Novel Role of Src in Priming Pyk2 Phosphorylation

<div><p>Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase (FAK) family of non-receptor tyrosine kinases and plays an important role in diverse cellular events downstream of the integrin-family of receptors, including cell migration, proliferation and survival. Here, we have identified a novel role for Src kinase in priming Pyk2 phosphorylation and subsequent activation upon cell attachment on the integrin-ligand fibronectin. By using complementary methods, we show that Src activity is indispensable for the initial Pyk2 phosphorylation on the Y402 site observed in response to cell attachment. In contrast, the initial fibronectin-induced autophosphorylation of FAK in the homologous Y397 site occurs in a Src-independent manner. We demonstrate that the SH2-domain of Src is required for Src binding to Pyk2 and for Pyk2 phosphorylation at sites Y402 and Y579. Moreover, Y402 phosphorylation is a prerequisite for the subsequent Y579 phosphorylation. While this initial phosphorylation of Pyk2 by Src is independent of Pyk2 kinase activity, subsequent autophosphorylation of Pyk2 <i>in trans</i> is required for full Pyk2 phosphorylation and activation. Collectively, our studies reveal a novel function of Src in priming Pyk2 (but not FAK) phosphorylation and subsequent activation downstream of integrins, and shed light on the signaling events that regulate the function of Pyk2.</p></div

Schematic representation of Pyk2 phosphorylation triggered by Src.

<p>In the inactive state, Pyk2 is in an unphosphorylated state. Upon integrin ligand binding, Src phosphorylates Y402, creating a binding site for the SH2-domain of Src, and enabling further phosphorylation of Pyk2 by Src, including at site Y579 (and likely at Y580, although not studied here). As shown by others [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149231#pone.0149231.ref021" target="_blank">21</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149231#pone.0149231.ref022" target="_blank">22</a>] phosphorylated and activated Pyk2 dimerizes or oligomerizes with itself, leading to further phosphorylation of Pyk2 via auto/trans-phosphorylation.</p

Src is required to initiate adhesion-induced Pyk2 phosphorylation.

<p>A, 293T cells were transfected with GFP-tagged Pyk2-WT, Pyk2-Y402F or Pyk2-KD, with or without Src, serum-starved, detached, and then reattached to fibronectin-coated dishes for 30 min. Cell lysates were collected and assayed by immunoblot to examine Pyk2 phosphorylation. B, SYF cells were transfected and assayed similarly as in A. C, SYF cells were co-transfected with Src and GFP-tagged Pyk2-WT, or Pyk2-Y402F, or Pyk2-KD as indicated. The expressed Src was immunoprecipitated, and the associated Pyk2 was examined by immunoblot. D, GST-Pyk2-WT and GST-Pyk2-Y402F were expressed in <i>E</i>. <i>coli</i>, purified, and employed in an in vitro kinase assay as described in Materials and Methods. Pyk2 phosphorylation by recombinant Src was examined by immunoblot analysis by using anti-phospho-Pyk2 (Y402) antibody.</p

Pyk2 transactivation is independent on Src activity.

<p>SYF cells (A) and 293T cells (B) were cultured in the presence of serum and transfected with Myc-tagged Pyk2 constructs and GFP-tagged Pyk2 constructs as indicated. Pyk2 phosphorylation was detected by immunoblot. The expression of GFP-Pyk2 and Myc-Pyk2 was also studied by immunoblot of Pyk2.</p

Analysis of proteins domains of Src involved in Pyk2 phosphorylation.

<p>A, SYF cells transiently transfected with Src-WT or the various Src mutant constructs were serum-starved, detached and kept in suspension in DMEM containing 0.5% BSA for 1 hr. Cells were then seeded on fibronectin-coated dishes, and allowed to attach for 30 min. Phosphorylation of Pyk2 and FAK was examined by immunoblot. B, Co-IP of Src and Pyk2. SYF cells were transiently transfected with Myc-Pyk2 along with Src-WT or Src mutant constructs, the expressed Src was immunoprecipitated and the associated Pyk2 was examined by immunoblot.</p

Src activity is required for Pyk2 phosphorylation in cells plated on fibronectin.

