Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M701797200 on July 9, 2007

J. Biol. Chem., Vol. 282, Issue 40, 29336-29347, October 5, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
282/40/29336    most recent
M701797200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Reddi, A. L.
Right arrow Articles by Band, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Reddi, A. L.
Right arrow Articles by Band, H.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Binding of Cbl to a Phospholipase C{gamma}1-docking Site on Platelet-derived Growth Factor Receptor beta Provides a Dual Mechanism of Negative Regulation*Formula

Alagarsamy Lakku Reddi{ddagger}, GuoGuang Ying{ddagger}, Lei Duan{ddagger}, Gengsheng Chen{ddagger}, Manjari Dimri{ddagger}, Patrice Douillard§, Brian J. Druker, Mayumi Naramura{ddagger}, Vimla Band||**, and Hamid Band{ddagger}**1

From the Divisions of {ddagger}Molecular Oncology and ||Cancer Biology, Evanston Northwestern Healthcare Research Institute, and Department of Medicine, Feinberg School of Medicine and **Department of Biochemistry, Molecular Biology and Cell Biology, Weinberg College of Arts and Science, Northwestern University, Evanston, Illinois 60201, the §Eucodis GmbH, Brunner Strasse 59, 1230 Vienna, Austria, and the Oregon Health and Science University Cancer Institute, Portland, Oregon 97239

Received for publication, March 1, 2007 , and in revised form, July 2, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ubiquitin conjugation to receptor tyrosine kinases is a critical biochemical step in attenuating their signaling through lysosomal degradation. Our previous studies have established Cbl as an E3 ubiquitin ligase for ubiquitinylation and degradation of platelet-derived growth factor receptor (PDGFR) {alpha} and PDGFRbeta. However, the role of endogenous Cbl in PDGFR regulation and the molecular mechanisms of this regulation remain unclear. Here, we demonstrate that endogenous Cbl is essential for ligand-induced ubiquitinylation and degradation of PDGFRbeta; this involves the Cbl TKB domain binding to PDGFRbeta phosphotyrosine 1021, a known phospholipase C (PLC) {gamma}1 SH2 domain-binding site. Lack of Cbl or ablation of the Cbl-binding site on PDGFRbeta impedes receptor sorting to the lysosome. Cbl-deficient cells also show more PDGF-induced PLC{gamma}1 association with PDGFRbeta and enhanced PLC-mediated cell migration. Thus, Cbl-dependent negative regulation of PDGFRbeta involves a dual mechanism that concurrently promotes ubiquitin-dependent lysosomal sorting of the receptor and competitively reduces the recruitment of a positive mediator of receptor signaling.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Receptor tyrosine kinases (RTKs)2 control cellular behavior in response to extracellular peptide growth factors. Platelet-derived growth factor receptors (PDGFRs) are prototypical RTKs that play critical biological roles in many species including mammals and are implicated in disease states such as oncogenesis (1-6). The two closely related PDGFRs ({alpha} and beta) function as homo- or hetero-dimers with differential affinities for homodimers or heterodimers of four distinct ligands (PDGF A-D). Deficiencies of PDGF ligands or receptors produce unique and often severe developmental defects or other pathological manifestations indicating the important physiological roles of the PDGF-PDGFR ligand-receptor signaling networks (1, 7-9). Notably, the overexpression or activation of PDGFRbeta correlates with the development of human tumors, including gliomas (10, 11), myelomonocytic leukemia (12), osteosarcoma (13), colon carcinoma (14, 15), prostate cancer, and breast cancer (16, 17). Given the critical physiological and pathogenic roles of PDGFRs, elucidating their regulatory mechanisms is of substantial interest.

