A Functional Nuclear EGFR, Src and Stat3 Complex Figure 2. Co-immunoprecipitation with immunoblotting analysis of EGFR, Src and Stat3 complex in the nucleus and the sub- cellular distribution of EGFR, Src and Stat3. (A and B) Immunoblotting analyses of immunecomplexes of EGFR (IP:EGFR), Src (IP:Src), Stat3 (IP:Stat3), EGFR/Src (IP:EGFR/IP:Src), or of non-specific IgG non-immuneprecpitate prepared from nuclear extracts of Panc-1 or Colo-357 cells and probing for Stat3, EGFR, Src, or the Tata-binding protein (TBP); and (C), immunoblotting analysis of membrane (mem) and cytosolic (cyto) fractions and of nuclear (nuc) extracts from Panc-1 cells probing for (i) EGFR, (ii) Stat3 and (iii) Src. Bands corresponding to proteins in gel are shown; input: except where indicated, represents the immunoblotting for the respective immunoprecipitated protein in the same amount of nuclear extract used in the assay; IP:EGFR/IP:Src, sequential immunoprecipitation with anti-EGFR and then anti-Src antibody; Data are representative of 3 independent studies. doi:10.1371/journal.pone.0019605.g002 whole-cell lysates analysis in Figure 1C, these data together indicate that the knockdown of one protein does not preclude the interaction between the other two protein partners. We next sought to determine if the heteromeric complex formation is dependent on the tyrosine kinase activities of EGFR and Src. Compared to IgG, immunoblotting of immunecomplex of EGFR showed no effect of 1 h-treatment of Panc-1 cells with the selective EGFR inhibitor, Iressa (ZD1839, ZD), the Src inhibitor, Dasatinib (Das), or the Stat3 dimerization disrupting inhibitor, S3I-201 [25] (Fig. 1G, IP:EGFR, lanes 3, 6, and 8, compared to lane 2). By contrast, immunoblotting analysis of EGFR immunecomplex showed decreased levels of associated Stat3 protein, but not Src, when cells were treated with ZD, Das, or S3I-201 for 24 h (Fig. 3B, lanes 4, 7, and 9). These findings together suggest the EGFR and Src kinase activities promote the association of Stat3 with EGFR in the nuclear heteromeric complex, while the inhibition of EGFR or Src kinase alone does not preclude the interaction between EGFR and Src. Further- more, the Stat3 dimerization disrupting inhibitor blocks Stat3 association in the complex; however, the disruption of Stat3 dimerization, and hence its binding to EGFR does not preclude EGFR interaction with Src. The observation that the nuclear heteromeric EGFR, Src, and Stat3 complex was not completely dissociated and that EGFR/Src complex persisted by 24-h inhibition of EGFR (ZD) or Src (Das) (Fig. 3B, lanes 4, and 9) has important clinical implications in regard to the responsiveness of pancreatic cancer cells to a monotherapy targeting EGFR or Src. During the stimulation of the EGFR signaling pathway, key phospho-tyrosine (pY) peptide motifs are induced, which recruit different signaling proteins, including Stat3 [26,27]. The Stat3 SH2 domain binds to the receptor pY motifs through pY-SH2 domain interactions [28]. We therefore sought to probe further the observed nuclear complex of the EGFR, Src and Stat3 with respect to the peptide motifs potentially involved in the interactions by using the known Stat3-binding EGFR motifs, pY1068 peptide and pY1086 peptide, and the newly reported Stat3 peptide inhibitor, SPI, which is derived from the Stat3 SH2 domain and is capable of blocking the binding of the Stat3 SH2 domain to pY peptide motifs [29]. Immunoblotting analysis of Stat3 immunoprecipitates from nuclear extracts that have been pre-incubated with the three peptides for 2 h at room temperature showed no detectable EGFR protein (Fig. 3C(i)). These results suggest the EGFR-Stat3 interaction is promoted by pY1068EGFR and pY1086EGFR, and also that in binding to EGFR, Stat3 utilizes the key amino acid residues, 588–615 of its SH2 domain, which make up the peptide inhibitor SPI [29]. By contrast, similar probing with the SPI peptide showed no significant effect on the immuno-detection of Src protein within the Stat3 immunecom- plex (Fig. 3C(ii)), suggesting the direct interaction between Src and Stat3 is independent of the amino acid residues 588–615 of the Stat3 SH2 domain. PLoS ONE | www.plosone.org 6 May 2011 | Volume 6 | Issue 5 | e19605