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S Atherton-Fessler

Publications and source records attributed to S Atherton-Fessler.

6 recordsLinked to original sources
PubMed1994

Cell cycle regulation of the p34cdc2 inhibitory kinases.

In cells of higher eukaryotic organisms the activity of the p34cdc2/cyclin B complex is inhibited by phosphorylation of p34cdc2 at two sites within its amino-terminus (threonine 14 and tyrosine 15). In this study, the cell cycle regulation of the kinases responsible for phosphorylating p34cdc2 on Thr14 and Tyr15 was examined in extracts prepared from both HeLa cells and Xenopus eggs. Both Thr14- and Tyr15- specific kinase activities were regulated in a cell cycle-dependent manner. The kinase activities were high throughout interphase and diminished coincident with entry of cells into mitosis. In HeLa cells delayed in G2 by the DNA-binding dye Hoechst 33342, Thr14- and Tyr15-specific kinase activities remained high, suggesting that a decrease in Thr14- and Tyr15- kinase activities may be required for entry of cells into mitosis. Similar cell cycle regulation was observed for the Thr14/Tyr15 kinase(s) in Xenopus egg extracts. These results indicate that activation of CDC2 and entry of cells into mitosis is not triggered solely by activation of the Cdc25 phosphatase but by the balance between Thr14/Tyr15 kinase and phosphatase activities. Finally, we have detected two activities capable of phosphorylating p34cdc2 on Thr14 and/or Tyr15 in interphase extracts prepared from Xenopus eggs. An activity capable of phosphorylating Tyr15 remained soluble after ultracentrifugation of interphase extracts whereas a second activity capable of phosphorylating both Thr14 and Tyr15 pelleted. The pelleted fraction contained activities that were detergent extractable and that phosphorylated p34cdc2 on both Thr14 and Tyr15. The Thr14- and Tyr15-specific kinase activities co-purified through three successive chromatographic steps indicating the presence of a dual-specificity protein kinase capable of acting on p34cdc2.

Animals↗
PubMed1993

Reversible tyrosine phosphorylation and cell cycle control.

In eukaryotic organisms, reversible tyrosine phosphorylation has been established as an important element in the regulation of cell growth and more recently as an essential element in the regulation of the cell division cycle. The activity of p34cdc2, a protein kinase whose activity is required for the entry of cells into mitosis, is tightly controlled by reversible phosphorylation at tyrosine 15. A complex network of interacting protein kinases and protein phosphatases regulate the state of p34cdc2 tyrosine phosphorylation and therefore the entry of cells into mitosis. In the fission yeast Schizosaccharomyces pombe, genes encoding several of these protein kinases and protein phosphatases have been obtained through genetic approaches. In this review, we will focus on the protein kinases encoded by wee1+, mik1+ and cdr1+/nim1+ and the protein phosphatases encoded by cdc25+ and pyp1+, pyp2+ and pyp3+. Homologs of many of these regulators have been identified and characterized in higher eukaryotes underscoring the importance of reversible tyrosine phosphorylation as a universal mechanism for the regulation of the cell division cycle.

CDC2 Protein Kinase↗
PubMed1993

Mechanisms of p34cdc2 regulation.

The kinase activity of human p34cdc2 is negatively regulated by phosphorylation at Thr-14 and Tyr-15. These residues lie within the putative nucleotide binding domain of p34cdc2. It has been proposed that phosphorylation within this motif ablates the binding of ATP to the active site of p34cdc2, thereby inhibiting p34cdc2 kinase activity (K. Gould and P. Nurse, Nature [London] 342:39-44, 1989). To understand the mechanism of this inactivation, various forms of p34cdc2 were tested for the ability to bind nucleotide. The active site of p34cdc2 was specifically modified by the MgATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA). The apparent Km for modification of wild-type, monomeric p34cdc2 was 148 microM FSBA and was not significantly affected by association with cyclin B. Tyrosine-phosphorylated p34cdc2 was modified by FSBA with a slightly higher Km (241 microM FSBA). FSBA modification of both tyrosine-phosphorylated and unphosphorylated p34cdc2 was competitively inhibited by ATP, and half-maximal inhibition in each case occurred at approximately 250 microM ATP. In addition to being negatively regulated by phosphorylation, the kinase activity of p34cdc2 was positively regulated by the cyclin-dependent phosphorylation of Thr-161. Mutation of p34cdc2 at Thr-161 resulted in the formation of an enzymatically inactive p34cdc2/cyclin B complex both in vivo and in vitro. However, mutation of Thr-161 did not significantly affect the ability of p34cdc2 to bind nucleotide (FSBA). Taken together, these results indicate that inhibition of p34cdc2 kinase activity by phosphorylation of Tyr-15 (within the putative ATP binding domain) or by mutation of Thr-161 involves a mechanism other than inhibition of nucleotide binding. We propose instead that the defect resides at the level of catalysis.

Adenosine↗
PubMed1992

p107wee1 is a dual-specificity kinase that phosphorylates p34cdc2 on tyrosine 15.

p107wee1 is a protein kinase that functions as a dose-dependent inhibitor of mitosis through its interactions with p34cdc2 in Schizosaccharomyces pombe. To characterize the kinase activity of p107wee1, its carboxyl-terminal catalytic domain was purified to homogeneity from overproducing insect cells. The apparent molecular mass of the purified protein (p37wee1KD) was determined to be approximately 37 kDa by gel filtration, consistent with it being a monomer. Serine and tyrosine kinase activities cofiltered with p37wee1KD, demonstrating that p107wee1 is a dual-specificity kinase. In vitro, p107wee1 phosphorylated p34cdc2 on Tyr-15 only when p34cdc2 was complexed with cyclin. Neither monomeric p34cdc2 nor a peptide containing Tyr-15 was able to substitute for the p34cdc2/cyclin complex in this assay. Furthermore, the phosphorylation of p34cdc2 by p107wee1 in vitro inhibited the histone H1 kinase activity of p34cdc2. These results indicate that p107wee1 functions as a mitotic inhibitor by directly phosphorylating p34cdc2 on Tyr-15 and that the preferred substrate for phosphorylation is the p34cdc2/cyclin complex.

CDC2 Protein Kinase↗
PubMed1991

Cyclin promotes the tyrosine phosphorylation of p34cdc2 in a wee1+ dependent manner.

The regulation of p34cdc2 was investigated by overproducing p34cdc2, cyclin (A and B) and the wee1+ gene product (p107wee1) using a baculoviral expression system. p34cdc2 formed a functional complex with both cyclins as judged by co-precipitation, phosphorylation of cyclin in vitro, and activation of p34cdc2 histone H1 kinase activity. Co-production of p34cdc2 and p107wee1 in insect cells resulted in a minor population of p34cdc2 that was phosphorylated on tyrosine and displayed an altered electrophoretic mobility. When p34cdc2 and p107wee1 were co-produced with cyclin (A or B) in insect cells, there was a dramatic increase in the population of p34cdc2 that was phosphorylated on tyrosine and that displayed a shift in electrophoretic mobility. The phosphorylation of p34cdc2 on tyrosine was absolutely dependent upon the presence of kinase-active p107wee1. Tyrosine-specific as well as serine/threonine-specific protein kinase activities co-immunoprecipitated with p107wee1. These results suggest that cyclin functions to facilitate tyrosine phosphorylation of p34cdc2 and that p107wee1 functions to regulate p34cdc2, either directly or indirectly, by tyrosine phosphorylation.

Animals↗