We have previously demonstrated that the ubiquitin ligase Cbl (Casitas B-lineage lymphoma) functions as a negative regulator of PDGFRs (18-20), and subsequent studies have extended the role of Cbl family proteins to a variety of other RTKs (21-28). Cbl associates with activated RTKs via binding of its tyrosine kinase-binding (TKB) domain to phosphopeptide motifs generated by autophosphorylation of the receptor. Cbl TKB domain-binding sites were initially identified in ZAP70 and Syk through phospho-peptide library screening (29) and further characterized as a phosphotyrosine within a consensus sequence of (D/E)XpYXXX{phi} (where {phi} is a hydrophobic amino acid; usually Pro) (56, 57). Additional distinct motifs, such as DpYR and RA(V/I)XNQpY(S/T), have been identified in the RTK c-MET and in the adaptor protein APS, respectively. Recruitment of Cbl results in RTK ubiquitinylation, which in turn serves as a signal for targeting the receptor to the lysosome for degradation via the evolutionarily conserved protein complexes called endosomal sorting complex required for transport 1-3 (30-32). Enhanced lysosomal degradation is thought to limit the biological half-life of the active pool of RTKs, thus serving as a global attenuation mechanism; however, differential attenuation of signals originating in specific endosomal compartments remains to be investigated (19, 20, 33, 34).

The Cbl RING finger domain is essential for the negative regulation of PDGFRs (18-20), suggesting that ubiquitination provides one mechanism for Cbl-mediated negative regulation of PDGFRs. However, the functional role of endogenous Cbl proteins has not been addressed, and the mechanisms underlying the Cbl-PDGFR interaction remain unidentified. Several previous findings suggest that Cbl may interact with PDGFRs through its TKB domain because a point mutation (G306E) within the TKB domain that abolishes the binding of Cbl to its phospho-peptide targets reduces Cbl-PDGFR association (18). An intact TKB domain was also required for Cbl protein to promote the ubiquitinylation of PDGFR as well as for negative regulation of PDGFR-mediated cell proliferation and survival (20). Thus, identification of Cbl TKB domain-binding motif(s) on PDGFR is crucial to elucidate the mechanism of Cbl-mediated PDGFR regulation.

PLCs play a crucial role downstream of RTKs by catalyzing the hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate and diacylglycerol, which in turn mediate intracellular Ca2+ mobilization and protein kinase C activation, respectively (35). Phospholipase C{gamma}1 (PLC{gamma}1) is important in directed cell migration (36), wound healing (37), neurite outgrowth (38), membrane ruffling (39), macropinocytosis (40), cytoskeletal reorganization, and cell adhesion (41). PLC{gamma}1 binds to phosphorylated Tyr1009 and Tyr1021 in PDGFRbeta and to Tyr988 and Tyr1018 in PDGFR{alpha} (42, 43). A PDGFRbeta mutant unable to bind and activate PLC{gamma}1 was impaired in its chemotactic response to PDGF stimulation (36). Overexpression of a catalytically inactive mutant of PLC{gamma}1 in NIH-3T3 cells reduced the chemotactic response to PDGF BB (36). Notably, the PLC{gamma}1-docking site tyrosine (Tyr1021) is crucial for PDFGRbeta-regulated chemotactic rather than mitogenic response of cells to PDGF BB stimulation (43, 44), suggesting a critical role for PLC{gamma}1 in PDGFR-regulated cell migration.

Here, we have identified the Cbl TKB domain-binding site on PDGFRbeta that overlaps with a previously known PLC{gamma}1 SH2 domain-binding site. We provide evidence that endogenous Cbl is not only an important determinant of targeting activated PDGFRbeta for ubiquitin-dependent lysosomal degradation, but a negative regulator of PLC{gamma}1-mediated chemotactic and migratory responses to PDGF. These findings reveal a novel dual mode of Cbl-mediated negative regulation of PDGFR via 1) ubiquitin-dependent degradation of the receptor and 2) competitive RTK binding with the positive signaling mediator PLC{gamma}1.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—The sources of reagents were as follows: PDGF AA and BB were obtained from Upstate%20Biotechnology">Upstate Biotechnology, Inc. (Upstate, NY); PLC{gamma} inhibitor U73122 [GenBank] and its inactive analog U73433 [GenBank] from Calbiochem; serum-free Opti-MEM I medium from Invitrogen; FuGENE 6 transfection reagent from Roche Applied Science; and Vectashield mounting medium containing 4',6-diamidino-2-phenylindole from Vector Laboratories (Burlingame, CA). Anti-phosphotyrosine antibody 4G10 (45) and 12CA5 antibody (anti-HA) (46) were purified from hybridomas. Anti-Cbl (C-15), anti-phospho-PDGFRbeta Tyr1009 and Tyr1021 (SC-12908 and SC-12909), and anti-PLC{gamma}1 (SC-81) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-PDGFRbeta C terminus was from PharMingen (San Diego, CA); anti-ubiquitin was from NovoCastra Laboratories (Newcastle, UK), and anti-phospho-PLC{gamma}1-pY783 and anti-phospho-MAPK (p42/44) were from Cell Signaling Technologies (Beverly, MA).

