Nucleo-Cytoplasmic Localization Domains Regulate Krüppel-Like Factor 6 (KLF6) Protein Stability and Tumor Suppressor Function

BACKGROUND: The tumor suppressor KLF6 and its oncogenic cytoplasmic splice variant KLF6-SV1 represent a paradigm in cancer biology in that their antagonistic cancer functions are encoded within the same gene. As a consequence of splicing, KLF6-SV1 loses both the C-terminus C(2)H(2) three zinc finger (ZF) domain, which characterizes all KLF proteins, as well as the adjacent 5′ basic region (5BR), a putative nuclear localization signal (NLS). It has been hypothesized that this NLS is a functional domain critical to direct the distinct subcellular localization of the tumor suppressor and its splice variant. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we demonstrate using EGFP fusion constructs that KLF6/KLF6-SV1 nucleo-cytoplasmic transport is not regulated by the 5′ basic region but activated by a novel NLS encoded within the ZF domain, and a nuclear export signal (NES) located in the first 16 amino acids of the shared N-terminus sequence. We demonstrate KLF6 nuclear export to be Crm1-dependent. The dysregulation of nucleo-cytoplasmic transport when disrupting the KLF6 NLS using site-directed mutagenesis showed that its integrity is necessary for appropriate protein stability. Moreover, these mutations impaired transcriptional induction of two KLF6 well-characterized target genes, E-cadherin and p21, as shown by RT-PCR and luciferase promoter assays. The addition of the ZF domain to KLF6-SV1 results in its nuclear localization and a markedly decreased half-life similar to wild type KLF6. CONCLUSIONS/SIGNIFICANCE: We describe the domains that control KLF6 nucleo-cytoplasmic shuttling and how these domains play a role in KLF6 protein half-life and tumor suppressor function. The results begin to mechanistically explain, at least in part, the opposing functions of KLF6 and KLF6-SV1 in cancer.