Interaction of Sp1 zinc finger with transport factor in the nuclear localization of transcription factor Sp1

doi:10.1016/j.bbrc.2010.10.036

Biochemical and Biophysical Research Communications

Volume 403, Issue 2, 10 December 2010, Pages 161-166

https://doi.org/10.1016/j.bbrc.2010.10.036 Get rights and content

Abstract

Transcription factor Sp1 is localized in the nucleus and regulates the expression of many cellular genes, but the nuclear transport mechanism of Sp1 is not well understood. In this study, we revealed that GST-fused Sp1 protein bound to endogenous importin α in HeLa cells via the Sp1 zinc finger domains, which comprise the DNA binding domain of Sp1. It was found that the Sp1 zinc finger domains directly interacted with a wide range of importin α including the armadillo (arm) repeat domain and the C-terminal acidic domain. Furthermore, it turned out that all three zinc fingers of Sp1 are essential for binding to importin α. Taken together, these results suggest that the Sp1 zinc finger domains play an essential role as a NLS and Sp1 can be transported into the nucleus in an importin-dependent manner even though it possesses no classical NLSs.

Research highlights

► Sp1 zinc fingers themselves interact with importin α. ► Sp1 zinc finger domains play an essential role as a nuclear localization signal. ► Sp1 can be transported into the nucleus in an importin-dependent manner.

Introduction

Proteins that function in the nucleus such as transcription factors are translocated into the nucleus after being synthesized in the cytoplasm. The translocation of proteins is performed through the nuclear pore complex (NPC) structure [1], [2]. Though small proteins of <20 kDa can freely diffuse through NPC, proteins larger than 40–60 kDa have a nuclear localization signal (NLS), which are generally divided into two groups, monopartite and bipartite NLSs, based on the number of basic amino acid clusters [3], [4]. However, NLSs without the basic amino acid clusters were recently reported [5], [6], [7], [8], [9]. Therefore, a NLS having basic amino acid clusters is designated as a classical NLS. The nuclear import of proteins containing a classical NLS is mediated by a specific complex with transport factors such as importin α and importin β, and then the NLS-importin α/β complex is transported into the nucleus through ATP-dependent active transport. The regulation of cargo binding and release of transport factors is carried out by the asymmetric distribution of two nucleotide states of the small GTPase Ran [10], [11].

Transcription factor Sp1 activates many mammalian genes containing the GC box sequence [12], [13] and requires the NLS to pass through the nuclear pore because of its relatively large molecular mass (105 kDa). However, a typical NLS has not been found within the Sp1 amino acid sequence and the nuclear transport mechanism is little understood. We previously found that the three contiguous Cys₂His₂-type zinc fingers known as the DNA binding domain functioned as the NLS, and the proper conformation of the first and third zinc finger domains was essential for the nuclear localization of Sp1 [14], [15], [16]. It seems that Sp1 zinc fingers, which comprise the NLS, are bound to the transport factor and Sp1 passes through the nuclear pore complex (NPC).

In this study, we examined the nuclear localization mechanism of Sp1, and showed the direct interaction of Sp1 with the transport factor importin α, not importin β. Our study suggests that the nuclear localization of Sp1 occurs through a typical importin α/importin β mediated mechanism.

Section snippets

Plasmid construction

The expression vector of GST-fused Sp1 zinc fingers (zf) was prepared by the following methods. Sp1 zinc finger cDNA was excised from the expression vector encoding GFP-fused Sp1 zinc fingers [16]. The resulting fragment was subcloned into pGEX-3X, GST-fused vector (GE Healthcare, Little Chalfont, UK). Deletion mutants of Sp1 fused with GST were prepared by similar methods using the expression vector encoding the GFP-fused Sp1 protein.

The expression vector of MBP-Rch1(Δ1–35) was prepared by the

The interaction of Sp1 and endogenous transport factor in HeLa cells

In order to investigate the mechanism of Sp1 nuclear translocation, we examined whether or not Sp1 interacts with transportation factors such as importin α (Rch1, an importin α) and importin β (Karyopherin β, an importin β) by means of the GST-pull down assay. Western blot analysis showed that both importin α and importin β were localized in both the nucleus and cytoplasm fractions of HeLa cells, and the result of immunofluorescence staining for the two transport factors was also identical to

Discussion

In this study, we determined that Sp1 zinc fingers tightly interacted with a nuclear transporters, importin α, and that the full binding of importin α to Sp1 zinc fingers required the whole of importin α except for the IBB domain.

Proteins are translocated to the nucleus after formation of the protein (cargo)/importin α/importin β complex in the cytoplasm through binding of the cargo to importin α through the cargo’s NLS, and binding of importin α to importin β through the IBB domain of importin

Acknowledgments

Sp1 cDNA was generous gift or professor R. Tjian and J. Kadonaga (University of California, Berkeley, USA). Plasmids containing Rch1 and Qip1 cDNAs, and importins α and β cDNAs were kindly gifted by professor T. Enomoto of Tohoku University, and, professor Y. Yoneda and Dr. M. Hieda of Osaka University, respectively. We thank Dr. Masayuki Shono (Support Center for Advanced Medical Sciences, School of Medicine, The University of Tokushima, Japan) for measurement of confocal laser scanning

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Interaction of Sp1 zinc finger with transport factor in the nuclear localization of transcription factor Sp1

Abstract

Research highlights

Introduction

Section snippets

Plasmid construction

The interaction of Sp1 and endogenous transport factor in HeLa cells

Discussion

Acknowledgments

Cell

Cell

Cell

Biochem. Biophys. Res. Commun.

Cell

Cell

Cell

Biochem. Biophys. Res. Commun.

Virology

FEBS Lett.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Cell

J. Mol. Biol.