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Originally published In Press as doi:10.1074/jbc.M404007200 on June 1, 2004

J. Biol. Chem., Vol. 279, Issue 36, 37662-37669, September 3, 2004
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Tyrosine-phosphorylated SOCS3 Interacts with the Nck and Crk-L Adapter Proteins and Regulates Nck Activation*

John C. Sitko, Claudia I. Guevara, and Nicholas A. Cacalano{ddagger}

From the Department of Radiation Oncology, University of California, Los Angeles, School of Medicine, Center for Health Sciences, Los Angeles, California 90095

Received for publication, April 12, 2004 , and in revised form, May 18, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Suppressors of cytokine signaling (SOCS) are negative feedback inhibitors of cytokine and growth factor signal transduction. Although the affect of SOCS proteins on the Jak-STAT pathway has been well characterized, their role in the regulation of other signaling modules is not well understood. In the present study, we demonstrate that SOCS3 physically interacts with the SH2/SH3-containing adapter proteins Nck and Crk-L, which are known to couple activated receptors to multiple downstream signaling pathways and the actin cytoskeleton. Our data show that the SOCS3/Nck and SOCS3/Crk-L interactions depend on tyrosine phosphorylation of SOCS3 Tyr221 within the conserved SOCS box motif and intact SH2 domains of Nck and Crk-L. Furthermore, SOCS3 Tyr221 forms a YXXP motif, which is a consensus binding site for the Nck and Crk-L SH2 domains. Expression of SOCS3 in NIH3T3 cells induces constitutive recruitment of a Nck-GFP fusion protein to the plasma membrane and constitutive tyrosine phosphorylation of endogenous Nck. Our findings suggest that SOCS3 regulates multiple cytokine and growth factor-activated signaling pathways by acting as a recruitment factor for adapter proteins.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The suppressors of cytokine signaling (SOCS)1 represent a heterogeneous family of proteins characterized by an N-terminal protein-protein interaction domain followed by a conserved 40 amino acid motif known as the SOCS box (1-4). The N termini of SOCS proteins are divergent and can contain SH2 domains, WD40 repeats, SPRY domains, or ankyrin repeats (3, 4). The most well characterized SOCS subfamily is represented by CIS, SOCS1, SOCS2, and SOCS3, which contain a short (~40 amino acids) N-terminal domain and an internal SH2 domain. These proteins have been shown to inhibit Janus kinase (Jak) signaling through SH2-dependent interactions with phosphotyrosine residues on cytokine receptors or in the catalytic loop of Jak kinases, to block downstream activation of the signal transducers and activators of transcription (STATs) (5-13).

The SOCS box is also a protein-protein interaction domain and mediates binding to elongin C, a component of a multisub-unit E3 ubiquitin ligase. It has been shown to regulate the stability of SOCS proteins as well as SOCS-associated signaling molecules (4, 14-15). The current models for the mechanisms of SOCS protein function include direct inhibition of Jak-STAT signaling as well as targeting of signal transduction molecules for degradation through the ubiquitination machinery via its association with elongin C (16-21). A well studied example of the latter mechanism is inhibition of transformation by the TEL-Jak2 oncogenic fusion protein by SOCS1. SOCS1 binds Tel-Jak2 through its SH2 domain and induces its proteasome-mediated destruction (16-18). Other signaling proteins that are known to be degraded by SOCS include insulin receptor substrate (IRS)-1 and IRS-2, the guanine nucleotide exchange factor (GEF) Vav, and focal adhesion kinase (FAK) (19-21).

Gene-targeting studies have demonstrated the essential role of SOCS1 and SOCS3 in cytokine responses (22-26). SOCS1 deficiency results in perinatal lethality due to hyper-responsiveness to interferon (IFN)-{gamma} (22, 23). Likewise, mice deficient in SOCS3 die embryonically because of combined defects in trophoblast implantation and uncontrolled proliferation of hematopoietic cells (24-26). Furthermore, signaling studies have shown that SOCS3 can regulate responses to many cytokines and growth factors including interleukin (IL)-2, erythropoietin (Epo), IL-6 family cytokines, granulocyte-macrophage colony stimulating factor (G-CSF), leptin, growth hormone, and IL-12 (11-13, 27-34). Most of these studies have focused on the role of SOCS3 as an inhibitor of the Jak-STAT pathway. However, the finding that SOCS1 and SOCS3 regulate the stability of other families of signaling proteins such as GEFs and IRS proteins suggest that they have a much broader spectrum of action.

