No abstract

Eukaryotic cell cycle progression requires the periodic activation and inactivation of a protein-serine/threonine kinase which in fission yeast is encoded by the cdc2+ gene. The activity of this gene product, p34cdc2, is controlled by numerous interactions with other proteins and by its phosphorylation state. In fission yeast, p34Cdc2 is phosphorylated on two sites, one of which has been identified as Tyrl5. Dephosphorylation of Tyrl5 regulates the initiation of mitosis. To understand more completely the regulation of p34""'2 kinase activity, we have identified the second site of phosphorylation as Thrl67, a residue conserved amongst all p34Cdc2 homologues. By analysing the phenotypes of cells expressing various position 167 mutations and performing in vitro experiments, we establish that Thrl67 phosphorylation is required for p34"""2 kinase activity at mitosis and is involved in the association of p34cdc2 with cyclin B. Dephosphorylation of Thrl67 might also play a role in the exit from mitosis.

Ezrin is a component of the microvilli of intestinal epithelial cells and serves as a major cytoplasmic substrate for certain protein‐tyrosine kinases. We have cloned and sequenced a human ezrin cDNA and report here the entire protein sequence derived from the nucleotide sequence of the cDNA as well as from partial direct protein sequencing. The deduced protein sequence indicates that ezrin is a highly charged protein with an overall pI of 6.1 and a calculated molecular mass of 69,000. The cDNA clone was used to survey the distribution of the ezrin transcript, and the 3.2 kb ezrin mRNA was found to be expressed in the same tissues that are known to express the protein and at the same relative levels. Highest expression was found in intestine, kidney and lung. The cDNA clone hybridized to DNAs from widely divergent organisms indicating that its sequence is highly conserved throughout evolution. The amino acid sequence of ezrin revealed a high degree of similarity within its N‐terminal domain to the erythrocyte cytoskeletal protein, band 4.1 and secondary structure predictions indicate that a second region of ezrin contains a long alpha‐helix, a feature also common to band 4.1. The structural similarity of ezrin to band 4.1 suggests a mechanism for the observed localization to the membrane, and a role for ezrin in modulating the association of the cortical cytoskeleton with the plasma membrane.

The Schizosaccharomyces pombe cdc5+ gene was identified in the first screen for cell division cycle mutants in this yeast. The cdc5+ gene was reported to be required for nuclear division but because of its modest elongation and leaky nature at the non‐permissive temperature, it was not investigated further. Here, we report the characterization of the single allele of this gene, cdc5‐120, in more detail. The mutant arrests with a 2N DNA content and a single interphase nucleus. Further genetic analyses suggest that cdc5+ gene function is essential in the G2 phase of the cell cycle. We have cloned and sequenced the cdc5+ gene. The deduced protein sequence predicts that Cdc5 is an 87 kDa protein and contains a region sharing significant homology with the DNA binding domain of the Myb family of transcription factors. Deletion mapping of the cdc5+ gene has shown that the N‐terminal 232 amino acids of the protein, which contain the Myb‐related region, are sufficient to complement the cdc5ts strain. A cdc5 null mutant was generated by homologous recombination. Haploid cells lacking cdc5+ are inviable, indicating that cdc5+ is an essential gene. A fusion protein consisting of bacterial glutathione S‐transferase joined in‐frame to the N‐terminal 127 amino acids of the Cdc5 protein is able to bind to DNA cellulose at low salt concentrations. This evidence suggests that cdc5+ might encode a transcription factor whose activity is required for cell cycle progression and growth during G2.

The gene encoding the actin‐related protein Arp3 was first identified in the fission yeast Schizosaccharomyces pombe and is a member of an evolutionarily conserved family of actin‐related proteins. Here we present several key findings that define an essential role for Arp3p in the functioning of the cortical actin cytoskeleton. First, mutants in arp3 interact specifically with profilin and actin mutants. Second, Arp3 localizes to cortical actin patches which are required for polarized cell growth. Third, the arp3 gene is required for the reorganization of the actin cytoskeleton during the cell cycle. Finally, the Arp3 protein is present in a large protein complex. We believe that this complex may mediate the cortical functions of profilin at actin patches in S. pombe.