Abstract
The tumor suppressor activity of maspin (mammary serine protease inhibitor) has been associated with its nuclear localization. In this study we explore the regulation of maspin nuclear translocation. An in vitro nuclear import assay suggested that maspin can passively enter the nucleus. However, in silico analysis identified a putative maspin nuclear localization signal (NLS), which was able to mediate the nuclear translocation of a chimeric protein containing this NLS fused to five green fluorescent protein molecules in tandem (5GFP). Dominant‐negative Ran‐GTPase mutants RanQ69L or RanT24N suppressed this process. Unexpectedly, the full‐length maspin fused to 5GFP failed to enter the nucleus. As maspin's putative NLS is partially hidden in its three‐dimensional structure, we suggest that maspin nuclear transport could be conformationally regulated. Our results suggest that maspin nuclear translocation involves both passive and active mechanisms.
Highlights
Maspin, known as SerpinB5, is a potential tumor suppressor first identified in breast tissue [1]
As active nuclear transport requires energy provided by Ran-GTPase-mediated GTP hydrolysis, we further investigate five green fluorescent proteins (5GFP)–maspinNLS nuclear transport in the presence of the RanQ69L and RanT24N mutants, which are deficient in GTP hydrolysis or do not bind to GTP, respectively, and act as dominant negative inhibitors of signal- and energy-dependent nuclear transport [24,25]
Recombinant maspin or BSA covalently linked to the nuclear localization signal (NLS) of SV40 T antigen was conjugated to Cy3 and assayed in digitonin-permeabilized HeLa cells in the presence or absence of an energy regenerating system (+ energy or À energy) and rabbit reticulocyte lysate (RRL), a source of cytoplasmic factors
Summary
Maspin (mammary serine protease inhibitor), known as SerpinB5, is a potential tumor suppressor first identified in breast tissue [1]. Abbreviations 5GFP, five green fluorescent proteins; DAPI, 40,6-diamidino-2-phenylindole; EGF, epidermal growth factor; Kabb, karyopherin-b; maspinFL, full-length maspin; maspin, mammary serine protease inhibitor; NLS, nuclear localization signal; RRL, rabbit reticulocyte lysates; WT, wild-type. Based on the observations described above, we hypothesized both passive and active/regulated mechanisms regulate maspin nucleocytoplasmic shuttling To test this hypothesis, we reconstituted maspin nuclear import in vitro using digitonin-permeabilized HeLa cells. Maspin NLS, but not maspin full length, was able to drive nuclear import of the 5GFP construct, indicating that this peptide sequence can mediate an active transport to the nucleus. We identified a buried NLS which is necessary and sufficient for nuclear import of a 5GFP construct in a Ran-GTPase-dependent manner This NLS was, unable to drive nuclear translocation of full length maspin in the tested conditions
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