Biochimica et Biophysica Acta 1833 (2013) 2639–2652

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Biochimica et Biophysica Acta

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

Savita Lochab a, Pooja Pal a, Isha Kapoor a, Jitendra Kumar Kanaujiya a, Sabyasachi Sanyal a,Gerhard Behre b, Arun Kumar Trivedi a,⁎a Drug Target Discovery and Development Division, CSIR-Central Drug Research Institute (CSIR-CDRI), Sector-10, Jankipuram Extension, Lucknow, 226031 UP, Indiab Division of Hematology and Oncology, University Hospital of Leipzig, Johannissallee 32A, 04103 Leipzig, Germany

⁎ Corresponding author at: LSS008, Drug Target DiscovCSIR-Central Drug Research Institute, New campus, SecSitapur Road, Lucknow, 226031, UP, India. Tel.: +91 9839

E-mail address: arun3vedi@yahoo.com (A.K. Trivedi

0167-4889/$ – see front matter © 2013 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.bbamcr.2013.06.018

a b s t r a c t

a r t i c l e i n f o

Article history:Received 8 February 2013Received in revised form 10 June 2013Accepted 21 June 2013Available online 29 June 2013

Keywords:Fbw7G-CSFRGSK3βUbiquitin–proteasome degradation

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 andubiquitin–proteasome mediated degradation. However, very few E3 ubiquitin ligases are known to targetG-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 associ-ates with G-CSFR and promotes its ubiquitin-mediated proteasomal degradation. Our data shows thatFbw7 also interacts with and degrades G-CSFR-T718 (a truncated mutant of G-CSFR found in severe congen-ital neutropenia/acute myeloid leukemia (SCN/AML patients)) though at a quite slower rate compared toG-CSFR. We further show that glycogen synthase kinase 3 beta (GSK3β), like Fbw7 also targets G-CSFRand 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 themis knocked down. We further show that Fbw7 mediated downregulation of G-CSFR inhibits signal transducerand activator of transcription 3 (STAT3) phosphorylation which is required for G-CSF dependent granulocyticdifferentiation. In addition, our data also shows that inhibition of Fbw7 restores G-CSFR signaling leadingto enhanced STAT3 activity resulting in massive granulocytic differentiation. These data indicate that Fbw7together with GSK3β negatively regulates G-CSFR expression and its downstream signaling.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Granulocyte colony stimulating factor receptor (G-CSFR) mediat-ed signaling critically regulates survival, proliferation and differentia-tion of myeloid progenitor cells. It is crucial for the hematopoieticdevelopment because G-CSF or G-CSFR deficient mice acquire severecongenital neutropenia (SCN) with impaired granulopoietic re-sponses to infection [1]. G-CSF binding to G-CSFR elicits the down-stream signaling by activating Janus tyrosine kinase (JAK)/signaltransducer and activator of transcription (STAT), Ras/Raf/MAP kinaseand PKB/AKT pathways. This initiates proliferation and survival ofmyeloid progenitors, subsequent cell cycle arrest and ultimatelyadvances to differentiation resulting in neutrophil formation [2]. How-ever, a balance between activation and attenuation of G-CSFR signalingis essential for maintaining homeostatic levels of neutrophils in blood

ery and Development Division,tor-10, Jankipuram Extension,790765; fax: +91 5222771941.).

rights reserved.

circulation. Mutations resulting in carboxy-terminal truncation inG-CSFR gene have been reported in patients with severe congenitalneutropenia (SCN) transformed to acute myeloid leukemia (AML).The truncated mutants of G-CSFR are constitutively active and trans-duce hyper proliferative responses upon G-CSF stimulation withoutneutrophil differentiation [3]. Therefore, downregulation of “activatedG-CSFR” is important to keep a check on G-CSFR mediated signaling inorder to protect cells from over-stimulation.

Attenuation of G-CSF signaling is controlled throughmultiplemech-anisms such as dephosphorylation of G-CSFR through tyrosine phos-phatases (SHP-1 and SHP-2) and inositol phosphatases (SHIP1) [4–6];G-CSFR protein-destabilization either by proteolytic cleavage throughneutrophil elastase or ubiquitination dependent degradation [7].Major-ity of the mechanisms explain the termination of G-CSFR signalingthrough SOCS proteins by inhibition of JAK kinase activity or by recruit-ment of elongins B and C to SOCS box to form an E3 ubiquitin ligasecomplex that directs G-CSFR to the endosomal–lysosomal degradationroute [8,9]. Moreover, recent studies have also shown ubiquitinmediat-ed proteasome-dependent degradation of G-CSFR [10]. However, theE3 ubiquitin ligases, regulating the rate-limiting step in ubiquitin–proteasome degradation of G-CSFR have largely remained unexplored.

2640 S. Lochab et al. / Biochimica et Biophysica Acta 1833 (2013) 2639–2652

This prompted us to identify the E3 ubiquitin ligases that may regulatethe constitutive distribution of G-CSFR.

F box protein Fbw7 (also known in cdc4) is a component of theSCF (Skp–Cullin–F box) ubiquitin ligase that recognizes and binds toits substrates through a consensus phospho-binding motif (T/S-P-X-X-S/T/D/E) known as Cdc4-Phospho Degron (CPD). The recognitionof CPDs by SCFFbw7 is regulated by phosphorylation of the substrateat threonine/serine residue within CPD motif. This phosphorylatedCPD binds with three conserved arginine residues within WD40 re-peats in the C-terminus of Fbw7 [11]. Majority of the known Fbw7substrates are phosphorylated by GSK3β such as c-myc [12,13],c-jun [14], MCL-1 [15], C/EBPα [16,17], KLF5 [18,19], SREBP1 [20], cy-clin E [21] and PGC-1 [22] (Table 1).

In this study for thefirst timewe show that Fbw7negatively regulatesG-CSFR by targeting it to degradation through the ubiquitin–proteasomepathway. Our data shows that Fbw7 facilitates ubiquitination and degra-dation of G-CSFR through multiple potential phosphodegrons present inG-CSFR (Table 1). This process is promoted by GSK3βwhich presumablyphosphorylates G-CSFR in its potential CPDs and thus facilitates its recog-nition by Fbw7 for subsequent ubiquitin–proteasome degradation. Weshow that Fbw7 also targets mutant G-CSFR-T718 for degradation,though at a relatively slower rate than the wild type G-CSFR. We furthershow that Fbw7 mediated downregulation of G-CSFR inhibits down-stream signaling and subsequent biological consequences. On the con-trary, inhibition of Fbw7 restores G-CSFR signaling leading to enhancedSTAT3 activity and granulocytic differentiation.

2. Materials and methods

2.1. Cell culture, transfection and siRNA introduction

Human embryonic kidney cell line HEK293T (293T) and IL-3 de-pendent murine myeloid 32Dcl3 cells were obtained from ATCC.293T cells were cultured as previously described [23]. 32Dcl3 cellswere maintained in RPMI-1640 medium supplemented with 10%

Table 1Putative CPD sequences in G-CSFr compared with CPD sequences in known substratesof FBW7. Few sequences do not exactly match with CPD consensus sequences howeverlike in mTOR they may also be targeted by Fbw7.

Consensus CPD sequence: (S/T)P-X-X-(S/T/D/E)

Potentialphosphorylationsite

Putative CPDsequence inG-CSFr

Other substratescontaining similarCPD sequences

Kinase Ref,

S322 S P S L E Cyclin E: pT P D K EKLF5: pT P D L D

not knownGSK3β (pT)

[21][18,19]

T620 T P E G S Cyclin E pT P P Q pS GSK3β(pT) [21]T802a T P A P S c-myc pT P P L S cdk2 (pS) [12,13]

c-jun pT P P L S GSK3β (pT) [14]SREBP 1: pT P P P pS GSK3β (pT) [20]KLF5: pS P P S SpT P P P S

GSK3β (pT,pS)GSK3β (pS)

[18,19]

PGC1: pT P E pS P GSK3β (pT)p38 MAPK (pT,pS)

[22]

T690 T P P I T C/EBPα: pT P P P pT GSK3β (pT, pT) [16,17]PGC1: pT P P T pT GSK3β (pT)

p38 MAPK (pT)[22]

S795a S P L G T MCL-1: pS L P S pT GSK3β (pS, pT) [15]S214 SPQLCb mTOR: T P S I H Not known [30]S238 SPEAAb

T420 TPVVFb

T505 TPLYQb

S557 SPLTHb

S651 SPNRKb

S758 SPTSPb

S761 SPGPGb

T781 TPSPKb

S783 SPKSYb

a CPD motifs absent in truncated mutant G-CSFr-T718.b These motifs could also act as CPD.

fetal bovine serum (FBS), 1% PenStrep (Gibco) and 10 ng/mL murineIL-3 (Prospec) at 37 °C and 5% CO2. Expression plasmids weretransfected using LTX (Invitrogen) while siRNAs were transfectedwith DhermaFECT (Dhermacon) as per manufacturer's protocol. ONTARGETplus SMARTpool siRNAs (Dhermacon) for hFbw7, mFbw7and hGSK3β were used, sicontrol non-targeting siRNA#1 was usedas control. Expression plasmids for pMX-IRES-GFP-HA-G-CSFR andpMX-IRES-GFP-HA-G-CSFR-T718 were kind gifts from Dr. Stefan N.Constantinescu [24]; pFLAG-Fbw7α, pFLAG-Fbw7β, pFLAG-Fbw7γ,pFLAG-Fbw7αΔF and pFLAG-dnFbw7αWD were kind gifts fromDr. E. Clurman [25]. V5-GSK3β and HA-GSK3βS9A were kind giftsfrom D. Helen Piwnica-Worms and Dr. William G. Kaelin respectively[14,26].