Our laboratory has identified a function for SOCS3 beyond its role as a STAT inhibitor. We have previously shown that SOCS3 is phosphorylated by activated Jaks, Src family kinases, and receptor-tyrosine kinases (RTKs) on Tyr204 and Tyr221 in the conserved SOCS box motif (35). Tyrosine 221 of SOCS3 forms part of a YXXP motif (where X is any amino acid), which is a consensus binding site for the SH2 domains of the Ras inhibitor p120 RasGAP. Phosphorylated SOCS3 inactivates RasGAP and allows for sustained MAP kinase activation (35). Tyrosine phosphorylation of SOCS3 is therefore important for its biological function. In the present study, we identify the Nck and Crk-L adapter proteins as SOCS3 binding partners. We demonstrate that phospho-SOCS3 recruits Nck to activated RTKs and modulates Nck tyrosine phosphorylation in fibroblasts, suggesting that SOCS3 regulates adapter protein signal transduction.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—All of the tissue culture media were supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), penicillin/streptomycin, L-glutamine, and 10 mM HEPES (Mediatech, Herndon, VA). 293T, NIH3T3, and the retroviral packaging cell line PlatE were grown in Dulbecco's modified Eagle's medium (Mediatech).

Plasmids, Antibodies, and Fusion Proteins—C-terminally FLAG-tagged or HA-tagged wild-type and mutant SOCS3 cDNAs cloned into the mammalian expression vector pME18S and the retroviral expression vector pMX-IRES-GFP have been described previously (36, 37). SOCS3 pY204/pY221 peptide spanning amino acids 200-225 (DSpYEKVTQLPGPIREFLDQpYDAPL) as well as pY204 and pY221 singly phosphorylated peptides and unphosphorylated controls were supplied by Research Genetics.

Mammalian expression vector pEBB encoding wild-type (WT) Nck{alpha} and R308K SH2 mutant Nck{alpha} cDNAs, tagged with the Myc epitope, were a generous gift from Dr. Bruce Mayer, University of Connecticut Health Center. GST-Nck fusion plasmids were constructed by engineering a 5'-NdeI site (CATATG) at the start codon, and a ClaI site at the 3'-end of WT Nck coding sequence, then cloning this fragment into the bacterial expression vector pGEX4T1 (Clontech, Palo Alto, CA). Transformation of DH5 competent Escherichia coli (Invitrogen), isopropyl-D-thiogalactopyranoside induction, and purification of GST fusion proteins were performed according to standard methods. The Nck coding sequence was also cloned into the green fluorescent protein fusion vector pEGFP-N1 (Clontech) to make the Nck-GFP expression vector. The Nck-GFP fusion coding sequence was then cloned into the retroviral expression vector pMX-IRES-Puro for production of stable cell lines. The Nck-GFP fusion expression plasmid was constructed by inserting a Nck cDNA PCR fragment into pEGFP-N1 (Clontech). Primers were designed to amplify the entire Nck cDNA sequence flanked by an EcoRI site on the 5'-end and a BamHI site in frame 3 immediately preceding the stop codon. PCR was carried out with Pfu polymerase, and the resulting fragment encoding full-length, WT Nck was ligated by the EcoRI and BamHI sites into pEGFP-N1, which contains the GFP coding sequence in-frame. All PCR products were sequenced in their entirety.

PCDNA-Crk-L expression vector was provided by Dr. Ronald Herbst, DNAX Research Inc., Palo Alto, CA. The Crk-L coding sequence was cloned by PCR amplification using a 5'-primer encoding a NdeI site at the start codon and a 3'-primer encoding a ClaI restriction site in place of the stop codon. The PCR product was cloned into our pME18S C-terminal FLAG tag mammalian expression vector, fusing the Crk-L cDNA sequence to the FLAG tag at its 3'-end (36). Crk-L R39A was generated using a fusion PCR approach with overlapping (sense and antisense) mutagenic oligonucleotide primers encoding the point mutation. The 5'-PCR primer spanned the 5'-coding sequence of Crk-L preceded by a NdeI site at the start codon. The 3'-primer spanned the unique NaeI site in the Crk-L cDNA. The mutant PCR product was swapped with the WT Crk-L NdeI-NaeI fragment from pME18S-Crk-L-FLAG vector.

Transfections and Infections—293T and PlatE cells were transfected with Effectene lipid-based transfection reagent (Qiagen, Crawley, UK) according to the manufacturer's instructions. For retroviral infections, 2 ml of supernatant from transfected PlatE cells was mixed with polybrene (8 mg/ml final concentration)(Sigma) and added to NIH3T3 cells in 2 ml of media at 48 and 72 h after PlatE transfections. After 48 h of infection, the cells were washed once and resuspended in fresh culture medium. Cells stably expressing the GFP fusion constructs were sorted by flow cytometry for green fluorescent protein expression.

