title: Destabilization of KLF10, a tumor suppressor, relies on thr93 phosphorylation and isomerase association.
abstract: KLF10 is now classified as a member of the Krüppel-like transcription factor family and acts as a tumor suppressor. Although KLF10 is originally named as TGF-β-inducible early gene-1 and mimicking the anti-proliferative effect of TGF-β in various carcinoma cells, the transcriptional upregulatory function of KLF10 has been described for a variety of cytokines and in many diseases. Through in vivo and in vitro phosphorylation assays, we identified that KLF10 is a phosphorylated protein in cells. Using yeast-two hybrid screening and site direct mutagenesis, we also identified PIN1 as a novel KLF10 associated protein. PIN1 is a peptidyl-prolyl isomerase enzyme belonging to the parvulin family, which specifically recognizes phosphorylated Ser/Thr-Pro containing substrates. Through protein-protein interaction assays, we showed that the Pro-directed Ser/Thr-Pro motif at Thr-93 in the KLF10 N-terminal region is essential for the interaction between KLF10 and PIN1. More importantly, PIN1 interacts with KLF10 in a phosphorylation-dependent manner and this interaction promotes KLF10 protein degradation in cells. Therefore, KLF10 shows shorter protein stability compared with mutant KLF10 that lacks PIN1 binding ability after cycloheximide treatments. The reversely correlated expression profile between KLF10 and PIN1 as observed in cell lines was also shown in clinic pancreatic cancer specimen. Using in vitro kinase assays and depletion assays, we were able to show that RAF-1 phosphorylates the Thr-93 of KLF10 and affects the KLF10 expression level in cells. Thus these findings as a whole indicate that RAF-1 phosphorylation and PIN1 isomerization together regulate KLF10 stability and further affect the role of KLF10 in tumor progression.
authors.affiliation: National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan.
authors.forename: Yu-Chyi
authors.initials: YC
authors.lastname: Hwang
authors.affiliation: National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan.
authors.forename: Chien-Hui
authors.initials: CH
authors.lastname: Yang
authors.affiliation: National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan.
authors.forename: Ching-Hui
authors.initials: CH
authors.lastname: Lin
authors.affiliation: National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan.
authors.forename: Hui-Ju
authors.initials: HJ
authors.lastname: Ch'ang
authors.affiliation: Program for Translation Medicine, College of Medical Science and Technology, Taipei Medical University, Taiwan. Electronic address: vhschang@gmail.com.
authors.forename: Vincent H S
authors.initials: VHS
authors.lastname: Chang
authors.affiliation: National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan; College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan. Electronic address: winston@nhri.org.tw.
authors.forename: Winston C Y
authors.initials: WCY
authors.lastname: Yu
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D045744:Cell Line, Tumor; D051765:Early Growth Response Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D019696:Peptidylprolyl Isomerase / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D055550:Protein Stability; D059748:Proteolysis; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D051765:Early Growth Response Transcription Factors; C098111:KLF10 protein, human; C569958:KLF10 protein, mouse; D051741:Kruppel-Like Transcription Factors; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D025521:Tumor Suppressor Proteins; D010769:Phosphothreonine; D010768:Phosphoserine; D019908:Proto-Oncogene Proteins c-raf; C000606846:PIN1 protein, human; D019696:Peptidylprolyl Isomerase; C000606850:Pin1 protein, mouse
keywords: KLF10; Krüppel-like factor 10; PIN1; RAF-1; TIEG1