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 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.


RESULTS
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
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.
Rodríguez
Estefanía
E
Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, New York, United States of America.
Aburjania
Nana
N
Priedigkeit
Nolan M
NM
DiFeo
Analisa
A
Martignetti
John A
JA
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006207:Half-Life; D006801:Humans; D000075723:Kruppel-Like Factor 6; D051741:Kruppel-Like Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D049790:Nuclear Export Signals; D019913:Nuclear Localization Signals; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
C105465:KLF6 protein, human; D000075723:Kruppel-Like Factor 6; D051741:Kruppel-Like Transcription Factors; D049790:Nuclear Export Signals; D019913:Nuclear Localization Signals; D011518:Proto-Oncogene Proteins