2.2. Cycloheximide half-life experiments

293T cells were transiently transfected with 0.5 μg HA-G-CSFR/G-CSFR-T718 alone or with 1 μg Flag-Fbw7α or Fbw7αΔF ordnFbw7αWD. 48 h post transfection, cycloheximide (CHX) (Enzo LifeSciences) was added to a final concentration of 80 μg/mL to inhibitnew protein synthesis for indicated time points followed by wholecell extracts (WCEs) preparation in RIPA buffer and immunoblottedwith anti-HA.

2.3. Peripheral blood mononuclear cells (PBMCs) isolation

PBMCs were isolated from healthy volunteer blood samples usinghistopaque-1077 (Sigma). Isolated PBMCs were grown in RPMI-1640supplemented with 10% FBS. PBMCs were transiently transfectedwith Fbw7α using Lipfectamine LTX (Invitrogen) according to themanufacturer's protocol. 48 h post transfection, WCEs were preparedand subjected to immunoblot against anti-G-CSFR antibody to assessendogenous levels of G-CSFR.

2.4. Morphology study

32Dcl3 cells were transfected with dnFbw7WD or siFbw7 in dupli-cates. 48 h post transfection one set was stimulated with 100 ng/mLG-CSF (Millipore) and the other was left untreated. Transfected32Dcl3 cells were then maintained in IL-3 deprived completeRPMI-1640 to assess the morphology. Cells after 3, 9 and 12 dayswere cytospun, air dried and stained with May-Grunwald and Giemsa(Sigma) and subsequently analyzed under microscope.

2.5. Co-immunoprecipitation and in vivo ubiquitination assay

Co-immunoprecipitation was performed as previously described[27–29]. Briefly, 293T cells were transfectedwith the indicated plasmids,namely G-CSFR/G-CSFR-T718 and Fbw7α, Fbw7β or Fbw7γ. 48 h posttransfection 10 μM MG132 or 10 μM Lactacystin (Sigma) was added tothe cells for 6 h, wherever used. Whole cell extracts (WCEs) were pre-pared using RIPA lysis buffer. Equal amounts of pre-cleared WCEs wereincubated at 4 °C for 4 h with pre-equilibrated Protein A/G agarosebeads (Millipore) and either anti-G-CSFR or anti-GFP antibody wasused to immunoprecipitate G-CSFR/G-CSFR-T718 or anti-cdc4 and anti-GSK3β antibody to immunoprecipitate endogenous Fbw7 andGSK3β re-spectively from 293T cells. Co-precipitates were further immunoblottedwith the indicated antibodies. PBMCs isolated from healthy volunteerbloodwere treatedwith orwithout 10 μMMG132.WCEswere preparedusing RIPA lysis buffer and endogenousG-CSFRwas immunoprecipitatedas described above.

In vivo ubiquitination was performed as described previously [30].Briefly, 293T cells were transfected with the indicated plasmids; 48 hpost transfection, 10 μM MG132 was added to the cells for 6 h. WCEswere subjected to co-immunoprecipitation with anti-GFP antibody topull out G-CSFR. Co-precipitates were then immunoblotted with

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anti-His antibody to assess the ubiquitinated G-CSFR. Antibody detailsare provided in the Appendix A.

2.6. Immunofluorescence

HEK293T (293T) cells grown on a coverslip in six well flasks weretransfected with HA-G-CSFR together with Fbw7α. 24 h post trans-fection, cells were fixed with 4% paraformaldehyde, permebealizedwith 0.5% Triton X-100 in 1× PBS and further blocked with 1% BSA.Cells were immunostained with rabbit G-CSFR (1:250) and mouseFlag M2 (1:250) antibody overnight at 4 °C. The next day, cellswere washed with 1× PBS and further incubated with anti-rabbit(alexafluor 488) and anti-mouse (alexafluor 594) secondary antibod-ies (1:500) respectively. Coverslips were washed, dried and mountedwith vectashield (vector, H-1000). Images were captured using LeicaDMI6000B microscope. PBMCs from the healthy volunteer's bloodwere isolated and cytospun on slides. Cells were subjected to similarprotocol described above and subsequently immunostained withanti-rabbit G-CSFR (1:250) and anti-mouse Fbw7 (1:250) antibodyovernight at 4 °C. The next day, cells were washed with 1× PBS andfurther incubated with anti-rabbit alexafluor 488 and anti-mousealexafluor 594 secondary antibodies (1:500) respectively. Imageswere captured using Leica DMI6000B microscope.

3. Results

3.1. Fbw7 negatively regulates steady state levels of G-CSFR

The apparent role of ubiquitin–proteasome pathway in negativeregulation of G-CSFR signaling prompted us to search for E3 ubiquitinligases that may target G-CSFR for degradation via the ubiquitin–proteasome pathway [10]. Thorough analysis of G-CSFR protein se-quence revealed the presence of several potential Cdc4-PhosphoDegron (CPD) motifs that may be recognized by Fbw7, a substraterecognizing component of SCF E3 ubiquitin ligase complex (SCFFbw7)for targeting them to ubiquitin–proteasome degradation [31]. Fivesuch potential CPDs starting at amino acid positions S322, T620,T690, S795 and T802 matching to the CPDs in other known substratesof Fbw7 are present in G-CSFR (Table 1). In addition few other motifsshowing modest homology to CPD consensus sequences are alsopresent in G-CSFR [32]. The presence of these scattered CPDs inG-CSFR prompted us to investigate whether G-CSFR is also a substratefor Fbw7. To address this, WCEs of HEK293T cells (here onwards293T) transfected with HA-G-CSFR and indicated amounts of Fbw7isoforms were immunoblotted with anti-HA antibody which showedthat all the three isoforms downregulated G-CSFR protein expression(Fig. 1A). Further, since Fbw7α is the most highly expressed and stableisoform of Fbw7 [33], we co-transfected HA-G-CSFR with increasingamounts of Fbw7α (0.5, 1.0 and 2.0 μg) in 293T and immunoblottedwith G-CSFR which showed an inverse correlation between G-CSFRand Fbw7α expression; persistent downregulation of G-CSFR proteinexpressionwith increasing doses of Fbw7αwas seen (Fig. 1B). This sug-gested that Fbw7might negatively regulate G-CSFR protein steady statelevels. To further verify that downregulation of G-CSFR is Fbw7α specif-ic, we over expressed HA-G-CSFR with Fbw7α deletion mutants:non-functional Fbw7αΔF (lacks the F box domain which binds directlyto the SKP1 component of SCF ubiquitin ligase) and dnFbw7αWD (con-tains only a stretch ofWD40 repeats, the protein interaction domains inFbw7 which bind to the substrate) in 293T cells. Contrary to the wildtype Fbw7α, G-CSFR expression was restored with Fbw7 mutants;confirming G-CSFR downregulation is Fbw7 specific (Fig. 1C). In fact,unlike Fbw7α, increasing amounts of Fbw7αΔF and dnFbw7αWD ratherstabilized the G-CSFR expression (Appendix A, Fig. S1). In order to as-certain these findings in a physiologically relevant model, we used32Dcl3 cells, a murine myeloid cell line that is used as a model tostudy G-CSFR signaling [34]. We co-transfected G-CSFR with different

isoforms of Fbw7 (Fbw7α, Fbw7β and Fbw7γ) in 32Dcl3, 24 h posttransfection, cells were lysed, resolved on 8% SDS-PAGE andimmunoblotted with HA antibody which affirmed that Fbw7 indeeddownregulates G-CSFR expression (Fig. 1D). Since G-CSFR generatesunique signals for the production of peripheral blood mononuclearcells (PBMCs), we assessed endogenous G-CSFR expression by tran-siently transfecting Fbw7α in PBMCs isolated from healthy volunteers.48 h post transfection, cells were harvested, resolved on 8% SDS-PAGEand immunoblottedwith anti-G-CSFR antibodywhich yielded three en-dogenous bands in the 80–130 kDa range, possibly representing differ-ent glycosylated states of G-CSFR [35]; as expected, the expression of allthe three bands are reduced in Fbw7α transfected PBMCs (Fig. 1E)which further strengthens our finding that Fbw7 negatively regulatessteady state levels of G-CSFR. We next asked if Fbw7 negatively regu-lates G-CSFR expression, should the knockdown of Fbw7 by siFbw7 re-store endogenous G-CSFR expression? To address this, we knockeddown endogenous Fbw7 by siFbw7 in a physiologically relevant cellsystem, 32Dcl3. We observed that siFbw7 mediated knockdown ofFbw7 led to increase in endogenous G-CSFR expression. Further, asexpected, IL3 replacement with G-CSF stimulated endogenous G-CSFRexpression in these cells and when Fbw7 was knocked down withsiFbw7, endogenous G-CSFR expression alleviated even in the absenceof G-CSF stimulation confirming an inverse relationship betweenG-CSFR and Fbw7 (Fig. 1F).

