The Fbw7/Human CDC4 Tumor Suppressor Targets Proproliferative Factor KLF5 for Ubiquitination and Degradation through Multiple Phosphodegron Motifs

Abstract

The proproliferative transcription factor KLF5 plays an important role in promoting cell proliferation and tumorigenesis. KLF5 is a short-lived protein that can be rapidly degraded through the ubiquitin-proteasome pathway in cancer cells. However, the mechanisms regulating protein stability remain poorly understood. In this study, the tumor suppressor Fbw7, a component of the SCF complex (SCF Fbw7 ) E3 ubiquitin ligase, specifically promoted the degradation and ubiquitination of KLF5 but had little effect on the stability of KLF4. Fbw7 interacted with KLF5 in a CDC4 phosphodegron (CPD)-dependent manner. Three CPDs were found in the KLF5 protein. Simultaneous mutation of these CPDs significantly abolished Fbw7-mediated ubiquitination and degradation. Furthermore, Fbw7 deficiency dramatically delayed KLF5 turnover and led to the accumulation of KLF5 protein in cancer cells. Glycogen synthase kinase-3␤ could phosphorylate and promote Fbw7-mediated KLF5 degradation. More importantly, Fbw7 negatively regulated the biological activity of KLF5 in gene regulation and cell proliferation. Taken together, these data indicate that Fbw7 is a key negative regulator controlling KLF5-mediated cell proliferation and suggest an additional mechanism linking the loss of Fbw7 function to tumorigenesis. Sp/Krüppel-like factor (KLF) 2 transcription factors are involved in various biological processes and human diseases (1, 2). KLF5 (also known as IKLF and BTEB2) is a basic KLF transcription factor that regulates cell proliferation and plays an important role in diverse physiological and pathological processes, including stemness, inflammation, and atherogenesis (3, 4). As a proproliferative factor, KLF5 also has essential functions in tumorigenesis (3). Increasing evidence indicates that KLF5 can function as an oncogenic protein by promoting cell proliferation in many cancers (5-10). For example, a high expression level of KLF5 correlates with a shorter survival time in breast cancer patients (11). Inhibition of KLF5 expression by pharmacological or genetic methods significantly reduces colorectal cancer cell proliferation (6, 12). However, under certain conditions, KLF5 can also act as a tumor suppressor in some cancers (13,14). The exact mechanisms underlying these apparently contradictory functions are not completely understood.The function of KLF5 is regulated at multiple levels. KLF5 transcription is regulated by several signaling molecules such as Wnt and lysophosphatidic acid (15,16). At the post-translational level, KLF5 function is modulated by phosphorylation, sumoylation, and acetylation (3). Phosphorylation of KLF5 by protein kinase C enhances its transactivation activity and its interaction with CBP (cAMP-responsive element-binding protein-binding protein) (17), whereas sumoylation regulates KLF5-mediated lipid metabolism and its subcellular localization (18,19).KLF5 is an unstable protein with a short half-life in cells. Its protein levels are regulated negatively by the ubiquitin-proteasome pathway (20). The E3 ubiquiti...

Regulation of c-Myc protein stability by proteasome activator REGγ

“…IPs were subjected to western blotting as described. 47 EdU incorporation assay. EdU, a thymidine analog, was used to detect cell proliferation by incorporating into cellular DNA during DNA replication.…”

Identification of Poly (ADP-ribose) Polymerase-1 (PARP-1) as a Novel Krüppel-like Factor 8-interacting and -regulating Protein

“…One KLF protein can be targeted by more than one ubiquitin E3 ligase, such as the homologous to the E6-AP carboxyl terminus domain E3-ubiquitin ligase WWP1 (29) and the F-box ubiquitin E3 ligase Fbw7 (30,33). On the other hand, one ubiquitin E3 ligase such as WWP1 can target more than one KLF protein for proteasomal degradation (29,31).…”

The E3 Ubiquitin Ligase SMAD Ubiquitination Regulatory Factor 2 Negatively Regulates Krüppel-like Factor 5 Protein

“…Cycloheximide Chase Assay-A cycloheximide chase assay was performed as described (5,7,17,19). Briefly, COS-1 cells were transfected with the indicated plasmids or vector alone, treated with 100 g/ml cycloheximide for the indicated time, lysed, boiled in Laemmli buffer containing complete protease inhibitor mixture, and subjected to SDS-PAGE and Western blotting with rabbit KLF5 and mouse Myc, FLAG (Sigma), and ␤-actin antibodies.…”

“…The expression and protein activity of KLF5 are tightly regulated at both transcriptional and posttranscriptional levels (5,7,(17)(18)(19)(20). A primary mechanism by which KLF5 is posttranslationally regulated is through ubiquitination and subsequent degradation of KLF5, as mediated by a number of E3 ubiquitin ligases (5,7,17,19), including WWP1 and FBW7 (5,7,19). The interaction between KLF5 and WWP1 involves the PPXY motif of KLF5 (PPPSY) and the WW domains in WWP1 (5).…”