Temporal inhibition of calmodulin in the nucleus

Calmodulin (CaM) acts as a primary mediator of calcium signaling by interac ting with target proteins. We have previously shown that nuclear CaM is cri tical 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) 22 3-228). To evaluate the role of CaM in the nucleus specifically during S ph ase of the cell cycle, a motif which stabilizes the mRNA only during S phas e 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 st ructure provides stability of the mRNA only during S phase, thereby restric ting CaM inhibitor expression to S phase. The inhibitor accumulates in the nucleus, particularly in the nucleoli. Flow cytometric analysis demonstrate d that the CaM inhibitor is expressed in S and G2. Transfected cells show g rowth inhibition and a reduction in DNA synthesis. The CaM inhibitor peptid e is a versatile reagent that allows spatial as well as temporal dissection of calmodulin function. (C) 1998 Elsevier Science B.V. All rights reserved .