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c-Cbl Is a Negative Regulator of GH-Stimulated STAT5-Mediated Transcription

Institute of Molecular and Cell Biology (E.L.K.G., T.Z., W.-Y.L., P.E.L.), National University of Singapore, Singapore 117609; and Department of Medicine (P.E.L.)., National University of Singapore, Singapore 119074, Republic of Singapore

Address all correspondence and requests for reprints to: Peter E. Lobie, M.D., Ph.D., Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Republic of Singapore. E-mail: mcbpel{at}imcb.nus.edu.sg.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have previously demonstrated that cellular stimulation with GH results in the formation of a multiprotein signaling complex. One component of this multiprotein signaling complex is the adapter molecule c-Cbl. Here we have examined the role of c-Cbl in the mechanism of GH signal transduction. Forced expression of c-Cbl in NIH3T3 cells did not alter GH-stimulated Janus kinase 2 tyrosine phosphorylation nor GH-stimulated p44/42 MAPK activation and consequent Elk-1- mediated transcription. c-Cbl overexpression did, however, result in enhanced and prolonged GH-stimulated activation of phosphatidylinositol 3-kinase. Forced expression of c-Cbl did not affect GH-stimulated STAT5 tyrosine phosphorylation, nuclear translocation, nor binding to DNA but markedly abrogated GH-stimulated STAT5-mediated transactivation. c-Cbl overexpression resulted in increased ubiquitination and proteosomal degradation of STAT5 and increased degradation of GH-stimulated tyrosine phosphorylated STAT5. Cellular pretreatment with the proteosomal inhibitor MG132 reversed the effect of c-Cbl overexpression with prolonged duration of GH-stimulated STAT5 tyrosine phosphorylation and restoration of STAT5-mediated transcription. Thus, c-Cbl is a negative regulator of GH-stimulated STAT5-mediated transcription by direction of STAT5 for proteosomal degradation.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
THE c-Cbl PROTO-ONCOGENE was originally identified as a cellular homolog of v-Cbl oncogene that was cloned from Cas NS-1murine leukemia virus (1). The c-Cbl gene product is a cytosolic protein that is expressed in a wide range of cell types, most abundantly in the testis, thymus, and in all hematopoietic cell lineages (2). It contains a highly conserved basic amino-terminal domain that contains a nuclear translocation signal, KKTK, followed by a RING finger domain, a proline-rich domain that has been shown to function as a ligand for SH3 domain of many signaling molecules, a putative leucine zipper for dimerization and several potential tyrosine phosphorylation sites that could interact with many SH2 containing molecules. The role of c-Cbl in the signal transduction mechanism of various extracellular ligands has been actively investigated; mainly pertaining to its rapidly and prominently phosphorylated tyrosine residues, as well as the ability of c-Cbl to interact with many signal transduction molecules after phosphorylation by cellular stimuli (3, 4, 5, 6, 7, 8, 9). The first evidence to indicate that Cbl proteins regulate tyrosine kinase signaling came from genetic studies in Caenorhabditis elegans (10). Loss-of-function mutations in sli-1 (c-Cbl homolog in C. elegans) restored signaling for vulval induction and survival through a weakly active epidermal growth factor (EGF) receptor homolog (LET-23), whereas additional copies of sli-1 suppressed vulval induction. Following this report, it has recently been demonstrated that c-Cbl is a negative regulator of various signaling pathways initiated by cell surface receptor tyrosine kinases such as colony-stimulating factor 1, EGF, and platelet-derived growth factor (PDGF) (11, 12, 13). As an E3 ligase, one mechanism whereby c-Cbl regulates signaling of growth factors is to modulate the endocytotic internalization, ubiquitination and subsequent proteosomal destruction of the respective receptor molecules (11, 12, 13). Various studies have indicated that c-Cbl also negatively regulates nonreceptor tyrosine kinases such as Lyn, ZAP-70, Syk, Fyn, and Src (14, 15, 16, 17) by enhancement of their ubiquitin-dependent degradation.

The GH receptor (GHR) is a member of the cytokine receptor superfamily that does not possess intrinsic tyrosine kinase activity (18). GH signal transduction has been postulated to be initiated by the binding of GH to the cell surface GHR, resulting in homodimerization of the receptor (18, 19). Dimerization of the GHR results in the association and activation of the associated Janus kinase 2 (JAK2). JAK2 is thought to subsequently tyrosine phosphorylate the GHR and molecules required to activate the various downstream signaling pathways (18, 20). The major groups of signaling molecules thus far identified to be activated by GH include: 1) other receptor (EGF receptor) (21) and nonreceptor (c-Src, c-Fyn (22), focal adhesion kinase (23) kinases, although as in the case of the EGF receptor it may be used simply as an adapter protein; 2) members of the MAPK family including p44/42 MAPK (24, 25, 26), p38 MAPK (27) and JNK/stress activated protein kinase (22) and the respective downstream pathways; 3) members of the insulin receptor substrate (IRS) group including IRS-1, 2, and 3, which may act as docking proteins for further activation of signaling molecules including phosphatidylinositol-3 kinase (PI3K) (28, 29, 30, 31, 32); 4) small Ras-like GTPases (33, 34); and 5) STAT family members including STATs 1,3, 5a, and 5b (35, 36, 37, 38), which constitute one major mechanism for transcriptional regulation by GH.

We have recently reported that GH stimulates the formation of a multiprotein signaling complex centered around CrkII and p130Cas (22). Other molecules contained in this complex include c-Src, c-Fyn, and focal adhesion kinase as well as c-Cbl, IRS-1, and Nck (22). We have subsequently analyzed the functional role of CrkII and determined that it specifically contributes to both the positive and negative modulation of the major pathways activated by GH (39). GH stimulates the tyrosine phosphorylation of c-Cbl and its association to the multiprotein signaling complex (22). However, the specific functional role of c-Cbl in the mechanism of GH signal transduction has not been defined. The expression of c-Cbl is ubiquitious and is expressed in multiple GH-responsive tissues such as adipose tissue (40), muscle (40), liver (41), and the mammary gland (42, 43, 44).

