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The RhoGTPase Rac1 is activated in a polarised fashion and controls cell motility. We previously showed that Rac1 binds the PP2A inhibitor SET and recruits nuclear SET to the cytosol. We show that a SET mutant, lacking a nuclear localization signal, SET(ΔNLS), promotes cell spreading and motility. This was accompanied by an increase in the number and frequency of membrane ruffles. Pharmacological inhibition of PP2A did not mimic the effects of SET(ΔNLS), however, we found that expression of SET and SET(ΔNLS) increases the levels of the MAP kinases ERK1 and ERK2., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

SET/I2PP2A is a nuclear protein that was initially identified as an oncogene in human undifferentiated acute myeloid leukemia, fused to the nuclear porin Nup-214. In addition, SET is a potent inhibitior of the phosphatase PP2A. Previously, we proposed a model in which the small GTPase Rac1 recruits SET from the nucleus to the plasma membrane to promote cell migration. This event represents an entirely novel concept in the field of cell migration. Now, fluorescent versions of the SET protein are generated to analyze its nucleo-cytoplasmic shuttling in live cells. Our studies showed that under steady-state conditions a fraction of the SET protein, which is primarily localized in the nucleus, translocates to the cytosol in an apparently random fashion. SET exiting the nucleus was also seen in spreading as well as dividing cells. We designed an image analysis method to quantify the frequency of nuclear exit of the SET proteins, based on 4D confocal imaging. This straightforward method was validated by analysis of SET wild-type and mutant proteins. This showed that the frequency of nuclear exit of a Ser-9 phosphomimetic mutant (S9E) is enhanced compared to wild-type SET or a S9A mutant. Thus, we have developed a novel method to analyze the nucleo-cytoplasmic shuttling of the proto-oncogene SET dynamics in live cells. This method will also be applicable to monitor dynamic localization of other nuclear and/or cytoplasmic signaling proteins., (Copyright © 2011 International Society for Advancement of Cytometry.)