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Temporal inhibition of calmodulin in the nucleus
K L King1, K M Moreira, G F Babcock
1Department of Molecular and Cellular Physiology, University of Cincinnati Medical Center, OH 45267-0576, USA.
Abstract:
Calmodulin (CaM) acts as a primary mediator of calcium signaling by interacting with target proteins. We have previously shown that nuclear CaM is critical for cell cycle progression using a transgene containing four repeats of a CaM inhibitor peptide and nuclear targeting signals (J. Wang et al., J. Biol. Chem. 270 (1995) 30245 30248; Biochim. Biophys. Acta 1313 (1996) 223-228). To evaluate the role of CaM in the nucleus specifically during S phase of the cell cycle, a motif which stabilizes the mRNA only during S phase was included in the transgene. The CaM inhibitor mRNA transcript contains a self-annealing stem-loop derived from histone H2B at the 3' end. This structure provides stability of the mRNA only during S phase, thereby restricting CaM inhibitor expression to S phase. The inhibitor accumulates in the nucleus, particularly in the nucleoli. Flow cytometric analysis demonstrated that the CaM inhibitor is expressed in S and G2. Transfected cells show growth inhibition and a reduction in DNA synthesis. The CaM inhibitor peptide is a versatile reagent that allows spatial as well as temporal dissection of calmodulin function.
Insights
Nuclear calmodulin (CaM) is crucial for cell cycle progression. A novel S-phase-specific CaM inhibitor peptide was developed to precisely study CaM
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Calmodulin (CaM) is a key calcium-binding protein mediating cellular signaling pathways.
- Nuclear CaM plays a critical role in cell cycle progression.
- Previous studies utilized a constitutively expressed nuclear CaM inhibitor peptide.
Purpose of the Study:
- To investigate the specific role of nuclear calmodulin during the S phase of the cell cycle.
- To develop a tool for temporal and spatial dissection of CaM function.
- To restrict CaM inhibitor expression to S phase using a novel mRNA stabilization motif.
Main Methods:
- Construction of a transgene encoding a CaM inhibitor peptide with an S-phase-specific mRNA stabilization element (histone H2B 3' stem-loop).
- Transfection of cells with the engineered transgene.
- Nuclear and nucleolar localization of the CaM inhibitor peptide.
- Flow cytometry to analyze cell cycle distribution and inhibitor expression.
- Assessment of cell growth and DNA synthesis.
Main Results:
- The engineered transgene restricted CaM inhibitor expression to S and G2 phases of the cell cycle.
- The CaM inhibitor accumulated in the nucleus, particularly in nucleoli.
- Transfected cells exhibited significant growth inhibition.
- A notable reduction in DNA synthesis was observed in cells expressing the CaM inhibitor.
Conclusions:
- The S-phase-specific CaM inhibitor peptide effectively targets nuclear CaM function during DNA replication.
- This tool allows for precise temporal and spatial analysis of calmodulin's role in cell cycle regulation.
- Nuclear CaM is essential for normal progression through S phase and DNA synthesis.