In this report, we have investigated the functional consequences of c-Cbl overexpression in NIH-3T3 cells on the ability of GH to activate its effector pathways. We demonstrate that c-Cbl overexpression is not involved in GH-stimulated JAK2 and p44/42 mitogen-activated protein activation and subsequent Elk-1-mediated transcription, despite enhancement of GH-stimulated PI3K activity. Interestingly, c-Cbl functions as a negative regulator of STAT5-mediated transcriptional activation. c-Cbl inhibition of GH-stimulated STAT5-mediated transcription is not mediated by interference with events leading up to DNA binding but rather is due to increased proteasomal degradation subsequent to ubiquitination of STAT5. Thus, we provide evidence for a novel mechanism to regulate STAT5-mediated transcription and therefore hormonal responsiveness of the cell.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Recombinant human GH (hGH) was a generous gift of Novo-Nordisk (Singapore). Sodium orthovanadate, phenylmethylsulfonyl fluoride (PMSF), and common chemicals and reagents were purchased from Sigma (St. Louis, MO). MG132 was purchased from Calbiochem (San Diego, CA). Polyclonal antisera against JAK2 and monoclonal antisera against phospho-STAT5a/b and phosphotyrosine (4G10) were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Monoclonal antiserum against STAT5a/b and polyclonal antisera against STAT5b and c-Cbl were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Polyclonal antiserum against ubiquitin was purchased from Novocastra Laboratories Ltd. (Newcastle Upon Tyne, UK). Phospho-Elk-1 (Ser383) polyclonal antibody, immobilized phospho-p44/42 MAPK (Thr202/Tyr204) monoclonal antibody and Elk-1 fusion protein were purchased from New England Biolabs, Inc. (Beverly, MA). Antirabbit IgG conjugated to fluorescein isothiocyanate (FITC) and poly-deoxyinosinic-deoxycytidylic acid were purchased from Roche Molecular Biochemicals (Mannheim, Germany). Secondary anti-IgG antibodies and the enhanced chemiluminescence (ECL) kit were from Amersham Life Sciences (Chicago, IL). Effectene transfection reagent was purchase from QIAGEN GmbH (Hilden, Germany). The empty vector, pUC-CAGGS and pUC-CAGGS containing c-Cbl cDNA (pUC-CAGGS-c-Cbl) used for transient transfection were kindly provided by Hisamaru Hirai (45). The fusion trans-activator plasmid, pFA-Elk-1 and the reporter plasmid, pFR-luciferase (Luc) were purchased from Stratagene (La Jolla, CA). The DNA 3' labeling kit was purchased from Roche Diagnostics GmbH (Mannheim, Germany), and Nck-associated protein-5 columns were purchased from Amersham Biosciences Inc. (Piscataway, NJ).

Cell lines
NIH3T3 cells stably transfected with pUC-CAGGS (designated NIH-3T3-vector) or pUC-CAGGS-c-Cbl (designated NIH-3T3-c-Cbl) were kindly provided by Hisamaru Hirai (45). The cells are maintained in DMEM supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, and 100 mg/ml streptomycin.

Treatment of cells with hGH
NIH3T3 cells were grown in medium containing 10% FCS for 48 h before complete serum deprivation for 15–18 h. Serum-deprived cells were treated with 50 nM hGH for the indicated time periods.

[¹²⁵I]-GH cell surface receptor binding assays
NIH3T3 cells were grown in six-well plates with medium containing 10% FCS for 48 h before serum deprivation for 18 h. The medium was replaced with 2 ml of fresh cold serum-free medium and incubated at 4 C for 2 h. A 10x higher concentration of hGH was added to the first three wells and I¹²⁵-labeled hGH (around 100,000 cpm) was added to all six wells. The plates were incubated at 4 C for another 4 h before washing twice with cold PBS. The cells were lysed in lysis buffer [0.1 M NaOH, 1% sodium dodecyl sulfate (SDS)] removed from the wells and counted using a -counter.

Immunoprecipitation, SDS-PAGE, and Western blot analysis
Cells were grown as described above and lysed in immunoprecipitation buffer (1% Triton X-100; 150 mM NaCl; 10 mM Tris, pH 7.4; 1 mM EDTA; 1 mM EGTA; 1% Nonidet P-40; 0.2 mM Na₃VO₄; 1 µg/ml protease inhibitor cocktail; and 0.1 mM PMSF). Samples were then immunoprecipitated with polyclonal antibody against JAK2 (Upstate Biotechnology, Inc.) or STAT5b (Santa Cruz Biotechnology, Inc.). SDS-PAGE sample buffer (50 mM Tris, pH 6.8; 2% SDS; 2% ß-mercaptoethanol and bromophenol blue) was added to each sample and the samples were boiled for 5 min. Samples were subjected to discontinuous SDS-PAGE with a 7.5% or 10% resolving gel and transferred to nitrocellulose membranes (Hybond C-extra) using standard electroblotting procedures. Membranes were blocked with 2% BSA overnight at 4 C and immunolabeled with monoclonal antibody against tyrosyl phosphorylated proteins (4G10) (1:1000 dilution), phospho-specific STAT5a/b antibody (1:1000 dilution) or polyclonal antibody against STAT5b (1:1000 dilution), JAK2 (1:1000 dilution) or ubiquitin (1:1000 dilution) for 1 h at room temperature. Immunolabeling was detected by the ECL kit according to the manufacturer’s instructions. Blots were stripped and reprobed with the same antibodies used for their immunoprecipitation to ensure equal loading of the immunoprecipitated proteins. Blots were stripped by incubation for 30 min at 50 C in a solution containing 62.5 mM Tris-HCl, pH 6.7; 2% SDS; and 0.7% ß-mercaptoethanol. Blots were then washed for 30 min with several changes of PBS, 0.1% Tween 20 at room temperature. Efficacy of stripping was determined by reexposure of the membranes to ECL. Thereafter, blots were reblocked and immunolabeled as described above.