Cell Lines—Immortal murine embryonic fibroblast (MEF) cell lines derived from two distinct Cbl-/- mouse lines (47, 48) and their littermate Cbl+/+ controls have been described (24). The MCF-7 cell line was obtained from ATCC. Cbl-/- MEFs were reconstituted with Cbl (HA-Cbl) in pMSCV-puro vector using retroviral infection, as described previously (24).

Construction and Expression of PDGFRbeta-GFP Chimeras— The PDGFRbeta-GFP chimeras, with a C-terminal GFP, were derived using the Gateway cloning system (Invitrogen). The PDGFRbeta coding sequences were PCR-amplified (primers 1 and 2; supplemental Table S1) from pcDNA3-PDGFRbeta (49) and Topo-cloned into entry vector pENTR/SD/D-TOPO. GFP coding sequences were cloned into destination vector pcDNA-DEST47 and pcDNA-PDGFRbeta-GFP chimera generated by recombineering according to the manufacturer's protocols. The chimeric sequences from this construct were PCR-amplified (primers 1 and 3 in supplemental Table S1) and Topo-cloned back into pENTR/SD/D-TOPO followed by LR Clonase recombination into lentiviral destination vector pLenti6-V5-DEST. Point mutants were generated using the site-directed mutagenesis kit (Stratagene, La Jolla, CA) (primers listed in supplemental Table S1). All of the constructs were sequence-verified. The PDGFRbeta-GFP chimera and its mutants (Y1009F, Y1021F, Y0009F/Y1021F, Y716F, Y716F/Y1009F, Y716F/Y1021F, and Y716F/Y1009F/Y1021F) were packaged and used to infect MCF-7 cells according to the vendor instructions (ViraPower lentiviral expression system; Invitrogen). Blasticidin-resistant (Invitrogen) (2 µg/ml) cells were analyzed using fluorescence-activated cell sorter (GFP) and anti-PDGFRbeta immunoblotting.

shRNA-mediated Cbl Knock-down—Three Cbl-specific shRNAs (supplemental Table S2) were designed using the S-fold software (sfold.wadsworth.org) and cloned into the pSUPERIOR-Retro vector (OligoEngine, Seattle, WA). The shRNAs were introduced into MCF-7 cells using retroviral infection (24). Puromycin-resistant (0.5 µg/ml) cells were analyzed for Cbl protein levels using Western blotting.

Wound Healing and Transwell Migration Assays—Serum-starved confluent cell monolayers were scratched with a plastic pipette tip and washed twice with phosphate-buffered saline. The plates were refed with starvation medium with 20 ng/ml PDGF-BB and allowed to heal at 37 °C. The cells were photographed at 0, 3, 6, 9, and 12 h with an inverted phase contrast microscope (Nikon, Eclipse TE2000-U) equipped with a CCD camera. For Boyden chamber Transwell migration assays, 105 cells in 400 µl of serum-free medium were added to the upper chamber coated with fibronectin (10 µg/ml), and the cells were allowed to adhere for 4 h. Subsequently, 400 µl of starvation medium containing various stimulants was added to the lower chamber, and cell migration was allowed for 4 h. The cells on the upper surface were removed with a cotton swab, and the migrated cells on the lower surface were fixed and stained using the Diff-Quick® stain kit (Dade Behring Inc., Newark, DE) as per the manufacturer's instructions. Each data point is a mean of six replicates, with average of three high power (400x) fields/filter. Statistical analysis used the Student's t test; p < 0.05 was considered significant. The experiments were repeated at least three times.

