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Calmodulin binds to p21(Cip1) and is involved in the regulation of its nuclear localization
M Taulés1, A Rodríguez-Vilarrupla, E Rius
1Departament de Biologia Cel.lular i Anatomia Patològica, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Facultat de Medicina, Universitat de Barcelona, 08036 Barcelona, Spain.
Abstract:
p21(Cip1), first described as an inhibitor of cyclin-dependent kinases, has recently been shown to have a function in the formation of cyclin D-Cdk4 complexes and in their nuclear translocation. The dual behavior of p21(Cip1) may be due to its association with other proteins. Different evidence presented here indicate an in vitro and in vivo interaction of p21(Cip1) with calmodulin: 1) purified p21(Cip1) is able to bind to calmodulin-Sepharose in a Ca(2+)-dependent manner, and this binding is inhibited by the calmodulin-binding domain of calmodulin-dependent kinase II; 2) both molecules coimmunoprecipitate when extracted from cellular lysates; and 3) colocalization of calmodulin and p21(Cip1) can be detected in vivo by electron microscopy immunogold analysis. The carboxyl-terminal domain of p21(Cip1) is responsible for the calmodulin interaction, since p21(145-164) peptide is also able to bind calmodulin and to compete with full-length p21(Cip1) for the calmodulin binding. Because treatment of cells with anti-calmodulin drugs decreases the nuclear accumulation of p21(Cip1), we hypothesize that calmodulin interaction with p21(Cip1) is important for p21(Cip1), and in consequence for cyclin D-Cdk4, translocation into the cell nucleus.
Insights
The protein p21 (also known as Cip1) interacts with calmodulin, a calcium-binding protein. This interaction is crucial for p21 and cyclin D-Cdk4 complex nuclear translocation, impacting cell cycle regulation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- p21 (Cip1) is a known inhibitor of cyclin-dependent kinases.
- p21 (Cip1) also plays a role in cyclin D-Cdk4 complex formation and nuclear translocation.
- The precise mechanisms governing p21 (Cip1)'s dual functions are under investigation.
Purpose of the Study:
- To investigate the potential interaction between p21 (Cip1) and calmodulin.
- To elucidate the role of calmodulin in the nuclear translocation of p21 (Cip1) and cyclin D-Cdk4 complexes.
Main Methods:
- In vitro binding assays using calmodulin-Sepharose.
- Coimmunoprecipitation from cellular lysates.
- In vivo colocalization studies using electron microscopy immunogold analysis.
- Peptide competition assays to identify the calmodulin-binding domain of p21 (Cip1).
Main Results:
- Purified p21 (Cip1) binds to calmodulin in a calcium-dependent manner, inhibited by calmodulin-dependent kinase II.
- p21 (Cip1) and calmodulin coimmunoprecipitate from cell extracts.
- Calmodulin and p21 (Cip1) colocalize within cells.
- The carboxyl-terminal domain of p21 (Cip1) mediates calmodulin binding.
- Anti-calmodulin drugs reduce nuclear accumulation of p21 (Cip1).
Conclusions:
- p21 (Cip1) directly interacts with calmodulin both in vitro and in vivo.
- Calmodulin binding is essential for the nuclear translocation of p21 (Cip1).
- Calmodulin-mediated nuclear import of p21 (Cip1) is critical for cyclin D-Cdk4 complex translocation and cell cycle regulation.