Confocal laser scanning microscopy
At the end of the respective treatment period, cells were rinsed with ice-cold PBS, fixed in ice-cold 4% paraformaldehyde, permeabilized for 10 min with 0.1% Triton X-100, blocked in 2% BSA, and incubated with polyclonal antibodies against c-Cbl (1:100 dilution) or STAT5b (1:100 dilution) followed by antirabbit IgG conjugated with FITC. Labeled cells were visualized with a Carl Zeiss (Jena, Germany) Axioplan microscope equipped with epifluorescence optics and a Bio-Rad Laboratories, Inc. (Hercules, CA) MRC1024 confocal laser system. Images were converted to the tagged-information-file format and processed with the Adobe Photoshop program.

p44/42 MAPK assays
p42/44 kinase assays were performed using the New England Biolabs, Inc. (Beverly, MA) assay kit according to the manufacturer’s instructions. Briefly, cells were serum deprived for 16 h, treated with 50 nM hGH, and lysed at 4 C in 1 ml of lysis buffer (20 mM Tris, pH 7.4; 150 mM NaCl; 1 mM EDTA; 1 mM EGTA; 1% Triton X-100; 2.5 mM sodium pyrophosphate; 1 mM glycerolphosphate, 1 mM Na₃VO₄, 0.1% PMSF, 1 mg/ml leupeptin) per sample. The lysates were centrifuged at 15,000 x g for 15 min at 4 C. The supernatant containing 200 µG protein per sample was incubated overnight at 4 C with 20 µl of immobilized phospho-specific p44/42 (Thr202/Tyr204) MAPK monoclonal antibody in a final volume of 500 µl in 1x lysis buffer. The mixture was incubated with gentle rocking overnight at 4 C. The pellets were washed twice with 500 µl of lysis buffer containing 0.1% PMSF and twice with 500 µl of kinase buffer (25 mM Tris, pH 7.5; 5 mM glycerolphosphate; 2 mM dithiothreitol; 0.1 mM Na₃VO₄; and 10 mM MgCl₂). The kinase reactions were performed in the presence of 2 µg of Elk-1 fusion protein and 200 µM ATP at 30 C for 30 min. Elk-1 phosphorylation was selectively detected by Western immunoblotting using a chemiluminescent detection system and a specific phospho-Elk1 (Ser383) antibody (1:1000 dilution).

PI3K assay
NIH3T3 cells were grown to 80% confluence, incubated for 15–18 h in serum-free medium, washed once in serum-free medium, and incubated with 50 nM hGH for the indicated time periods. Cells were lysed with lysis buffer (10 mM Tris-HCl, pH 7.4; 0.25 M sucrose;1 mM EDTA; 0.1 mM sodium orthovanadate (Na₃VO₄); 0.1 mM diisopropyl fluorophosphate; 1% Triton X-100; 10 µg/ml protease inhibitor cocktail; and 0.1 mM PMSF) for 1 h at 4 C, scraped, and spun for 10 min at 4 C. The supernatants were collected and the proteins in the whole cell extracts were assayed using a Bio-Rad Laboratories, Inc. protein assay kit with BSA as standards. Eight hundred micrograms of each sample was immunoprecipitated overnight with 3 µg of polyclonal antibody against PI3K. Twenty-five milliliters of protein G plus/protein A agarose (Calbiochem) were added for 2 h and the agarose beads were washed twice with lysis buffer, twice with PBS and twice with assay buffer (40 mM Tris-HCl, pH 7.4; 5 mM MgCl₂; and 0.5 mM EGTA). A 50-µl assay mix (0.01 mg/ml phosphatidylserine, 0.2 mg/ml phosphatidylinositol, and 0.2 µM [³²P] ATP) was added to each sample and incubated at 30 C for 10 min. The reaction was terminated by adding 200 µl 1 M HCl and 400 µl MeOH:CHCl₃ (1:1). The organic phase was dried, redissolved in 20 µl MeOH:CHCl₃ (5:95), spotted onto Silica Gel 60 plates (Merck, Darmstadt, Germany), and developed in MeOH:CHCl₃:H₂O:NH₄OH (35:45:7.5:2.8).

Densitometric analysis of band intensities
The intensities of the respective bands of proteins on the autoradiographs were quantified using a Bio-Rad Laboratories, Inc. GS-700 imaging densitometer and analyzed with the MultiAnalyst (version 1.0.1) program (Bio-Rad Laboratories, Inc.).

Oligonucleotides
Double-stranded serine protease inhibitor-GAS-like response element (SPI-GLE1), TGTTCTGAGAAATA (46) was used as the probe in gel EMSA (GEMSA). The DNA fragment was labeled with [-³²P] 2',3'-dideoxyadenosine 5'-phosphate using the DNA 3' labeling kit purchased from Roche Diagnostics GmbH. The labeled probe was purified through Nck-associated protein-5 column (Amersham Biosciences Inc.)

GEMSA
GEMSA was performed according to standard protocol. The binding reactions were performed by preincubating 10 µg of nuclear extracts of both GH treated and untreated NIH3T3-vector and NIH3T3-c-Cbl cells with 3 µg of poly-deoxyinosinic-deoxycytidylic acid (Roche) in 15 ml buffer containing 20% Ficoll; 60 mM HEPES, pH 7.9; 20 mM Tris, pH 7.9; 0.5 mM EDTA; and 5 mM dithiothreitol for 15 min on ice. For supershift analysis, the extracts were incubated with the antibodies recognizing STAT5B (1 µg), c-Cbl (1 µg), or control antibodies (1 µg) for another 10 min on ice. ³²P-3'-End-labeled double-stranded SPI-GLE1 probe (50 ng) was added, and the mixture was incubated for 5 min on ice followed by another 10 min at room temperature. The samples were electrophoresed on 4.5% nondenaturing polyacrylamide gels in 0.25x TBE buffer (22.5 mM Tris borate, pH 8.0; 0.5 mM EDTA) at 250 V at 4 C for 2 h. The gel was dried and visualized by autoradiography.

