Role of rab phosphorylation in cellular architecture changes during mitosis
Contact
Dr. Peter van der Sluijs, Departement of Cell Biology, UMC Utrecht, p.vandersluijs@umcutrecht.nl,
088-7557580
Background
The function of eukaryotic cells depends on the compartmentalization of biochemical reactions in distinct intracellular compartments that provide optimal environments for enzymes with often distinct pH, redox and substrate requirements. Critical to the generation and maintenance of these compartments is their ability to communicate with each other via vesicular transport carriers to exchange content such as substrates and co-factors. A key principle in this process is the GTPase switch of rab proteins that controls specificity in vesicular transport.
Many proteins controlling membrane traffic and organelle integrity are phosphorylated during mitosis and it is thought that this reversible modification triggers downregulation of transport and architectural reorganization of several intracellular compartments. The activity of the mitotic kinase cdk1 is required for the inhibition of intracellular transport pathways at the onset of mitosis in higher eukaryotes. Since rabs are critically involved in membrane transport, their activity might be controlled by mitotic phosphorylation, as we orginally showed for rab4a and others for rab1a. To begin to address this hypothesis, we undertook a bioinformatic screen for conserved S/T-P sites in rab proteins as these residues are invariably present in the consensus motif for substrates of proline-directed kinases.
Using in vitro- and in vivo-phosphorylation assays we identified rab1b, rab7b, rab8a and rab40 as novel targets for mitosis-specific phosphorylation by cdk1. We then selected rab8a to investigate the function of phosphorylation. Rab8 is required for membrane delivery from endosomes to the plasma membrane and it serves an important role in the biogenesis of cilia. We created GFP-rab8a and the GFP-tagged rab8aS181A mutant that can not be phosphorylated by cdk1. In preliminary live cell imaging experiments we discovered that expression of the mutant
(but not of GFP-rab8a) gave rise to multinucleated cells and abnormal cytokinesis.
Objective

The aim of this project which is to investigate the function of rab8a phosphorylation in dividing cells using live cell imaging.
Methods
- In silico sequence analysis to design expression constructs
- Recombinant DNA methods to prepare expression constructs
- Protein methods like SDS-PAGE, Western blot, immunoprecipitation
- Cell culture and transfections
- Knock down in tissue culture cells with RNAi
- Analysis of living cells with (dual label) fluorescence microscopy
- Fluorescence Activated Cell Sorting
Selected papers from the group
- van Vlijmen* T, Rojas* R, Mardones G, Mohammed S, Heck AJR, Raposo G, van der Sluijs P and
Bonifacino JS. 2008. Regulation of retromer recruitment to endosomes by sequential action of rab5 and rab7. (* denotes equal contributions). J. Cell Biol.183: 513-526.
- Fila C, Metz C, and van der Sluijs P. 2008. Juglone inactivates cysteine-rich proteins required for progression through mitosis. J. Biol. Chem. 283: 21714-21724.
- Kamsteeg IBM, Oorschot V, van der Sluijs P, Klumperman J, and Deen EJ, Hendriks G, Konings PMT. 2006.
Short-chain ubiquitination mediates the regulated endocytosis of the aquaporin-2 water channel. Proc. Natl. Acad.
Sci. USA. 103, 18344-18349.
- Linder MD, Uronen RL, Vuori MH, van der Sluijs P, Peränen J, and Ikonen E. 2006. Rab8-dependent recycling promotes endosomal cholesterol removal in normal and sphingolipidosis cells. Mol. Biol. Cell, 18, 47-56, 2007.
- Neeft M, Wieffer M, de Jong AS, Negroiu G, Metz C, van Loon A, Krijgsveld J, Griffith J, Wulffraat N, Koch H,
Heck A, Brose N, Kleijmeer M, and van der Sluijs P. 2004. Munc13-4 is an effector of rab27a and controls secretion of lysosomes in haematopoietic cells. Mol. Biol. Cell 15, 731-741.
- Roberts M, Woods AJ, van der Sluijs P, and Norman JC. 2004. PKB/Akt acts via GSK-3 to regulate recycling of  v  3 and  5  1 integrins. Mol. Cell Biol. 24, 1505-1515.
- Deneka M, Neeft M, Popa I, van Oort M, Sprong H, Oorschot V, Klumperman J, Schu P, and van der Sluijs P.
2003. rabaptin-5  /rabaptin-4 serves as a linker between rab4 and  1-adaptin in membrane recycling from endosomes. EMBO J. 22, 2645-2657.