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

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

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

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

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