Immunoprecipitation (IP) and Immunoblotting (IB)—IPs and IBs were carried as described previously (18).

GST Fusion Protein Pulldown Assays—MCF-7 cells expressing PDGFRbeta-GFP or its mutants were serum-starved and stimulated with PDGF BB (20 ng/ml) for 10 min prior to lysis. GST fusion protein pulldowns from 1-mg aliquots of lysate protein and 20 µg of GST or GST-Cbl (Cbl-N, amino acids 1-357; and Cbl-N/G306E) fusion proteins were carried out as described (29), and bound proteins were visualized by Western blotting, as above.


Figure 1
Figure 1
View larger version (72K):
[in this window]
[in a new window]

  		FIGURE 1.
Impaired ligand-induced PDGFRbeta ubiquitinylation and degradation in MEFs lacking endogenous Cbl expression. A and B, the HG (A) or DB (B) Cbl+/+ and Cbl-/- MEF lines were serum-starved and stimulated with PDGF BB (20 ng/ml) for the indicated time points (in min) prior to lysis. Anti-PDGFRbeta IPs from 1-mg aliquots of lysate protein were subjected to serial IB with anti-PDGFRbeta (top panel), anti-ubiquitin (middle panel), and anti-Tyr(P) (bottom panel) antibodies. The relative PDGFRbeta signals were quantified by densitometry using the Scion Image software (Scion Corp., Frederick, MD) and are depicted as a proportion of signals observed with unstimulated cell lysates (set as 1). C-E, retro-viral infection was used to express HA-tagged human Cbl (HA-Cbl) or vector alone in Cbl+/+ and Cbl-/- MEFs (HG or DB as indicated). 200-µg aliquots of lysate protein were subjected to anti-Cbl IP followed by anti-Cbl IB. The levels of HA-Cbl protein in reconstituted Cbl-/- (DB and HG) MEFs is comparable with that in Cbl+/+ cells (C). D and E, The vector-transfected or Cbl-reconstituted Cbl-/- MEFs (HG, D; DB, E) were serum-starved and stimulated with 20 ng/ml PDGF BB for the indicated times prior to cell lysis. 1-mg aliquots of lysate protein were used for anti-PDGFRbeta IP followed by serial anti-PDGFRbeta (top panel), anti-ubiquitin (middle panel), and anti-Tyr(P) (bottom panel) immunoblotting. Relative PDGFRbeta signals were determined by densitometry.

 
Competition Between GST-PLC{gamma}1 SH2-C and Cbl-N for PDGFR Binding—The GST fusion of the PLC{gamma}1 C-terminal SH2 domain (residues 663-759) (PLC{gamma}1 SH2-C) was generated by reverse transcription-PCR amplification from a human cDNA using primers (listed in supplemental Table S3) that incorporated BamHI and NotI sites for cloning into the pGEX-6P-1 bacterial expression vector (Pharmacia-LKB). The sequence-verified construct was used for expression and purification of the GST fusion protein as described (29). The GST tag was cleaved from fusion proteins using the PreScission protease, according to the manufacturer's protocols (GE Healthcare). For Cbl-N/Cbl-N-G306E proteins, the GST tag was cleaved with human thrombin; contaminating thrombin was removed by incubating proteins with p-aminobenzamidine beads (Sigma) for 30 min at 4 °C. Cleaved proteins were dialyzed against phosphate-buffered saline prior to binding assays.

For competitive binding assays, various concentrations of isolated PLC{gamma}1SH2 domain protein were added to lysates of nonstimulated or PDGF BB-stimulated MEFs for 45 min at 4 °C followed by pulldown using 20 µg of GST or GST-Cbl-N (or Cbl-N/G306E) fusion proteins as above.

