Two Potent Nuclear Localization Signals in the Gut-enriched Krüppel-like Factor Define a Subfamily of Closely Related Krüppel Proteins

Abstract

The gut-enriched Krü ppel-like factor (GKLF) is a newly identified transcription factor that contains three C 2 H 2 Krü ppel-type zinc fingers. Previous immunocytochemical studies indicate that GKLF is exclusively localized to the nucleus. To identify the nuclear localization signal (NLS) within GKLF, cDNA constructs with various deletions in the coding region of GKLF were generated and analyzed by indirect immunofluorescence in transfected COS-1 cells. In addition, constructs fusing regions representing putative NLSs of GKLF to green fluorescent protein (GFP) were generated and examined by fluorescence microscopy in similarly transfected cells. The results indicate that GKLF contains two potent, independent NLSs: one within the zinc fingers and the other in a cluster of basic amino acids (called 5 basic region) immediately preceding the first zinc finger. In comparison, putative NLSs within the zinc fingers and the 5 basic region of a related Krü ppel protein, zif268/Egr-1, are relatively less efficient in their ability to translocate GFP into the nucleus. A search in the protein sequence data base revealed that despite the existence of numerous Krü ppel proteins, only two, the lung Krü ppel-like factor (LKLF) and the erythroid Krü ppel-like factor (EKLF), exhibit similar NLSs to those of GKLF. These findings indicate that GKLF, LKLF, and EKLF are members of a subfamily of closely related Krü ppel proteins.Various mechanisms that are responsible for nuclear localization of eukaryotic transcription factors have been proposed. Most transcription factors contain one or more nuclear localization signal (NLS), 1 which, when recognized by nuclear transport proteins, results in the translocation of the transcription factor to the nuclear pore complex. Subsequent translocation across the nuclear membrane occurs in an ATP-dependent fashion (1). By inspecting the amino acid sequences of a large number of transcription factors, two types of NLSs have been defined (2, 3). The first type, called a "core" NLS, contains four or more arginine and lysine residues within a hexapeptide and is frequently flanked by acidic residues or "helix-breakers" such as proline and glycine (3). The second type of NLS is "bipartite" and consists of two clusters of basic amino acids separated by a short nonbasic peptide. It is hypothesized that the two clusters of basic amino acids in a bipartite NLS are brought to a juxtaposed position due to protein folding and are subsequently recognized by the nuclear import machinery (2). In an analysis of the sequences of 117 transcription factors, 106 were found to contain one or more core NLS, whereas relatively few contain a bipartite NLS (2, 3). Interestingly, many putative NLSs are present in close proximity to the DNA-binding domains of transcription factors, exemplified by the bZIP proteins c-Fos and c-Jun, and the bHLH proteins Myc, Max, and Myo D1 (3). This conserved arrangement seems to suggest that DNA-binding motifs and nuclear localization signals may have coevolved.We recently identified a n...

A unique sequence in the N-terminal regulatory region controls the nuclear localization of KLF8 by cooperating with the C-terminal zinc-fingers

“…Among these transcription factors are the C 2 H 2 -type ZF transcription factors including KLF1 [27,28], KLF4 [29] and Zif268 [35]. Our results suggest that the presence of two consecutive ZFs is required for the nuclear localization of KLF8, since removal of more than one and a half ZFs inhibited the nuclear localization of KLF8 and removal of any of the ZFs individually did not affect the nuclear localization of KLF8.…”

“…Both the mNLS1 and the ZFs are well conserved among all the KLF members [30,31] and have been demonstrated to control the nuclear localization of KLF1 and KLF4 [27][28][29], suggesting a possibility that these regions may also be required for the nuclear localization of KLF8. To test this possibility, we deleted each of the mNLS sequences and the ZFs from the Cterminus ( Figure 1B, dC8 to dC96).…”

“…Similar to most other members of the KLF family, KLF8 contains, in the C-terminus, three C 2 H 2 -type ZF motifs in tandem flanked by two mNLSs [25,26]. Previous studies have demonstrated that the conserved mNLS and ZF regions are functional NLSs for both KLF1 [27,28] and KLF4 [29].…”

Krüppel-like factors 4 and 5: the yin and yang regulators of cellular proliferation

“…The carboxyl terminus of KLF4 contains three C2H2-zinc fingers that are most closely related to another member of the family, KLF2 [4]. KLF4 is a nuclear protein whose cellular address depends on two nuclear localization signals [6]. A survey of the tissue distribution in adult mice revealed that KLF4 is highly expressed in terminally differentiated, post-mitotic epithelial cells of the intestinal tract [5], a finding consistent with the anti-proliferative effect of KLF4 (see below).…”

Krüppel-like factor 4 exhibits antiapoptotic activity following γ-radiation-induced DNA damage

“…To explore the possibility that KLF4 negatively regulates BAX expression at a transcriptional level, we examined the immediate 1000 base pair (bp) upstream sequence from the translation initiation site of the human BAX gene (Miyashita and Reed, 1995) for possible KLF4-binding sites based on the established consensus KLF4-binding sequence (Shields and Yang, 1997). A total of nine potential binding sites were identified (Supplementary Figure 4), with site number 5 located at nucleotide positions À464 and À458, immediately adjacent to an established p53-binding site (Miyashita and Reed, 1995).…”