E3 ubiquitin ligase Fbw7 negatively regulates granulocytic differentiation by targeting G-CSFR for degradation

Abstract

Tight control between activation and attenuation of granulocyte colony stimulating factor receptor (G-CSFR) signaling is essential to regulate survival, proliferation and differentiation of myeloid progenitor cells. Previous studies demonstrated negative regulation of G-CSFR through endosomal-lysosomal routing and ubiquitin-proteasome mediated degradation. However, very few E3 ubiquitin ligases are known to target G-CSFR for ubiquitin-proteasome pathway. Here we identified F-box and WD repeat domain-containing 7 (Fbw7), a substrate recognizing component of Skp-Cullin-F box (SCF) E3 ubiquitin Ligase physically associates with G-CSFR and promotes its ubiquitin-mediated proteasomal degradation. Our data shows that Fbw7 also interacts with and degrades G-CSFR-T718 (a truncated mutant of G-CSFR found in severe congenital neutropenia/acute myeloid leukemia (SCN/AML patients)) though at a quite slower rate compared to G-CSFR. We further show that glycogen synthase kinase 3 beta (GSK3β), like Fbw7 also targets G-CSFR and G-CSFR-T718 for degradation; however, Fbw7 and GSK3β are interdependent in targeting G-CSFR/G-CSFR-T718 for degradation because they are unable to degrade G-CSFR individually when either of them is knocked down. We further show that Fbw7 mediated downregulation of G-CSFR inhibits signal transducer and activator of transcription 3 (STAT3) phosphorylation which is required for G-CSF dependent granulocytic differentiation. In addition, our data also shows that inhibition of Fbw7 restores G-CSFR signaling leading to enhanced STAT3 activity resulting in massive granulocytic differentiation. These data indicate that Fbw7 together with GSK3β negatively regulates G-CSFR expression and its downstream signaling.

E3 Ubiquitin Ligase Fbw7 Negatively Regulates Osteoblast Differentiation by Targeting Runx2 for Degradation

“…Runx2-(1-376) was a kind gift from Dr. G. Stein (30). Runx2-specific reporter construct p6OSE2-luc containing six copies of OSE2 oligonucleotides cloned upstream of the osteocalcin basal promoter was kindly provided by Dr. P. Ducy (1), while GSK3␤, GSK3␤S9A, and ubiquitin constructs have been described previously (21).…”

“…Protein samples from femur bone of different experimental groups were isolated as described by Wejheden et al (34). Immunoblotting was performed as described previously (21,35 Immunohistochemistry-Bones of different groups were decalcified in EDTA followed by block preparation and sectioning. After de-waxing of the samples, 1% rat serum in PBS with 0.1% Triton X-100 (Sigma) was used for antigen retrieval for 90 min.…”

“…This phosphorylated CPD binds with three conserved arginine residues within WD40 repeats in the C terminus of Fbw7 and is subsequently targeted for degradation by SCF Fbw7 . The majority of the known Fbw7 substrates are phosphorylated by GSK3␤ within their CPD including c-Myc (17), MCL-1 (18), , cyclin E (20), and PGC-1␣ (21,22). These studies suggest Fbw7 to be an important protein involved in the development and regulation of cellular differentiation by targeting multiple substrate molecules for degradation.…”

Aberrant regulation of FBW7 in cancer

“…Recently, multiple new targets of FBW7 including MED13 (Mediator 13), KLF2 (Krüppel-like factor 2), NF-κB2 [47, 48], and G-CSFR (Granulocyte colony stimulating factor receptor) [49] have been also discovered. Since several excellent review articles have already summarized the roles of FBW7 in human cancers [20, 22, 50, 51], we will briefly discuss these newly identified FBW7 substrates that would help us to further understand the critical role of FBW7 in tumorigenesis.…”

“…G-CSFR mutations could disrupt its ubiquitination and subsequently cause aberrant receptor signaling, leading to leukemic transformation [87]. Recently, Lochab et al found that FBW7 negatively controlled the granulocytic differentiation in part by targeting G-CSFR for degradation [49]. Furthermore, it has been shown that both FBW7 and GSK3β are required for G-CSFR degradation.…”