Confocal Immunofluorescence Microscopy—The cells were seeded on sterilized coverslips in regular growth medium for 24 h, serum-starved for 48 h, and stimulated with PDGF-BB as described above. The cells were fixed in 3.8% paraformaldehyde and immunostained as described (24) with anti-LAMP-1 antibody (1:1000) followed by Alexa Flour 594-conjugated goat anti-mouse antibody. Coverslips were mounted in Vectashield mounting medium containing 4',6-diamidino-2-phenylindole for visualizing the nuclei (Vector Laboratories). The images were acquired with a Nikon C1 confocal microscope system under 600x magnification.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Endogenous Cbl Is Required for Efficient Ligand-induced Ubiquitinylation and Degradation of PDGFRbeta—To address the role of endogenous Cbl in PDGFR regulation, we first assessed the extent of ligand-induced PDGFRbeta ubiquitinylation in Cbl+/+ and Cbl-/- MEF cell lines. Ligand-dependent ubiquitinylation of PDGFRbeta was detected in both of the independently derived Cbl+/+ MEF lines (HG and DB; Fig. 1, A and B); however, the extent of ligand-induced PDGFRbeta ubiquitinylation was greatly reduced in both Cbl-/- MEFs (HG and DB), indicating that endogenous Cbl is the major E3 mediating PDGFRbeta ubiquitinylation in MEFs. Anti-PDGFRbeta IP followed by anti-PDGFRbeta IB (Fig. 1, A and B) revealed that ligand-induced loss of the PDGFRbeta protein was substantially slower in both Cbl-/- MEFs; concomitantly, we observed a slower and less pronounced decrease in tyrosine-phosphorylated PDGFRbeta (Fig. 1, A and B). The pattern of PDGFRbeta phosphorylation indicated that impaired PDGFRbeta ubiquitinylation in Cbl-/- cells was not due to defective PDGFRbeta activation.

To exclude that defective PDGFRbeta ubiquitinylation and degradation in Cbl-/- MEFs was not an artifact of cell line derivation, we reconstituted the Cbl expression in both (HG and DB) Cbl-/- MEF lines (Fig. 1C). Comparison of the Cbl-/-/HA-Cbl versus Cbl-/-/vector cells demonstrated that Cbl reconstitution fully restored the PDGF-BB-induced ubiquitinylation and degradation of PDGFRbeta (Fig. 1, D and E). Correlating with a higher level of Cbl, PDGF-induced loss of phosphorylated and total PDGFRbeta was faster in Cbl+/+/HA-Cbl MEFs versus Cbl+/+/vector MEFs (supplemental Fig. S1). Thus, the defect in PDGFRbeta ubiquitinylation and degradation in Cbl-/- MEFs is solely due to a lack of Cbl expression. The role of endogenous Cbl in ubiquitinylation and degradation of PDGFRbeta was independently verified in MCF-7 cells with stable knock-down of Cbl (supplemental Fig. S5).


Figure 2
View larger version (41K):
[in this window]
[in a new window]

  		FIGURE 2.
Identification of phosphorylated Y1021 as the predominant Cbl TKB domain-binding site on PDGFRbeta. A, MCF7 derivatives expressing C-terminally GFP-tagged chimeras of wild type PDGFRbeta (WT) or its mutants (Y716F, Y716F/Y21F, Y09F/Y21F, Y716F/Y09F, Y716F/Y21F, Y09F/Y21F, or Y716F/Y09F/Y21F) were serum-starved and stimulated with PDGF BB (20 ng/ml) prior to lysis. 100-µg aliquots of lysate protein were subjected to anti-PDGFRbeta (upper panel) followed by anti-Tyr(P) IB (lower panel). B and C, 1-mg aliquots of protein lysate of MCF7 cells expressing the indicated PDGFR-GFP chimeras were used for binding to GST (control) or GST-Grb2-SH2 fusion protein and phosphorylated PDGFR chimeras visualized using anti-Tyr(P) IB. C, in vitro binding to GST, GST-Cbl-N (TKB domain) or GST-Cbl-N-G306E nonbinding mutant control fusion proteins was carried out as in B followed by anti-Tyr(P) immunoblotting.