STAT5 reporter assay
NIH3T3 cells were cultured to 50% confluence in six-well plates. Transient transfection was performed in serum-free DMEM with Effectene according to the manufacturer’s instructions (QIAGEN GmbH). Cytomegalovirus-luciferase (CMV-LUC) (0.2 µg) of together with either 0.2 µg of reporter plasmid (SPI-GLE1-CAT) or 0.2 µg of reporter plasmid (SPI-GLE1-CAT) and 0.2 µg of pUC-CAGGS or pUC-CAGGS-c-Cbl were transfected per well in serum-free DMEM. Cells were incubated with Effectene/DNA for 12 h before. After a further 24 h, cells were washed in PBS and scraped into lysis buffer (250 mM Tris-HCl, pH 8.0; 1 mM dithiothreitol). The protein contents of the samples were normalized and chloramphenicol acetyl transferase assays were performed. Results were normalized to the level of luciferase activity to control for transfection efficiency and calculated as the fold stimulation of unstimulated (nonhormone treated) cells.

Elk-1 reporter assay
NIH-3T3 cells were cultured to 60–80% confluence for transfection experiments in 6-well plates. Reporter plasmid pFR-Luc (1.0 µg) was transfected together with 0.02 µg of fusion trans-activator plasmid, pFA-Elk-1. Twenty-five microliters of Effectene for each microgram of DNA was used as per the manufacturer’s instructions. DNA-lipid complex was diluted to a final volume of 2 ml/well with serum- free medium, added to the cells and the cells were left in the 37 C incubator for 12–16 h. The media were then changed to fresh serum-free DMEM with or without 50 nM hGH for an additional 24 h. The cells were washed in PBS and lysed with 200 µl of 1x lysis buffer (25 mM Tris-phosphate, pH 7.8; 2 mM EDTA; 2 mM dithiothreitol; 10% glycerol; 1% Triton X-100) by a freeze-thaw cycle, and lysate was collected by centrifugation at 14,000 rpm for 15 min. The supernatant was used for the assay of luciferase activity. The luciferase activities were normalized on the basis of protein contents well as on the ß-galactosidase activity of pCMV-ß-galactosidase vector.

Statistical analysis and presentation of data
All experiments were performed at least three times. Numerical data are expressed as mean ± SD. Data were analyzed using the two-tailed t test or ANOVA.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Characterization of c-Cbl overexpressing NIH3T3 cells
We have previously demonstrated that c-Cbl is tyrosyl-phosphorylated upon GH stimulation and is a component of a GH-stimulated multiprotein signaling complex (22). To determine the role of c-Cbl in the cellular effects of GH, we simply overexpressed c-Cbl in NIH3T3 cells by stable transfection with either pUC-CAGGS-c-Cbl (NIH3T3-c-Cbl) or with pUC-CAGGS vector alone (NIH3T3-vector). Confocal laser scanning microscopic analysis of c-Cbl expression in NIH3T3-c-Cbl and NIH3T3-vector cells clearly demonstrated overexpression of c-Cbl in NIH3T3-c-Cbl cells compared with vector-transfected control cells (Fig. 1A). c-Cbl was predominantly localized to the cytoplasm in both vector-transfected and c-Cbl-overexpressing NIH3T3 cells. Western blot analysis in Fig. 1B also confirmed the overexpression of c-Cbl in NIH3T3-c-Cbl cells. The level of c-Cbl expression in NIH3T3-c-Cbl cells was approximately 10 times that of vector-transfected control cells (NIH3T3-vector) as determined by densitometric analysis of the Western blot for c-Cbl (not shown).

View larger version (24K): [in this window] [in a new window]   Figure 1. Stable transfection of c-Cbl cDNA results in c-Cbl overexpression in NIH3T3 cells. A, Confocal laser scanning microscopic analysis of c-Cbl expression in NIH3T3 cells stably transfected with either pUC-CAGGS-c-Cbl (NIH3T3-c-Cbl) or pUC-CAGGS (NIH3T3-vector). NIH3T3 cells were grown on coverslips, serum deprived for 18 h, fixed with 4% paraformaldehyde and processed for confocal laser scanning microscopy using polyclonal antibody against c-Cbl followed by antirabbit antibody conjugated with FITC (see Materials and Methods). Bar, 10 µm. B, Western blot analysis of NIH3T3-vector (stably transfected with empty pUC-CAGGS vector) and NIH3T3-c-Cbl (stably transfected with pUC-CAGGS-c-Cbl) cells. Cells were grown to 80% confluence, serum-deprived and total cell extracts were prepared and processed as described in Materials and Methods. The immunoblot was detected by the enhanced chemiluminescence method. C, c-Cbl overexpression does not alter cell surface expression of the GHR. NIH3T3 cells were cultured, serum deprived for 18 h, and processed for cell surface I125-labeled GH binding as described in Materials and Methods. The data presented are from at least three independently performed experiments.

c-Cbl overexpression does not affect the ability of GH to stimulate JAK2 phosphorylation
Phosphorylation and activation of JAK2 after GH binding to the receptor has been postulated to be required for subsequent transduction of the GH signal into the cell (18). We therefore first examined the effect of c-Cbl overexpression in NIH-3T3 cells on the ability of GH to stimulate tyrosine phosphorylation of JAK2. As is observed in Fig. 2, GH stimulated the tyrosine phosphorylation of JAK2 in vector transfected cells at both 5 and 15 min after exposure of the cells to GH. Overexpression of c-Cbl in NIH-3T3-c-Cbl cells did not significantly alter the ability of GH to stimulate the tyrosine phosphorylation of JAK2 (Fig. 2) with a slight but inconsistent increase in the tyrosine phosphorylation of JAK2 observed. Thus, c-Cbl overexpression does not significantly influence JAK2 stimulation by GH.

