Platelet-Derived Growth Factor Receptor B–Mediated Phosphorylation of MUC1 Enhances Invasiveness in Pancreatic Adenocarcinoma Cells Pankaj K. Singh, 1,2 Yunfei Wen, 1,2 Benjamin J. Swanson, 1 Kandavel Shanmugam, 3 Andrius Kazlauskas, 4 Ronald L. Cerny, 5 Sandra J. Gendler, 3 and Michael A. Hollingsworth 1,2 1 Eppley Institute for Research in Cancer and Allied Diseases and 2 Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska; 3 Department of Biochemistry/Molecular Biology Program, Mayo Clinic College of Medicine, Scottsdale, Arizona; 4 Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts; and 5 University of Nebraska-Lincoln, Lincoln, Nebraska Abstract MUC1 is a heterodimeric transmembrane glycoprotein that is overexpressed and aberrantly glycosylated in ductal adeno- carcinomas. Differential phosphorylation of the MUC1 cyto- plasmic tail (MUC1CT) has been associated with signaling events that influence the proliferation and metastasis of cancer cells. We identified a novel tyrosine phosphorylation site (HGRYVPP) in the MUC1CT by mass spectrometric analysis of MUC1 from human pancreatic adenocarcinoma cell lines. Analyses in vitro and in vivo showed that platelet- derived growth factor receptor B (PDGFRB) catalyzed phos- phorylation of this site and of tyrosine in the RDTYHPM site. Stimulation of S2-013.MUC1F cells with PDGF-BB increased nuclear colocalization of MUC1CT and B-catenin. PDGF-BB stimulation had no significant effect on cell proliferation rate; however, it enhanced invasion in vitro through Matrigel and in vivo tumor growth and metastases. Invasive properties of the cells were significantly altered on expression of phosphor- ylation-abrogating or phosphorylation-mimicking mutations at these sites. We propose that interactions of MUC1 and PDGFRB induce signal transduction events that influence the metastatic properties of pancreatic adenocarcinoma. [Cancer Res 2007;67(11):5201–10] Introduction Pancreatic cancer is associated with high mortality and poor prognosis, in part because it is known to metastasize early during disease progression. Several studies have shown that the MUC1 glycoprotein contributes to growth and metastasis of pancreatic adenocarcinomas. MUC1 protein has been detected in >90% of pancreatic tumors examined by immunohistochemistry (1), in the pancreatic juice of pancreatic adenocarcinoma patients by proteomic analysis, and in most pancreatic cancer cell lines (2, 3). Sialylated MUC1 is overexpressed by invading and metastatic pancreatic cancer cells but not by normal pancreas or in cases of chronic pancreatitis or pancreatic ductal hyperplasia (4). Previous studies from our laboratory have established a role for MUC1 in invasion and metastasis in pancreatic cancer (5, 6). MUC1 is a type I transmembrane protein that consists of a large extracellular subunit comprised by a mucin-type tandem repeat and a smaller subunit that includes a small extracellular domain and a transmembrane domain plus a 72–amino acid cytoplasmic tail (MUC1CT). These two subunits are generated from the intracellular autocatalytic proteolytic cleavage of a single precursor polypeptide chain (7–9). The larger extracellular subunit consists of a heavily O -glycosylated tandem repeat unit of 20 amino acids that is repeated from 18 to over 100 times in different alleles (10–12). The MUC1CT is phosphorylated and involved in different signaling pathways (13). Initial studies suggested that changes in phosphorylation of MUC1CT were correlated with differences in cell adhesion (14–17). An SXXXXXSSL motif in the MUC1CT interacts with h-catenin and may compete with E-cadherin for binding with h-catenin (18, 19). This interaction is abrogated by mutation of a tyrosine to phenylalanine at the DRSPYEKV sequence, a site that is phosphorylated by c-Src, epidermal growth factor receptor (EGFR), or Lyn kinases. A fragment of MUC1CT is translocated to the nucleus in association with h-catenin and may influence its activity as a transcriptional coactivator (19). Phosphorylated YEKV is believed to serve as a binding site for Src SH2 domains (20). Phosphorylation of tyrosine in the YTNP sequence in the MUC1CT is postulated to enhance binding to the adaptor protein Grb2 and its associated small G protein activator SOS, providing a potential activation mechanism for Ras and its effectors (21, 22). Thus, there is substantial evidence that phosphorylation of the MUC1CT modulates signaling related to proliferation and metastasis. Previous investigations into the phosphorylation of MUC1 have been based on expression of constructs harboring mutations at specific tyrosine residues and analysis of changes in degrees of phosphorylation of all tyrosine residues on the MUC1CT by Western blot analysis with anti-phosphotyrosine antibodies. Here, we directly evaluated the phosphorylation status of MUC1 in pancreatic cancer cell lines by mass spectrometry (MS)-based sequence analysis of the MUC1CT, which revealed a novel tyrosine phosphorylation site at the HGRYVPP sequence in MUC1CT. We showed that platelet-derived growth factor receptor h (PDGFRh)is a kinase for the site and that stimulation of the pancreatic cancer cell line S2-013.MUC1F with PDGF-BB enhanced phosphorylation of MUC1CT, increased nuclear localization of MUC1CT and its association partner, h-catenin, and enhanced the invasiveness of S2-013.MUC1F cells without significantly affecting their prolifera- tion rate. Nuclear localization of MUC1CT and invasiveness of the cells were significantly diminished by tyrosine to phenylalanine mutations at these sites, whereas mutations of the tyrosine Requests for reprints: Michael A. Hollingsworth, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198-6805. Phone: 402-559-8343; Fax: 402-559-3339; E-mail: mahollin@unmc.edu. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-4647 www.aacrjournals.org 5201 CancerRes2007;67:(11).June1,2007 Research Article Research. on January 17, 2015. © 2007 American Association for Cancer cancerres.aacrjournals.org Downloaded from