 
Identification of the Cbl TKB Domain-binding Site on PDGFRbeta—To identify the TKB domain-binding site(s) on PDGFRbeta, we used GST fusions of Cbl TKB domain (GST-Cbl-N) or its nonbinding point mutant (GST-Cbl-N-G306E) to pull down wild type (WT) PDGFRbeta or its potential Cbl TKB domain-binding site Tyr -> Phe mutants ectopically expressed in PDGFRbeta-null MCF-7 cells (Fig. 2A). The previously described consensus sequence (N/D)XpYXXX{phi} (29) suggested the sequence around Tyr1021 in PDGFRbeta (NDpYIIPL) as a potential Cbl TKB domain-binding motif (supplemental Fig. S6). We also included a PDGFRbeta mutant with a Tyr -> Phe mutation in the Grb2 SH2 domain-binding site (Y716F) and the double mutants as controls. Analysis of cell lysates demonstrated that WT and mutant receptors were expressed at comparable levels and underwent similar levels of overall tyrosine phosphorylation upon PDGF BB stimulation (Fig. 2A). The control GST-Grb2 SH2 domain pulled down WT or mutant PDGFRs as long as the Grb2-binding site Tyr716 was intact (Fig. 2B). The GST-Cbl-TKB fusion protein could pull down the PDGF-stimulated WT PDGFRbeta, whereas the G306E mutation abolished the TKB interaction with PDGFRbeta (Fig. 2C). Importantly, GST-Cbl-TKB failed to pull down the Y1021F mutant but could pull down the PDGFRbeta Y716F and Y1009F mutants (Fig. 2C). The inability to pull down Y1021F PDGFRbeta indicates that Tyr1021 is the predominant Cbl TKB domain-binding site on PDGFRbeta.

Competition between Cbl TKB and PLC{gamma}1 SH2 Domain Binding to Phosphorylated PDGFRbeta—Binding of Cbl TKB domain to a known PLC{gamma}1 SH2 domain-binding site (42, 43, 50, 51) raised the possibility that these proteins may compete for binding to PDGFR. We carried out in vitro competition studies with purified recombinant proteins to explore this possibility. As expected, the GST fusion of the C-terminal SH2 domain of PLC{gamma}1 (GST-PLC{gamma}1 SH2-C) pulled down the phosphorylated PDGFRbeta from lysates of PDGF-stimulated cells (Fig. 3A), and this interaction was dose-dependently reduced by incorporating increasing concentrations of purified Cbl-N in the pulldown assays, whereas the Cbl-N-G306E protein failed to do so (Fig. 3B). Conversely, GST-Cbl-N pull down of the phosphorylated PDGFRbeta from lysates of PDGF-stimulated cells was dose-dependently abrogated by incorporating increasing concentrations of recombinant PLC{gamma}1 SH2-C. These results clearly demonstrate that Cbl TKB and PLC{gamma}1 SH2 domains compete for binding to phosphorylated PDGFRbeta.

