In vitro phosphorylation of the focal adhesion targeting domain of focal adhesion kinase by Src kinase

Abstract

Focal adhesion kinase (FAK), a key regulator of cell adhesion and migration, is overexpressed in many types of cancer. The C-terminal focal adhesion targeting (FAT) domain of FAK is necessary for proper localization of FAK to focal adhesions and subsequent activation. Phosphorylation of Y926 in the FAT domain by the tyrosine kinase Src has been shown to promote metastasis and invasion in vivo by linking the FAT domain to the MAPK pathway via its interaction with growth factor receptor-bound protein 2. Several groups have reported that inherent conformational dynamics in the FAT domain likely regulate phosphorylation of Y926; however, what regulates these dynamics is unknown. In this paper, we demonstrate that there are two sites of in vitro Src-mediated phosphorylation in the FAT domain: Y926, which has been shown to affect FAK function in vivo, and Y1008, which has no known biological role. The phosphorylation of these two tyrosine residues is pH-dependent, but this does not reflect the pH dependence of Src kinase activity. Circular dichroism and nuclear magnetic resonance data indicate that the stability and conformational dynamics of the FAT domain are sensitive to changes in pH over a physiological pH range. In particular, regions of the FAT domain previously shown to regulate phosphorylation of Y926 as well as regions near Y1008 show pH-dependent dynamics on the microsecond to millisecond time scale.

FIGURE 1

MS-based absolute quantification of the phosphopeptide containing Y926 (z=+2). MS spectra of the phosphopeptide containing Y926 with corresponding sample peptide (solid oval) and heavy internal standard peptide (empty oval) spiked in at A. 0.2 μg/mL, B. 0.4 μg/mL and C. 1.0 μg/mL. D. Extracted ion chromatogram (XIC) of sample and internal standard spiked in at 0.2 μg.mL⁻¹. E. Linear regression fit of the normalized peak area of the sample as a function of spiked-in standard concentration. Dashed lines represent 95% confidence intervals.

FIGURE 2

Site-specific quantitation of phosphorylation. A. Full-length FAT domain and B. FAT domain peptides were phosphorylated in vitro with Src kinase at pH 5.5, 6.0, 6.5, 7.0, and 7.5. After phosphorylation, the full-length FAT domain was trypsinized, and the levels of the Y926 phosphorylated peptide (● z=+2, ■ z=+3) and Y1008 (▲ z=+2, ▼ z=+3) were quantified as described in the text. Comparison of the phosphorylation profiles of the full-length FAT domain and of FAT domain peptides reveals that the specificity of phosphorylation is different in the peptides than in the full-length protein. Y1008 is preferred in the full-length FAT domain whereas Y926 is preferred in the peptides. Furthermore, the pH dependence of phosphorylation of the peptides is different than that of the full-length protein.

FIGURE 3

pH-dependent stability of the FAT domain. The thermal stability of the FAT domain was measured at pH 5.5, 6.0, 6.5, 7.0, and 7.5 by monitoring the CD signal at 221 nm from 25–95°C. The FAT domain is significantly more stable at pH 5.5 compared to pH 7.5, as indicated by the melting temperature.

FIGURE 4

¹H-¹⁵N HSQC spectra as a function of pH. A. Overlay of ¹H-¹⁵N HSQC spectra collected at pH 5.5 (black), 6.0 (green), 6.5 (blue), 7.0 (orange), and 7.5 (red). The chemical shifts of several peaks are affected by pH whereas others do not change. The chemical shifts of the three histidine residues in the FAT domain (H981, H1026, and H1053) are circled. B. The weighted changes in chemical shift from pH 5.5 to 7.5 were calculated. Changes greater than 0.05 ppm (dashed line) were considered significant. The histidine residues are shown in red.

FIGURE 5

pH-dependent changes in intensity derived from ¹H-¹⁵N HSQC spectra. A. Peaks that broaden in the ¹⁵N-¹H HSQC spectra from pH 5.5 to pH 7.5 are shown in red. These residues localize to the N and C-termini, the turn between helices 3 and 4 and the N-terminus of helix-4. B. Peaks that narrow from pH 5.5 to pH 7.5 are shown in red. These residues localize to the “hinge region” between helices 1 and 2. The two sites of phosphorylation, Y926 and Y1008, are shown in magenta, and the histidine residues are shown in blue.

FIGURE 6

Backbone dynamics as a function of pH derived from 2D T1, T2, and HetNOE data as described in the Experimental Procedures. A. S² order parameters at pH 6.0 and pH 7.5 mapped onto the sequence of the FAT domain. B. Rex values at pH 6.0.