Immunoprecipitations, GST Pull-down Experiments, and Western Blotting—Cells were lysed in buffer containing 150 mM NaCl, 50 mM Tris-HCl, 2 mM EDTA, 0.875% Brij 97 (Sigma), 0.125% Nonidet P-40 (British Drug Houses, Poole, UK), 10 µg/ml aprotinin (ICN, Aurora, OH), 10 µg/ml leupeptin (ICN), 1 mM phenylmethylsulfonyl fluoride (Sigma), 1 mM Na3VO4 (Sigma). The lysates were centrifuged at 12,000 x g for 5 min at 4 °C to remove nuclei. The lysates were immunoprecipitated with appropriate antibodies as described in the figure legends. Rabbit polyclonal anti-FLAG antibodies and Sepharoseconjugated M2 monoclonal antibody were purchased from Sigma. Monoclonal anti-Myc 9E10, rabbit anti-hemagglutinin tag, and rabbit anti-Myc were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphotyrosine 4G10 was purchased from Upstate Biotechnology (Lake Placid, NY). For GST pull-down experiments, the cells were lysed as described above, and 2 µg of the appropriate GST fusion protein were added to the lysates in combination with 30 µl of a 50% slurry of glutathione-Sepharose beads (Sigma). The lysates were incubated with fusion proteins for 4 h at 4 °C. The precipitates were washed three times in lysis buffer, boiled, and resolved by SDS-PAGE. The proteins were electroblotted onto nylon membranes (Immobilon-P, Millipore, Bradford, MA). The membranes were probed with appropriate antibodies, as described in the figure legends, followed by incubation with horseradish peroxidase-labeled anti-mouse or anti-rabbit secondary antibodies (Amersham Biosciences). The proteins were detected with chemiluminescent substrate (Pierce).

Confocal Visualization—GFP fusion protein expressing NIH3T3 cells were plated in glass bottom microwell dishes (MatTek, Ashland, MA) 24 h before visualization. Cells were serum-starved for 6 h in 0.1% serum and stimulated with 50 ng/ml PDGF BB. Microscopy was preformed on a Leica TCS SP MP Inverted Confocal Microscope with a 488 nm excitation line provided by an argon laser. Image analysis was preformed with Leica Confocal Software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
In a previous study, we determined that SOCS3 Tyr221 is phosphorylated by Jak1, Jak2, EGF receptor, and PDGF receptor and interacts with p120 RasGAP in IL-2-stimulated T cells (35). We have extended these findings to demonstrate that phosphorylated SOCS3 interacts with p120 RasGAP in PDGF-stimulated fibroblasts as well. As shown in Fig. 1, panels B and C, stimulation of murine NIH3T3 fibroblasts with 50 ng/ml PDGF BB resulted in robust SOCS3 expression and tyrosine phosphorylation at the 1- and 2-h time points. Furthermore, p120 RasGAP was found in SOCS3 immunoprecipitates from PDGF-treated cells (Fig. 1, panel A). We examined the sequences surrounding SOCS3 Tyr204 and Tyr221 and found that Tyr221 forms part of a YXXP motif, which is a consensus binding site for the RasGAP SH2 domains (Table I). The YXXP motif has also been shown to interact with the SH2 domains of the Nck and Crk-L adapter proteins (38-43). Therefore, we examined the ability of phosphorylated SOCS3 to interact with Nck and Crk-L. Lysates from NIH3T3 cells were precipitated with N-terminal biotinylated peptides spanning both SOCS3 phosphorylation sites (amino acids 200-225). In order to map the Nck and Crk-L binding sites, we measured Nck and Crk-L binding to peptides containing pY204/pY221, single pY204 or pY221 phosphorylation sites, or a control unphosphorylated peptide of identical sequence. As shown in Fig. 2A, both Nck and Crk-L bound the pY204/pY221 peptide (lanes 2 and 6), as well as the pY221 singly phosphorylated peptide (lanes 4 and 8). However, both molecules failed to interact with the pY204 peptide (lanes 3 and 7) or the unphosphorylated control (lanes 1 and 5).



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  		FIG. 1.
SOCS3 is tyrosine-phosphorylated and binds p120 Ras-GAP in PDGF-stimulated fibroblasts. Approximately 5 x 106 NIH3T3 fibroblasts were incubated in 0.2% fetal calf serum for 4 h, and then stimulated with 50 ng/ml PDGF-BB for a time course of 4 h. Cell lysates were immunoprecipitated (IP) with rabbit anti-SOCS3 antibodies, followed by Western blotting with anti-RasGAP (panel A), anti-phosphotyrosine antibody 4G10 (panel B), or anti-SOCS3 (panel C).

 


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  		TABLE I
Putative RasGAP binding sites

 



