Analysis of molecular determinants of PRL-3

Abstract

In order to analyse whether a C-terminal polybasic sequence represents a nuclear localization signal (NLS) we obtained several truncated and mutant forms of protein of regerating liver (PRL)-3 and evaluated their subcellular localization as compared to the wild-type form. Our results invalidate the hypothesis that this is an NLS. We also analysed the influence of the C- and N-terminal residues on the phosphatase activity of PRL-3. Our results provide in vitro evidence that the C-terminal CAAX motif, besides directing the protein farnesylation, plays an additional regulatory role by inhibiting the catalytic efficiency of PRL-3. Taking into account the results we obtained, as well as reported data, we propose a hypothetical molecular mechanism for the nucleocytoplasmic localization and transfer of PRL-3.

Figure 1

Schematic representation of wild-type and mutant forms of PRL-3. 6xHis – 6 histidine tag at N-terminal of the proteins expressed in prokaryotic system; CAAX box – C-terminal prenylation box; EGFP – enhanced green fluorescent protein at N-terminal of the proteins expressed in eukaryotic system; KRR/QLQ – substitution of KRR amino acids with QLQ amino acids in the putative nuclear localization sequence.

Figure 2

Cellular localization of wild-type and mutant forms of PRL-3, fused with EGFP. EGFP-tagged PRL-3 constructs were introduced by Lipofectamine transfection in HeLa (A), CHO (B) and COS (C) cell lines and visualized after approximately 24 hrs. DAPI stain was used for the visualization of the cell nucleus in fixed cells (second columns). Fluorescence of PRL-3 constructs is shown in the first columns while merge of DAPI staining in EGFP- constructs are in the third columns. Scalebar, 30 μM

Figure 2

Cellular localization of wild-type and mutant forms of PRL-3, fused with EGFP. EGFP-tagged PRL-3 constructs were introduced by Lipofectamine transfection in HeLa (A), CHO (B) and COS (C) cell lines and visualized after approximately 24 hrs. DAPI stain was used for the visualization of the cell nucleus in fixed cells (second columns). Fluorescence of PRL-3 constructs is shown in the first columns while merge of DAPI staining in EGFP- constructs are in the third columns. Scalebar, 30 μM

Figure 2

Cellular localization of wild-type and mutant forms of PRL-3, fused with EGFP. EGFP-tagged PRL-3 constructs were introduced by Lipofectamine transfection in HeLa (A), CHO (B) and COS (C) cell lines and visualized after approximately 24 hrs. DAPI stain was used for the visualization of the cell nucleus in fixed cells (second columns). Fluorescence of PRL-3 constructs is shown in the first columns while merge of DAPI staining in EGFP- constructs are in the third columns. Scalebar, 30 μM

Figure 3

Electrophoretic analysis of PRL-3 proteins. (A) 12% SDS-PAGE of the purified preparations of the PRL-3 constructs, under reducing conditions. (B) Evaluation of PRL-3 protein dimerization. For all PRL-3 constructs 5μg protein was applied on each lane of the 12% SDS – PAGE gel. Samples were processed under non-reducing condition. We refer to non-reducing conditions in terms of presence or absence of β-mercaptoethanol or DTT in the loading buffer. WT – wild type PRL-3, ΔN10 – PRL-3 without first 10 amino acids, ΔC4 –PRL-3 without CAAX box, ΔN10C4 – PRL-3 without first 10 and the last 4 amino acids.

Figure 4

Mass spectrometric analysis of prenylated and non-prenylated PRL-3 forms. MALDI-TOF analysis of wild-type (WT) (A), farnesylated (B) and geranyl-geranylated forms of PRL-3. Farnesylated and geranyl-geranylated forms were obtained by in vitro prenylation of the purified, WT form of PRL-3.