Related Experiment Videos
Regulation of the retinoblastoma protein-related p107 by G1 cyclin complexes
R L Beijersbergen1, L Carlée, R M Kerkhoven
1Division of Molecular Carcinogenesis, Netherlands Cancer Institute, Amsterdam.
Abstract:
The orderly progression through the cell cycle is mediated by the sequential activation of several cyclin/cyclin-dependent kinase (cdk) complexes. These kinases phosphorylate a number of cellular substrates, among which is the product of the retinoblastoma gene, pRb. Phosphorylation of pRb in late G1 causes the release of the transcription factor E2F from pRb, resulting in the transcriptional activation of E2F-responsive genes. We show here that phosphorylation of the pRb-related p107 is also cell cycle regulated. p107 is first phosphorylated at 8 hr following serum stimulation of quiescent fibroblasts, which coincides with an increase in cyclin D1 protein levels. Consistent with this, we show that a cyclin D1/cdk4 complex, but not a cyclin E/cdk2 complex, can phosphorylate p107 in vivo. Furthermore, phosphorylation of p107 can be abolished by the overexpression of a dominant-negative form of cdk4. Phosphorylation of p107 results in the loss of the ability to associate with E2F-4, a transcription factor with growth-promoting and oncogenic activity. A p107-induced cell cycle block can be released by cyclin D1/cdk4 but not by cyclin E/cdk2. These data indicate that the activity of p107 is regulated by phosphorylation through D-type cyclins.
Insights
Cell cycle progression involves cyclin/cyclin-dependent kinase (cdk) complexes. This study shows that D-type cyclins regulate p107 phosphorylation, impacting its association with transcription factor E2F-4 and cell cycle control.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Cell cycle progression relies on sequential activation of cyclin/cyclin-dependent kinase (cdk) complexes.
- Phosphorylation of the retinoblastoma gene product (pRb) by these complexes releases transcription factor E2F, activating target genes.
- The pRb-related protein p107 also plays a role in cell cycle regulation.
Purpose of the Study:
- To investigate the cell cycle regulation of p107 phosphorylation.
- To identify the specific cyclin/cdk complexes involved in p107 phosphorylation.
- To determine the functional consequences of p107 phosphorylation on its interaction with transcription factors and cell cycle progression.
Main Methods:
- Serum stimulation of quiescent fibroblasts to induce cell cycle progression.
- Western blotting to detect protein levels of cyclin D1 and phosphorylated p107.
- In vivo phosphorylation assays using cyclin D1/cdk4 and cyclin E/cdk2 complexes.
- Overexpression of dominant-negative cdk4 to inhibit p107 phosphorylation.
- Co-immunoprecipitation to assess the association between p107 and E2F-4.
- Cell cycle analysis to evaluate the effect of p107 phosphorylation on cell cycle progression.
Main Results:
- p107 phosphorylation is cell cycle regulated, occurring 8 hours after serum stimulation, coinciding with increased cyclin D1 levels.
- Cyclin D1/cdk4 complexes, but not cyclin E/cdk2, phosphorylate p107 in vivo.
- Overexpression of dominant-negative cdk4 abolishes p107 phosphorylation.
- Phosphorylation of p107 leads to the dissociation of the transcription factor E2F-4.
- A p107-induced cell cycle arrest is released by cyclin D1/cdk4, but not by cyclin E/cdk2.
Conclusions:
- p107 phosphorylation is regulated by D-type cyclins, specifically through the cyclin D1/cdk4 complex.
- Phosphorylation of p107 by cyclin D1/cdk4 disrupts its association with E2F-4, influencing cell cycle progression.
- These findings highlight a novel regulatory mechanism for p107 activity in controlling cell cycle progression.