Mechanism of Ca2+-dependent nuclear accumulation of calmodulin.
The intracellular Ca2+ 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 Ca2+-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. Ca2+-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 Ca2+-dependence and affinity. At high [Ca2+], 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 Ca2+-CaM, resulting in accumulation of CaM in the nucleus on elevation of intracellular free Ca2+.
Liao
B
B
Department of Physiology, University of Texas Southwestern Medical Center at Dallas, TX 75235, USA.
Paschal
B M
BM
Luby-Phelps
K
K
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002460:Cell Line; D002463:Cell Membrane Permeability; D002467:Cell Nucleus / Q000378:metabolism*; D003911:Dextrans; D004533:Egtazic Acid / Q000494:pharmacology; D005456:Fluorescent Dyes; D007700:Kinetics; D008856:Microscopy, Fluorescence; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012235:Rhodamines
D002147:Calmodulin; D003911:Dextrans; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D012235:Rhodamines; C051311:rhodamine dextran; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D009219:Myosin-Light-Chain Kinase; D002118:Calcium