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

Abstract

BackgroundThe 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 C2H2 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 FindingsIn 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/SignificanceWe 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.

Krüppel-like Factor 7 engineered for transcriptional activation promotes axon regeneration in the adult corticospinal tract

“…Expression of KLF7 in cultured neurons was not increased by using smaller epitope tags, such as Flag or myc, or by attaching tags to the C terminus, suggesting that poor expression of KLF7 was not an artifact of tag size or location. Other KLF family members are subject to rapid degradation under the control of posttranslational modifications and protein-protein interactions; notably, the stability of both KLF5 and of KLF6 (which has high sequence homology to KLF7) is increased by deleting N-terminal motifs (28,29). We found that deletion of the KLF7 N terminus, a previously identified 76-aa acidic transactivation domain, increased KLF7 expression more than threefold (Fig.…”

“…The fact that two evolutionarily divergent domains, with well-characterized and opposite effects on transcription, give opposite effects on neurite outgrowth when fused to KLF7's DNA binding domain strongly favors transcriptional activation as KLF7's mechanism of action. Second, N-terminal sequences keep expression low in neurons even after attempted overexpression, possibly by targeting KLF7 protein for degradation (28,29). We developed VP16-KLF7 as a means to enhance expression, but cannot rule out the possibility that VP16-KLF7 could activate nonphysiological targets, which would confound conclusions regarding endogenous KLF7.…”

Inhibition of the Nuclear Export Receptor XPO1 as a Therapeutic Target for Platinum-Resistant Ovarian Cancer

“…XPO1 overexpression has been suggested to be a general feature of cellular transformation (9) and has been observed in a number of hematologic and solid tumors (9-13), including ovarian cancer (10). Most relevant to this study, XPO1 is the only known transporter for a number of well-characterized ovarian cancerassociated proteins, including p53 (11)(12), BRCA1 (13), IkBa (14), KLF6 (15), sequestosome 1 (SQSTM1; also known as p62), and PP2A (CIP2A) (16), all of which play roles in ovarian cancer initiation, cell-cycle progression, the DNA damage response, apoptosis, autophagy, and chemoresistance. Therefore, XPO1 seems an excellent ovarian cancer therapeutic target.…”

Krüppel-like Factor 6 Is a Co-activator of NF-κB That Mediates p65-dependent Transcription of Selected Downstream Genes

“…4C). It has been reported that KLF6 shuttles between the cytoplasm and nucleus (24). We thus performed cellular fractionation assays to isolate the cytosol and nuclear fractions and examined whether KLF6 interacts with p65 in the nucleus or cytoplasm by coimmunoprecipitation.…”