FBW7 targets KLF10 for ubiquitin-dependent degradation.

FBW7, a key component of SCFFBW7 E3 ubiquitin ligase, targets various proteins for degradation via the conserved Cdc4 phosphodegron (CPD) in substrates. In this study, we report that KLF10 is degraded by FBW7 via a conserved CPD. Through systematic analysis of the degradation of KLF transcription factors by FBW7, we identified KLF10 as a novel degradation target of FBW7. Ectopic expression of FBW7 markedly promoted the degradation of KLF10 while knockdown of endogenous FBW7 increased the protein levels of KLF10. In addition, simultaneous mutations of both threonine 82 (T82) and serine 86 (S86) significantly reduced the FBW7-mediated KLF10 degradation. Moreover, KLF10 containing a conserved putative CPD (TPPXSP) from amino acids 82 to 87, directly interacted with WD40 domain of FBW7 in a phosphorylation-dependent manner. Importantly, FBW7 could reverse the KLF10-mediated inhibition of Smad7 activity. Thus, our study uncovers a novel regulatory mechanism underlying which KLF10 stability and its biological function are mediated by FBW7.

CDK2 phosphorylation regulates the protein stability of KLF10 by interfering with binding of the E3 ligase SIAH1.

Downregulation of multiple cell cycle-regulatory molecules is a dominant event in TGF-ß1-mediated growth inhibition of human carcinoma cells. It is known that KLF10 mimics the anti-proliferative and apoptotic effects that TGF-ß1 has on epithelial cell growth and the growth of various tumor cells; based on these findings it is considered as a tumor suppressor. KLF10 protein expression is tightly associated with cell cycle-dependent events. However, the regulatory mechanism and its biological meaning have not been identified. In this study, we have demonstrated that KLF10 is a substrate of CDK2/cyclin E and can be phosphorylated. We also have shown that KLF10 efficiently binds to CDK2, while binding much less to CDK4, and displaying no binding to Cdk6. Using mass spectrometry, site direct mutagenesis, in vitro kinase assays and depletion assays, we have established that CDK2 phosphorylates Ser206, which subsequently affects the steady state level of KLF10 in cells. Our studies have also proved that CDK2 up-regulates the protein level of KLF10 through reducing its association with SIAH1, a KLF10 E3-ubiqutin ligase involved in proteasomal degradation. Taken all together, these findings indicate that CDK2-dependent phosphorylation regulates KLF10 stability and that this affects the role of KLF10 in cell.

Differential coupling of KLF10 to Sin3-HDAC and PCAF regulates the inducibility of the FOXP3 gene.

Inducible gene expression, which requires chromatin remodeling on gene promoters, underlies the epigenetically inherited differentiation program of most immune cells. However, chromatin-mediated mechanisms that underlie these events in T regulatory cells remain to be fully characterized. Here, we report that inducibility of FOXP3, a key transcription factor for the development of T regulatory cells, depends upon Kruppel-like factor 10 (KLF10) interacting with two antagonistic histone-modifying systems. We utilized chromatin immunoprecipitation, genome-integrated reporter assays, and functional domain KLF10 mutant proteins, to characterize reciprocal interactions between this transcription factor and either the Sin3-histone deacetylase complex or the histone acetyltransferase, p300/CBP-associated factor (PCAF). We characterize a Sin3-interacting repressor domain on the NH2 terminus of KLF10, which works to limit the activating function of this transcription factor. Indeed, inactivation of this Sin3-interacting domain renders KLF10 able to physically associate with PCAF as to induce FOXP3 gene transcription. We show that this biochemical data derived from studying our genome-integrated reporter cell system are recapitulated in primary murine lymphocytes. Collectively, these results advance our understanding of how a single transcription factor, namely KLF10, functions as a toggle to integrate antagonistic signals regulating FOXP3 and, thus, immune activation.

Destabilization of KLF10, a tumor suppressor, relies on thr93 phosphorylation and isomerase association.

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-ß-inducible early gene-1 and mimicking the anti-proliferative effect of TGF-ß 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.

Klf10 and Klf11 as mediators of TGF-beta superfamily signaling.

Klf10 and Klf11 belong to the family of Sp1/Krüppel-like zinc finger transcription factors that play important roles in a variety of cell types and tissues. Although Klf10 and Klf11 were initially introduced as transforming growth factor-beta (TGF-beta)-inducible genes, several studies have described their upregulation by a plethora of growth factors, cytokines and hormones. Here, we review the current knowledge of the inductive cues for Klf10 and Klf11 and focus on their transcriptional regulation by members of the TGF-beta superfamily. We further summarize their involvement in the regulation of the TGF-beta signaling pathway and discuss their possible role as molecules mediating crosstalk between various signaling pathways. Finally, we provide an overview of the pro-apoptotic and anti-proliferative functions of Klf10 and Klf11.

Experimentally based contact energies decode interactions responsible for protein-DNA affinity and the role of molecular waters at the binding interface.

A major obstacle towards understanding the molecular basis of transcriptional regulation is the lack of a recognition code for protein-DNA interactions. Using high-quality crystal structures and binding data on the promiscuous family of C(2)H(2) zinc fingers (ZF), we decode 10 fundamental specific interactions responsible for protein-DNA recognition. The interactions include five hydrogen bond types, three atomic desolvation penalties, a favorable non-polar energy, and a novel water accessibility factor. We apply this code to three large datasets containing a total of 89 C(2)H(2) transcription factor (TF) mutants on the three ZFs of EGR. Guided by molecular dynamics simulations of individual ZFs, we map the interactions into homology models that embody all feasible intra- and intermolecular bonds, selecting for each sequence the structure with the lowest free energy. These interactions reproduce the change in affinity of 35 mutants of finger I (R(2) = 0.998), 23 mutants of finger II (R(2) = 0.96) and 31 finger III human domains (R(2) = 0.94). Our findings reveal recognition rules that depend on DNA sequence/structure, molecular water at the interface and induced fit of the C(2)H(2) TFs. Collectively, our method provides the first robust framework to decode the molecular basis of TFs binding to DNA.

Elucidation of the substrate binding site of Siah ubiquitin ligase.

The Siah family of RING proteins function as ubiquitin ligase components, contributing to the degradation of multiple targets involved in cell growth, differentiation, angiogenesis, oncogenesis, and inflammation. Previously, a binding motif (degron) was recognized in many of the Siah degradation targets, suggesting that Siah itself may facilitate substrate recognition. We report the crystal structure of the Siah in complex with a peptide containing the degron motif. Binding is within a groove formed in part by the zinc fingers and the first two beta strands of the TRAF-C domain of Siah. We show that residues in the degron, previously described to facilitate binding to Siah, interact with the protein. Mutagenesis of Siah at sites of interaction also abrogates both in vitro peptide binding and destabilization of a known Siah target.