<p>A, serum-starved HeLa cells were detached and kept in suspension in DMEM containing 0.5% BSA for 1 hr. Cells were then seeded on fibronectin- or poly-l-lysine (PLL)-coated dishes, and allowed to attach for the indicated times. Phosphorylation of Pyk2 and FAK was examined by immunoblot, as indicated. B, HeLa cells were kept in suspension with or without indicated Src inhibitors for 1 hr before the attachment to fibronectin-coated dishes for 15 min, and phosphorylation of Pyk2 and FAK was examined as above. C, HeLa cells transfected with mock, wild-type Src (Src-WT) or kinase-dead mutant (Src-KD) were kept in suspension for 1 hr then were attached to fibronectin-coated dishes for 15 min, and the phosphorylation of Pyk2 and FAK was examined by immunoblot as indicated. D, serum-starved SYF and Src⁺⁺ MEFs were kept in suspension for 1 hr, then were attached to fibronectin-coated dishes for 15 or 30 min as indicated, and the phosphorylation of Pyk2 and FAK was examined as in above.</p

3-Dimensional Culture Systems for Anti-Cancer Compound Profiling and High-Throughput Screening Reveal Increases in EGFR Inhibitor-Mediated Cytotoxicity Compared to Monolayer Culture Systems

<div><p>3-dimensional (3D) culture models have the potential to bridge the gap between monolayer cell culture and <i>in vivo</i> studies. To benefit anti-cancer drug discovery from 3D models, new techniques are needed that enable their use in high-throughput (HT) screening amenable formats. We have established miniaturized 3D culture methods robust enough for automated HT screens. We have applied these methods to evaluate the sensitivity of normal and tumorigenic breast epithelial cell lines against a panel of oncology drugs when cultured as monolayers (2D) and spheroids (3D). We have identified two classes of compounds that exhibit preferential cytotoxicity against cancer cells over normal cells when cultured as 3D spheroids: microtubule-targeting agents and epidermal growth factor receptor (EGFR) inhibitors. Further improving upon our 3D model, superior differentiation of EC50 values in the proof-of-concept screens was obtained by co-culturing the breast cancer cells with normal human fibroblasts and endothelial cells. Further, the selective sensitivity of the cancer cells towards chemotherapeutics was observed in 3D co-culture conditions, rather than as 2D co-culture monolayers, highlighting the importance of 3D cultures. Finally, we examined the putative mechanisms that drive the differing potency displayed by EGFR inhibitors. In summary, our studies establish robust 3D culture models of human cells for HT assessment of tumor cell-selective agents. This methodology is anticipated to provide a useful tool for the study of biological differences within 2D and 3D culture conditions in HT format, and an important platform for novel anti-cancer drug discovery.</p></div

Description of the ADC library screen over four concentrations in 3D vs. 2D co-cultures.

<p><b>A</b>, immunofluorescent images of fixed and stained 2D (left panels) and fixed, sectioned, and stained 3D (right panels) co-cultures stained for the endothelial cell marker CD31 (green), the fibroblast-rich protein vimentin (red), and all the cells’ nuclei (blue), <i>bars,</i> 200 microns. The top most panels are the BT-474+HF+HE co-cultures, seeded with 1500 BT-474+750 fibroblasts+750 endothelial cells per well. The bottom panels are the HF+HE co-cultures, seeded with 750 fibroblasts+750 endothelial cells. <b>B</b>, schematic of the four-concentration screening protocol in BT-474+HF+HE and HF+HE cells cultured in 3D or 2D. <b>C</b>, Z’ values generated from one or two 96-well plates set, incubated, and handled as described for the screen for the 2D BT-474+HF+HE cells (top left panel), 2D HF+HE cells (top right panel), 3D BT-474+HF+HE cells (bottom left panel), 3D HF+HE cells (bottom right panel).</p

Screening hits and secondary confirmation concentration response assays.

<p><b>A–E</b>, concentration-response curves generated from the data obtained in the screen (top panels) compared to those curves generated from cherry-picked wells containing ixabepilone (A), paclitaxel (B), gefitinib (C) or dasatinib (D) or lapatinib (E). For all curves, the cells were plated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108283#pone-0108283-g003" target="_blank">Fig. 3A</a> and treated as described in 3B. <b>F</b>, Concentration-response curve generated by treating 2D BT-474+HF+HE (“2T”), 3D BT-474+HF+HE (“3T”), 2D HF+HE (“2N”), or 3D HF+HE (“3N”) co-cultures with concentrations of lapatinib ranging over 5 logs for 48 hours. Cell viability was assessed using the CellTiterGLO assay, as described for the screens, and normalized to vehicle control. Experiment was performed in triplicate, <i>bars</i>, standard deviation.</p

Spheroid and monolayer plating schema and screen design.

<p><b>A</b>, one spheroid per well was formed in 96-well round bottom ultra-low attachment plates. Brightfield micrographs of typical BT-474 and MCF-10A spheroids are shown 48 hours after plating, <i>bar,</i> 200 microns. <b>B</b>, the 2D monolayer cultures were set using the same cell number as in the round bottom plates (3000 cells/well for BT-474 or 1000 cells/well for MCF-10A) in flat bottom, tissue culture-coated 96-well plates. Fluorescence images are shown of typical BT-474 and MCF-10A monolayers 48 hours after plating, stained for actin (red) and DNA (blue), <i>bar</i>, 200 microns. <b>C</b>, schematic of screen design.</p