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The Motif of SPARC that Inhibits DNA Synthesis Is not a Nuclear Localization Signal
- Source :
- Journal of Molecular Biology. 371:883-901
- Publication Year :
- 2007
- Publisher :
- Elsevier BV, 2007.
-
Abstract
- SPARC (secreted protein acidic and rich in cysteine), although primarily known as a secreted, matricellular protein, has also been identified in urothelial cell nuclei. Many biological activities, including inhibition of cell adhesion and repression of DNA synthesis, have been ascribed to SPARC, but the influence of its intracellular localization on each of these activities is unknown. When exposed by epitope retrieval and nuclear matrix unmasking techniques, endogenous SPARC was found to localize strongly to the nuclei and the nuclear matrix of cultured urothelial cells. Live-cell time-lapse imaging revealed that exogenous fluorescently labeled recombinant (r) SPARC was taken up from medium over a 16 h period and accumulated inside cells. Two variants of rSPARC with alterations in its putative nuclear localization signal (NLS) were generated to investigate the existence and effects of the NLS. These variants demonstrated similar biophysical characteristics as the wild-type protein. Visualization by a variety of techniques, including live-cell imaging, deconvolution microscopy, and cell fractionation, all concurred that exogenous rSPARC was not able to localize to cell nuclei, but instead accumulated as perinuclear clusters. Localization of the rSPARC NLS variants was no different than wild-type, arguing against the presence of an active NLS in rSPARC. Imaging experiments showed that only permeabilized, dead cells avidly took up rSPARC into their nuclei. The rSPARC(no NLS) variant proved ineffective at inhibiting DNA synthesis, whereas the rSPARC(strong NLS) variant was a more potent inhibitor of DNA synthesis than was wild-type rSPARC. The motif of SPARC that inhibits the synthesis of urothelial cell DNA is therefore not a nuclear localization signal, but its manipulation holds therapeutic potential to generate a "Super-SPARC" that can quiesce proliferative tissues.
- Subjects :
- Models, Molecular
Amino Acid Motifs
Nuclear Localization Signals
Biology
law.invention
Structural Biology
law
Live cell imaging
Humans
NLS
Nuclear Matrix
Osteonectin
Amino Acid Sequence
Cell adhesion
Molecular Biology
Cells, Cultured
Cell Nucleus
DNA synthesis
DNA
Nuclear matrix
Recombinant Proteins
Protein Structure, Tertiary
Cell biology
Microscopy, Fluorescence
Biochemistry
Mutation
Recombinant DNA
Urothelium
Cell fractionation
Nuclear localization sequence
Subjects
Details
- ISSN :
- 00222836
- Volume :
- 371
- Database :
- OpenAIRE
- Journal :
- Journal of Molecular Biology
- Accession number :
- edsair.doi.dedup.....8538ce9ec714b94195bc6eda95301993