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  		FIG. 2.
Nck and Crk-L SH2 domains bind phosphorylated SOCS3 Tyr221. A, N-terminal biotinylated peptides spanning the SOCS3 phosphorylation sites (amino acids 200-225) were prebound to streptavidin beads. Lysates from 5 x 106 NIH3T3 cells were precipitated with streptavidin beads bound to pY(204, 221) double-phosphorylated peptides, singly phosphorylated pY204 or pY221 peptides, or an unphosphorylated control peptide of the same amino acid sequence. The precipitates were analyzed by SDS-PAGE followed by Western blotting for Nck (lanes 1-4) or Crk-L (lanes 5-8). B, 293T cells were transiently transfected with mammalian expression vector pEBB encoding N-terminal Myc-tagged WT Nck (lane 1); a Nck SH2 mutant, R308A (lane 2); or WT Nck SH2 domain (lane 3). 293T cells were also transfected with pME18S encoding C-terminal FLAG-tagged WT Crk-L (lane 5) or the Crk-L SH2 mutant R39A (lane 6). Cell lysates were precipitated with biotinylated pY221 SOCS3 phosphopeptide bound to streptavidinagarose beads, and the precipitates were analyzed in Western blotting with anti-Myc antibodies to detect Nck (lanes 1-4) or anti-FLAG antibodies to detect Crk-L (lanes 5-6). C, GST-Nck fusion protein binds to phosphorylated full-length SOCS3. 293T cells were transiently transfected with SOCS3-HA tag pME18S either alone or in combination with pME18S encoding Jak1. Cell lysates were split and precipitated with GST-Nck or GST proteins bound to glutathione-agarose beads, and the precipitates were analyzed in Western blotting with anti-HA antibodies, to detect SOCS3 (panel a). Expression controls for SOCS3 (panel b), tyrosine-phosphorylated Jak1 (panel c), and phosphorylated SOCS3 (panel d) are shown.

 
To further characterize the requirements for SOCS3/Nck and SOCS3/Crk-L interactions, we examined the binding of SOCS3 pY221 phosphopeptide to WT and SH2 domain mutants of Nck and Crk-L. 293T cells were transfected with Myc-tagged Nck or FLAG-tagged Crk-L expression vectors. Included were WT full-length Nck, a Nck SH2 domain mutant (R308A), WT Nck SH2 domain alone (lacking the N-terminal SH3 domains), as well as WT Crk-L and a Crk-L SH2 mutant (R39A). Lysates from transfected cells were precipitated with the SOCS3 phosphopeptide and analyzed by Western blotting with anti-Myc or anti-FLAG antibodies to detect Nck or Crk-L. As shown in Fig. 2B, the binding of Nck and Crk-L to the SOCS3 phosphopeptide was dependent on intact SH2 domains. WT Nck and Crk-L bound strongly to the peptide (lanes 2 and 5), while both SH2 mutants failed to bind (lanes 3 and 6). In addition, strong binding of the Nck SH2 domain to the SOCS3 peptide demonstrates that an intact SH2 domain is sufficient for the interaction, and the 3 N-terminal SH3 domains of Nck are not required for binding (lane 4).

We next determined whether full-length phosphorylated SOCS3 was capable of interacting with Nck. 293T cells were transiently transfected with a pME18S HA-tagged SOCS3 expression construct either alone or in combination with pME18S-Jak1 vector, to induce SOCS3 tyrosine phosphorylation. Cell lysates were split and precipitated with either GST or GST-Nck proteins coupled to glutathione beads, followed by anti-HA tag Western blotting to detect SOCS3. As shown in Fig. 2C, full-length SOCS3 interacted with the GST-Nck fusion protein when tyrosine phosphorylated (lane 4), whereas unphosphorylated SOCS3 failed to interact with Nck (lane 3).

In a previous report, we determined that stimulation of fibroblasts with PDGF for 1 h induces SOCS3 expression and tyrosine phosphorylation (35). Thus, in order to determine whether endogenous SOCS3/Nck and SOCS3/Crk-L complexes form in ligand-activated cells, we stimulated NIH3T3 cells with PDGF-BB for 1 h to induce SOCS3 expression. Cell lysates were immunoprecipitated with anti-SOCS3 antiserum followed by Western blotting with anti-Nck or anti-Crk-L antibodies. As shown in Fig. 3, PDGF stimulation of NIH3T3 cells induces SOCS3 expression and tyrosine phosphorylation (panels B and C). In addition, SOCS3 immunoprecipitates from stimulated cells contain Nck and Crk-L (panel A), demonstrating that endogenous SOCS3 interacts with these adapter proteins in a ligand-dependent manner.



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  		FIG. 3.
Endogenous phosphorylated SOCS3 interacts with Nck and Crk-L in PDGF-stimulated fibroblasts. Approximately 5 x 106 NIH3T3 fibroblasts were serum-starved in 0.2% fetal calf serum for 4 h, then stimulated with 50 ng of PDGF-BB for 1 h to induce SOCS3 expression. Cell lysates were immunoprecipitated with anti-SOCS3 antibodies, followed by Western blotting with anti-Nck or anti-Crk-L antibodies (panel A), anti-SOCS3 (panel B), or anti-phosphotyrosine (panel C).