View larger version (18K): [in this window] [in a new window]   Figure 2. c-Cbl overexpression does not affect GH-stimulated tyrosine phosphorylation of JAK2. Western blot analysis of tyrosine phosphorylation of JAK2 stimulated by GH in NIH3T3-vector and NIH3T3-c-Cbl cells. NIH3T3 cells were grown to 80% confluence and serum deprived for 18 h. Cells were treated with 50 nM hGH and processed for immunoprecipitation with a polyclonal antibody recognizing JAK2 and subsequently western blotting with a monoclonal antibody recognizing tyrosine phosphorylated proteins as described in Materials and Methods. The membranes were then stripped and reblotted to demonstrate the equal loading of JAK2 protein. The data presented are representative of at least three independently performed experiments.

View larger version (22K): [in this window] [in a new window]   Figure 3. c-Cbl overexpression does not affect the GH-stimulated activation of p44/42 MAPK or Elk-1-mediated transcription. A, Western blot analysis with anti-phospho-Elk1 to demonstrate the activity of p44/42 MAPK upon GH stimulation of NIH3T3 cells stably transfected with the empty vector, pUC-CAGGS (NIH3T3-vector) or NIH3T3 cells stably transfected with pUC-CAGGS-c-Cbl (NIH3T3-c-Cbl). NIH3T3 cells were grown to 80% confluence and serum deprived for 18 h. Cells were treated with 50 nM hGH and processed for p44/42 MAPK assay as described in Materials and Methods. The autoradiograph is representative for at least three independent experiments. B, Effect of GH on Elk-1-mediated transcription in NIH3T3-vector and NIH3T3-c-Cbl cells. NIH3T3 cells were grown to 60–80% confluence and transiently transfected with the reporter system for Elk-1. Cells were treated with 50 nM hGH and processed for luciferase assay as described in Materials and Methods. The luciferase activities were normalized on the basis of protein contents well as on the ß-galactosidase activity of pCMV-ß-galactosidase vector. The data are presented as the mean ± SD of triplicate determinations. The data presented are from at least three independently performed experiments. C, Effect of GH on Elk-1-mediated transcription in NIH3T3-vector cells transiently transfected with either empty vector (pUC-GAGGS) or c-Cbl cDNA (pUC-GAGGS-c-Cbl). NIH3T3 cells were grown to 60–80% confluence and transiently transfected with the reporter system for Elk-1 with either pUC-GAGGS or pUC-GAGGS-c-Cbl. Cells were treated with 50 nM hGH and processed for luciferase assay as described in Materials and Methods. The luciferase activities were normalized on the basis of protein contents well as on the ß-galactosidase activity of pCMV-ß-galactosidase vector. The data are presented as the mean ± SD of triplicate determinations. The data presented are from at least three independently performed experiments.

View larger version (23K): [in this window] [in a new window]   Figure 4. Effect of c-Cbl overexpression on hGH-stimulated PI3K activity. A, Autoradiograph demonstrating the formation of PIP by PI3K activity upon GH stimulation of NIH3T3 cells stably transfected with either empty vector, pUC-CAGGS (NIH3T3-vector) or c-Cbl cDNA (NIH3T3-c-Cbl). The autoradiograph is representative of at least three independent experiments. B, Quantitative analysis by densitometry of the level of PI3K activity upon GH stimulation of NIH3T3-vector and NIH3T3-c-Cbl cells. The level of PI3K activity observed in unstimulated NIH3T3-vector has been arbitrarily assigned a value of 1, and the other values are plotted as the fold-increase over the unstimulated value resulting in the bar chart.

View larger version (48K): [in this window] [in a new window]   Figure 5. c-Cbl overexpression does not affect GH-stimulated tyrosine phosphorylation of STAT5, translocation of STAT5 into the nucleus, nor binding of STAT5 to DNA. A, Western blot analysis of GH-stimulated tyrosine phosphorylation of STAT5 in NIH3T3-vector and NIH3T3-c-Cbl cells. NIH3T3 cells were grown to 80% confluence and serum deprived for 18 h. Cells were treated with 50 nM hGH and processed for Western blotting with monoclonal antibody recognizing tyrosine phosphorylated STAT5a/b proteins as described in Materials and Methods. Membranes were then stripped and reblotted to demonstrate the equal loading of STAT5 protein. The data presented are representative of at least three independently performed experiments. B, GH-stimulated STAT5 translocation in NIH3T3-vector and NIH3T3-c-Cbl cells. Confocal laser-scanning photomicrographs of STAT5 nuclear translocation upon GH stimulation in NIH3T3-vector and NIH3T3-c-Cbl cells. NIH3T3 cells were grown on coverslips, serum deprived for 18 h, fixed with 4% paraformaldehyde, and processed for confocal laser scanning microscopy using monoclonal antibody against STAT5a/b followed by antimouse antibody conjugated with FITC (see Materials and Methods). The photomicrographs presented are representatives of at least three independently performed experiments. Bar, 10 µm. C, GEMSA demonstrating the DNA binding activity of STAT5 in both NIH3T3-vector and NIH3T3-c-Cbl cells. GEMSA was performed by preincubating 10 µg of nuclear extracts from NIH3T3-vector and NIH3T3-c-Cbl cells treated as indicated. For supershift analysis, the extracts were incubated with antibodies against STAT5, c-Cbl or control antibodies for another 10 min on ice. 32P-3'end-labeled double-stranded SPI-GLE1 probe was added and samples were electrophoresed on 4.5% nondenaturing polyacrylamide gels. The gel was dried and the radioactive pattern was then visualized by autoradiography.

