Regulation of the Rab5 GTPase-activating protein RN-tre by the dual specificity phosphatase Cdc14A in human cells.
The Cdc14 family of dual specificity phosphatases regulates key mitotic events in the eukaryotic cell cycle. Although extensively characterized in yeast, little is known about the function of mammalian Cdc14 family members. Here we report a genetic substrate-trapping system designed to identify substrates of the human Cdc14A (hCdc14A) phosphatase. Using this approach, we identify RN-tre, a GTPase-activating protein for the Rab5 GTPase, as a novel physiological target of hCdc14A. As a Rab5 GTPase-activating protein, RN-tre has previously been implicated in control of intracellular membrane trafficking. We find that RN-tre forms a stable complex with the catalytically inactive hCdc14A C278S mutant but not with the wild type protein in human cells, indicative of a substrate/enzyme interaction. In support, we show that RN-tre is regulated by cell cycle-dependent phosphorylation peaking at mitosis, which can be antagonized by hCdc14A activity in vitro as well as in vivo. Furthermore, we show that RN-tre phosphorylation is critical for efficient hCdc14A association and that RN-tre binding can be displaced by tungstate, a competitive inhibitor that binds to the active site of hCdc14A. Consistent with the preference of hCdc14A for phosphorylations mediated by proline-directed kinases, we find that RN-tre is a direct substrate of cyclin-dependent kinase. Finally, phosphorylation of RN-tre appears to finely modulate its catalytic activity. Our findings reveal a novel connection between the cell cycle machinery and the endocytic pathway.
Lanzetti
Letizia
L
Dipartimento di Scienze Oncologiche, Università degli Studi di Torino, Istituto per la Ricerca e la Cura del Cancro, Strada Provinciale 142, 10060 Candiolo, Torino, Italy.
Margaria
Valentina
V
Melander
Fredrik
F
Virgili
Laura
L
Lee
Myung-Hee
MH
Bartek
Jiri
J
Jensen
Sanne
S
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism*; D004705:Endocytosis; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000502:physiology*; D017027:Protein Tyrosine Phosphatases; D013379:Substrate Specificity / Q000187:drug effects / Q000235:genetics; D017973:Tungsten Compounds / Q000494:pharmacology; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D020690:GTPase-Activating Proteins; D046912:Multiprotein Complexes; D047428:Protein Kinase Inhibitors; D017973:Tungsten Compounds; C100665:USP6NL protein, human; D010744:Phosphoric Monoester Hydrolases; C120902:CDC14A protein, human; D017027:Protein Tyrosine Phosphatases; D020696:rab5 GTP-Binding Proteins; C045951:tungstate