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Cell cycle regulation of retinoblastoma protein phosphorylation
Abstract:
The product of the retinoblastoma susceptibility gene (Rb) is a substrate of the cell cycle-regulated cdc2 and cdk kinases. The Rb protein is phosphorylated from S through M phases of the cell cycle and is dephosphorylated in G1. In in vivo phosphorylated Rb protein, we have found ten phosphotryptic peptides, all of which could be phosphorylated by cdc2 kinase, p34cdc2, in vitro. The sites of phosphorylation for eight of the ten peptides have been mapped and they conform to the known p34cdc2 phosphorylation consensus. Although the activated p34cdc2 in mitotic cells is the major phosphorylation enzyme for Rb, the Rb kinase activity of p34cdc2 is not activated at G1/S transition. A cyclin A/p33 complex is activated at G1/S. We have assembled active cyclin B1/p34cdc2 complex in insect cells. The insect cell-derived kinase complex phosphorylates histone H1 well but exhibits a poor Rb kinase activity. These results indicate that the retinoblastoma protein is phosphorylated by distinct cyclin/kinase complexes in the cell cycle and suggest a regulation of the substrate specificity of the p34cdc2/cyclin complex.
Insights
The retinoblastoma protein (Rb) is phosphorylated by cell cycle kinases. Distinct cyclin/kinase complexes regulate Rb phosphorylation, suggesting substrate specificity controls this process.
Area of Science:
- Molecular Biology
- Cell Cycle Regulation
- Biochemistry
Background:
- The retinoblastoma protein (Rb) is a key regulator of the cell cycle.
- Rb protein phosphorylation is a critical event during cell cycle progression.
- Rb is phosphorylated by cyclin-dependent kinases (CDKs) throughout the cell cycle.
Purpose of the Study:
- To investigate the specific cyclin/kinase complexes responsible for Rb phosphorylation.
- To map the phosphorylation sites on Rb protein.
- To understand the regulation of Rb kinase activity by different cyclin/kinase complexes.
Main Methods:
- In vivo and in vitro phosphorylation assays.
- Peptide mapping and site identification.
- Expression and assembly of cyclin/kinase complexes in insect cells.
Main Results:
- Ten phosphotryptic peptides from in vivo phosphorylated Rb were identified.
- Eight of these peptides were phosphorylated by p34cdc2 kinase in vitro, conforming to the known consensus site.
- While p34cdc2 is the major Rb kinase in mitosis, its activity is not activated at G1/S transition.
- A cyclin A/p33 complex is activated at G1/S.
- Insect cell-derived cyclin B1/p34cdc2 complex showed poor Rb kinase activity but robust histone H1 kinase activity.
Conclusions:
- Rb protein is phosphorylated by distinct cyclin/kinase complexes during the cell cycle.
- Substrate specificity of p34cdc2/cyclin complexes is regulated.
- This suggests a complex regulatory mechanism for Rb phosphorylation controlling cell cycle progression.