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Mechanism of Ca 2+ -dependent nuclear accumulation of calmodulin

1999; National Academy of Sciences; Volume: 96; Issue: 11 Linguagem: Inglês

10.1073/pnas.96.11.6217

1091-6490

Birong Liao, Bryce M. Paschal, Katherine Luby‐Phelps,

Toxin Mechanisms and Immunotoxins

The intracellular Ca 2+ receptor calmodulin (CaM) coordinates responses to extracellular stimuli by modulating the activities of its various binding proteins. Recent reports suggest that, in addition to its familiar functions in the cytoplasm, CaM may be directly involved in rapid signaling between cytoplasm and nucleus. Here we show that Ca 2+ -dependent nuclear accumulation of CaM can be reconstituted in permeabilized cells. Accumulation was blocked by M13, a CaM antagonist peptide, but did not require cytosolic factors or an ATP regenerating system. Ca 2+ -dependent influx of CaM into nuclei was not blocked by inhibitors of nuclear localization signal-mediated nuclear import in either permeabilized or intact cells. Fluorescence recovery after photobleaching studies of CaM in intact cells showed that influx is a first-order process with a rate constant similar to that of a freely diffusible control molecule (20-kDa dextran). Studies of CaM efflux from preloaded nuclei in permeablized cells revealed the existence of three classes of nuclear binding sites that are distinguished by their Ca 2+ -dependence and affinity. At high [Ca 2+ ], efflux was enhanced by addition of a high affinity CaM-binding protein outside the nucleus. These data suggest that CaM diffuses freely through nuclear pores and that CaM-binding proteins in the nucleus act as a sink for Ca 2+ -CaM, resulting in accumulation of CaM in the nucleus on elevation of intracellular free Ca 2+ .