A Functional Nuclear Epidermal Growth Factor Receptor, Src and Stat3 Heteromeric Complex in Pancreatic Cancer Cells Soumya Jaganathan1, Peibin Yue1, David C. Paladino1, Jelena Bogdanovic2,3, Qun Huo2,3, James 1 Turkson * 1 Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, Florida, United States of America, 2 NanoScience Technology Center, University of Central Florida, Orlando, Florida, United States of America, 3 Department of Chemistry, University of Central Florida, Orlando, Florida, United States of America Abstract Evidence is presented for the nuclear presence of a functional heteromeric complex of epidermal growth factor (EGFR), Src and the Signal Transducer and Activator of Transcription (Stat)3 proteins in pancreatic cancer cells. Stat3 remains nuclear and associated with Src or EGFR, respectively, upon the siRNA knockdown of EGFR or Src, demonstrating the resistance of the complex to the modulation of EGFR or Src alone. Significantly, chromatin immunoprecipitation (ChIP) analyses reveal the nuclear EGFR, Src and Stat3 complex is bound to the c-Myc promoter. The siRNA knockdown of EGFR or Src, or the pharmacological inhibition of Stat3 activity only marginally suppressed c-Myc expression. By contrast, the concurrent modulation of Stat3 and EGFR, or Stat3 and Src, or EGFR and Src strongly suppressed c-Myc expression, demonstrating that the novel nuclear heteromeric complex intricately regulates the c-Myc gene. The prevalence of the transcriptionally functional EGFR, Src, and Stat3 nuclear complex provides an additional and novel mechanism for supporting the pancreatic cancer phenotype and explains in part the insensitivity of pancreatic cancer cells to the inhibition of EGFR, Src or Stat3 alone. Citation: Jaganathan S, Yue P, Paladino DC, Bogdanovic J, Huo Q, et al. (2011) A Functional Nuclear Epidermal Growth Factor Receptor, Src and Stat3 Heteromeric Complex in Pancreatic Cancer Cells. PLoS ONE 6(5): e19605. doi:10.1371/journal.pone.0019605 Editor: Marcelo G. Bonini, University of Illinois at Chicago, United States of America Received November 30, 2010; Accepted April 12, 2011; Published May 5, 2011 Copyright: 2011 Jaganathan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Cancer Institute Grants CA106439 (JT) and CA128865 (JT), Florida Department of Health Bankhead-Coley Foundation (Bridge Grant) (HQ), partial support from World Gold Council (GROW Program) (HQ), and the National Natural Science Foundation of China Overseas Young Investigator Award (20828006) (HQ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: james.turkson@.ucf.edu Introduction Many intracellular biochemical processes are triggered by the assembly of proteins into macromolecular complexes. The association between proteins or of proteins with other molecular entities modulates protein conformation, providing a means to regulate the myriad of biochemical processes that serve to efficiently manage vital biological responses. Protein dynamics and trafficking, and protein stability are also processes that can be modulated by the association of proteins with others. In the broader sense, inter- molecular associations allow specialty proteins, such as receptors, adapters, enzymes, and transcription factors to differentially modulate intracellular events, thereby creating the diversity in physiological responses and promoting context dependency. During the induction of signal transduction, there is assembly of different proteins, each of which has specific functions important for the signal transduction and the accompanying biological response. The traditional epidermal growth factor receptor (EGFR) signal transduction pathway incorporates the activation of the mitogen-activated protein kinase kinase (MEK)-mitogen- activated protein kinase/extracellular signal-regulated kinase (ErkMAPK) and promotes mitogenic responses [1,2]. The EGFR induction also promotes the activation of the Signal Transducer and Activator of Transcription (STAT) family of proteins, which similarly have a central role in EGF-induced biological responses [1]. The STAT proteins are latent cytoplasmic transcription factors that are activated in response to cellular stimulation by cytokines and growth factors [3] via the phosphorylation of a critical tyrosyl residue (Tyr705 for Stat3). The tyrosine phosphor- ylation of STATs is mediated by tyrosine kinases of growth factor receptors and by cytoplasmic tyrosine kinases, such as Src and Janus kinase (Jaks) families. Activated STATs as dimers in the nucleus bind to specific DNA response elements in the promoters of target genes to induce gene transcription. The nuclear translocation mechanism for STATs has been the subject of recent intense investigation. Stat3 nuclear translocation has been reported to be mediated by the recognition and transport by importin-a and the Ran-GTPase [4], and by mechanisms involving the chaperoning by MgcRacGAP [5], EGF receptor- mediated endocytosis [6], and by plasma membrane-associated lipid rafts trafficking [7]. The prevalence of many hyperactive signal transduction pathways that support the cancer phenotype is a major challenge to therapy. Further to the classical way of promoting crosstalks PLoS ONE | www.plosone.org 1 May 2011 | Volume 6 | Issue 5 | e19605