Krit 1 interactions with microtubules and membranes are regulated by Rap1 and integrin cytoplasmic domain associated protein-1.
The small G protein Rap1 regulates diverse cellular processes such as integrin activation, cell adhesion, cell-cell junction formation and cell polarity. It is crucial to identify Rap1 effectors to better understand the signalling pathways controlling these processes. Krev interaction trapped 1 (Krit1), a protein with FERM (band four-point-one/ezrin/radixin/moesin) domain, was identified as a Rap1 partner in a yeast two-hybrid screen, but this interaction was not confirmed in subsequent studies. As the evidence suggests a role for Krit1 in Rap1-dependent pathways, we readdressed this question. In the present study, we demonstrate by biochemical assays that Krit1 interacts with Rap1A, preferentially its GTP-bound form. We show that, like other FERM proteins, Krit1 adopts two conformations: a closed conformation in which its N-terminal NPAY motif interacts with its C-terminus and an opened conformation bound to integrin cytoplasmic domain associated protein (ICAP)-1, a negative regulator of focal adhesion assembly. We show that a ternary complex can form in vitro between Krit1, Rap1 and ICAP-1 and that Rap1 binds the Krit1 FERM domain in both closed and opened conformations. Unlike ICAP-1, Rap1 does not open Krit1. Using sedimentation assays, we show that Krit1 binds in vitro to microtubules through its N- and C-termini and that Rap1 and ICAP-1 inhibit Krit1 binding to microtubules. Consistently, YFP-Krit1 localizes on cyan fluorescent protein-labelled microtubules in baby hamster kidney cells and is delocalized from microtubules upon coexpression with activated Rap1V12. Finally, we show that Krit1 binds to phosphatidylinositol 4,5-P(2)-containing liposomes and that Rap1 enhances this binding. Based on these results, we propose a model in which Krit1 would be delivered by microtubules to the plasma membrane where it would be captured by Rap1 and ICAP-1.
Béraud-Dufour
Sophie
S
UMR 6097 CNRS-UNSA, Institut de Pharmacologie Moléculaire et Cellulaire, Vabonne, France.
Gautier
Romain
R
Albiges-Rizo
Corinne
C
Chardin
Pierre
P
Faurobert
Eva
E
D048868:Adaptor Proteins, Signal Transducing; D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D006224:Cricetinae; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008566:Membranes / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D011487:Protein Conformation; D014162:Transfection; D020798:Two-Hybrid System Techniques; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; C108257:ITGB1BP1 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D020827:rap1 GTP-Binding Proteins