Abstract
We have previously identified the scaffold protein liprin-α1 as an important regulator of integrin-mediated cell motility and tumor cell invasion. Liprin-α1 may interact with different proteins, and the functional significance of these interactions in the regulation of cell motility is poorly known. Here we have addressed the involvement of the liprin-α1 partner GIT1 in liprin-α1-mediated effects on cell spreading and migration. GIT1 depletion inhibited spreading by affecting the lamellipodia, and prevented liprin-α1-enhanced spreading. Conversely inhibition of the formation of the liprin-α1-GIT complex by expression of liprin-ΔCC3 could still enhance spreading, although to a lesser extent compared to full length liprin-α1. No cumulative effects were observed after depletion of both liprin-α1 and GIT1, suggesting that the two proteins belong to the same signaling network in the regulation of cell spreading. Our data suggest that liprin-α1 may compete with paxillin for binding to GIT1, while binding of βPIX to GIT1 was unaffected by the presence of liprin-α1. Interestingly, GIT and liprin-α1 reciprocally regulated their subcellular localization, since liprin-α1 overexpression, but not the GIT binding-defective liprin-ΔCC3 mutant, affected the localization of endogenous GIT at peripheral and mature central focal adhesions, while the expression of a truncated, active form of GIT1 enhanced the localization of endogenous liprin-α1 at the edge of spreading cells. Moreover, GIT1 was required for liprin-α1-enhanced haptotatic migration, although the direct interaction between liprin-α1 and GIT1 was not needed. Our findings show that the functional interaction between liprin-α1 and GIT1 cooperate in the regulation of integrin-dependent cell spreading and motility on extracellular matrix. These findings and the possible competition of liprin-α1 with paxillin for binding to GIT1 suggest that alternative binding of GIT1 to either liprin-α1 or paxillin plays distinct roles in different phases of the protrusive activity in the cell.
Highlights
Cell migration requires complex molecular events that need to be finely regulated in time and space [1]
Our findings show that the functional interaction between liprin-a1 and GIT1 cooperate in the regulation of integrin-dependent cell spreading and motility on extracellular matrix
During cell spreading and migration on extracellular matrix, continuous reorganization of FAs and actin dynamics at the cell front are necessary for effective protrusion [33]
Summary
Cell migration requires complex molecular events that need to be finely regulated in time and space [1]. GIT1 (G protein-coupled receptor kinase-interacting protein 1) and GIT2/PKL form a family of multi-domain ArfGAP proteins with scaffolding activity, which are implicated in the regulation of cell adhesion and migration on extracellular matrix [2]. They interact via an SHD (Spa homology domain) with the components of the PIX (p21activated kinase-interacting exchange factor) family of guanine nucleotide exchanging factors for Rac and Cdc GTPases [3,4,5]. GIT proteins are involved in different pathways that regulate cell motility. GIT1 is involved in EGF-dependent vascular smooth muscle cell migration [12], while the second member of the family, GIT2 is a key player for chemotactic directionality in stimulated neutrophils [13], and is required for PDGF-dependent directional cell migration and cell polarity, but not for random migration [14]
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