IDENTIFICATION AND CHARACTERIZATION OF THE TUMOR-SPECIFIC P1A GENE-PRODUCT

In murine mastocytoma P815, gene PIA 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 tissues except testis and placenta; its activation is thus possibly related to malignant transformation. An anti-synthetic peptide rabbit antiserum 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 P815A: P1.HTR mastocytoma cells and, after transfection with cosmids carrying the P1A gene, the antigen-loss variant PO.HTR cells and DAP-3 H-2L(d) fibroblasts. The identity of this protein as the PIA gene product was confirmed by cell-free transcription-translation of the P1A cDNA, 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 vesicles, 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 [P-32]-orthophosphate. When immunoisolated from cells lysed with Nonidet 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.