3.2. Fbw7 promotes G-CSFR degradation primarily throughubiquitin–proteasome pathway

Because Fbw7 is an E3 ubiquitin ligase, we sought to assess ifdownregulation of G-CSFR expression by Fbw7 is mediated by its ligaseactivity leading to ubiquitin–proteasome-dependent degradation. Sub-stantial restoration of G-CSFR protein expression even in the presenceof Fbw7α by active proteasome inhibitor MG132 (Fig. 2A) endorsedour assumption that Fbw7 (through its E3 ubiquitin ligase activity) ap-parently targets G-CSFR for ubiquitin–proteasome mediated degrada-tion [36]. Further, cells were also treated with lysosomal inhibitorBafilomycin A1 either alone or together with MG132 to test whetherdegradation of ubiquitinated G-CSFR also occurs through lysosomal-pathway in addition to ubiquitin–proteasome pathway [37]. Immuno-blot of the lysates revealed that MG132 majorly restored the G-CSFRprotein level in 293T cells. Although Bafilomycin A1 also slightly miti-gated Fbw7mediated G-CSFR downregulation,much enhanced restora-tion of G-CSFR expression was observed with MG132, suggestingG-CSFR downregulation through Fbw7 is primarily mediated throughubiquitin–proteasome pathway (Fig. 2A). To further strengthen thesefindings, we performed in vivo ubiquitination assay using WCEs from293T cells co-transfected with plasmids expressing Fbw7α, G-CSFRand His-Ubiquitin. G-CSFR was immunoprecipitated with anti-GFPwhile the co-precipitates were immunoblotted with anti-His antibodyafter resolving on 8% SDS-PAGE. Heavy G-CSFR ubiquitination as a lad-der was observed in Fbw7α, G-CSFR and His-Ubiquitin co-transfectedcells while almost no ubiquitination was observed in cells co-transfected with G-CSFR, His-Ubiquitin and either of the deletion mu-tants of Fbw7 (Fbw7αΔF and dnFbw7αWD) suggesting Fbw7 indeedpromotes ubiquitination of G-CSFR. Interestingly, a rather more intenseubiquitinated ladder pattern was observed in cells treated with MG132which apparently stabilized the ubiquitinated G-CSFR by inhibitingproteasome machinery (Fig. 2B). In order to further consolidate ourfinding that Fbw7 is affecting steady state levels of G-CSFR protein, wedetermined the half-life of G-CSFR after inhibiting new protein synthe-sis by cycloheximide (CHX). Immunoblot as shown in Fig. 2Ci clearlydemonstrates that Fbw7α markedly reduces the half-life of G-CSFR,while Fbw7 deletion mutants have no effect. The graphical analysis(Fig. 2Cii) indicates that Fbw7α led to a significant decrease in thehalf-life of G-CSFR from 120 min to less than 60 min. Detailed analysis

B

IB: HA

IB: Flag

IB: β-actin

Fbw7α (µg) 2 0.5 1 2 0.5µg G-CSFR

42kDa

130kDa

100kDa

A0.5 1.0 0.5 1.0 0.5 1.0 (µg)

G-CSFR (0.5µg) + + + + + + +

Fbw7α Fbw7β Fbw7γ

IB: HA

IB: Flag

IB: β-actin

* αβ

γ

42kDa

48kDa

75kDa100kDa

130kDa

G-CSFR + + + +

IB: HA

IB: Flag

IB: β-actin

dnFbw7αWD

C

130kDa

100kDa

42kDa

D

EFbw7α - +

PBMCs

IB: G

-CS

FR

IB: Flag

IB: β-actin

En

do

gen

ou

s G

-CS

FR

85kDa

100kDa130kDa

100kDa

42kDa

Fbw7 γ (1.0µg) - - - - +Fbw7β (1.0µg) - - - + -Fbw7α (1.0µg) - - + - -G-CSFR (0.5µg) - + + + +

α

βγ

IB: HA

IB: Flag

IB: β-actin 42kDa48kDa

75kDa100kDa

130kDa

+GCSF

D2 D3

siFBW7- GCSF

D2 D3

siFBW7+ GCSF

D2 D3

IB: G

-CS

FR

En

do

gen

ou

s G

-CS

FR

IB: FBW7

130kDa100kDa

75kDa

100kDa

F

Fig. 1. Fbw7 negatively regulates steady state levels of G-CSFR. (A) 293T was transiently transfected with 0.5 μg HA tagged G-CSFR with indicated amount of Flag-Fbw7α,Flag-Fbw7β and Flag-Fbw7γ. Post 48 h of transfection WCEs were prepared, resolved on 8% SDS-PAGE and immunoblotted with anti-HA and anti-Flag-M2 antibodies to detectG-CSFR, Fbw7α, Fbw7β, and Fbw7γ expression respectively. β-actin was probed as loading control. (*indicates non specific band, possibly unstriped G-CSFR). (B) 0.5 μg ofG-CSFR co-transfected with increasing amounts of Flag-Fbw7α (0.5, 1.0 and 2.0 μg). 48 h post transfection, WCEs were prepared, resolved on SD-PAGE and immunoblottedwith anti-HA and anti-Flag-M2 antibodies respectively. β-actin was probed as loading control (C) 0.5 μg of G-CSFR was co-transfected with 1.0 μg of Flag-Fbw7α or Fbw7αΔFor dnFbw7αWD in 293T cells. WCEs were prepared after 48 h of transfection, resolved on SDS-PAGE and immunoblotted with anti-HA and anti-Flag-M2 antibodies respectively(D) 0.5 μg of G-CSFR was transfected with or without 1.0 μg of Flag-Fbw7α, Flag-Fbw7β or Flag-Fbw7γ in 32Dcl3 cells. 48 h post transfection, WCEs were prepared andimmunoblotted with anti-HA and anti-Flag-M2 antibodies to detect G-CSFR and Fbw7 respectively. β-actin was probed as loading control. (E) PBMCs isolated from healthy volun-teers were transfected with 2.0 μg of Fbw7α. 48 h post transfection, WCEs were prepared, resolved on SDS-PAGE and immunoblotted with anti-G-CSFR antibody for endogenousG-CSFR and anti-Flag-M2 antibody for Fbw7α. The arrows indicate the multiple bands of endogenous G-CSFR between 80 and 130 kDa. β-actin was probed as loading control.(F). 32Dcl3 cells were transfected with siFbw7 and were either left unstimulated or stimulated with 100 ng/mL G-CSF for 2 (d2) and 3 days (d3) followed by WCEs preparation,separation on SDS-PAGE and immunoblotted with anti-G-CSFR and anti-cdc4 antibodies to detect G-CSFR and Fbw7 respectively.

2642 S. Lochab et al. / Biochimica et Biophysica Acta 1833 (2013) 2639–2652

of expressions of Fbw7α, Fbw7αΔF and dnFbw7αWD alongwithβ-actinis provided in Appendix A, Fig. S2.

Collectively, these results suggest that Fbw7 inhibits steady statelevels of G-CSFR by promoting its proteasomal degradation.

3.3. Fbw7α physically interacts with G-CSFR

Fbw7 is the substrate binding moiety of SCF ubiquitin ligase com-plex and because G-CSFR is being targeted by Fbw7, we asked

whether Fbw7 physically associates with G-CSFR. To determine this,we co-immunoprecipitated ectopically expressed G-CSFR fromWCEs of 293T transfected with G-CSFR and Fbw7α, Fbw7β orFbw7γ (Fig. 3A). We found that the three isoforms co-precipitatedwith G-CSFR, verifying it to be a probable substrate of Fbw7. More-over, to further identify the region of Fbw7 that interacts withG-CSFR in vivo, we co-transfected G-CSFR with Fbw7α and itsdeletion mutants Fbw7αΔF and dnFbw7αWD in 293T cells. We ob-served that Fbw7α, Fbw7αΔF and dnFbw7αWD co-precipitated with

0

20

40

60

80

100

120

0 30 60 120 240

G-CSFR

Fbw7αdnFbw7WD

Fbw7αΔF

% G

-CS

FR

Minutes

A

iiCHX (min) 0 30 60 120 240

0.5µg G-CSFR

IB:

HA

dnFbw7αWD

Fbw7α

Fbw7αΔF

C i

130kDa

130kDa

130kDa

130kDa

B

dnFbw7αWD - - - - - + -Fbw7αΔF - - - - + - -Fbw7α - - - + - - +

His -Ubi - - + + + + + G -CSFR - - + + + + +

IP: GFP

IB: His

IB: HA 130kDa

Fbw7α - - + ++ ++ + + ++ G -CSFR - + + + + + +

IB: HA

IB: β -actin

IB: Flag

130kDa

100kDa

42kDa

Fig. 2. Fbw7 promotes G-CSFR degradation through ubiquitin–proteasome pathway. (A) 293T cells were co-transfected with 0.5 μg G-CSFR and either 1.0 μg (+) or 2.0 μg (++) ofFbw7α. 48 h post transfection, MG132 (10 μM) and Bafilomycin A (300 nM) were added in indicated lanes for 6 h before harvesting cells. Expression of G-CSFR and Fbw7α wasassessed with anti-HA and anti-Flag-M2 antibodies respectively. β-actin was probed as loading control. (B) 293T cells were co-transfected with G-CSFR (1.0 μg) and His-Ubi eitheralone or together with Fbw7 or its deletion mutants as indicated. After 48 h of transfection, MG132 (10 μM) was added in indicated condition 6 h before harvesting cells. Post 48 h,cells were harvested, and G-CSFR was co-immunoprecipitated using anti-GFP antibody (G-CSFR is cloned in pMX-GFP vector). Co-precipitates were immunoblotted with anti-Hisand anti-HA antibodies as indicated. (Ci) 293T cells were co-transfected with either 0.5 μg HA-G-CSFR or together with Fbw7α, or Fbw7αΔF or dnFbw7αWD. After 48 h, cells weretreated with 80 μg/mL of CHX andWCEs were prepared after 30, 60, 120 and 240 min and immunoblotted with anti-HA (Cii). The percentage of remaining G-CSFR calculated usingdensitometry after various time points is graphically represented. Protein expression levels of Fbw7α, Fbw7αΔF or and dnFbw7αWD and their respective β-actin loading controlsare shown in Appendix A Fig. S2.

2643S. Lochab et al. / Biochimica et Biophysica Acta 1833 (2013) 2639–2652

G-CSFR from the lysates (Fig. 3B) suggesting G-CSFR interacts to Fbw7in its WD domain. Our finding that Fbw7 interacts with G-CSFRthrough its WD domain further approves the notion that G-CSFRis a substrate of Fbw7. We observed an intensified interactionbetween G-CSFR and Fbw7α in the presence of proteasome inhibi-tors, MG132 and Lactacystin (Fig. 3C) which confirms that Fbw7binding with G-CSFR is prerequisite for ubiquitin mediatedproteasome degradation of G-CSFR. Furthermore, endogenous Fbw7was also found to be interacting with exogenously expressedG-CSFR (Appendix A, Fig. S3) that verified the direct interaction be-tween the two. To further validate the in vivo interaction betweenG-CSFR and Fbw7, we co-immunoprecipitated endogenous G-CSFRfrom 32Dcl3 cells after G-CSF stimulation for 1 and 3 days and simulta-neously from PBMCs treated with and without MG132 (to restore en-dogenous G-CSFR). Fbw7 was detected when co-immunoprecipitateswere immunoblotted with Fbw7 antibody (Fig. 3D–E) which stronglyconfirms our finding that there is a physical interaction betweenendogenous G-CSFR and Fbw7. Consistent with the physical inter-action, endogenous G-CSFR and Fbw7 also co-localize together inPBMCs (Fig. 3F) as well as in ectopically expressed 293T cells(Fig. 3G). We observed that Fbw7α being localized in the nucleus andnuclear periphery apparently co-localizes with G-CSFR at the nuclearperiphery.

Taken together, these data suggest that Fbw7 through its WDdomain physically associates and co-localizes with G-CSFR.

3.4. Fbw7 interacts and degrades G-CSFR-T718 but at a quite slower rate

While there are several scattered CPDs in the G-CSFR protein se-quence, many of them aremissing in G-CSFR-T718, reported in patientswith severe congenital neutropenia (SCN) transforming to AML [38].This inspired us to investigate whether Fbw7 can also target thistruncated G-CSFR. To answer this, we co-transfected G-CSFR-T718with Fbw7α, Fbw7β or Fbw7γ in 293T and found that Fbw7 alsodownregulates G-CSFR-T718; however, G-CSFR-T718 downregulationwas quite slow compared to G-CSFR in the presence of Fbw7 isoforms(Fig. 4A). We further assessed the down-modulation of G-CSFR-T718with increasing amounts (0.5, 1.0 and 2.0 μg) of Fbw7α (Fig. 4B). Wefound a slight decrease in G-CSFR-T718 with 2.0 μg of Fbw7αsuggesting Fbw7 indeed targets G-CSFR-T718 but not as efficiently asthe wild type G-CSFR. This is likely due to the absence of two fully con-sensus putative CPDs from G-CSFR-T718. The above experiments whenperformed in 32Dcl3 cells showed similar results; Fbw7 isoformsdownregulated G-CSFR-T718 however, at a slower rate as in Fig. 4C. In-deed, CHX half-life determination of G-CSFR-T718 in the presence ofFbw7α evidently confirmed that Fbw7 degrades G-CSFR-T718 at aslower rate compared to G-CSFR wild type (Fig. 4Di–ii). Clearly, thesefindings indicate the importance of CPDs present in the carboxy-terminal of G-CSFR after aa718. The reduced rate of degradation ofG-CSFR-T718 propelled us to examine if Fbw7 also interacts withG-CSFR-T718. For this, we co-transfected 293T cells either with

A

B

IP: GFP, IB: HA

IB: Flag

MG132 - + - + - +0.5 µg G-CSFR + + + + + + +

dnFbw7WD 35kDa

48kDa

100kDa

130kDa

IP: G-CSFR

Fbw7α - - + - - + - + - -Fbw7β - - - + - - - - + -Fbw7γ - - - - + - - - - +

G-CSFR - + + + + - + + + +

INPUTS

IB: HA

IB: Flag

IgG Heavy Chain 48kDa

75kDa100kDa

130kDa

Fbw7α + + + + - +0.5 µg G-CSFR + + + + - - +

IP: GFP

IB: Flag

G-CSFR*

C

IB:HA

IB: G-CSFR

MG132 - + -

IB: FBW7

IP: G-CSFR

100kDa

75kDa

130kDaG-CSFR

D

E

100kDa

75kDa

130kDa

IB:

G-C

SF

R

G-CSF d0 d1 d3 d0 d1 d2

IP: G-CSFR INPUTS

IB: FBW7 100kDa

100kDaF

G-CSFR Fbw7α

DAPI Merged

G-CSFR Fbw7

DAPIMerged

G

Fig. 3. Fbw7α physically interacts with G-CSFR. (A) Fbw7α, Fbw7β and Fbw7γ were co-transfected with G-CSFR. 48 h post transfection, whole cell extracts were prepared andHA-G-CSFR was co-immunoprecipitated using anti-GFP antibody. Immunoblotting with anti-Flag-M2 antibody shows in vivo interaction between G-CSFR and all isoforms offbw7 (Fbw7α, Fbw7β and Fbw7γ). (B) 293T cells were co-transfected with 1.0 μg of G-CSFR and 1.0 μg of Fbw7α or its deletion mutants (Fbw7αΔF or dnFbw7αWD). 48 h aftertransfection, MG132 (10 μM) was added in indicated lanes 6 h before WCEs were prepared. G-CSFR co-immunoprecipitation was performed with anti-GFP antibody followed byimmunoblotting of co-immunoprecipitates with anti-Flag-M and anti-HA antibodies. (C) 293T cells were co-transfected with 0.5 μg G-CSFR and 1.0 μg (+) of Fbw7α. 48 h posttransfection, MG132 (10 μM) and Lactacystin (10 μM) were added to 293T for 6 h before harvesting cells. WCEs were subjected to immunoprecipitation with anti-GFP antibodyand immunoprecipitates were probed first with anti-Flag-M2 antibody followed by anti-HA antibody after stripping the same blot. In vivo interaction between G-CSFR andFbw7α is intensified in the presence of MG132 and Lactacystin (*unstripped Fbw7α below G-CSFR as the blot was first immunoblotted with anti-Flag and then anti-HA; mild strip-ping was performed to capture G-CSFR) (D) 32Dcl3 cells were stimulated with 100 ng/mL G-CSF for 2 (d2) and 3 days (d3). Endogenous G-CSFR was co-immunoprecipitated usinganti-G-CSFR antibody and co-immunoprecipitates were immunoblotted with anti-G-CSFR and anti-cdc4 antibodies respectively. (E) PBMCs isolated from volunteer's blood weretreated with 10 μM MG132 for 6 h. Endogenous G-CSFR was immunoprecipitated using anti-G-CSFR antibody and immunoprecipitates were immunoblotted with anti-cdc4 anti-body. (F) PBMCs isolated from volunteer's blood were cytospun on slides. Immunofluorescence (IFC) assay was performed as indicated in the Materials and methods usinganti-G-CSFR (rabbit) and anti-cdc4 (mouse) primary antibodies and anti-rabbit (Alexafluor 488) and anti-mouse (Alexafluor 594) secondary antibodies. Cells were air dried andmounted to capture the images. (G) 293T cells were grown on coverslips and transfected with 0.5 μg G-CSFR and 0.5 μg Fbw7α. In order to avoid heavier degradation ofG-CSFR, IFC was performed after 24 h of transfection. Similar procedure as described in Fig. 3F was used.

