Tyrosine phosphorylation of type Igamma phosphatidylinositol phosphate kinase by Src regulates an integrin-talin switch

Abstract

Engagement of integrin receptors with the extracellular matrix induces the formation of focal adhesions (FAs). Dynamic regulation of FAs is necessary for cells to polarize and migrate. Key interactions between FA scaffolding and signaling proteins are dependent on tyrosine phosphorylation. However, the precise role of tyrosine phosphorylation in FA development and maturation is poorly defined. Here, we show that phosphorylation of type Igamma phosphatidylinositol phosphate kinase (PIPKIgamma661) on tyrosine 644 (Y644) is critical for its interaction with talin, and consequently, localization to FAs. PIPKIgamma661 is specifically phosphorylated on Y644 by Src. Phosphorylation is regulated by focal adhesion kinase, which enhances the association between PIPKIgamma661 and Src. The phosphorylation of Y644 results in an approximately 15-fold increase in binding affinity to the talin head domain and blocks beta-integrin binding to talin. This defines a novel phosphotyrosine-binding site on the talin F3 domain and a "molecular switch" for talin binding between PIPKIgamma661 and beta-integrin that may regulate dynamic FA turnover.

Figure 1.

FAK signaling results in phosphorylation of PIPKIγ661 at Y644. (A) PIPKIγ661 was coexpressed with pcDNA3 (control), wild-type FAK (FAKwt), Y397F FAK (FAKY397F), or kinase-dead FAK (FAKkd) in HEK293T cells as indicated. After 48 h, tyrosine phosphorylation of PIPKIγ661 was analyzed by immunoprecipitation and Western blot as indicated. pY, phosphotyrosine. (B) The epitope-tagged PIPKIα/γ chimeras c-Myc-PIPKIα/γ635 (Iα/Iγ635), c-Myc-PIPKIα/γ661 (Iα/Iγ661), c-Myc-PIPKIα/γ636–661 (Iα/Iγ636–661), and wild-type HA-tagged PIPKIγ661 (Iγ661) were cotransfected with pcDNA3 (1) or wild-type FAK (2) in HEK293 cells for 48 h. Tyrosine phosphorylation of the PIPKI constructs was examined by immunoprecipitation and Western blot as indicated. (C) The wild-type (Iγ661) and Y to F mutant (Iγ661Y644F, Iγ661Y649F, Iγ661Y644/649F) PIPKIγ661 constructs were co-overexpressed in HEK293T cells with pcDNA3 (1) or wild-type FAK (2) for 48 h, and tyrosine phosphorylation of the PIPKIγ661 constructs was examined using immunoprecipitation and Western blot as indicated.

Figure 2.

c-Src phosphorylates PIPKIγ661 at Y644. (A) HEK293T cells, transfected with PIPKIγ661 and the indicated constructs, were treated with 1 μM PP2 or PP3 for 30 min at 37°C. Then, cells were lysed and tyrosine phosphorylation of immunoprecipitated PIPKIγ661 was examined. (B) 2 mg lysate of c-Src/Fyn/Yes triple-knockout (SFY^−/−), SFY^−/− cells reintroduced with c-Src (Src^+/+), FAK knockout (FAK^−/−), or the FAK^−/− parent (FAK^+/+) cells were used for immunoprecipitation using purified anti-PIPKIγ antibody. Then, tyrosine phosphorylation of immunoprecipitated PIPKIγ was analyzed by Western blot as indicated. (C) In vitro Src kinase assay with His-tagged wild-type (wt-T) or mutant (Y644F-T, Y649F-T, or Y644/649F-T) PIPKIγ661 tails as substrates. Phosphorylation data were analyzed using SigmaPlot 4.0 from at least three independent experiments. (D) Analysis of tyrosine phosphorylation by tryptic phosphopeptide mapping. In vitro Src kinase assays using the substrates described in C were resolved by SDS-PAGE, and the tryptic digests of the tyrosine-phosphorylated substrates were analyzed on two-dimensional maps (exposure for 7 d at −70°C). Arrows indicate the spots corresponding to the loss of phosphorylation in the Y644F mutant. The large horizontal arrow points to the cathode and the large vertical arrow indicates the direction of chromatographic resolution.

