We have determined how the phosphorylation of the retinoblastoma family (pRb, p107, and p130) is governed in individual cell cycle phases of Daudi B-cells during cell cycle exit triggered by ␣-interferon (␣-IFN). ␣-IFN causes dephosphorylation of pRb and loss of p130 phosphorylated Form 3. However, the change in p130 phosphorylation in response to ␣-IFN occurs before dephosphorylation of pRb is complete because loss of p130 Form 3 occurs throughout the cell cycle prior to complete arrest in G 1 , whereas pRb is dephosphorylated only in G 1 . In contrast, p107 is dephosphorylated and is then depleted from cells as they exit the cell cycle. p130, predominantly in Form 1, and hypophosphorylated pRb bind an E2F DNA binding site; p130 complexes E2F-4, whereas pRb binds both E2F-4 and E2F-1. The phosphorylated forms of E2F-4 that bind to the E2F DNA site are different from hyperphosphorylated E2F-4, which predominates in primary hemopoietic cells in G 0 . We conclude that although cell cycle arrest induced by ␣-IFN may be mediated in part by formation of a complex containing p130 and E2F-4, ␣-IFN does not induce hyperphosphorylation of E2F-4, which characterizes primary hemopoietic cells in G 0 .

We have recently shown that induction of the p53 tumour suppressor protein by the small-molecule RITA (reactivation of p53 and induction of tumour cell apoptosis; 2,5-bis(5-hydroxymethyl-2-thienyl)furan) inhibits hypoxia-inducible factor-1α and vascular endothelial growth factor expression in vivo and induces p53-dependent tumour cell apoptosis in normoxia and hypoxia. Here, we demonstrate that RITA activates the canonical ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related DNA damage response pathway. Interestingly, phosphorylation of checkpoint kinase (CHK)-1 induced in response to RITA was influenced by p53 status. We found that induction of p53, phosphorylated CHK-1 and γH2AX proteins was significantly increased in S-phase. Furthermore, we found that RITA stalled replication fork elongation, prolonged S-phase progression and induced DNA damage in p53 positive cells. Although CHK-1 knockdown did not significantly affect p53-dependent DNA damage or apoptosis induced by RITA, it did block the ability for DNA integrity to be maintained during the immediate response to RITA. These data reveal the existence of a novel p53-dependent S-phase DNA maintenance checkpoint involving CHK-1.

A putative protein kinase gene (PfPK2) has been isolated from the human parasite Plasmodium fakiparum by using a mixed oligonucleotide pool which corresponds to a highly conserved region of serinelthreonine protein kinases. The complete nucleotide sequence of 5 kb suggests the existence of a second transcriptional unit besides that of the PfPK2 gene, separated by a highly (A + T)-rich region and transcribed in a different orientation. No intron sequence exists in PfPK2. The predicted amino acid sequence of PfPK2 contains features characteristic of eukaryotic serinelthreonine protein kinases. Within its putative catalytic domain it shares 33%, 30%, and 28% amino acid identities with rat calcium-calmodulin-dependent protein kinase, human protein kinase C, and bovine CAMP-dependent protein kinase, respectively. Outside the catalytic domain, however, PfPK2 has no homology with regulatory domains of other protein kinases, indicating PfPK2 might be modulated by signals different from those of higher eukaryotes or might be associated with other regulatory subunits. Using a specific antiserum raised in rabbits against a recombinant fragment of the protein expressed in Escherichiu coli, PfPK2 was found to be expressed in a stage-specific fashion and mainly localized in the parasitic membrane.Phosphorylation of specific proteins by protein kinases has been recognized as one of the most important strategies for the regulation of protein and enzyme activity in the transduction of environmental, developmental, and metabolic signals in animals as well as in lower-order eukaryotes [l, 21. There are few, if any, physiological processes in eukaryotes that are not dependent on protein phosphorylation. Protein kinases represent a large family of over a hundred regulatory proteins. In spite of the tremendous diversity of these enzymes, all share a common catalytic domain which typically extends about 260 amino acid residues [3], including the regions for ATP binding, protein substrate recognition, and catalysis [4, 51. The majority of protein kinases fall into two classes depending on their ability to phosphorylate either serine/threonine or tyrosine. These kinases can be further grouped into subfamilies based on characteristic structural features, regulatory ligand, and cellular function [6].

Cloning and sequencing of the pho2 gene which codes for a specific p‐nitrophenylphosphatase from Schizosaccharomyces pombe is described. The gene has an open contiguous reading frame of 269 amino acids corresponding to a protein with a molecular mass of 29.5 kDa and a calculated pI of 6.6. The sequence reveals four regions that share significant sequence similarity with the corresponding gene PHO13 of Saccharomyces cerevisiae. Purification of the enzyme to apparent homogeneity is reported. The amino acid composition of the purified protein matches well the values predicted from the nucleotide sequence. On SDS/polyacrylamide gels, the enzyme runs as a protein with a molecular mass of 33 kDa, and by Sephadex chromatography under non‐denaturing conditions as 70 kDa. This indicates that the enzyme is a homodimer in its native form. The enzyme is not glycosylated. Its activity is stimulated by Mg2+ and inhibited by Zn2+. The available data on p‐nitrophenylphosphatase do not give any clues to its biological role and its physiological substrates.