Polo-like kinases (PLKs) have an important role in several stages of mitosis. They contribute to the activation of cyclin B/Cdc2 and are involved in centrosome maturation and bipolar spindle formation at the onset of mitosis. PLKs also control mitotic exit by regulating the anaphase-promoting complex (APC) and have been implicated in the temporal and spatial coordination of cytokinesis. Experiments in budding yeast have shown that the PLK Cdc5 may be controlled by the DNA damage checkpoint. Here we report the effects of DNA damage on Polo-like kinase-1 (Plk1) in a variety of human cell lines. We show that Plk1 is inhibited by DNA damage in G2 and in mitosis. In line with this, we show that DNA damage blocks mitotic exit. DNA damage does not inhibit the kinase activity of Plk1 mutants in which the conserved threonine residue in the T-loop has been changed to aspartic acid, suggesting that DNA damage interferes with the activation of Plk1. Significantly, expression of these mutants can override the G2 arrest induced by DNA damage. On the basis of these data we propose that Plk1 is an important target of the DNA damage checkpoint, enabling cell-cycle arrests at multiple points in G2 and mitosis.

The cyclin-dependent kinase inhibitor p21 is required for a sustained G 2 arrest after activation of the DNA damage checkpoint. Here we have addressed the mechanism by which p21 can contribute to this arrest in G 2 . We show that p21 blocks the activating phosphorylation of Cdc2 on Thr 161 . p21 does not interfere with the dephosphorylation of two inhibitory phosphorylation sites on Cdc2, Thr 14 and Tyr 15 , indicating that p21 targets a different event in Cdc2 activation as the well described DNA damage checkpoint pathway involving Chk1 and Cdc25C. Taken together our data show that a cell is equipped with at least two independent pathways to ensure efficient inhibition of Cdc2 activity in response to DNA damage, influencing both positive and negative regulatory phosphorylation events on Cdc2.

In human breast cancer, c‐Src activity is elevated compared to normal breast tissue. It is not yet known whether this increase in c‐Src activity is accompanied by an increase in c‐Src protein expression. In this study, c‐Src activity and protein expression were determined in a series of human breast cancers and in normal breast tissue, using immune complex kinase assays and immunoblotting. As the heterogeneity of breast cancer is not taken into account in these biochemical experiments, immunohistochemistry was also used to distinguish between normal and malignant cells. In human breast cancers, the c‐Src activity is increased 4‐ to 30‐fold, compared with normal breast tissue. This enhanced activity is accompanied by an increase in c‐Src protein expression, as shown by both immunoblotting and immunohistochemistry. Immunohistochemistry indicates that the majority of c‐Src appears to be concentrated around the nucleus in malignant cells, whereas in normal cells, it is distributed more evenly in the cytoplasm. These data confirm that c‐Src activity is increased in human breast cancer. In addition, this study provides strong immunohistochemical evidence that the c‐Src protein is also overexpressed, enabling a distinction to be made between normal and malignant cells.

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Overexpression of EGFR and c-erbB2 frequently occurs in human breast cancers, correlating with poor prognosis. Here we show that overexpression of EGFR and c-erbB2 in cell lines increases cell migration, an important step in metastasis formation. The effect of EGFR on migration is dependent on the addition of EGF to the cells. In contrast, c-erbB2 seems to act independently of its ligand in these assays. Overexpression of this receptor is sufficient to induce cell migration. In addition, we investigated the involvement of a number of signal transduction pathways known to be activated by the EGFR. We found that inactivation of MAPKK results in a decreased migration, while inactivation of PI3K increases migration.z 1998 Federation of European Biochemical Societies.