Figure 3.

Src association with PIPKIγ661 is enhanced by FAK in vivo. (A) 48 h after cotransfection with c-Src and PIPKIγ661 constructs, HEK293T cells were immunoprecipitated using normal rabbit IgG (rIgG) or purified anti-PIPKIγ antibody. The immunoprecipitates and cell lysate were analyzed by Western blot as indicated. (B) HEK293T cells were transfected with PIPKIγ661, FAK, and/or Src constructs as indicated for 48 h, and then immunoprecipitated using the indicated antibodies. Immunoprecipitates were analyzed by Western blot as indicated. (C) A431 cells were lifted by trypsin, washed three times, and kept in suspension for 1 h at 37°C in serum-free DME. Cells were lysed (sus.) or plated on type I collagen-coated (10 μg/ml) plates in serum-free DME for the indicated time and then lysed, immunoprecipitated, and analyzed by Western blot as indicated.

Figure 4.

Y644 of PIPKIγ661 is required for talin association. (A) Immunoprecipitations were performed with HEK293T cell lysate with normal rabbit IgG (rIgG), purified anti-PIPKIγ antibody, normal mouse IgG (mIgG), or monoclonal anti-talin antibody. The immunoprecipitates were analyzed by Western blot as indicated. (B) Top, HEK293T cells were cotransfected with PIPKIγ661 and indicated FAK construct, and then used for immunoprecipitation with purified anti-PIPKIγ antibody after 48 h. The immunoprecipitates were analyzed by Western blot as indicated. Bottom, quantification of talin association. (C) Top, HEK293 cells were transfected with wild-type (Iγ661), last 20 amino acids truncated (Iγ661Δ20), last 13 amino acids truncated (Iγ661Δ13), last 3 amino acids truncated (Iγ661Δ3), or Y to F mutant (Iγ661Y644F, Iγ661Y649F, Iγ661Y644/649F) PIPKIγ constructs for 48 h. Talin association with wild-type or mutant PIPKIγ661 was examined by immunoprecipitation and Western blot as indicated. Bottom, quantification of talin association. Data were quantified using NIH image 1.62 and plotted using SigmaPlot 4.0 from at least three independent experiments.

Figure 5.

Tyrosine phosphorylation of PIPKIγ661 is required for focal adhesion targeting. (A) NRK cells were transfected with wild-type or mutant PIPKIγ constructs using FuGENE™ 6 for 24 h. Overexpressed PIPKIγ constructs and endogenous talin in NRK cells were visualized by anti-PIPKIγ antibody (green) and anti-talin antibody (red). Bars, 10 μm. (B) Quantification of A. 200 transfected NRK cells were counted per slide. Percentage of FA targeting positive cells on each slide were calculated and plotted using SigmaPlot 4.0 from at least three independent experiments. (C) Src, Fyn, and Yes triple-knockout cells (SFY^−/−) or c-Src–reintroduced SFY^−/− cells (Src^+/+) were transfected with wild-type PIPKIγ661 using FuGENE™ 6 for 24 h. Overexpressed PIPKIγ661 and pY were visualized by anti-PIPKIγ antibody (red) and anti-pY (PY99) antibody (green). Bars, 10 μm.

Figure 6.