2644 S. Lochab et al. / Biochimica et Biophysica Acta 1833 (2013) 2639–2652

A

C

B

100kDa

Fbw7 α Fbw7 β Fbw7 γ

IB: HA

IB: Flag

IB:β-actin

0.5 1.0 0.5 1.0 0.5 1.0 (µg)

G-CSFR-T718 + + + + + + +

α

βγ

42kDa

75kDa

100kDa

IB: HA

IB: Flag

Fbw7α (µg) - + - 0.5 1 2 2+MG132

0.5µg G-CSFR-T718

IB: β-actin

100kDa

100kDa

42kDa

Fbw7γ (1.0µg) - - - - +Fbw7β (1.0µg) - - - + -Fbw7α (1.0µg) - - + - -G-CSFR-T718 (0.5µg) - + + + +

α

βγ

IB: HA

IB: Flag

IB: β-actin

100kDa

100kDa

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75kDa

E

Di

ii

0

20

40

60

80

100

120

0 30 60 120 240

G-CSFR

G-CSFR+Fbw7α

G-CSFR-T718

G-CSFR-T718+Fbw7α

% G

-CS

FR

/G-C

SF

R-T

718

Minutes

CHX(min) 0 30 60 120 240

0.5µg G-CSFR-T718

IB:

HA

Fbw7α

100kDa

MG132 - - + - - + -Fbw7α - + + - + + +G-CSFR + + + - - - -G-CSFR-T718 - - - + + + -

IB: Flag

IB: HA, G-CSFRG-CSFR-T718

IP: GFP

Fbw7α

130kDa100kDa

100kDa

48kDa

IgG Heavy Chain

Fig. 4. Rate of degradation of mutant G-CSFR-T718 by Fbw7 is relatively slower than G-CSFR. (A) 293T cells were transiently co-transfected with 0.5 μg HA tagged G-CSFR-T718 andindicated amounts of Fbws. After 48 h of transfection, WCEs were prepared, resolved on 8% SDS-PAGE and probed with anti-HA and anti-Flag antibodies to detect expression ofG-CSFR-T718 and Fbw7 isoforms (Fbw7α, Fbw7β and Fbw7γ). β-actin was probed as loading control. (B) 293T cells were co-transfected with 0.5 μg G-CSFR-T718 and increasingamounts of Flag-Fbw7α (0.5, 1.0 and 2.0 μg). 48 h post transfection, lysates were prepared, resolved on SDS-PAGE and immunoblotted with anti-HA and anti-Flag-M2 antibodies. βactin was probed as loading control. (C) 32Dcl3 cells were co-transfected with 0.5 μg HA tagged G-CSFR-T718 and indicated isoforms of Fbw7 (1.0 μg Fbw7α, Fbw7β and Fbw7γ)separately. 48 h post transfection, WCEs were immunoblotted with anti-HA and anti-Flag-M2 (Di–ii) 293T cells were co-transfected with 0.5 μg HA-G-CSFR-T718 and Fbw7α. Post48 h of transfection, cells were treated with 80 μg/mL of CHX and lysates were prepared after 30, 60, 120 and 240 min followed by immunoblotting with anti-HA antibody. Thepercentage of remaining G-CSFR-T718 after various time points is depicted in the graph. Immunoblots for Fbw7α and β actin are shown in Appendix A Fig. S2. (E) 293T cellswere co-transfected with Fbw7 and either G-CSFR or G-CSFR-T718; 48 h post transfection, G-CSFR and G-CSFR-T718 were immunoprecipiated from lysates using anti-GFP antibodyand co-immunoprecipitates were probed with anti-HA and anti-Flag antibodies.

2645S. Lochab et al. / Biochimica et Biophysica Acta 1833 (2013) 2639–2652

G-CSFR or G-CSFR-T718 and Fbw7α. MG132 was added to cells as indi-cated prior to WCE preparation in order to stabilize G-CSFR in thepresence of Fbw7α. Anti-GFP antibody immunoprecipitated G-CSFR/G-CSFR-T718 from WCEs and anti-Flag immunoblotting showed thatthey still interactwith each other which probably is due to the presenceof the remaining CPDs in G-CSFR-T718 however, the observed slowdegradation of GSCFr-T718may be attributed to the absence of two po-tential CPDs (Fig. 4E). To further corroborate our findings, we show thatendogenous Fbw7 interacts with ectopically expressed G-CSFR-T718suggesting the direct in vivo interaction between the two (Fig. S3). Infact, we also determined that besides Fbw7α, other two isoformsFbw7β and Fbw7γ also interact with G-CSFR-T718 (Fig. S3) which sug-gests that region of G-CSFR required to interact with Fbw7 is also pres-ent before aa718 in G-CSFR. Taken together, these data indicate thatFbw7 also interacts with G-CSFR-T718 and targets it for degradation;

however, its rate of degradation is relatively slower owing to the ab-sence of some of CPD motifs which are otherwise present in wild typeG-CSFR.

3.5. Fbw7α mediated degradation of G-CSFR is GSK3β-dependent

Several groups have previously reported that substrates like c-jun,c-myc, cyclin E and PGC-1α contain CPD motifs similar to the onespresent in G-CSFR and are phosphorylated by GSK3β within CPDmotifs prior to recognition and subsequent degradation by Fbw7(Table 1). This inspired us to assess if GSK3β also instigates Fbw7αmediated degradation of G-CSFR. To answer this, we transfected293T cells with G-CSFR and Fbw7 and treated them with GSK3β in-hibitor Lithium Chloride (LiCl) [39]. Immunoblotting with G-CSFRshowed that, like MG132, LiCl also restored G-CSFR expression in

2646 S. Lochab et al. / Biochimica et Biophysica Acta 1833 (2013) 2639–2652

Fbw7α co-transfected condition (Fig. 5A), suggesting a probable roleof GSK3β in G-CSFR downregulation mediated through Fbw7. In linewith this, we examined G-CSFR half-life by inhibiting new proteinsynthesis by CHX to determine the rate of degradation of G-CSFRwith Fbw7α in the presence of LiCl (Fig. 5B), we observed that asexpected, the rate of degradation of G-CSFR reduced in the presenceof LiCl. To substantiate further, we separately co-transfected G-CSFR

A

C

GSK3β (µg) - 0.5 1 2 - - 0.5 1 2

IB: HA

IB: GSK3β

IB: β -actin

0.5µg G -CSFR

*

0.5µg G -CSFR -T718

130kDa100kDa

48kDa

42kDa

Fbw7α (2µg) - - +G -CSFR (0.5µg ) - + +

++

+ ++ -

IB: HA

IB: Flag

IB: β -actin 42kDa

100kDa

130kDa

E

G

G -CSFR - + + + + + + +Fbw7α - -

siGSK3β

IB: Flag

IB: HA

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IB: GSK3β 48kDa

100kDa

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42kDa

G -CSFR - + + + + + +GSK3β - -

siFbw7

IB: Fbw7

IB: HA

IB: β -actin

GSK3β48kDa

42kDa

75kDa

130kDa

100kDa

and G-CSFR-T718 with increasing amounts (0.5, 1.0 and 2.0 μg) ofGSK3β and found that both G-CSFR and G-CSFR-T718 expression is no-tably reduced in the presence of GSK3β in a dose-dependent manner(Fig. 5C). To consolidate further, we performed similar experimentswith constitutively active GSK3β (GSK3βS9A); like GSK3β, it also mas-sively downregulated both G-CSFR and G-CSFR-T718, however, the rateof degradation of G-CSFR-T718was again relatively slower than G-CSFR

B

D

IB: HA

IB: GSK3β

IB: β -actin

GSK3βS9A - - + - +

42kDa

48kDa

100kDa130kDa

+ + +

CHX for 60 min

IB: HA

IB: Flag

IB: β -actin

LiCl (20mM) - - - - - +Fbw7α (2µg) - - + - + +

+ +G -CSFR (0.5 µg ) -

42kDa

100kDa

130kDa

F

*

G -CSFR - + + + + ++ + +GSK3β - - +

Fbw7α - - - + - -Fbw7αΔF - - - - + -dnFbw7 WD - - - - - +

IB: Flag

IB: HA

IB: β-actin

IB: GSK3β 48kDa

63kDa

130kDa

100kDa

35kDa

42kDa

H

GSK3βS9A - - + - + + - + - + G -CSFR - + + - - - + + - -

G-CSFR-T718 - - - + + - - - + +

INPUTS

*

IP: G-CSFR

IB: HA, G-CSFR

G-CSFR-T718130kDa100kDa

47kDaIB: GSK3β

i.

ii.

