Biol Cell (1994) 81,195-203 © Elsevier, Paris 195 Original article Identification and characterization of the tumor-specific PIA gene product Alain Amar-Costesec a, Dani~le Godelaine ~, Benolt Van den Eynde b, Henri Beaufay ~ alnternational Institute of Cellular and Molecular Pathology, Universitd Catholique de Louvain, UCL 75-39, avenue Hippocrate 75, B-1200 Brussels; bLudwig Institute for Cancer Research, Brussels, Belgium (Received 6 May 1994; accepted 29 September 1994) Summary - In murine mastocytoma P815, gene P1A directs the expression of antigens P815A and B which are the target of a T cell- mediated rejection response in syngeneic animals. This gene is expressed at a high level in various tumors, but is silent in normal tis- sues except testis and placenta; its activation is thus possibly related to malignant transformation. An anti-synthetic peptide rabbit anti- serum reacted by immunoblotting with a cellular protein migrating near 40 kDa on SDS-PAGE. The immunoreactive protein was detected only in lysates from cells which express antigen PS15A: P1.HTR mastoeytoma cells and, after transfection with cosmids car- rying the P1A gene, the antigen-loss variant P0.HTR cells and DAP-3 H-2L d fibroblasts. The identity of this protein as the P1A gene product was confirmed by cell-free transcription-translation of the P1A eDNA, the product of which also migrated near 40 kDa in SDS-PAGE and was captured by protein A-Sepharose in the presence of the antiserum. Subcellular fractionation by differential and isopycnic centrifugation indicated that the P1A protein is associated with cytoplasmic membranes demonstrating a broad distribution with respect to size and density. Immunofluorescence microscopy also revealed a cytoplasmic signal, particularly intense in small vesi- cles, which coincides with that produced by an anti-mouse type I collagen guinea pig antiserum except near the cell periphery where the P1A signal is weaker. We conclude that the P1A protein is bound to membranes of the secretory pathway, at a concentration which goes increasing from the endoplasmic reticulum to secretion vesicles. The N-terminal portion of the protein was readily removed by proteolytic enzymes in the absence of detergent, suggesting a localization at the cytoplasmic surface. The P1A protein is renewed with a half-life of 50 min and is readily phosphorylated upon metabolic labeling of mastocytoma cells with [32p]-orthophosphate. When immunoisolated from cells lysed with Nonldet P-40, the P1A protein is accompanied by a 62-kDa protein which also exists in a phosphorylated form. Thus, it is probably associated with another phosphoprotein, either as a stable functional unit, or as a dissociable complex. mouse mastocytoma P815 / tumor rejection antigen / subcellular topology / phosphorylation Introduction Gene PIA codes for tumor rejection antigens P815A and B [18] of mouse mastocytoma P815, a tumor induced by methylcholanthrene in a DBA/2 mouse. These antigens are recognized by DBA/2 cytolytic T lymphocytes (CTL). They play a major role in the immune rejection of tumor P815, since tumor cells that escape immune rejection in vivo are often found to be resistant to anti-P815A and B CTL clones [16]. An antigenic nonapeptide derived from the P1A pro- tein is presented by H-2 molecule L a to the anti-P815A and B CTL clones. These two CTL clones appear to recognize two distinct epitopes borne by the same peptide. Indeed, a mutation in the peptide-coding sequence renders P815 cells resistant to anti-A but not anti-B CTL [10]. Gene P1A is highly expressed in different tumors, but is silent in most normal tissues, with the exceptiotl of tes- tis and placenta. Activation of gene P1A is linked to tumoral transformation of mast cells since a P1A-negative mast cell line can be transformed by transfection of onco- gene v-Ha-ras, with 4/8 of the tumors obtained in mice expressing gene P1A (manuscript in preparation). The function of the P1A protein remains unknown. Its associa- tion with the transformed phenotype suggests a function in growth regulation, and a possible involvement in the process of oncogenesis. We attempted to gain insight into the function by defining the biochemical characteristics of the P1A protein in the cell and investigating its subcellu- lax distribution. Materials and methods Cell lines P1 is a clonai cell line derived from mouse mastocytoma P815 [17]. The variant Pl.ist A-B-, isolated from ascite of a mouse injected intraperitoneally with P1 cells [16], underwent a dele- tion covering the entire P1A gene [10]. The variant Pl.isc A-B-, isolated from cell line P1 by in vitro selection with a CTL clone directed against antigen P815A [16], carries a deletion that cov- ers exon 1 and part of the promoter of gene PIA, but leaves exons 2 and 3 intact [10]. The highly transfectable variant PI.HTR was obtained from P1 [19]. The variant P0.HTR derives from P1.HTR by immunoselection with CTL clones directed against antigen P815A [18]; a mutation in exon 1 in gene P1A at nucleotide 390 transforms a TGG codon into TAG stop codon [10]. The L fibroblastic cells DAP-3 transfected with a plasmid coding for H-2L d (abbreviated DAP-3) were kindly provided by K Ozato (NIH, Bethesda, MD) [11]. These cells were transfected with a cosmid containing the P1A gene and then designated DAP-3 PIA [18].