 
The finding that endogenous phosphorylated SOCS3 interacts with Nck and Crk-L suggested that SOCS3 may regulate Nck and Crk-L signal transduction. To address this issue, NIH3T3 cells stably expressing FLAG-tagged WT SOCS3 were stimulated with PDGF-AA or PDGF-BB for a 6-h time course. SOCS3 was precipitated from cell lysates with anti-FLAG antibodies, followed by anti-phosphotyrosine Western blotting. As shown in Fig. 4, both PDGF-AA and PDGF-BB induced rapid SOCS3 tyrosine phosphorylation, which peaks from 0.5 to 1 h post-stimulation, but persists throughout the time course (top panel). Interestingly, we observed a 47 kDa phosphoprotein that co-precipitated with SOCS3, which comigrates with Nck. We confirmed the identity of the 47kDa protein by blotting the SOCS3 immunoprecipitates with anti-Nck antibodies (second panel). The kinetics of SOCS3/Nck complex formation parallels the appearance of the 47 kDa phosphoprotein in SOCS3 immunoprecipitates. In contrast, SOCS3/Nck complexes were not observed in cells expressing the Y204F/Y221F SOCS3 mutant (not shown). Interestingly, the SOCS3/Nck complex appears to be transient, as it was only detected in unstimulated cells and cells activated by PDGF for 30 min, but cannot be detected at later time points.



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  		FIG. 4.
Tyrosine-phosphorylated SOCS3forms a regulated complex with Nck in PDGF-stimulated fibroblasts. 5 x 106 NIH3T3 cells stably expressing FLAG-tagged WT SOCS3 were serum-starved for 4 h, then stimulated with 50 ng/ml PDGF-AA (left panels) or PDGF-BB (right panels) for a 6-h time course. Cell lysates were immunoprecipitated with anti-FLAG antibody followed by anti-phosphotyrosine Western blotting (top panels). The same immunoprecipitates were also analyzed by Western blotting with anti-Nck antibodies (second panel).

 
The presence of a SOCS3/Nck complex in serum-starved fibroblasts, which is regulated by ligand activation suggested that Nck may be recruited to activated receptors by phosphorylated SOCS3. Therefore, we analyzed the subcellular localization of a Nck-GFP fusion protein in NIH3T3 cells either alone or in combination with SOCS3. The cells were stimulated with PDGF-BB for 30 min and the subcellular localization of Nck-GFP was determined by confocal microscopy. As shown in Fig. 5, the Nck-GFP fusion protein was distributed diffusely throughout the cytoplasm of unstimulated NIH3T3 cells (panel A). Upon PDGF stimulation, the fusion protein is translocated to the plasma membrane (panel B). In contrast, Nck-GFP was constitutively membrane-associated in cells expressing exogenous SOCS3 (panel C), and was further recruited to the membrane following PDGF stimulation (panel D). In order to determine whether constitutive membrane association of Nck-GFP was dependent on SOCS3 tyrosine phosphorylation, we analyzed Nck-GFP localization in cells expressing the Y204F/Y221F phosphorylation-deficient SOCS3 mutant (panels E and F). SOCS3 Y(204,221)F failed to recruit Nck-GFP to the plasma membrane in unstimulated cells (panel E), suggesting that SOCS3 tyrosine phosphorylation was required for its ability to translocate Nck to the membrane.



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  		FIG. 5.
SOCS3 induces constitutive membrane association of a Nck-GFP fusion protein. NIH3T3 cells stably expressing a Nck-GFP fusion protein either alone (panels A and B) or in combination with WT SOCS3 (panels C and D) or SOCS3 Y204F/Y221F (panels E and F) were serum-starved for 4 h and then stimulated with 50 ng/ml PDGF-BB for 30 min. Subcellular localization of Nck-GFP was visualized by confocal microscopy.

 
Our imaging results suggested that SOCS3 may regulate the activation level of Nck. Therefore, we compared Nck tyrosine phosphorylation in parental NIH3T3 cells to NIH3T3 expressing WT SOCS3 or the Y204F/Y221F SOCS3 mutant. The cells were stimulated over a 2-h time course, and lysates were precipitated with anti-Nck antibodies, followed by anti-phosphotyrosine Western blotting- As shown in Fig. 6, we did not detect Nck tyrosine phosphorylation in serum-starved NIH3T3 cells (lane 1). Upon PDGF stimulation, Nck phosphorylation was markedly increased (lanes 2-4). In cells expressing WT SOCS3, we detected constitutive Nck tyrosine phosphorylation, which was increased following addition of PDGF (lanes 5-8). In contrast, SOCS3 Y204F/Y221F, which lacks the Nck interaction site, failed to induce constitutive Nck phosphorylation (lanes 9-12).



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  		FIG. 6.
Expression of WT SOCS3 induces constitutive Nck tyrosine phosphorylation. 5 x 106 parental NIH3T3 cells, or NIH3T3 stably expressing FLAG-tagged WT SOCS3 or the Y204F/Y221F SOCS3 mutant were serum-starved for 4 h and then stimulated with 50 ng/ml PDGF-BB for a 2-h time course. Cell lysates were immunoprecipitated with anti-Nck antibodies, followed by anti-phosphotyrosine 4G10 Western blotting (panel A). Nck and SOCS3 levels were determined by Western blotting with anti-Nck or anti-FLAG antibodies, respectively (panels B and C).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we present evidence that SOCS3 expression and tyrosine phosphorylation are induced by activated RTKs, and that endogenous phosphorylated SOCS3 interacts with the Nck and Crk-L adapter proteins in growth factor-stimulated cells. Our data also suggest that SOCS3 regulates the activation and subcellular localization of Nck in response to PDGFR{alpha} and PDGFR{beta} stimulation through a physical interaction between a SOCS box phosphotyrosine residue, pY221 and the SH2 domain of Nck. Our findings suggest that SOCS3 is not only a signal transduction inhibitor but also functions to recruit adapter proteins to activated receptors. Our data expand the potential targets of SOCS proteins and the signaling pathways that they may regulate.