GSK3β

IB: GSK3β

GSK3β - - + - + + - + - + G -CSFR - + + - - - + + - -

G -CSFR -T718 - - - + + - - - + +

INPUTS

*

IP: G-CSFR

IB: HA, G-CSFR

G-CSFR-T718

IgG Heavy Chain

130kDa100kDa

48kDa

INPUTSIP: GSK3β

IP: GSK3βIB: GSK3β

48kDa

IB: HA, G-CSFR

G-CSFR-T718130kDa100kDa

IgG Heavy Chain

IgG Heavy Chain

I

Fig. 5. G-CSFR degradation by Fbw7 isGSK3β-dependent. (A) 293T cellswere co-transfectedwith G-CSFR (0.5 μg) and Fbw7α (2.0 μg). 10 μMMG132was added for 6 h (36 h post trans-fection) and 20 mM LiCl was added for 24 h (post 24 h transfection) in indicated lanes; thus after total of 48 h, lysates were prepared, resolved and immunoblotted with anti-HA andanti-Flag-M2 antibodies. β-actin was probed as loading control. (B) 293T cells were transfected with 0.5 μg of each HA-G-CSFR and Fbw7α. 20 mM LiCl was added to transfected cellsin indicated lanes for 24 h. Cells were lysed 60 min after addition of CHX (80 μg/mL). G-CSFR and Fbw7 expression was compared to the conditions without CHX using anti-HA andanti-Flag antibodies respectively; β-actin was probed as loading control. (C) 293T cells were co-transfected with 0.5 μg G-CSFR or G-CSFR-T718 together with increasing amounts ofGSK3β (0.5, 1.0 and 2.0 μg). 48 h post transfection, WCEs were prepared and subjected to immunoblotting with anti-HA and anti-GSK3β antibodies. Slow migrating band is V5 taggedGSK3β while *marked faster migrating band is endogenous GSK3β. β-actin was used as loading control. (D). 293T cells were co-transfected with 0.5 μg of G-CSFR or G-CSFR-T718 and0.5 μg of GSK3β S9A. 48 h post transfection, WCEs were subjected to immunoblotting with anti-HA and anti-GSK3β antibodies. (E) Increasing amounts of Fbw7 (0.5, 1.0 and 2.0 μg)was transfected in 293T cells either alone or togetherwith siGSK3β (25 nM). 24 h post transfection, cellswere lysed, resolved on8%SDS-PAGE and immunoblottedwith anti-HA, anti-Flagand anti-GSK3β antibodies. β-actin was immunoblotted as loading control. (F) 293T cells were co-transfected with G-CSFR and GSK3βwith/without Fbw7α, Fbw7αΔF or dnFbw7αWD.WCEs prepared after 48 h of transfectionwere subjected to immunoblottingwith anti-HA and anti-Flag antibody. Inhibition of endogenous Fbw7with Fbw7αΔF or dnFbw7αWD inhibitedGSK3β instigated G-CSFR degradation; suggesting the inter dependence of both Fbw7 and GSK3β in degrading G-CSFR. (G) 293T cells were co-transfected with G-CSFR and increasingamounts of either GSK3β alone (0.5, 1.0 and 2.0 μg) or together with siFbw7 (25 nM), lysates were prepared after 24 h of transfection and subjected to immunoblotting with anti-HAand anti-Fbw7 antibodies; siFbw7 effectively knocked down endogenous Fbw7 as shown in Fig. S6. (H) 293T cells were transiently co-transfectedwith G-CSFR andG-CSFR-T718 togetherwith either GSK3β (H-i) or GSK3βS9A (H-ii) as indicated. 24 h post transfection, lysateswere prepared and G-CSFRwas co-immunoprecipitatedwith anti-G-CSFR antibody. Immunopre-cipitates were subsequently probed with anti-GSK3β and anti-HA antibody. Endogenous GSK3β* and ectopically expressed V5-tagged GSK3β both co-precipitated with G-CSFR andG-CSFR-T718 verifying an intense interaction between G-CSFR and GSK3β. GSK3βS9A has slightly higher molecular weight than endogenous GSK3β due to HA tagging. Therefore,GSK3βS9A and endogenous GSK3β bands appear as doublet. (I) Endogenous GSK3β was immunoprecipitated using anti-GSK3β antibody from WCEs of 293 T cells transfected withG-CSFR or G-CSFR-T718. Immunoblot with anti-HA shows in vivo interaction between ectopically expressed G-CSFR and/or G-CSFR-T718 with endogenous GSK3β.

2647S. Lochab et al. / Biochimica et Biophysica Acta 1833 (2013) 2639–2652

(Fig. 5D). GSK3βS9A led to massive downregulation of G-CSFR expres-sion even in the absence of exogenous Fbw7. In fact G-CSFR completelyvanished with increasing doses of GSK3βS9A alone (Appendix A,Fig. S4). We next asked whether Fbw7 driven degradation of G-CSFRis GSK3β-dependent.We therefore, transfected 293T cells with increas-ing amounts of Fbw7α (0.5, 1.0 and 2.0 μg) and silenced endogenousGSK3β in 293T with siGSK3β. It showed that silencing of endogenousGSK3β crippled Fbw7α driven degradation of G-CSFR (Fig. 5E);suggesting GSK3β probably facilitates its kinase activity to G-CSFR for

Fbw7 mediated degradation. Because alleviated levels of GSK3β elimi-nated G-CSFR and we hypothesized that GSK3 may phosphorylateG-CSFR in its CPDs for its recognition by Fbw7, we therefore assessedif GSK3β mediated downregulation of G-CSFR is dependent on Fbw7.To answer this, we co-transfected G-CSFR or GSK3β either with Fbw7or its deletion mutants (Fbw7αΔF and dnFbw7αWD) as indicatedin Fig. 5E. 24 h post transfection, WCEs were prepared, resolved on10% SDS-PAGE and immunoblotted with the indicated antibodies.We observed that GSK3β furthered degradation of G-CSFR when

2648 S. Lochab et al. / Biochimica et Biophysica Acta 1833 (2013) 2639–2652

co-transfected with Fbw7; however, G-CSFR expression was ratherrestored and stabilized when co-transfected with deletion mutantseven in the presence of exogenous GSK3β (Fig. 5F). Silencing endoge-nous Fbw7 with siRNA also inhibited G-CSFR degradation even inthe presence of increasing doses of GSK3β (Fig. 5G) which furtherconfirms the notion that Fbw7 and GSK3β are indispensible forG-CSFR degradation.

To further substantiate the role of GSK3β in G-CSFR degradationthrough Fbw7, we determined the in vivo interaction of GSK3βwith G-CSFR and G-CSFR-T718. For this, 293T cells were co-transfected with G-CSFR or G-CSFR-T718 with GSK3β or GSK3βS9A.Co-immunoprecipitation with G-CSFR antibody showed that en-dogenous GSK3β also co-precipitated with ectopically expressedGSK3β (V5-tagged-GSK3β migrates slowly compared to endogenousGSK3β) and GSK3βS9A (band could be seen slightly above the en-dogenous GSK3β due HA tagging) with G-CSFR and G-CSFR-T718.The results showed that GSK3β efficiently interacts with bothG-CSFR and G-CSFR-T718 (Fig. 5Hi–ii). Further, we also performed co-immunoprecipitation with endogenous GSK3β from 293T cells andfound that ectopically expressed G-CSFR and G-CSFR-T718 also co-precipitated with endogenous GSK3β which again verifies a direct in-teraction between the two (Fig. 5I). To further provide substantial evi-dence that GSK3β binds to G-CSFR and mediates its Fbw7 dependentdegradation, we show that all three proteins (GSK3β, G-CSFR/G-CSFR-T718 and Fbw7) are present in the same complex by showing thatGSK3β co-precipitates with Fbw7 isoforms when G-CSFR and G-CSFR-T718 were immunoprecipitated (Appendix A, Fig. S3).

Collectively, these results suggest that Fbw7 mediated degrada-tion of G-CSFR and G-CSFR-T718 is GSK3β dependent.

3.6. Fbw7 inhibits STAT3 activation while knockdown of Fbw7 orectopically expressed dnFbw7αWD enhances STAT3 activation

Previous studies have shown that STAT3 activation is required forG-CSFR dependent normal proliferation and granulocytic differentia-tion [24,40]. Therefore, we reasoned that degradation of G-CSFR byFbw7 also inhibits STAT3 activation and subsequent downstreamG-CSFR signaling. To address this, we ectopically over expressedFbw7α in murine myeloid cell line 32Dcl3 (a model system to studyneutrophil functions [41]) and post 24 h transfection, treated cellswith G-CSF for 10, 30 and 60 min respectively, As expected, STAT3phosphorylation dramatically decreased upon Fbw7α over expres-sion (Fig. 6A). Moreover, when 32Dcl3 cells were transfected with in-creasing amounts of Fbw7α and induced with G-CSF for shorterduration (10 min), G-CSF mediated STAT3 activation was againinhibited suggesting Fbw7 mediated downregulation of G-CSFR in-deed negatively influences its downstream signaling (Fig. 6B). Toconsolidate these findings further, we assessed STAT3 phosphoryla-tion by inhibiting endogenous Fbw7 either by knocking down Fbw7through siFbw7 (Fig. 6D) or by over expressing dnFbw7αWD

(Fig. 6C) again in 32Dcl3 cells. Cells were induced with G-CSF for in-dicated time points and lysates were immunoblotted with pSTAT3.Massive phosphorylation of STAT3 in both conditions was observedsuggesting that inhibiting Fbw7 targeted degradation of G-CSFRmay restore G-CSFR signaling. To substantiate further, we performedrescue experiment by expressing either DN-Fbw7 (dnFbw7αWD) orknocking down Fbw7 by siFbw7 with simultaneous knock down ofG-CSFR by siG-CSFR and then assessed pSTAT3 expression. Asshown in the immunoblot in Fig. 6E and F; upon G-CSF treatment,STAT3 phosphorylation (pSTAT3) are enhanced in both conditionswhen dnFbw7αWD was expressed or Fbw7 was knocked down;however this pSTAT3 substantially reduced when G-CSFR was alsoknocked down simultaneously in the rescue experiment suggestingSTAT3 activation and subsequent enhanced granulocytic differentia-tion are really because Fbw7 functions through G-CSFR.

