Polarized cell movement is an essential requisite for cancer metastasis; thus, interference with the tumor cell motility machinery would significantly modify its metastatic behavior. Protein kinase C␣ (PKC␣) has been implicated in the promotion of a migratory cell phenotype. We report that the phorbol ester-induced cell polarization and directional motility in breast carcinoma cells is determined by a 12-amino-acid motif (amino acids 313 to 325) within the PKC␣ V3 hinge domain. This motif is also required for a direct association between PKC␣ and ␤1 integrin. Efficient binding of ␤1 integrin to PKC␣ requires the presence of both NPXY motifs (Cyto-2 and Cyto-3) in the integrin distal cytoplasmic domains. A cell-permeant inhibitor based on the PKC-binding sequence of ␤1 integrin was shown to block both PKC␣-driven and epidermal growth factor (EGF)-induced chemotaxis. When introduced as a minigene by retroviral transduction into human breast carcinoma cells, this inhibitor caused a striking reduction in chemotaxis towards an EGF gradient. Taken together, these findings identify a direct link between PKC␣ and ␤1 integrin that is critical for directed tumor cell migration. Importantly, our findings outline a new concept as to how carcinoma cell chemotaxis is enhanced and provide a conceptual basis for interfering with tumor cell dissemination.An early critical step within the metastatic cascade involves cell polarization and directed cell movement. The mechanisms controlling these events, however, are poorly understood. Cell polarization signals can be triggered by the binding of cells to extracellular matrix (ECM) proteins, cytokines or chemoattractants, or ligands that are expressed on the surface of target tissues or vessels. Sensing of these extracellular signals results in a redistribution and trafficking of membrane receptors (1, 19) and/or intracellular signaling proteins, as well as the localized formation of protein complexes which can be stable or transient.Members of the integrin receptor family are vital in mediating cell matrix adhesion and have been implicated in providing the persistence and directionality for cell motility, as in wound healing (7). Specifically, a number of studies have demonstrated that the ␤-subunit cytoplasmic domains are involved in the control of cell migration. Thus, the tyrosine residues of the two NPXY motifs in ␤1 are important for directed cell migration through integrin substrate-coated filters in response to growth factor chemotactic gradients (22,23). In fact, when expressed as chimeric receptors connected to heterologous extracellular domains, such as the interleukin-2 receptor (IL-2R) tac subunit, the ␤1 integrin cytoplasmic tail acts as a dominant negative inhibitor of endogenous integrin function (4). Moreover, inhibition of integrin-ECM interactions by GRGDS peptides or inhibitory antibodies was also shown to abolish the persistence or directionality of neural crest cell movement (7).Protein kinase C (PKC) may be seen as part of the molecular machinery that deciphers...

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Previous investigations of a Li ± Fraumeni like family (Barnes et al., 1992) demonstrated that both the proband and her mother had elevated p53 protein levels in both tumour tissue and normal tissue at sites distant from the tumour, although no mutation was found in the p53 gene. In the present study two recently described functional assays for p53, an apoptotic assay and the functional assay for the separation of alleles in yeast (FASAY), have been employed to study the functional activity of p53 from this patient. The results of the apoptotic assay demonstrated that this patient had a p53 functional defect and the FASAY result suggested that this defect was in fact a germline mutation of the p53 gene. A point mutation of codon 337, which results in an amino acid substitution of a cysteine for an arginine, was demonstrated initially in cDNA and was con®rmed by sequencing of genomic DNA. This is an unusual mutation as it is in the oligomerisation domain of p53, in contrast to the majority of p53 mutations which are in the core DNA binding domain. This mutation results in a protein which still retains partial transactivational activity in the FASAY. The mutation of codon 337 is only the second reported case of a germline missense mutation occurring in the oligomerisation domain of p53.Keywords: p53; Li ± Fraumeni syndrome; oligomerisation domain; functional assaysThe tumour suppressor gene, p53, was ®rst discovered in 1979 (Lane and Crawford, 1979;Linzer and Levine, 1979). p53 is a 393 amino acid nuclear phosphoprotein which plays a central role in the cellular processes involved in the response to DNA damage (reviewed Kastan et al., 1995). It is made up of at least four domains, a transactivation domain, a central DNA binding domain, an oligomerisation domain and a regulatory domain, the latter two residing at the carboxy end of the protein.Following initial studies by Malkin et al. (1990) it is now established that germline p53 mutations are associated with Li ± Fraumeni syndrome (LFS), a dominantly inherited syndrome ®rst proposed by Fraumeni in 1969 (Li andFraumeni, 1969). However mutations in the coding region of p53 have only been con®rmed in about 50% of families with LFS (Birch et al., 1994). Families with this syndrome exhibit multiple primary neoplasms in childhood and early adulthood and the spectrum of cancers which commonly occurs in LFS families includes brain tumours, breast tumours, sarcomas, leukaemia and adrenal cortical tumours (Li et al., 1988). Families have now been recognised in which there is a similar spectrum of malignancies to LFS but the families do not ®t exactly into the criteria of LFS. These families have been named Li ± Fraumeni like (LFL) (Birch et al., 1994) and p53 germline mutations have been found in 7 ± 20% of these families (Eeles, 1995; Varley et al. manuscript in preparation). The patient who is the subject of this study is a member of a LFL family and meets the criteria for LFL families as set out by Birch et al. (1994).In 1992 Barnes et al. described a member of a LFL ...

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