c-Cbl overexpression inhibits GH-stimulated STAT5-mediated transcription
STAT5 binding to the -interferon-activated site-like element in the promoter of the SPI 2.1 gene subsequently results in transcription (60, 61, 62). Concordantly, GH-stimulated STAT5-mediated transactivation via SPI-GLE1 in NIH3T3-vector cells (Fig. 6). Surprisingly, c-Cbl overexpression in NIH3T3-c-Cbl cells resulted in a dramatically reduced GH-stimulated STAT5-mediated transcription to between 5 and 30% of that seen in the NIH3T3-vector cells (Fig. 6A). To exclude the possibility of clonal selection artifact giving rise to such a result, we therefore transiently transfected either the vector or c-Cbl cDNA into NIH-3T3 cells and examined the level of GH-stimulated STAT5-mediated transcription (Fig. 6B). We observed the same inhibition of GH-stimulated STAT5- mediated transcription in cells transiently transfected with c-Cbl cDNA as we observed for NIH3T3-c-Cbl cells stably transfected with c-Cbl cDNA (Fig. 6B). Thus overexpression of c-Cbl in NIH3T3 cells dramatically decreases STAT5-mediated transcription in response to GH.

View larger version (15K): [in this window] [in a new window]   Figure 6. Effect of c-Cbl overexpression on hGH-stimulated STAT5-mediated transcription. A, Effect of GH on STAT5 (Spi2.1-GLE1-CAT) mediated transcription in NIH3T3-c-Cbl and NIH3T3-vector cells. NIH3T3 cells were grown to 60–80% confluence and transiently transfected with Spi2.1-GLE1-CAT. Cells were treated with 50 nM hGH and processed for CAT assay as described in Materials and Methods. The CAT activities were normalized on the basis of protein contents well as on the luciferase activity of pCMV-LUC vector. The data are presented as the mean ± SD of triplicate determinations. The data presented are from at least three independently performed experiments. B, Effect of c-Cbl overexpression on hGH-stimulated STAT5 (Spi2.1-GLE1-CAT)-mediated transcriptional activation in NIH3T3-vector cells. NIH3T3-vector cells were grown to 60–80% confluence and transiently transfected with pUC-GAGGS and Spi2.1-GLE1-CAT or pUC-GAGGS-c-Cbl and Spi2.1-GLE1-CAT. Cells were treated with 50 nM hGH and processed for CAT assay as described in Materials and Methods. The CAT activities were normalized on the basis of protein contents well as on the luciferase activity of pCMV-LUC vector. The data are presented as the mean ± SD of triplicate determinations. The data presented are from at least three independently performed experiments.

View larger version (34K): [in this window] [in a new window]   Figure 7. STAT5 is ubiquitinated in NIH3T3-c-Cbl cells. Western blot analysis of ubiquitination of STAT5 in NIH3T3-vector and NIH3T3-c-Cbl cells. NIH3T3 cells were grown to 80% confluence and serum deprived for 18 h. The cells were either untreated or pretreated with 50 µM MG132 for 1 h before stimulation with 50 nM hGH. Immunoprecipitation on cell extracts was performed with monoclonal antibody recognizing STAT5a/b and processed for Western blotting with polyclonal antibody recognizing ubiquitin as described in Materials and Methods. Membranes were then stripped and reblotted to demonstrate the equal loading of STAT5b protein. The data presented are representative of at least three independently performed experiments.

View larger version (35K): [in this window] [in a new window]   Figure 8. The level of GH-stimulated tyrosyl-phosphorylated STAT5B is increased by pretreatment with the proteosome inhibitor, MG132. A, Western blot analysis of the GH-stimulated tyrosine phosphorylation of STAT5 in NIH3T3-vector and NIH3T3-c-Cbl cells. Cells were either untreated or pretreated with 50 µM MG132 for 1 h and then stimulated with 50 nM hGH for 0, 2, and 6 h and processed for Western blotting with polyclonal antibody recognizing tyrosine-phosphorylated STAT5a/b protein as described in Materials and Methods. The data presented are representative of at least three independently performed experiments. B, Quantitative analysis by densitometry of the level of tyrosine-phosphorylated STAT5a/b upon hGH stimulation of NIH3T3-vector and NIH3T3-c-Cbl cells. The graph is representative of the experiment in A. The level of tyrosine-phosphorylated STAT5a/b are plotted as arbitrary units relative to the arbitrary units of untreated NIH3T3-vector cells at 0 h, thus resulting in the bar chart.

View larger version (15K): [in this window] [in a new window]   Figure 9. The proteosomal inhibitor MG132 prevented c-Cbl inhibition of GH-stimulated STAT5-mediated transcriptional activation. Effect of pretreatment with MG132 on GH-stimulated STAT5-mediated transcription in NIH3T3-vector and NIH3T3-c-Cbl cells. NIH3T3 cells were grown and transiently transfected with Spi2.1-GLE1-CAT. Cells were pretreated 1 h with 50 µM MG132 prior stimulation with 50 nM hGH and processed for CAT assay as described in Materials and Methods. The CAT activities were normalized on the basis of protein contents well as on the luciferase activity of pCMV-LUC vector. The data are presented as the mean ± SD of triplicate determinations. The data presented are from at least three independently performed experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The evidence for a regulatory function of c-Cbl in receptor tyrosine kinase signal transduction came from genetic studies in C. elegans (10, 73). c-Cbl was later found to be able to down-regulate signaling pathways including those used by EGF, PDGF, and colony stimulating factor-1 by promoting multiubiquitination of the respective receptor molecule and thus targeting these molecules for degradation (11, 12, 13). This negative regulation is dependent on the integrity of both the TKB and the RING finger domain of c-Cbl (70, 74). The c-Cbl RING finger has intrinsic E3 ligase activity and can independently recruit ubiquitin-conjugating enzymes and direct ubiquitin transfer to substrates (48, 75, 76). Many nonreceptor tyrosine kinases are also negatively regulated by c-Cbl. However, the mechanism of regulation is not as straight forward as those for receptor tyrosine kinases. Evidence has shown that c-Cbl overexpression down-regulated c-Fyn by targeting c-Fyn for degradation (17), but the protein levels of spleen tyrosine kinase (Syk) and 70-kDa -associated protein (ZAP-70) are not altered although their activity is suppressed (14, 16). Moreover, the regulation of Src by c-Cbl is independent of the E3 ligase activity of c-Cbl.