The SH2-containing SOCS subfamily, which includes SOCS1 and SOCS3 was initially characterized as specific inhibitors of the Jak-STAT pathway. Through their SH2 domains and N-terminal kinase inhibitory regions (KIR), both molecules have been shown to bind to a phosphotyrosine residue in the catalytic loop of Jak1 and Jak2, blocking kinase activity and downstream STAT signaling (5-10). In addition, SOCS3 can also form inhibitory complexes with cytokine receptors by directly interacting with phosphorylated tyrosines on the receptor cytoplasmic domains, blocking downstream STAT activation (10-13, 27-33).

More recently, SOCS proteins have been shown to regulate additional signaling pathways by a distinct mechanism involving proteasome-mediated protein destabilization. The SOCS box has been demonstrated to regulate the stability of SOCS proteins as well as SOCS-interacting molecules (4, 14, 15, 37). SOCS1 accelerates the degradation of Jak1, Jak2, a TEL-Jak2 oncogenic fusion protein, the GEF Vav, and IRS-1 and IRS-2 (16-20). Likewise, SOCS3 also destabilizes IRS-1 and IRS-2, modulating insulin signaling and Akt activation, and also induces the degradation of the integrin-associated focal adhesion kinase (FAK) (19, 21).

In addition, our laboratory has shown that SOCS3 modulates the ERK MAP kinase pathway. We have shown that SOCS3 is phosphorylated on Tyr204 and Tyr221 within the conserved SOCS box. In IL-2-stimulated T cells, SOCS3 binds the SH2 domains of the Ras inhibitor p120 RasGAP through pY221 and sustains activation of the ERK MAP kinase pathway (35). In agreement with this model, it has been shown that macrophages from the SOCS3 knockout very poorly activated ERK in response to IL-6 (44). Thus, there is significant evidence that SOCS proteins have important functions beyond the Jak-STAT pathway.

In the present study, we have extended our original findings and demonstrate that tyrosine phosphorylated SOCS3 binds p120 RasGAP in PDGF-stimulated NIH3T3 fibroblasts as well. Upon examination of the amino acid sequence of the RasGAP binding site, we found that SOCS3 pY221 lies within a YXXP (YDAP) motif, which not only binds RasGAP, but is a consensus binding site for the Nck and Crk-L adapter proteins. Both Nck and Crk-L bound SOCS3 phosphopeptides that contained pY221, which forms a consensus binding site, but failed to bind a peptide containing only pY204, which does not form a YXXP motif. Our mapping studies have also shown that the pYSOCS3/Nck and pYSOCS3/Crk-L interactions depend on intact Nck and Crk-L SH2 domains, and that the Nck SH2 domain alone is sufficient for phosphopeptide binding. Furthermore, we have demonstrated that full-length pYSOCS3 bound a GSTNck fusion protein and endogenous phosphorylated SOCS3 co-precipitates with Nck and Crk-L in growth factor-stimulated cells implicating SOCS3 in the control of Nck and Crk-L signal transduction.

Nck and Crk-L are adapter molecules that are composed entirely of protein-protein interaction motifs (SH2 and SH3 domains) in the configuration SH3-SH3-SH3-SH2 (Nck) and SH2-SH3-SH3 (Crk-L) (39-43). Both of these phosphoproteins lack endogenous catalytic or DNA binding activities. However, they form signaling complexes by docking to phosphorylated receptors through their SH2 domains and activating signaling proteins that are constitutively bound to their SH3 domains, thus linking receptors to downstream signaling pathways. Our findings suggest that SOCS3 may play a role in adapter protein signaling, as expression of SOCS3 in NIH3T3 cells alters the subcellular localization and tyrosine phosphorylation levels of Nck.

A Nck-GFP fusion protein was diffusely expressed throughout the cytoplasm of serum-starved fibroblasts, but was translocated to the plasma membrane following PDGF stimulation, in agreement with previous findings that Nck forms a complex with tyrosine-phosphorylated PDGF receptors (45-47). However, Nck-GFP was constitutively localized to the plasma membrane in cells expressing WT SOCS3, but not the Y204F/Y221F SOCS3 mutant. This finding argues that SOCS3 functions to recruit Nck to the PDGF receptor, and that recruitment of Nck requires SOCS3 tyrosine phosphorylation. In support of this model, we have shown that SOCS3 can physically interact with phosphorylated PDGFR{alpha} and PDGFR{beta},2 demonstrating that SOCS3 may indeed bridge these receptors to downstream signaling molecules.

