Two potent nuclear localization signals in the gut-enriched Krüppel-like factor define a subfamily of closely related Krüppel proteins

Abstract

The gut-enriched Krüppel-like factor (GKLF) is a newly identified transcription factor that contains three C2H2 Krüppel-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 Krüppel 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 Krüppel proteins, only two, the lung Krüppel-like factor (LKLF) and the erythroid Krüppel-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 Krüppel proteins.

Fig. 1. Immunolocalization of GKLF in transfected COS-1 cells

a depicts the various constructs used in the experiment. The full-length GKLF is 483 aa. The 5′ basic region is shown as a filled box. The three zinc fingers are sequentially numbered. All constructs were generated in the expression vector, PMT3. b is the result of indirect immunofluorescence analysis of COS-1 cells transfected with the various constructs shown in a. F is a negative control showing cells transfected with an empty PMT3 vector.

Fig. 2. Cellular localization of green fluorescent protein (GFP) fusion proteins

a shows the various GFP fusion constructs cloned in the expression vector, pEGFP-C3. The 5′ basic region of GKLF is shown as a filled box and that of zif268/Egr-1 is shown as a shaded box. Construct E is GFP fused to a peptide containing a core NLS within the third zinc finger of zif268/Egr-1. b shows the amino acid sequences of peptides included in constructs C, D, and E. Basic amino acid residues are underlined. c shows the results of fluorescence microscopy of COS-1 cells transfected with the depicted constructs.

Fig. 3. Amino acid sequence alignment of the 5 ′ basic regions of Krüppel-like proteins

The position of the first amino acid residue in the 5′ basic region is shown for each protein. The basic amino acid residues are boxed. The first cysteine residue of the first zinc finger of each protein is identified by an asterisk. The h and m preceding the name of each protein indicates human and mouse, respectively. The study describing mouse GC box-binding protein (mGCBP) has not been published, although its sequence has been deposited in the GenBank^™ data base under the accession number Z36270.