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A method for the isolation of tyrosine kinases substrates was developed. The method takes advantage of immuno-affinity purification of an entire set of proteins phosphorylated by tyrosine kinases, followed by generation of antisera against the purified protein pool and immunological screening of bacterial expression libraries with these antisera. By applying this methodology to the study of the phosphorylation events triggered by activation of the epidermal growth factor receptors, we have isolated several cDNAs encoding novel putative tyrosine kinase substrates. One of these cDNAs encodes radixin, a protein belonging to the band 4.1 family of proteins and highly related to ezrin and moesin. We demonstrated that, despite a high degree of relatedness, these three proteins exhibit a distinct receptor-specific pattern of phosphorylation, raising the possibility that they might mediate receptor-specific cellular changes. In addition the generation of antibodies specific for either radixin, ezrin or moesin allowed us to show that a previously described tumor transplantation antigen is indeed ezrin, thus implicating this protein in the determination of the biological phenotype of certain tumors.

The nuclear phosphoprotein p53 is an important regulator of cell proliferation in normal cells. Interestingly, the gene encoding p53 has usually undergone mutations in a wide range of tumor types. Recent studies of the p53 gene in Burkitt's lymphomas have demonstrated that mutations are extremely common, and in fact it is rare that both alleles of the p53 gene in these tumors are not inactivated by mutation or deletion. We present here genetic data regarding the status of the p53 gene in the Burkitt lymphoma cell line, Raji. As is typical for this type of tumor, both alleles have undergone point mutations. Further, statistical analysis of available data from a large number of Burkitt's lymphomas indicates an apparent tumor-specific distribution of p53 mutations. The possibility that specific mutations of the p53 gene may be important for different tumor types is discussed.

The wild-type (wt) human tumor-suppressor gene product, p53, and its mutant form have been analysed in an in vivo system in which the inducible expression of wt p53 results in growth arrest in the G1 phase of the cell cycle. Two major pools of p53 are detected after wt p53 expression by their differential reactivity with the p53 monoclonal antibodies PAb 421 and 1801 as well as the mutant and wt-specific monoclonal antibodies PAb 240 and 1620; one pool contains wt and mutant p53 and is characterized as having a mutant conformation, whereas the other pool contains only wt p53 with a wt conformation. As G1 arrest is entered, the amount of wt p53 associated with the mutant pool decreases, such that by 12 h free wt and mutant p53 are the major pools. Two-dimensional gel analysis of the p53 pools revealed that free wt p53 is phosphorylated to a greater degree than mutant p53, which correlated with the loss of the PAb 421 epitope on wt p53. In summary, the ability of wt p53 to exert an antiproliferative effect correlates with the presence of a unique conformational state of wt p53 characterized by increased phosphorylation and the loss of both the PAb 421 epitope and association with mutant p53 pool, whereas mutant p53 is unable to assume this conformational state.

The transformation related protein p53 has been implicated in the process of normal cell proliferation and neoplastic transformation. In this study, the influence of wild type human p53 on cell proliferation was examined. Plasmid constructs encoding the wild type human p53 and various mutant p53 cDNAs, driven by the mouse mammary tumor virus (MMTV) promoter linked to the dominant biochemical selection marker gpt, were used in a colony forming assay employing SV40 transformed HR8 hamster cells. Plasmids encoding wild type p53 drastically reduced the number of gpt+ colonies obtained after transfection, whereas the mutant forms of p53 had no effect. Stable clonal hamster cell lines that constitutively express wild type p53 were isolated and found to have altered growth characteristics (i.e. lower saturation densities, increased doubling times). These findings are consistent with the notion that wild type p53 protein could function as a growth suppressor. The potential role of p53 in the normal cell cycle and in the transformation process is discussed.

The nuclear phosphoprotein p53 occurs at elevated levels in many transformed cells. Mutant forms of mouse p53 possess enhanced transforming activity compared with wild type p53. Mutant mouse p53 proteins form complexes with the 70 kDa family of heat shock proteins (HSPs). We previously demonstrated an association between p53 and the 70 kDa HSPs in the human osteosarcoma (HOS) derived cell line HOS-SL. We report here the molecular cloning and sequencing of the p53 gene from HOS-SL cells, and demonstrate that it is in fact mutant. Further, analysis of similar HOS-derived cell lines demonstrates that they also encode the same mutant form of p53, whereas the wild type form of p53 appears to be lost in these cells. Stability studies demonstrate an increased half life of the p53 protein in these cells, in keeping with its association with the HSP 70 proteins. A potential role for this p53 mutant in the transformation process is discussed.

We report here immunological evidence for the specific association between p53 and hsp72/hsc73 heat shock proteins in a human cell line derived from an osteosarcoma. The same association between p53 and hsp72/hsc73 was observed in HOS-TE85 clone 5 from which the HOS-SL cell line was derived. This association was indicated by the co-immunoprecipitation from HOS-SL of both p53 and hsp72/hsc73 proteins observed with either an anti-p53 monoclonal antibody (PAb421) or an antiserum specifically reacting with hsp72/hsc73 heat shock proteins. Furthermore, Western blot analysis allowed us to show that hsp72/hsc73 proteins did not share an epitope with p53, confirming that the co-immunoprecipitation of p53 and hsp72/hsc73 was attributable to the physical association of the proteins. Data obtained from SDS-PAGE show that the HOS-SL cells expressed two forms of p53 with distinct molecular weights. Both forms contained several species with different isoelectric points ranging between pH 6.0 and 6.5. The data obtained from both 1D and 2D gel analyses consistently show that the p53 proteins involved in the association with hsp72/hsc73 were mainly the species that migrated with the slower mobility in the SDS dimension. The possibility is discussed that the HOS-SL p53 variant forming a complex with hsp72/hsc73 contains an activating mutation for transformation.