Inability to Interact with Cbl Alters the Lysosomal Trafficking of PDGFRbeta—To assess whether Cbl regulates PDGFR through lysosomal degradation, a PDGFRbeta-GFP chimera was generated and stably expressed in non-PDGFR-expressing MCF-7 cells (as in Fig. 2A). We then examined the endocytic trafficking of the WT versus Y1021F PDGFRbeta-GFP chimeras using confocal immunofluorescence microscopy. Upon ligand stimulation, WT PDGFRbeta-GFP was internalized and initially colocalized with transferrin, indicating its entry into early and sorting endosomes (Fig. 4A, 10 min) and subsequently showed a lack of colocalization with transferrin (Fig. 4A, 30 min), as expected from trafficking of transferrin to the recycling endosomes and that of PDGFRbeta-GFP chimera to the lysosomes (52). Indeed, PDGFRbeta-GFP showed a nearly complete colocalization with the lysosomal marker LAMP-1 at 30 min, with only minor colocalization at 10 min (Fig. 4C). The signals of WT PDGFRbeta-GFP chimera were essentially undetectable 90 min after PDGF stimulation (Fig. 4, A and C), consistent with its ligand-induced lysosomal degradation (19, 20, 33, 34). In contrast, Y1021F PDGFRbeta-GFP showed prolonged colocalization with transferrin (seen at 10 and 30 min) and only partial colocalization with LAMP-1 at 30 min (Fig. 4B). Furthermore, a substantial proportion of vesicles containing mutant PDGFRbeta-GFP chimera were seen after 90 min, consistent with delayed degradation (Fig. 4, B and D). Confirming these results, biochemical analyses also showed impaired ligand-induced degradation of the non-Cbl-binding mutant Y1021F compared with WT PDGFRbeta (supplemental Fig. S2). Thus, the inability of Cbl recruitment via Tyr1021 indeed leads to delayed sorting to the lysosome, whereas entry into early/sorting endosomes was unaltered.


Figure 3
View larger version (39K):
[in this window]
[in a new window]

  		FIGURE 3.
Competition between Cbl TKB and PLC{gamma}1 SH2 domains for binding to PDGFRbeta. A, 1-mg aliquots of lysate proteins from unstimulated or PDGF BB-stimulated (20 ng/ml; 10 min) Cbl+/+ MEF cells were incubated with 20 µg of GST or GST-PLC{gamma}1 SH2-C fusion proteins on beads, and bound phosphorylated PDGFRbeta was visualized using anti-Tyr(P) IB. 100-µg aliquots of lysate protein were subjected to direct anti-Tyr(P) IB. The relative PDGFRbeta signals were quantified by densitometry using the Scion Image software (Scion Corp.) and are depicted as a proportion of signals observed with unstimulated cell lysates (set as 1). B, pulldown assay was carried out as in A except that indicated concentrations of purified Cbl-N or Cbl-N G306E proteins (cleaved from GST tags) were added for 45 min before the pulldown. C, purified recombinant PLC{gamma}1 SH2-C at various concentrations was added to lysates prior to pulldowns with GST-Cbl-N (Cbl TKB domain) or GST-Cbl-N-G306E (nonbinding mutant) fusion proteins. Relative PDGFRbeta signals were determined by densitometry.

 
Cells Expressing the Cbl TKB Domain Nonbinding Y1021F Mutant PDGFRbeta Display Reduced Cell Migration—Mutation of the Cbl TKB domain-binding site on Cbl targets has been shown to enhance downstream signaling (18-20). However, because phosphorylated Tyr1021 has been established as a PLC{gamma}1 SH2 domain-binding site in PDGFRbeta (42, 43, 50, 51), Tyr1021 mutation in PDGFRbeta could impair PLC{gamma}1-mediated functions, as indicated by a knock-in mutation in mice (53). To assess the functional consequences of the Y1021F mutation of PDGFRbeta, we examined its impact on PLC{gamma}1-mediated cell migratory response in our cell lines using a wound healing assay. PDGFRbeta-null cells reconstituted with WT PDGFRbeta showed complete wound healing response within 12 h of culture in the presence of PDGF BB (Fig. 5). In contrast, the wound remained unclosed at 12 h in cells expressing PDGFRbeta Tyr1021 (Fig. 5), or cells expressing the Y1009F/Y1021F or Y716F/Y1009F/Y1021F mutants (supplemental Fig. S3). Thus, mutation of the Cbl TKB domain-binding site on PDGFRbeta, which coincides with a major PLC{gamma}1-binding site, induces a loss of cell migration function. These results suggest that the major role of the Tyr1021 phosphorylation site is to recruit PLC{gamma}1, and direct binding of Cbl TKB domain and the ensuing Cbl recruitment could negate PLC{gamma}1 signaling in addition to facilitating receptor ubiquitinylation and degradation. This possibility is further addressed below.