Our imaging results are supported by biochemical data which show that after serum starvation, cells expressing exogenous WT SOCS3 contained phosphorylated SOCS3/Nck complexes and constitutively phosphorylated Nck. The abundance of the SOCS3/Nck complexes was increased after 30 min of PDGF stimulation and correlated with a marked increase of SOCS3 tyrosine phosphorylation. In contrast, control NIH3T3 cells and NIH3T3 expressing the Y204F/Y221F SOCS3 mutant did not contain SOCS3/Nck complexes and did not demonstrate constitutive Nck tyrosine phosphorylation in the absence of PDGF stimulation. These findings suggest that SOCS3 can bind Nck and induce its tyrosine phosphorylation (probably by recruiting it to the PDGFR), and correlate well with our data showing constitutive membrane association of Nck-GFP in the presence of WT SOCS3. Our finding that low levels of phosphorylated SOCS3/Nck complexes are present in serum-starved cells was somewhat surprising. However, we have consistently found that after 4 h of serum starvation, low but detectable levels of phosphorylated SOCS3 persisted in the cells, which was likely sufficient to bind Nck and activate its tyrosine phosphorylation.

Our data support a model in which Nck binds to the PDGF receptor indirectly through interactions with phosphorylated SOCS3. Indirect recruitment of Nck and Crk-L to activated receptors by signaling proteins has been demonstrated in several systems. Although Nck docks directly to phosphotyrosine residues on VEGFR1, VEGFR2, and PDGFR{beta} (45-48), it also binds receptors indirectly via interactions with phosphorylated docking proteins DOK1 and DOK2. Nck is targeted to several RTKs, including EGFR, Tek/Tie2, EphB1, EphB2, and insulin receptor, as well as fc{epsilon} by binding to phosphorylated tyrosine residues on receptor-associated DOK1 or DOK2 (49-55). Likewise, Crk-L is recruited to the EGF receptor via its interaction with tyrosine phosphorylated p120 c-Cbl (56). Indirect binding of Nck and Crk-L to receptor chains has been demonstrated to be critical for the activation of downstream signaling pathways in these systems. Thus, the translocation of Nck to PDGF receptors via phosphorylated SOCS3 may represent another important mechanism to regulate Nck-dependent signal transduction.

A related finding in our studies was that SOCS3/Nck complexes were transient and appeared to be regulated by PDGF receptor activation. Specifically, SOCS3 tyrosine phosphorylation persisted throughout a 6-h PDGF time course, but SOCS3/Nck complexes were detected only in unstimulated cells and up to 30 min following PDGF stimulation.

The mechanisms regulating the SOCS3/Nck interaction are unknown, but may be related to phosphorylation-dependent SOCS3 protein destabilization. It has been shown by our laboratory and others that the SOCS box, which is a protein-protein interaction domain, regulates the stability of SOCS proteins by physically coupling to elongin C, a component of an E3 ubiquitin ligase containing a cullin (Cul-2 or Cul-5), elongin B, a ring finger protein (Rbx-1), and an E2 ligase (4, 14, 15, 37). Our previous data have shown that the SOCS3/elongin C interaction stabilizes SOCS3. Phosphorylation of the SOCS box tyrosines Tyr204 and Tyr221 disrupts the SOCS3/elongin C complex and accelerates SOCS3 degradation (37). Thus, a possible explanation for the transient nature of the SOCS3/Nck complexes in fibroblasts is that SOCS3 is destabilized by tyrosine phosphorylation and the complex is targeted for proteasome-mediated degradation. However, we cannot exclude the possibility that the complex is disrupted by a different mechanism, such as binding of competing signaling proteins to Nck or SOCS3, or decreased SOCS3 tyrosine phosphorylation at late time points.

Our current data as well as a previous study from our laboratory (35) have demonstrated that RasGAP, Nck, and Crk-L can all bind to a single phosphotyrosine residue on SOCS3. Our findings raise the possibility that these proteins compete for phosphorylated SOCS3 during a cytokine or growth factor response, with consequences for downstream signaling. We may postulate that engagement of one of these proteins would block or reduce the effects of SOCS3 on the other two binding proteins. The extent and downstream effects of competition for SOCS3 would likely depend on the abundance of phosphorylated SOCS3 within the cell as well as the relative expression levels, accessibility, and affinities of RasGAP, Nck, and Crk-L for SOCS3. Thus, we might expect that the extent of competition would depend on cellular context and may also be ligand-specific (i.e. different ligands may preferentially induce the formation of specific complexes). On the other hand, in a previous study (37) we analyzed the distribution of SOCS3 in fractionated lysates from cytokine-stimulated cells. We found that SOCS3 is present in fractions representing a wide range of molecular weights, which is consistent with the presence of multiple SOCS3-containing signaling complexes. Thus, although adapter proteins may compete for SOCS3 binding, it is also possible that pools of pYSOCS3 bound to RasGAP, Nck, and Crk-L may co-exist within the cell, resulting in coordinate regulation of RasGAP, Nck, and Crk-L signaling. We are currently investigating whether SOCS3 forms distinct complexes at different time points during a cytokine or growth factor response, or in response to specific ligands.

