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Because of the broad clinical interest which tissue polypeptide antigen (TPA) has attracted as a tumor marker, human cell lines and human tissues have been analyzed for TPA expression using immunofluorescence microscopy. Epithelial cell lines including HeLa, MCF‐7, and A‐431 are recognized by TPA antibodies whereas human lines of non‐epithelial origin are not. The positive staining patterns coincide with keratin‐type intermediate filaments of the cytoskeleton. On tissue sections a subset of epithelial cells including uterine epithelium, bile duct cells in liver and tumor cells in breast carcinoma are strongly positive; cells of the squamous epithelia of skin and tongue as well as cells of non‐epithelial origin are negative. In immunoblots of human epidermis, human tongue mucosa, human hair follicles, Detroit 562 cells, HeLa cells, MCF‐7 and RT‐4 cells, only keratins 8, 18 and 19 show TPA antigenicity. Conversely a TPA preparation is recognized by various antibodies known to react with keratins, including alpha‐IFA, KG 8.13.2 and two antibodies which recognize keratins 18 (CK2) and 19, respectively. Our results thus relate TPA to human keratins 8, 18 and 19 which are known cytoskeletal components in both normal and malignant epithelial cells of simple and non‐squamous origin. We speculate that the elevated levels of circulating TPA antigenicity present in the sera of patients with carcinoma, which are often used to monitor tumor progression, correspond to soluble proteolytic fragments originating from this particular keratin subgroup.

Abstract. We describe a novel approach to study tyrosine-phosphorylated (PY) integrins in cells transformed by virally encoded tyrosine kinases. We have synthesized a peptide (PY/~ peptide) that represents a portion of the cytoplasmic domain of the/~1 integrin subunit and is phosphorylated on the tyrosine residue known to be the target of oncogenic tyrosine kinases. Antibodies prepared against the PY/3~ peptide, after removal of cross-reacting antibodies by absorption and affinity purification, recognized the PYfll peptide and the tyrosine-phosphorylated form of the intact/~ subunit, but did not bind the nonphosphorylated/~ peptide, the nonphosphorylated/~ subunit or other unrelated tyrosine-phosphorylated proteins. The anti-PY/~I antibodies labeled the podosomes of Rous sarcoma virus-transformed fibroblasts, but did not detectably stain nontransformed fibroblasts. The localization of the tyrosine phosphorylated ~ subunit appeared distinct from that of the/~1 subunit. Adhesion plaques were stained by the anti-/~l subunit antibodies in Rous sarcoma virus-transformed fibroblasts plated on fibronectin, whereas neither podosomes nor adhesion plaques were labeled on vitronectin or on uncoated plates. Anti-phosphotyrosine antibodies labeled podosomes, adhesion plaques and cell-cell boundaries regardless of the substratum. One of the SH2 domains of the p85 subunit of phosphatidylinositol-3-kinase bound to the PY~I peptide, but not to the nonphosphorylated ~1 cytoplasmic peptide. Other SH2 domains did not bind to the PY~1 peptide. These results show that the phosphorylated form of the ~ integrin subunit is detected in a different subcellular localization than the nonphosphorylated form and suggest that the phosphorylation on tyrosine of the ~ subunit cytoplasmic domain may affect cellular signaling pathways. INTEGRINS are a family of transmembrane proteins composed of an c~ and/3 subunit. Many of the integrins are receptors for extracellular matrix proteins and mediate cell adhesion to matrices (for reviews, see ' Ruoslahti, 1991;Hynes, 1992). The//~ integrin subunit combines with several c~ subunits to form receptors for extracelhlar matrix proteins such as fibronectin, laminin, and collagens. The cytoplasmic domains of the integrins are thought to bind cytoskeletal and other intraceilular components. The ~1 integrin subunit has been shown to interact with talin ) and ce-actinin (Otey et al., 1990(Otey et al., , 1993 in vitro. Furthermore, the cytoplasmic tail is necessary and sufficient for ~1 integrins to localize in adhesion plaques (Solowska et al., 1989; l_aFlamme et al., 1992). Cell transformation by vitally encoded tyrosine kinases has been shown to cause increased phosphorylation on tyrosine of the ~1 subunit cytoplasmic domain (Hirst et al., 1986). The transforming protein of Rous sarcoma virus (RSV),I p60 v~'c, is a cytoplasmic tyrosine kinase with constitutive activity . Fibroblasts transformed by RSV express high levels of p60v-s% which causes extensive protein tyrosine phosphorylation of cellular proteins ...

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