A novel ER-retention motif within KIAA1199 that is required for its ER localization, BiP conversation, and enhanced cell migration was recognized. differences. All statistical assessments were two-sided. Results KIAA1199 was upregulated in invasive breast malignancy specimens and inversely associated with patient survival rate. Silencing of KIAA1199 in MDA-MB-435 malignancy cells resulted in a mesenchymal-to-epithelial transition that reduced cell migratory ability in vitro (75% reduction; < .001) and decreased metastasis in vivo (80% reduction; < .001). Gain-of-function assays further exhibited the role of KIAA1199 in cell migration. KIAA1199-enhanced cell migration required endoplasmic reticulum (ER) localization, where it forms a stable complex with the chaperone binding immunoglobulin protein (BiP). A novel ER-retention motif within KIAA1199 AZ5104 that is required for its ER localization, BiP conversation, and enhanced cell migration was recognized. Mechanistically, KIAA1199 was found to mediate ER calcium leakage, and the resultant increase in cytosolic calcium ultimately led to protein kinase C alpha activation and cell migration. Conclusions serves as a novel cell migrationCpromoting gene and plays a critical role in maintaining malignancy mesenchymal status. Cell migration is usually a complicated and incompletely comprehended process required for malignancy invasion (1). Cell migration is often a result of epithelial-to-mesenchymal transition (EMT) of malignancy cells, which leads to a more aggressive phenotype. Reversal of EMT (mesenchymal-to-epithelial-transition) results in decreased cell migration (2). Identification of specific genes involved in malignancy cell migration is usually critically important in preventing malignancy dissemination (3). To identify novel genes involved in malignancy cell invasion, we used a polymerase chain reactionCbased suppression subtractive hybridization method, which has been demonstrated to be effective in isolating, normalizing, and enriching differentially expressed genes >1000-fold in a single round of hybridization (4). Because concanavalin A enhances cell surface proteolytic activity AZ5104 and cell migratory ability (3,5), differential gene expression in concanavalin ACtreated HT-1080 human fibrosarcoma cells was examined. This approach resulted in the identification of a marked upregulation of a previously obscure gene, in families with nonsyndromic hearing loss, this gene appears to be essential for auditory function (6), even though function was not investigated. Clinical relevance of KIAA1199 in cancers has been highlighted by reports of increased KIAA1199 mRNA expression in human gastric and colorectal cancers; an association was shown between KIAA1199 expression level and disease stage/5-12 months survival rates (7,8). However, the Mmp7 function of KIAA1199 in malignancy remains unknown. In this study, we discovered that KIAA1199 is usually a novel endoplasmic reticulum (ER) resident protein that plays a critical role in malignancy cell migration and invasion. Moreover, KIAA1199 enhances cell migration through its conversation with ER glucose-regulated protein 78/binding immunoglobulin protein (GRP-78/BiP), leading to ER calcium release. Increased cytosolic calcium results in the activation of protein kinase C alpha (PKC), ultimately leading to enhanced cell migration. Methods Materials Oligo primers were synthesized by Operon. RNAi-Ready pSIREN Retro-Q vector for specific gene silencing AZ5104 and pQCXIP retroviral vector for generation of stable cells were purchased from Clontech (Mountain View, CA). D1ER expression plasmid was kindly provided by Dr Roger Tsien (University or college of CaliforniaCSan Diego) (9). Mouse anti-Myc monoclonal antibody was purchased from Roche (Indianapolis, IN). The pcDNA3.1-myc expression vector, rabbit anti-PKC pT674 polyclonal antibody, and Organelle Lights reagents were purchased from Invitrogen (Grand Island, NY). Rabbit anti-KIAA1199 polyclonal antibody was produced by PrimmBiotech (Cambridge, MA) using the C-terminus of the KIAA1199 protein between Gly1108-Thr1340 as an antigen. Mouse antiCprotein disulfide isomerase monoclonal antibody was purchased from AssayDesign (Farmingdale, NY). Rabbit anti-BiP monoclonal, -/-tubulin polyclonal, –actin monoclonal, and -Twist-1 polyclonal were purchased from Cell Signaling Technology (Danvers, MA). Rabbit anti-XBP-1 polyclonal, -pan-PKC polyclonal, and mouse anti-cytokeratin 8/18 monoclonal were purchased from Santa Cruz Biotechnology (Dallas, TX). Mouse anti-PKC monoclonal and rabbit anti-PKCI monoclonal were purchased from Enzo Life Sciences (Farmingdale, NY). Mouse anti-N-cadherin monoclonal antibody was purchased from BD Transduction Laboratories (San Jose, CA). Mouse anti-vimentin monoclonal antibody, concanavalin A, and phalloidin were purchased from Sigma (St. Louis, MO). SNAP-capture beads and rabbit anti-SNAP polyclonal antibody were purchased from New England Biolabs (Ipswich, MA). PKCK380R cDNA (Addgene plasmid 21239) and PKCI cDNA (Addgene plasmid 16378) (10) were purchased from Addgene (Cambridge, MA). All antibodies were used at 1:1000 dilution unless normally specified in each individual experiment. The human breast cancer tissue microarray samples (BR804, BC08013a, and AZ5104 BR10010a) were purchased from US AZ5104 Biomax Inc (Rockville, MD). SNAP Pull-Down Assay and Proteomic Analysis COS-1 cells transiently transfected with a control plasmid made up of a SNAP tag or KIAA1199-SNAP plasmids were lysed, followed by incubation with SNAP beads (New England.