to their role in regulating the cell cycle and protein trafficking, 14-3-3 proteins are prevalent in human diseases such as cancer, diabetes, and neurodegeneration. 14-3-3 proteins are expressed in all eukaryotic cells, suggesting that they mediate their biological functions through evolutionarily conserved protein interactions. To identify these core 14-3-3 client proteins, we used an affinity-based proteomics approach to characterize and compare the human and Drosophila 14-3-3 interactomes. Using this approach, we identified a group of Rab11 effector proteins, termed class I Rab11 family interacting proteins (Rab11-FIPs), or Rip11 in Drosophila. We found that 14-3-3 binds to Rip11 in a phospho-dependent manner to ensure its proper subcellular distribution during cell division. Our results indicate that Rip11 plays an essential role in the regulation of cytokinesis and that this function requires its association with 14-3-3 but not with Rab11. Together, our results suggest an evolutionarily conserved role for 14-3-3 in controlling Rip11-dependent protein transport during cytokinesis.

KEYWORDS:

• 14-3-3
• cell division
• cytokinesis
• proteomics
• vesicular trafficking

Supplemental material for this article may be found at https://doi.org/10.1128/MCB.00278-16.

ACKNOWLEDGMENTS

J.A.G. developed, performed, and analyzed the proteomics experiments related to the fly and human 14-3-3 interactomes, as well as all 14-3-3 pulldown assays and the far-Western analysis. C.L. performed the molecular biology assays and Drosophila S2 cell culture, as well as the immunoprecipitation assays and Rab11 pulldown assays, together with C.Z. C.L. performed live-cell microscopy of Rip11 vesicles. K.B.E.K. performed live-cell imaging of Ani/Sqh cells, quantified multinucleated cells, and characterized the localization of the Rip11 mutants in rescue experiments. A.M. performed the FIP5 coimmunoprecipitations with HA-tagged 14-3-3. C.L., J.A.G., S.C., P.P.R., and G.E. wrote the manuscript.

We thank Jordi Casanova for the Rip11 antibody. We thank Gilles Hickson for helpful discussion and for reagents. We thank Vincent Archambault for Gateway plasmid reagents. We thank James Goldenring and Mary McCaffrey for providing mammalian FIP cDNAs.

This work was supported by Canadian Institutes of Health Research (CIHR) grants MOP133683 (to S.C.), MOP123408 (to P.P.R.), and MOP114899 (to G.E.), a grant from the Human Frontier Science Program (to P.P.R.), a Cancer Research Society grant (to G.E.), and an NSERC grant (to G.E.). S.C. holds a New Investigator Award from the CIHR. P.P.R. holds a Canada Research Chair in Cell Signaling and Proteomics and a Chercheur-Boursier award from the Fonds de la Recherche du Québec en Santé (FRQS). G.E. holds a Canada Research Chair in Vesicular Trafficking and Cell Signaling. C.L., K.B.E.K., and C.Z. held Ph.D. training awards from the FRQS. K.B.E.K. also held a Ph.D. fellowship from the Fondation Desjardins and a Ph.D. fellowship from the Fondation du Grand Défi Pierre Lavoie. J.A.G. held postdoctoral fellowships from the CIHR and FRQS.