3.7. Knockdown of Fbw7 or ectopically expressed dnFbw7αWD triggersdifferentiation in 32Dcl3 cells

The above assessment of the STAT3 activation/inhibition uponFbw7 knockdown or over expression in 32Dcl3 cells encouraged usto verify the biological response of 32Dcl3 cells upon Fbw7 inhibition.We therefore, examined the morphological changes in 32Dcl3 byinhibiting endogenous Fbw7 expression/function with siFbw7 ordnFbw7αWD over expression. Notably, IL-3 replacement with G-CSFstimulates proliferation of these 32Dcl3 cells for 4–5 days followedby growth arrest and apparent neutrophil like morphology by day12. Moreover, G-CSF alone can drive 32Dcl3 cells to differentiation[42,43]. We assessed if inhibition of Fbw7 triggers early and robustdifferentiation in 32Dcl3 cells. For this, 32Dcl3 was either mocktransfected or transfected with siFbw7 and dnFbw7αWD separately;grown in IL3 free medium containing G-CSF. Post 3, 9 and 15 daysof culture, Giemsa stained cells were assessed for their morphologicalchanges. Interestingly, on day 3 few granules like nuclear morphologywere observed in cells transfected with siFbw7 and dnFbw7αWD evenin the cells not treated with G-CSF (Fig. 7A). Moreover, by day 9 cellsattained myelocyte morphology with oval shaped nucleus with obvi-ous differentiation like morphology even in G-CSF untreated mocktransfected cells in addition to siFbw7 and dnFbw7αWD transfectedcells (Fig. 7B). At day 15, cells appeared more like metamyelocyteswith nucleus being elongated, dense horseshoe or ring shaped andfew cells demonstrated polymorphonuclear neutrophil like morphol-ogy with segmented nucleus (Fig. 7C) [34]. The fact that G-CSF stim-ulation with siFbw7 or dnFbw7αWD transfection shows additiveeffect andmore interestingly siFbw7 and dnFbw7αWD alone triggeredprofound granulocytic differentiation in 32Dcl3 cells affirms our find-ing that Fbw7 obstructs granulocytic differentiation apparently bynegatively regulating G-CSFR expression and functions.

3.8. siFbw7 or ectopically expressed dnFbw7αWD with simultaneousknock down of G-CSFR by siG-CSFR is unable to induce granulocyticdifferentiation

After validation of STAT activity by assessing pSTAT3 expression insiFbw7 or ectopically expressed dnFbw7αWD with simultaneousknock down of G-CSFR by siG-CSFR in Fig. 6E, F; we assessed granulo-cyte differentiation like morphological changes in cells expressingeither dnFbw7αWD or siFbw7 alone or together with siG-CSFR in32Dcl3 cells treated or untreated with G-CSF (Fig. 8). In line with re-duced pSTAT3, almost no differentiation was seen even after 15 dayswhich further corroborates our claim that Fbw7 negatively regulatesG-CSFR expression and its downstream functions by targeting it fordegradation through the ubiquitin–proteasome pathway.

4. Discussion

In this study we have identified G-CSFR as a novel substrate ofFbw7. Fbw7 has been shown to regulate the abundance of proteinssuch as cyclin E, c-Myc, Notch, c-Jun, MCL-1, SREBP1, mTOR andPGC-1α (Table 1). Our present study extends this list to G-CSFRwhere we show that Fbw7 regulates G-CSFR expression. Previousstudies have demonstrated attenuation of G-CSFR signaling viaubiquitin–proteasome pathway; however, the E3 ubiquitin ligases in-volved have largely remained unexplored. The presence of severalputative CPD motifs in G-CSFR (Table 1) indicated that Fbw7 couldgovern G-CSFR expression by targeting it for degradation. We foundthat all the three isoforms of Fbw7 downregulated G-CSFR both innon-myeloid (HEK293T) and myeloid cells (32Dcl3 and peripheralblood mononuclear cells) (Fig. 1). Although, these isoforms are locat-ed in different sub-cellular locations, they may still negativelyregulate G-CSFR by interacting with each other as reported in thecase of another substrate cyclin E1 [44]. Nonetheless, using the most

A

B

G-CSF(min) - - - 10 10 10

Fbw7α Fbw7α

IB: pSTAT3

IB: STAT3

G-CSF(min) - 10 30 60 - 10 30 60

FBW7α

IB: pSTAT3

IB: STAT3

IB: Flag

IB:β-actin

100kDa

75kDa

100kDa

100kDa

75kDa

42kDa

G-CSF(min) - 10 30 60 - 10 30 60

siFbw7

IB: pSTAT3

IB: STAT3

IB: Fbw7 100kDa

75kDa

100kDa

100kDa

75kDa

IB: pSTAT3

IB: STAT3

IB: Flag

IB:β-actin

G-CSF(min) - 10 30 60 - 10 30 60

dnFbw7αWD

dnFbw7αWD

100kDa

75kDa

100kDa

35kDa

42kDa

C

D

G-CSF(min) - 10 60 - 10 60 - 10 60

siFbw7 +siG-CSFR

IB: pSTAT3

IB: STAT3

IB: Fbw7

100kDa

75kDa

100kDa

100kDa

siFbw7

IB: G-CSFR

IB: β-actin

E

42kDa

130kDa

100kDa

G-CSF(min) - 10 60 - 10 60 - 10 60

dnFbw7αWD+siG-CSFR

IB: pSTAT3

IB: STAT3

IB: Flag

100kDa

75kDa

100kDa

IB: G-CSFR

IB: β-actin

dnFbw7αWDF

42kDa

100kDa130kDa

Fig. 6. siFbw7 or dnFbw7αWD transfection enhances STAT3 activation in 32Dcl3 cells. (A) 32Dcl3 cells were transfected with Fbw7α and 48 h post transfection, cells were stimu-lated with 100 ng/mL G-CSF for 10, 30 and 60 min. WCEs were prepared and subjected to immunoblotting with anti-pSTAT3, anti-STAT3, anti-Flag and anti-β-actin antibodies.(B) Increasing amounts of Fbw7α (0.5, 1.0 and 2.0 μg) were transfected in 32Dcl3, 24 h post transfection, cells were stimulated with 100 ng/mL G-CSF for 10 min followed byWCE preparation. Lysates were resolved on 8% SDS-PAGE and probed with anti-pSTAT3 and anti-STAT3 antibody. (C) 2.0 μg dnFbw7αWD was transfected in 32Dcl23 and treatedwith 100 ng/mL G-CSF for 10, 30 and 60 min followed by cell harvesting. Lysates were resolved on 8% SDS-PAGE and probed with pSTAT3 and STAT3 antibody (D) EndogenousFbw7 was silenced by transfecting siFbw7 (50 nM); post 24 h transfection, cells were stimulated with 100 ng/mL G-CSF followed by WCE preparation. WCEs were resolved on8% SDS-PAGE and probed with anti-pSTAT3 and anti-STAT3 antibody. (E) Endogenous Fbw7 and G-CSFR were silenced by transfecting siFbw7 (50 nM) and siG-CSFR (50 nM) re-spectively. Post 24 h transfection, cells were stimulated with 100 ng/mL G-CSF for 10 and 60 min followed by WCE preparation. WCEs were resolved on 8% SDS-PAGE and probedwith pSTAT3 and STAT3 antibody. (F) 2.0 μg dnFbw7αWD was co-transfected with 50 nM of siG-CSFR in 32Dcl23 and treated with 100 ng/mL G-CSF for 10 and 60 min followed bycell harvesting. Lysates were resolved on 8% SDS-PAGE and probed with anti-pSTAT3 and anti-STAT3 antibody.