One issue that must be addressed is the potential biological function of SOCS3/Nck and SOCS3/Crk-L complexes. We have observed that SOCS3 shares signaling properties with two other adapter proteins, p62 DOK-1 and p56 DOK-2. As shown in Table I, DOK-1 and DOK-2 are phosphorylated on multiple tyrosine residues as a result of their recruitment to phosphorylated RTKs via their phosphotyrosine binding (PTB) domains. Both DOK-1 and DOK-2 contain several YXXP motifs and have been shown to bind RasGAP, Nck, and Crk-L (49-60). DOK and DOK-2 regulate RasGAP function and the Ras/Raf/MEK/ERK signaling pathway, and target Nck and Crk-L to phosphorylated receptors, activating downstream signal transduction. Thus, SOCS3 may also function to recruit adapter proteins to phosphorylated receptors and augment signaling. The finding that SOCS3, DOK-1 and DOK-2 all have similar protein-protein interaction motifs and bind an overlapping subset of signaling proteins suggests that there may be a requirement for coordinate regulation of RasGAP, Nck, and Crk-L. The fact that SOCS3 is an inducible protein suggests that such coordinate regulation can be accomplished rapidly and transiently in response to growth factors.

An alternative explanation for our data is that SOCS3 may function to attenuate Nck and Crk-L signaling by activating the degradation of signaling complexes. Although our data argue that SOCS3 enhances Nck receptor binding and tyrosine phosphorylation, the observation that the SOCS3/Nck complex is transient suggests that SOCS3 can rapidly down-regulate Nck activation. Thus, SOCS3 may cause Nck-dependent signaling to decay over an extended growth factor response by inducing the turnover of activated signaling complexes.

Gene targeting studies have shown that both Nck and Crk-L are critical signaling proteins that are indispensable for proper organogenesis and embryonic development (61, 62). Both of these proteins regulate MAP kinases, actin cytoskeleton rearrangement, as well as cell migration, growth and differentiation. There are two highly homologous Nck proteins, Nck{alpha} and Nck{beta}, both of which are involved in signaling from activated receptors (61, 63-65). The Nck SH3 domains can bind p21-activated kinases (PAK), the actin-associated Wiscott-Aldrich syndrome protein (WASP), PAK-related kinases (PRK), and the Nck-interacting kinase (NIK), and can activate the Jnk and p38 MAP kinase cascades (54, 66-73). Likewise, Crk-L SH3 domains couple to the BCR-Abl oncogene, the Rap1 GEF C3G, as well as germinal center kinase-related and hematopoietic progenitor kinase-1 (74-82), and can activate the ERK and Jnk pathways (74-84, 86-89). Therefore, through its phosphorylation-dependent interactions with Nck and Crk-L, SOCS3 can potentially target multiple Nck- and Crk-L-dependent signaling pathways and biological responses.

It is of note that both Nck and Crk-L are oncogenic. Overexpression of Nck transforms NIH3T3 fibroblasts (47, 90). Crk-L is a major phosphoprotein in cells derived from chronic myeloid leukemia (CML), and has been shown to be critical for myeloid cell transformation by the BCR-Abl oncogene (80, 91-95). Considering that SOCS3 gene expression is aberrantly regulated in CML cells (85, 96), we may speculate that SOCS3 affects Nck and Crk-L tumorigenesis as well.

Presently, however, the biological role of SOCS3/Nck and SOCS3/Crk-L complexes is not understood. Further studies in SOCS3-deficient cells and cells transformed by Nck and Crk-L should clarify the role of SOCS3 in the regulation of normal and oncogenic signal transduction by adapter proteins.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: UCLA Center for Health Sciences, Dept. of Radiation Oncology, Rm. B3-133, Los Angeles, CA 90095. Tel.: 310-267-2803; Fax: 310-206-1260; E-mail: ncacalano{at}mednet.ucla.edu.

1 The abbreviations used are: SOCS, suppressors of cytokine signaling; STAT, signal transducers and activators of transcription; GST, glutathione S-transferase; WT, wild type; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; Jak, Janus kinase; PDGF, platelet-derived growth factor; IRS, insulin receptor substrate; GFP, green fluorescent protein; RTK, receptor-tyrosine kinase; SH, Src homology domain; GEF, guanine nucleotide exchange factor; IL, interleukin; HA, hemagglutinin; pY, phosphorylated tyrosine. Back

2 J. Sitko and N. Cacalano, unpublished observation. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Bruce Mayer for the WT Nck and K308A Nck expression constructs and the GST-Nck bacterial expression vector, and Dr. Ronald Herbst of DNAX Research, Inc. for the WT Crk-L cDNA. We also acknowledge Dr. Matthew Schibler of the UCLA Brain Research Institute for his expert assistance with our imaging experiments.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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