Control of the nuclear-cytoplasmic partitioning of annexin II by a nuclearexport signal and by p11 binding

This study investigated mechanisms controlling the nuclear-cytoplasmic part itioning of annexin If (AnxII). AnxII and its ligand, p11, were localized b y immunofluorescence to the cytoplasmic compartment of U1242MG cells, with minimal AnxII or p11 detected within nuclei. Similarly, GFP-AnxII and GFP-p 11 chimeras localized to the endogenous proteins. Likewise, GFP-AnxII(1-22) was excluded from nuclei, whereas GFP-AnxII(23-338) and GFP alone were dis tributed throughout the cells. Immunoprecipitation and biochemical studies showed that GFP-AnxII did not form heteromeric complexes with endogenous p1 1 and AnxII. Thus, the AnxII N-tail is necessary and sufficient to cause nu clear exclusion of the GFP fusion protein but this does not involve p11 bin ding. A nuclear export signal consensus sequence was found in the AnxII 3-1 2 region. The consensus mutant GFP-AnxII(L10A/L12A) confirmed that these re sidues are necessary for nuclear exclusion. The nuclear exclusion of GFP-An xII(1-22) was temperature-dependent and reversible, and the nuclear export inhibitor leptomycin B (LmB) caused GFP-AnxII or overexpressed AnxII monome r to accumulate in nuclei. Therefore, AnxII monomer can enter the nucleus a nd is actively exported. However, LmB had little effect on the localization of AnxII/p11 complex in U1242MG cells, indicating that the complex is sequ estered in the cytoplasm. By contrast, LmB treatment of v-src-transformed f ibroblasts caused endogenous AnxII to accumulate in nuclei. The LmB-induced nuclear accumulation of AnxII was accelerated by pervanadate and inhibited by genistein, suggesting that phosphorylation promotes nuclear entry of An xII. Thus, nuclear exclusion of AnxII results from nuclear export of the mo nomer and sequestration of AnxII/p11 complex, and may be modulated by phosp horylation.