Identification of an intramolecular interaction important for the regulation of GIT1 functions.
G-protein coupled receptor kinase-interacting protein (GIT) proteins include an N-terminal Arf GTPase-activating protein domain, and a C terminus that binds proteins regulating adhesion and motility. Given their ability to form large molecular assemblies, the GIT1 protein must be tightly regulated. However, the mechanisms regulating GIT1 functions are poorly characterized. We found that carboxy-terminal-truncated fragments of GIT1 bind their partners with higher efficiency compared with the full-length GIT1. We have explored the hypothesis that GIT1 is regulated by an intramolecular mechanism, and we identified two distinct intramolecular interactions between the N and C terminus of GIT1. The release of these interactions increases binding of GIT1 to paxillin and liprin-alpha, and it correlates with effects on cell spreading. Analysis of cells plated on fibronectin has shown that different deletion mutants of GIT1 either enhance or inhibit spreading, depending on their subcellular localization. Moreover, although the association between betaPIX and GIT1 is insufficient to activate GIT1 binding to paxillin, binding of a PAK1 fragment including the betaPIX-binding domain enhances paxillin binding to betaPIX/GIT1, indicating that p21-activated kinase can activate the binding of paxillin to GIT1 by a kinase-independent mechanism. The release of the identified intramolecular interaction seems to be an important mechanism for the regulation of GIT1 functions.
Totaro
Antonio
A
Cell Adhesion Unit, Dibit, San Raffaele Scientific Institute, 20132 Milan, Italy.
Paris
Simona
S
Asperti
Claudia
C
de Curtis
Ivan
I
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002448:Cell Adhesion; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002522:Chlorocebus aethiops; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D009154:Mutation / Q000235:genetics; D051419:Paxillin / Q000378:metabolism; D011485:Protein Binding
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D005353:Fibronectins; D051419:Paxillin