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Biochemical characterization of p16INK4- and p18-containing complexes in human cell lines
F D Ragione1, G L Russo, A Oliva
1Institute of Biochemistry of Macromolecules, Medical School, Second University of Naples, Italy.
Abstract:
The regulation of the D-type cyclin-dependent kinase (CDK4 and CDK6) activity appears to be the key step in the progression of eukaryotic cells through the G1 cell cycle phase. One of the mechanisms involved in this process is the binding of some small proteic inhibitors, with a molecular mass ranging between 14 and 20 kDa, to these CDKs. We have evaluated the amount of two such inhibitors, namely p16(INK4) and p18, in normal and transformed cells, as well as the biochemical features of the macromolecular complexes containing these proteins. The results obtained indicated that (i) p18 gene expression, unlike p16(INK4) gene, is not regulated by pRb status, (ii) no evident relationship exists between the expression of p16(INK4) and p18 genes, (iii) significant amounts of the two proteins are not bound to CDKs but occur as free molecules, (iv) each inhibitor forms a complex with the CDK protein with a 1:1 stoichiometry, and (v) a competition exists between cyclin D and the inhibitor protein toward the CDK protein resulting in the absence of detectable cellular free kinase. Moreover, employing the human native partially purified p16(INK4)or the pure recombinant protein, we have been able to demonstrate in vitro the dissociation of CDK4-cyclin D1 complex and the formation of CDK4-p16(INK4) bimolecular complex. Our findings suggest that during the cell division cycle the members of the p16(INK4) protein family and cyclin Ds compete for binding to CDK4/CDK6 and that their quantitative ratio is essential for G1 --> S transition.
Insights
Cell cycle regulators p16(INK4) and p18 compete with cyclin D for CDK4/CDK6 binding. Their quantitative ratio is crucial for G1 to S phase transition in eukaryotic cells.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Cell cycle progression is tightly regulated, with D-type cyclin-dependent kinases (CDK4 and CDK6) playing a critical role in the G1 phase.
- Small proteic inhibitors (14-20 kDa) bind to CDKs, modulating their activity.
Purpose of the Study:
- To evaluate the levels of p16(INK4) and p18 inhibitors in normal and transformed cells.
- To characterize the biochemical properties of macromolecular complexes involving these inhibitors and CDKs.
Main Methods:
- Quantitative analysis of p16(INK4) and p18 protein levels.
- Biochemical characterization of protein complexes.
- In vitro studies using purified p16(INK4) and CDK4-cyclin D1 complexes.
Main Results:
- p18 gene expression is independent of pRb status, unlike p16(INK4).
- No direct correlation exists between p16(INK4) and p18 gene expression.
- Both free and CDK-bound forms of p16(INK4) and p18 exist, with inhibitors forming 1:1 complexes with CDKs.
- Competition between cyclin D and inhibitors for CDK binding was observed, leading to the absence of free kinase.
- In vitro, p16(INK4) dissociates CDK4-cyclin D1 complexes and forms a bimolecular complex with CDK4.
Conclusions:
- Members of the p16(INK4) family and cyclin Ds compete for binding to CDK4/CDK6 during the cell cycle.
- The relative abundance of these competing proteins is essential for regulating the G1 to S phase transition.