Phosphorylation of paxillin LD4 destabilizes helix formation and inhibits binding to focal adhesion kinase

Abstract

Cell migration is a dynamic process that requires the coordinated formation and disassembly of focal adhesions (FAs). Several proteins such as paxillin, focal adhesion kinase (FAK), and G protein-coupled receptor kinase-interacting protein 1 (GIT1) are known to play a regulatory role in FA disassembly and turnover. However, the mechanisms by which this occurs remain to be elucidated. Paxillin has been shown to bind the C-terminal domain of FAK in FAs, and an increasing number of studies have linked paxillin association with GIT1 during focal adhesion disassembly. It has been reported recently that phosphorylation of serine 273 in the LD4 motif of paxillin leads to an increased association with Git1 and focal adhesion turnover. In the present study, we examined the effects of phosphorylation of the LD4 peptide on its binding affinity to the C-terminal domain of FAK. We show that phosphorylation of LD4 results in a reduction of binding affinity to FAK. This reduction in binding affinity is not due to the introduction of electrostatic repulsion or steric effects but rather by a destabilization of the helical propensity of the LD4 motif. These results further our understanding of the focal adhesion turnover mechanism as well as identify a novel process by which phosphorylation can modulate intracellular signaling.

Figure 1

Binding curves of fluorescein-tagged LD4 peptides titrated with increasing concentrations of FAT-LD2, as measured by fluorescence anisotropy in 10-mM phosphate, pH 8.0. The y-axis indicates the fraction of maximal binding. a-c LD4, LD4-S273p, and LD4-S275p, respectively. d LD4 in 15% TFE.

Figure 2

a Circular dichroism scans of 5 µM FAT-LD2 in 0% TFE (blue), 5% TFE (purple), 10% TFE (green), and 15% TFE (red).b Helix-formation propensity of LD4 (shaded), LD4-S273p (wide diagonal), and LD4-S275p (narrow diagonal) as measured by circular dichroism in increasing concentrations of TFE.

Figure 3

a-c Chemical shift perturbation plots of the FAT domain residues in the FAT-LD2 construct titrated with LD4, LD4-S273p, and LD4-S275p, respectively. Helices (H) 1–4 of the FAT domain are indicated in plot A. The largest chemical shifts correspond to residues in the LD4-binding pocket. d-f Binding curves derived from weighted chemical shift values for FAT residues M954 (■), L965 (▼), and N992 (▲).

Figure 4

a Ribbon diagram of the FAT domain of FAK (Protein Data Bank accession code 1KTM).(17) The LD4 binding pocket (shaded area) is formed between helices H2 and H3 of the four helix bundl. b Lypophilic surface map of the LD4 binding pocket generated using SYBYL v7.2.5 (Tripos Inc.). The hydrophobic surface interacts with hydrophobic leucine residues on LD4. c Electrostatic surface of the LD4 binding pocket generated using Pymol (DeLano Scientific LLC, San Carlos, CA). Only a positively charged lysine, K1003, is in a position to interact with the phosphate moiety on LD4-S273p. d The LD4-S273p peptide modeled as an α-helix by using ICM-Pro v3.4 (Molsoft Inc.). Top view shows leucine residues that form the hydrophobic binding surface (shaded tan) and the phosphorylated Ser 273 (shaded rose). Bottom view shows the relative orientation of the peptide as it binds to the LD4-binding pocket of FAT.