Genetic studies in C. elegans indicate that Sli-1, a p120cbl homolog, plays a negative regulatory role in control of the Ras signaling pathway initiated by the C. elegans EGF receptor homolog (10). However, other studies in NIH3T3 cells overexpressing c-Cbl have shown that c-Cbl suppresses EGF stimulated activation of the JAK/STAT pathway but has no effect on the Ras pathway in EGF receptor signaling (45). Our data also support this observation in as much that overexpression of c-Cbl in NIH3T3 cells does not affect the activation of p44/42 MAPK by GH. These data partially contradict the results expected from the role of Sli-1 protein in the let-23-mediated vulval induction pathway (10). One possibility is that the EGF receptor-mediated signaling pathways are differently regulated by c-Cbl between nematodes and mammals. In mammalian cells, only the EGFR of the c-ErbB (product of oncogene of avian erythroblastosis virus) family kinases is able to bind c-Cbl (77), indicating that c-Cbl only regulates the kinase activity of EGF receptor but not that of the other c-ErbB proteins. This redundancy may not exist in nematodes; therefore, Sli-1 can directly regulate the let-23-, sem-5-, and let-60-mediated pathway (10).

We have demonstrated that overexpression of c-Cbl in NIH-3T3 cells resulted in enhanced GH stimulation of PI3K activity. This is concordant with previous studies which have demonstrated that c-Cbl is involved in the regulation of cell morphology and functional organization of the actin cytoskeleton (78, 79, 80, 81, 82). Cytoskeletal reorganization is generally PI3K dependent, including actin cytoskeletal reorganization stimulated by GH (83), and c-Cbl has been demonstrated to possess PI3K interacting regions. c-Cbl is able to associate with both the SH2 and SH3 domains of p85 (53, 54, 55, 57). Separate studies have identified a specific tyrosine residue on c-Cbl (tyrosine 731) that is essential for direct interaction with the SH2 domain of p85 (56, 84), and this interaction has been demonstrated to be required for c-Cbl-mediated PI3K activation (54, 56, 58). Members of the cytokine receptor superfamily such as the IL-4 receptor and the prolactin receptor activate the PI3K pathway through stimulation of the tyrosine phosphorylation of c-Cbl and its association with the p85 subunit of PI3K (54, 58). We have also demonstrated here that c-Cbl overexpression enhances GH-stimulated activation of PI3K activity and GH may therefore use a similar c-Cbl-dependent mechanism for activation of PI3K as reported for other cytokine receptor superfamily members (54, 56, 58, 69). We have also previously demonstrated that GH stimulates actin cytoskeletal reorganization and this reorganization is PI3K dependent (83). We have also observed that c-Cbl overexpressing cells exhibit altered actin cytoskeletal morphology compared with NIH3T3-vector 3T3 cells (Goh, E. L. K., and P. E. Lobie, unpublished observations) consistent with the higher basal and GH-stimulated levels of PI3K activity in c-Cbl overexpressing cells.

We have demonstrated here that both the initial activation of STAT5 and the ability of STAT5 to bind to its DNA-responsive element are not affected by the overexpression of c-Cbl. However, GH-dependent STAT5-mediated transcriptional activation is diminished by c-Cbl overexpression which we demonstrated was due to enhanced proteosomal degradation of STAT5 in NIH3T3-c-Cbl cells. Separate studies have shown that the proteosome inhibitors, MG132 and lactacystin, affected STAT4, STAT5, and STAT6 turnover but not that of STAT1, STAT2, and STAT3 by significantly stabilizing the tyrosine phosphorylated form (85). Recent studies have also demonstrated that murine embryonic fibroblasts derived from c-Cbl-deficient mice exhibit significantly increased levels of STAT1 and STAT5 protein (72). The increased levels of STAT1 in c-Cbl-deficient mice was not due to decreased degradation but rather due to increased synthesis (72). As such, the proteosomal inhibitor MG132 had no effect on the level of STAT1 in the c-Cbl replete or c-Cbl-deficient cells. This is apparently not the case with STAT5 as equal levels of STAT5 were observed here in the unstimulated state and also STAT5 was equipotently activated by GH in the vector and c-Cbl transfected cells. In contrast, STAT2 and STAT3 expression and activation appear to be regulated independently of c-Cbl (72). Further, we could prevent the c-Cbl enhanced degradation of tyrosine phosphorylated STAT5 and inhibition of GH-stimulated STAT5-mediated transcription by use of the proteosomal inhibitor MG132. Thus c-Cbl regulates STAT5 levels, and therefore activity, by promotion of its degradation. We have also demonstrated in this study that STAT5 is more heavily ubiquitinated in the cell line overexpressing c-Cbl and marked accumulation of ubiquitinated STAT5 is observed in c-Cbl overexpressing cells treated with MG132. To our knowledge, this is the first demonstration that a transcription factor (level and activity) is being directly regulated by c-Cbl through ubiquitination and proteosomal degradation. c-Cbl mediated multiubiquitination of STAT5 would therefore result in degradation and subsequent diminished STAT5-mediated transcriptional activity. We demonstrated that the proteosomal inhibitor MG132 greatly enhanced the GH-stimulated STAT5-mediated transcriptional activation in both NIH3T3 cells as well as the NIH3T3 cells overexpressing c-Cbl. Therefore, by reducing the degradation of ubiquitin-conjugated STAT5, MG132 is able to enhance STAT5-mediated transcriptional activation, either directly by the consequent increased pool of phosphorylated STAT5, or through other unidentified mechanisms. Use of the proteosome inhibitor MG132 has previously been demonstrated to prolong signaling from JAK2 to STAT5b in the rat liver cell line (CWSV-1) (84). In this study, the mechanism of the MG132 effect was not investigated and was postulated to be an indirect stabilizing effect on the GHR-JAK2 complex (86). We have, however, demonstrated the normal activation of JAK2 and certain JAK2 activated downstream signaling pathways. Thus, the effect of c-Cbl on the level of STAT5-mediated transcription is presumably due to direct c-Cbl mediated ubiquitination and subsequent proteosomal degradation. We have therefore identified a novel mechanism for regulation of STAT5- mediated transcription. Cellular stimuli resulting in increased levels of c-Cbl in the cell would therefore preferentially direct the GH response to be mediated by transcriptional pathways other than STAT5, such as p44/42 MAPK stimulated Elk-1-mediated transcription.