2649S. Lochab et al. / Biochimica et Biophysica Acta 1833 (2013) 2639–2652

abundant and stable isoform Fbw7αwe further show that through itsWD domain, Fbw7 interacts with G-CSFR (Fig. 3) and targets it forproteasome-dependent degradation (Fig. 2). Several independentmutations in the gene encoding the G-CSFR have been reported inmyeloid diseases, including severe congenital neutropenia (SCN).The most frequent mutations in SCN result in the introduction ofstop codons in the G-CSFR C-terminal region, the region implicatedin the induction of maturation and growth arrest [45]. The expressionof these truncated forms of G-CSFR in SCN patients are associatedwith an increased risk of developing acute myeloid leukemia (AML)due to hyperactivation of G-CSFR signaling [46]. C-terminal truncatedG-CSFR-T718 (deletion of c-terminal after aa 718) is one such mutantthat is frequently detected in SCN/AML patients. We observed thatFbw7s like with G-CSFR, also interact with G-CSFR-T718 and targetit for degradation (Fig. 4 and Appendix A, Fig. S3); however, therate of G-CSFR-T718 degradation is far slower compared to G-CSFR.Our finding may explain why cells expressing this truncated G-CSFR

do exhibit increased receptor expression at the membrane surfacebut are unable to undergo granulocytic differentiation.

Literature search through known substrates of Fbw7 indicatedthat prior phosphorylation of CPD motifs within substrates is a pre-requisite for their recognition and subsequent degradation by Fbw7.Previous studies have shown that GSK3β, a serine/threonine kinaseacts as an upstream modifying enzyme that phosphorylates the CPDmotifs present in some of the known Fbw7 substrates and triggerstheir Fbw7 mediated degradation [47]. The presence of five such sim-ilar motifs in G-CSFR encouraged us to hypothesize that GSK3βmightalso phosphorylate G-CSFR within putative CPD motifs for Fbw7 rec-ognition and subsequent degradation. The fact that increasing dosesof GSK3β or GSK3S9A persistently inhibits the expression of bothG-CSFR and G-CSFR-T718 suggests that GSK3β may phosphorylateG-CSFR probably in its CPDs marking it to be recognized by Fbw7for efficient degradation. Interestingly, even with GSK3β, the rate ofdegradation of G-CSFR-T718 was again slower compared to G-CSFR.

DAY 3MOCK siFbw7 dnFbw7αWD

G-C

SF

(+)

G

-CS

F (

-)

A

DAY 9MOCK siFbw7 dnFbw7αWD

G-C

SF

(+)

G

-CS

F (

-)

B

DAY 15MOCK siFbw7 dnFbw7αWD

G-C

SF

(+)

G

-CS

F (

-)

C

Fig. 7. Knockdown of Fbw7 or ectopically expressed dnFbw7αWD triggers differentiation in 32Dcl3 cells: 32Dcl3 cells in duplicates were either mock transfected or with siFbw7 ordnFbw7αWD. One set was induced with 100 ng/mL of G-CSF while the other was left untreated. Cells were deprived of IL3. After 3, 9 and 15 days both G-CSF and untreated cellswere collected, cytospun and air dried. The next day cells were Giemsa stained and images were captured under microscope (Leica DMI6000B). (A) Day 3: few granules were ob-served on G-CSF treated cells and cells transfected with siFbw7, dnFbw7αWD. (B) Day 9: G-CSF treated cells and siFbw7, dnFbw7αWD transfected cells attained myelocyte morphol-ogy with oval shaped and elongated nucleus. (C) Day 15: differentiated cells show nucleus being elongated, dense with horseshoe or ring shaped. Few cells also demonstratedpolymorphonuclear neutrophil like morphology with segmented nucleus. Notably, as expected, cells treated with G-CSF showed neutrophil differentiation however surprisinglyG-CSF untreated cells transfected with siFbw7 and dnFbw7αWD alone also triggered massive neutrophil differentiation in 32Dcl3 cells. Arrows indicate differentiated cells.

2650 S. Lochab et al. / Biochimica et Biophysica Acta 1833 (2013) 2639–2652

Although it needs further study to specify the exact CPDs and theamino acid residues phosphorylated by GSK3β (creation of pointand deletion mutants is already underway; Appendix A, Fig. S5);

nonetheless, restoration of G-CSFR expression in the presence ofLiCl or upon siRNA mediated knock down of either GSKβ or Fbw7confirms their indispensible interdependence for targeting G-CSFR

ADAY 15

MOCK siFbw7+ siG-CSFR dnFbw7αWD+ siG-CSFR

G-C

SF

(+)

G

-CS

F (

-)

Fig. 8. siFbw7 or ectopically expressed dnFbw7αWD with simultaneous knock down of G-CSFR by siG-CSFR is unable to induce granulocytic differentiation: 32Dcl3 cells wereco-transfected with siFbw7 or dnFbw7αWD together with siG-CSFR. 48 h post transfection, cells were stimulated with 100 ng/mL G-CSF. After 15 days, cells were cytospun, Giemsastained, air dried and photographed. Arrows indicate differentiated cells.

2651S. Lochab et al. / Biochimica et Biophysica Acta 1833 (2013) 2639–2652

for degradation (Fig. 5). Their interdependence was further con-firmed by the fact that these proteins G-CSFR, G-CSFR-T718, Fbw7sand GSK3β are present in the same complex as demonstrated bytheir in vivo interaction in co-immunoprecipitation study (Appendix A,Fig. S3).

G-CSF mediates its biological effects by binding to its cognate re-ceptor G-CSFR. Upon activation by G-CSF, G-CSFR is phosphorylatedat four conserved tyrosines that serve as docking sites for SH2domain-containing signaling proteins such as signal transducer andactivator of transcription-3 (STAT3) and a variety of other down-stream signaling proteins [48]. Activation of STAT3 is critical for nor-mal G-CSF dependent proliferation and granulocytic differentiation ofmyeloid progenitors [42]. Therefore, we asked if G-CSFR inhibition byFbw7 hampers its downstream signaling. To this end, over expressing32Dcl3 cells with Fbw7α followed by treatment with G-CSF post 24 htransfection showed regression of STAT3 phosphorylation suggestingFbw7 indeed hampers G-CSFR signaling. The negative effects ofFbw7 on G-CSFR signaling were corroborated further by inhibitingendogenous Fbw7 either with siFbw7 or dnFbw7αWD, which resultedin enhanced STAT3 activation upon G-CSF stimulation in 32Dcl3 cells(Fig. 6). Following Fbw7 mediated inhibition of G-CSFR downstreamsignaling, we were now curious to know if targeting Fbw7 bysiFbw7 or dnFbw7αWD restores G-CSFR and its downstream signalsare transduced further in the form of granulocytic differentiation.Therefore, we studied the morphology of 32Dcl3 cells transfectedwith dnFbw7αWD or siFbw7 followed by treatment with G-CSF.After 3, 9 and 15 days, Giemsa stained cells were analyzed for mor-phological changes which showed enhanced granulocytic differentia-tion. Cells appeared to be metamyelocytes with elongated nucleus,horseshoe or ring shaped. In fact few cells possessed polymorphonu-clear neutrophil like morphology with segmented nucleus. Although,G-CSF is known to stimulate such morphological changes but surpris-ingly siFbw7 and dnFbw7αWD alone also triggered dramatic differen-tiation in 32Dcl3 even in the absence of G-CSF (Fig. 7). Additionallywhen 32Dcl3 cells were supplemented with G-CSF along withsiFbw7 or dnFbw7αWD transfection, a rather additive differentiationeffect was seen. On the contrary, the fact that pSTAT3 and subsequentgranulocytic differentiation substantially mitigated when G-CSFR andFbw7 were simultaneously knocked down in a rescue experimentfurther affirms our finding that pSTAT3 activation and subsequent

enhanced granulocytic differentiation is because Fbw7 functionsthrough G-CSFR to activate pSTAT3.

5. Conclusion

Our study demonstrates that Fbw7 in cooperation with GSK3βnegativelymodulates G-CSFR protein steady state levels by promotingits degradation and thus regulates granulocytic differentiation. Likeother known substrates, through its WD domain Fbw7 physically in-teracts with CPDmotifs of G-CSFR that are apparently phosphorylatedby GSK3β. Although detailed molecular study is underway to pull outthe exact CPD motifs and its GSK3β phosphomodified amino acidsamong several CPDs present in G-CSFR. Nonetheless, siFbw7 anddnFbw7αWD mediated inhibition of Fbw7 leading to activation ofSTAT3 and subsequent induction of enhanced granulocytic differenti-ation in 32Dcl3 even in the absence of G-CSF strongly vouches for ourfinding that Fbw7 negatively regulates G-CSFR signaling. More impor-tantly, our finding that Fbw7 andGSKβ induced degradation of G-CSFRis faster than that of G-CSFR-T718 mutant lacking some of consensusCPD motifs may be significant for understanding pathophysiology ofSCN/AML patients and developing better therapeutics. Overall, thesefindings suggest that Fbw7 with G-SK3β negatively regulates G-CSFRsignaling through ubiquitin–proteasome pathway.

Conflict of interest statement

None declared.

Acknowledgements

This study was supported by a grant in aid from the Department ofBiotechnology, India (GAP0068) and Council of Scientific and indus-trial Research, CSIR (Center for Research on Anabolic Skeletal Targetsin Health and Illness (ASTHI-BSC0201)) to AKT, SL and JKK. Institu-tional communication number for this article is 8481.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.bbamcr.2013.06.018.

2652 S. Lochab et al. / Biochimica et Biophysica Acta 1833 (2013) 2639–2652

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