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Mechanisms of p34cdc2 regulation
S Atherton-Fessler1, L L Parker, R L Geahlen
1Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Molecular and Cellular Biology
|March 1, 1993
Summary
Phosphorylation of human p34cdc2 at Tyr-15 inhibits kinase activity, but not ATP binding. Inactivation by Thr-161 mutation also does not affect nucleotide binding, suggesting a catalytic defect.
Area of Science:
- Molecular Biology
- Enzymology
- Cell Cycle Regulation
Background:
- Human p34cdc2 kinase activity is regulated by phosphorylation.
- Negative regulation occurs at Thr-14 and Tyr-15 within the nucleotide binding domain.
- Positive regulation involves cyclin-dependent phosphorylation at Thr-161.
Purpose of the Study:
- To investigate the mechanism of p34cdc2 kinase inactivation.
- To determine if phosphorylation or mutation affects nucleotide binding.
- To elucidate the role of Thr-161 in p34cdc2/cyclin B complex activity.
Main Methods:
- Site-directed mutagenesis of p34cdc2 at Thr-161.
- Modification of p34cdc2 active site with MgATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA).
- Assessing competitive inhibition of FSBA modification by ATP.
Main Results:
- Tyrosine-phosphorylated p34cdc2 showed a slightly higher Km for FSBA modification (241 microM) compared to wild-type (148 microM).
- ATP competitively inhibited FSBA modification of both phosphorylated and unphosphorylated p34cdc2.
- Mutation of Thr-161 did not significantly affect p34cdc2 nucleotide binding ability.
Conclusions:
- Inhibition of p34cdc2 kinase activity by Tyr-15 phosphorylation or Thr-161 mutation is not due to impaired nucleotide binding.
- The findings suggest the defect in kinase activity resides at the catalytic step.
- Regulation of p34cdc2 kinase activity involves mechanisms beyond simple nucleotide binding inhibition.