Direct c-Cbl-mediated ubiquitination and subsequent proteosomal degradation of STATs may be a common mechanism employed by members of the cytokine receptor superfamily It has been reported that IL-2-induced DNA-binding activity and tyrosine phosphorylation of STAT5 are stabilized by MG132 (87). However, no detectable ubiquitination of the STAT proteins was observed (87). Interpretation of their results suggested that proteosome-mediated protein degradation modulated the protein-tyrosine phosphatase activity that negatively regulated the JAK/STAT pathway stimulated by IL-2 (87). Similarly, IL-6 stimulation of Hep3B hepatoma cells stably expressing p53 demonstrated that IL-6 caused a rapid and marked loss of cellular immunostaining for STAT3 and STAT5 (88). However, the loss was blocked by MG132 and the level of both proteins subsequently remained unchanged, suggesting that IL-6 triggered proteosomal degradation and/or p53-dependent masking but not degradation of STAT3 and STAT5 (88).

The mechanism by which c-Cbl promotes ubiquitination of STAT5 remains to be determined. Recent evidence derived from studies performed in Cbl-b overexpressing cells have demonstrated that the c-Cbl regulation of protein levels by its E3 ligase activity is not solely restricted to receptor tyrosine kinase or nonreceptor tyrosine kinases (89). Multiubiquitination of the p85 subunit of PI3K can also occur through interaction of the proline rich domain in Cbl-b with the SH3 domain of p85 (89). STAT5 does not possess a tyrosine kinase domain for binding to the TKB domain of c-Cbl, nor does it possess any SH3 domain for binding to the proline-rich region of c-Cbl. It is possible that STAT5 either interacts with c-Cbl directly or indirectly through another adapter protein. This would be possible upon formation of the multiprotein signaling complex stimulated by GH centered around CrkII (22). Indeed, we have previously demonstrated that overexpression of CrkII, which would enhance the formation of the multiprotein signaling complex that includes c-Cbl, also results in inhibition of GH-stimulated STAT5-mediated transcription (39).

c-Cbl is expressed in multiple GH-responsive tissues (40, 41, 42, 43, 44), suggestive that the level of c-Cbl in these different cell types will regulate the STAT5-mediated response to GH or to other cytokines using STAT5. For example, c-Cbl deficient (c-Cbl^-/-) mice exhibit lymphoid hyperplasia with changes in T-cell behavior, marked splenomegaly due to splenic extramedullary hematopoiesis and the mammary gland of c-Cbl^-/- female mice exhibits increased ductal density and branching (44). These features are also shared by GH transgenic mice that exhibit altered thymocytic responses (90), marked splenomegaly (e.g. Ref. 91) and mammary gland hyperplasia (92). Interestingly, these cellular responses are also presumably mediated by STAT5 as indicated by recent studies in STAT5 deficient animals (93, 94, 95, 96). Thus, physiological or pathological conditions which result in decreased cellular c-Cbl would allow for increased STAT5-mediated proliferation resulting in tissue hyperplasia and potential oncogenic transformation. Given the demonstration that STAT5 can be both necessary and sufficient for malignant transformation (97), and that v-Cbl exerts its transforming effects by acting as a dominant negative inhibitor of c-Cbl, then regulated control of STAT5 activity by c-Cbl may constitute a major mechanism for tissue homeostasis.

In summary, we have demonstrated here that c-Cbl is a negative regulator of GH-stimulated STAT5-mediated transcription by the direction of STAT5 for proteosomal degradation. We therefore provide the first evidence that c-Cbl directly regulates the level and activity of a transcription factor through ubiquitination and proteosomal degradation. Thus, the extent to which GH stimulates STAT5-mediated transcription in any particular cell type would depend on the cellular level of c-Cbl and the factors regulating such. c-Cbl, together with other identified negative regulators of GH-stimulated STAT-mediated transcription such as the suppressor of cytokine signaling proteins (98), therefore constitute an array of mechanisms by which the cell can limit or prevent STAT- mediated transcriptional events in response to GH or other stimuli.

	Acknowledgments

 

	Footnotes

 
This work was supported by grants from the Agency for Science, Technology and Research of Singapore (A*STAR) (to P.E.L.).

Abbreviations: CMV, Cytomegalovirus; ECL, enhanced chemiluminescence; EGF, epidermal growth factor; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; GEMSA, gel EMSA; GHR, GH receptor; hGH, human GH; IRS, insulin receptor substrate; JAK2, Janus kinase 2; Luc, luciferease; PDGF, platelet-derived growth factor; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate; SPI-GLE1, serine protease inhibitor-GAS-like response element; TKB, tyrosine kinase binding.

Received April 3, 2002.

Accepted for publication May 9, 2002.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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