Tyrosine phosphorylation of PIPKIγ661 at Y644 and Y649 enhances its interaction with talin. (A) Top, effect of the Y to F mutation on PIPKIγ661 binding to talin. 50 nM GST talin head (GST-TaH) was incubated with indicated amount of His-tagged wild-type (Iγ661T) or mutant PIPKIγ661 tails (Iγ661Y644F-T, Iγ661Y649F-T, or Iγ661Y644/649F-T). Bottom, bound PIPKIγ661 tail bands were scanned, and the gray scales were quantified by NIH image 1.62 and then plotted using SigmaPlot 4.0 from at least three independent experiments. (B) Inhibition of PIPKIγ661–talin interaction by phosphorylated PIPKIγ661 peptides. Top, sequences and nomenclature of synthesized PIPKIγ661 peptides. Bottom, immunoprecipitation was performed using PIPKIγ661 overexpressing HEK293T lysates supplemented with 5 μM indicated PIPKIγ661 peptide. The talin–PIPKIγ661 association was analyzed from the immunoprecipitates by Western blot as indicated. (C) Direct binding curves of synthesized PIPKIγ661 peptides with wild-type GST fusion talin head domain. 20 nM fluorescein-labeled PIPKIγ661 peptides were incubated respectively with increased concentration of talin head in 0.1% BSA-PBS for 30 min at RT, and then anisotropy values were measured and analyzed using SigmaPlot 4.0 from three independent experiments. K_d values of wild-type talin head binding to each PIPKIγ661 peptide are shown as insets.

Figure 7.

Phosphorylation of PIPKIγ661 disrupts integrin binding to talin. (A) 100 nM GST-TaH and 100 nM His-β1-integrin tail (His-β1InT) were used for GST pull-down assays with indicated amount of His-PIPKIγ635 tail (Iγ635T) or His-Iγ661T (Iγ661T). (B) The interactions between 100 nM GST-TaH and 100 nM His-β1InT, with indicated amounts of synthesized PIPKIγ661 peptides, were examined by GST pull-down assay and analyzed by Western blot as indicated. (C) The displacement curves of synthesized PIPKIγ661 peptides binding to GST-TaH in competition with His-β1InT were obtained by GST pull-down and Western blot. Both talin head bands and integrin tail bands were quantified by NIH image 1.62, and data were plotted by SigmaPlot 4.0 from at least three independent experiments.

Figure 8.

PIPKIγ661 and β1-integrin bind to distinct sites on the talin head region. (A) The interactions between 50 nM wild-type His-Iγ661T or His-β1InT and 50 nM wild-type (GST-TaH), K357Q mutant (GST-TaHK357Q), R358Q mutant (GST-TaHR358Q) talin head region, or GST alone. (B) Binding curves of His-Iγ661T with 20 nM GST-TaH, GST-TaHK357Q, or GST-TaHR358Q were obtained by GST pull-down, Western blot, and quantification using NIH image 1.62, and were plotted using SigmaPlot 4.0 from at least three independent experiments. (C) Interactions between 20 nM His-Iγ661T and 20 nM GST-TaH, GST-TaHK357Q, or GST-TaHR358Q, with or without 500 nM synthesized PIPKIγ661 peptide as indicated, were analyzed by GST pull-down and Western blot as indicated. (D) Direct binding curves of synthesized PIPKIγ661 peptides with mutant GST fusion talin head domains were determined by fluorescence anisotropy. K_d values of wild-type or mutant talin head binding to each PIPKIγ661 peptide are shown as insets.

Figure 9.

Phosphorylation of β1-integrin tail does not increase interaction with talin. Top, sequences and nomenclature of the synthesized β1-integrin peptides. Bottom, the interactions between 100 nM GST-TaH and 100 nM His-β1InT with indicated amount of indicated β1-integrin peptides.

Figure 10.

Models of talin–PIPKIγ661 interaction and PIPKIγ661-mediated regulation of focal adhesion organization. (A) Structure model of talin F3 lobe with dual phosphorylated PIPKIγ661 peptide (green) bound (phosphates are highlighted as red). Structure model was generated with the Sybyl molecular modeling program using the crystal structure as guide to dock the phosphorylated peptides, which were then energy minimized with the talin F3 lobe. (B) Signaling mechanism depicting PIPKIγ661 regulation of FAs and the interplay with β-integrin.