No abstract

In the fission yeast Schizosaccharomyces pombe, the protein kinase Chk1 has an essential role in transducing a delay signal to the cell cycle machinery in the presence of DNA damage. Fission yeast cells lacking the chk1 gene do not delay progression of the cell cycle in response to damage and are thus sensitive to DNA damaging agents. We have previously shown that Chk1 is phosphorylated following DNA damage induced by a variety of agents and that this is dependent on the integrity of the DNA damage checkpoint pathway, including Rad3, the ATR homolog. Through a combination of mutagenesis and phospho-specific antibodies, we have shown that serine at position 345 (S345) is phosphorylated in vivo in response to DNA damage, and that S345 phosphorylation is required for an intact checkpoint response. We have developed a kinase assay for Chk1, and have shown that basal Chk1 kinase activity is increased in response to DNA damage and that this increase, but not the basal activity, is dependent on S345. Furthermore, we show that S345 phosphorylation is required for Chk1 to associate with Rad24, a 14-3-3 protein, upon DNA damage. These results are consistent with a model whereby Chk1 phosphorylation results in increased Chk1 kinase activity that is necessary for both checkpoint delay and cellular survival following damage to the genome. These data are similar to observations made in mammalian cells and Xenopus oocyte extracts, suggesting that mechanisms leading to Chk1 activation have been conserved in evolution.

Heparanase is an endo-␤-D-glucuronidase that degrades heparan sulfate in the extracellular matrix and on the cell surface. Human proheparanase is produced as a latent protein of 543 amino acids whose activation involves excision of an internal linker segment (Ser 110 -Gln 157 ), yielding the active heterodimer composed of 8-and 50-kDa subunits. Applying cathepsin L knock-out tissues and cultured fibroblasts, as well as cathepsin L gene silencing and overexpression strategies, we demonstrate, for the first time, that removal of the linker peptide and conversion of proheparanase into its active 8 ؉ 50-kDa form is brought about predominantly by cathepsin L. Excision of a 10-amino acid peptide located at the C terminus of the linker segment between two functional cathepsin L cleavage sites (Y156Q and Y146Q) was critical for activation of proheparanase. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry demonstrates that the entire linker segment is susceptible to multiple endocleavages by cathepsin L, generating small peptides. Mass spectrometry demonstrated further that an active 8-kDa subunit can be generated by several alternative adjacent endocleavages, yielding the precise 8-kDa subunit and/or slightly elongated forms. Altogether, the mode of action presented here demonstrates that processing and activation of proheparanase can be brought about solely by cathepsin L. The critical involvement of cathepsin L in proheparanase processing and activation offers new strategies for inhibiting the prometastatic, proangiogenic, and proinflammatory activities of heparanase.

The conserved protein kinase Chk1 mediates cell cycle progression and consequently the ability of cells to survive when exposed to DNA damaging agents. Cells deficient in Chk1 are hypersensitive to such agents and enter mitosis in the presence of damaged DNA, whereas checkpoint-proficient cells delay mitotic entry to permit time for DNA repair. In a search for proteins that can improve the survival of Chk1-deficient cells exposed to DNA damage, we identified fission yeast Msc1, which is homologous to a mammalian protein that binds to the tumor suppressor Rb (RBP2). Msc1 and RBP2 each possess three PHD fingers, domains commonly found in proteins that influence the structure of chromatin. Msc1 is chromatin associated and coprecipitates a histone deacetylase activity, a property that requires the PHD fingers. Cells lacking Msc1 have a dramatically altered histone acetylation pattern, exhibit a 20-fold increase in global acetylation of histone H3 tails, and are readily killed by trichostatin A, an inhibitor of histone deacetylases. We postulate that Msc1 plays an important role in regulating chromatin structure and that this function modulates the cellular response to DNA damage.