Reversible phosphorylation--dephosphorylation determines the localization of rab4 during the cell cycle.

Abstract

The ras‐like GTP binding protein rab4 is the only known rab protein on endosomes that is phosphorylated during mitosis. Since a large fraction of rab4 accumulates in the cytosol in mitotic cells, we investigated the molecular mechanism controlling membrane association of rab4. We first show that human rab4 is phosphorylated by recombinant mammalian p34cdc2 kinase in vitro. Next, the actual site of phosphorylation and its functional significance were determined using stably transfected CHO cell lines producing high levels of wild type rab4 or rab4 mutants bearing alterations at Ser196, which occurs within a consensus site for p34cdc2 kinase phosphorylation (S196PRR). Mutation of Ser196 to glutamine or aspartic acid completely prevented rab4 phosphorylation in mitotic cells and also blocked its appearance in the cytosol. Neither C‐terminal isoprenylation nor carboxymethylation of rab4 was affected by the mutations or by phosphorylation. Finally, dephosphorylation and reassociation of soluble rab4 with membranes occurred upon exit of cells from mitosis. Thus, phosphorylation of Ser196 is directly responsible for the reversible translocation of rab4 into the cytosol of mitotic cells.

Posttranslational modifications of Rab proteins cause effective displacement of GDP dissociation inhibitor

“…Rab1a and Rab4 are modified during mitosis by Cdk1 (20). The cytoplasmic concentration of Rab4 is increased upon phosphorylation (20,21) whereas phosphorylation of Rab1 leads to a decrease in cytoplasmic protein (20). Phosphorylation of Rab6 by Protein Kinase C also decreases the cytoplasmic protein concentration (22).…”

Co‐operative regulation of endocytosis by three RAB5 isoforms

“…In the case of the Rab4 subgroup, Rab4a and Rab4b may have a similar role in endocytosis [17] (Bucci and Zerial, unpublished data). However, whereas Rab4a is phosphorylated in the C-terminal domain by the cdc2 kinase during mitosis and upon phosphorylation becomes cytosolic [42], Rab4b does not contain this phosphorylation site. Among the three Rab5 isoforms, only Rab5c contains a putative phosphorylation site for cdc2 kinase (S 196) in its C-terminus.…”

Posttranslational modifications of Rab GTPases help their insertion into membranes

“…Posttranslational modifications of Rab GTPases represent a tentative mechanism to explain, at least in part, how Rab GTPases can be targeted to membranes. Evidence exists that phosphorylation can affect the membrane/cytosol distribution of a few Rabs (18)(19)(20). However, it remains to be established whether posttranslational modifications can be considered as a general regulatory mechanism.…”