Cbl-deficient Cells Exhibit Increased PDGFR-PLC{gamma}1 Association and Elevated Levels of Phosphorylated PLC{gamma}1 in Response to PDGF—The overlapping Cbl TKB domain- and PLC{gamma}1-SH2 domain-binding sites on PDGFRbeta suggested that Cbl deficiency might enhance PLC{gamma}1 recruitment to PDGFR and its activation. To assess whether Cbl deficiency promotes increased PLC{gamma}1 association with PDGFRbeta, we coimmunoprecipitated PDGFRbeta with PLC{gamma}1 in Cbl-/-/vector or Cbl-/-/HA-Cbl MEFs. Indeed, the level of association of PDGFRbeta with PLC{gamma}1 was higher in Cbl-/- cells compared with the reconstituted cells (Fig. 6A). Anti-Tyr(P) immunoblotting also demonstrated a higher level of phosphorylated PDGFR in anti-PLC{gamma}1 IPs in Cbl-/- MEFs. Use of phospho-specific antibodies indicated that PDGFRbeta Tyr1021 was indeed hyperphosphorylated as were other sites (e.g. Tyr1009) (supplemental Fig. S4).


Figure 4
View larger version (114K):
[in this window]
[in a new window]

  		FIGURE 4.
Cbl TKB domain nonbinding PDGFRbeta Y1021F mutant is inefficiently sorted to the lysosome. A and B, MCF-7 cells expressing PDGFRbeta-GFP or PDGFRbeta Y1021F-GFP were serum-starved for 72 h and stimulated with 20 ng/ml PDGF BB for the indicated times. The cells were loaded with Alexa Fluor 546-conjugated transferrin for 10 min prior to fixation to visualize the early endosome/recycling endosomal compartment. The cells were imaged by confocal microscopy to visualize the colocalization of PDGFRbeta-GFP (green) and Alexa 546-transferrin (red), seen as yellow in merged images. C and D, the cells were serum-starved and stimulated as in A and B and fixed, permeabilized, and immunostained for LAMP1 (H4A3 mouse monoclonal followed by Alexa Flour 594-conjugated goat anti-mouse antibody; red) followed by confocal microscopy.

 
PDGF stimulation induced rapid PLC{gamma}1 phosphorylation in Cbl+/+ MEFs (seen at 3 and 10 min), which declined subsequently to reach nearly undetectable levels by 90 min (Fig. 6B, lanes 13-18). In contrast, phospho-PLC{gamma}1 levels were undiminished at 30 min in PDGF-stimulated Cbl-/- MEFs, started to decline by 90 min, and were still detectable at 270 min (Fig. 6B, lanes 1-6). The pattern of prolonged PLC{gamma}1 phosphorylation was reversed by HA-Cbl reconstitution of Cbl-/- MEFs (Fig. 6B, lanes 7-12). Notably, total PLC{gamma}1 levels were comparable in all cell lines (Fig. 6B, upper panel). To confirm this result, we further used MCF-7 cells with ectopically expressed PDGFRbeta. As expected, PDGF BB induced PLC{gamma}1 phosphorylation only in the cells with PDGFRbeta expression (Fig. 6D). When Cbl was knocked down in PDGFRbeta-expressing MCF-7 cells using three distinct shRNAs (Fig. 6C, lanes 4 and 5), ligand-induced PDGFRbeta ubiquitinylation and degradation were impaired (supplemental Fig. S5). More importantly, Cbl knockdown prolonged the PLC{gamma}1 phosphorylation induced by PDGF BB stimulation in PDGFRbeta-expressing MCF-7 cells (Fig. 6D, lanes 7-18), whereas the total PLC{gamma}1 levels were unaltered in all cell lines (Fig. 6D, upper panel). Thus, in two distinct cellular systems, deficiency of Cbl protein prolonged the PLC{gamma}1 phosphorylation upon PDGFR activation.


Figure 5
View larger version (101K):
[in this window]
[in a new window]

  		FIGURE 5.
PDGFRbeta Tyr1021 mutant is impaired in mediating PDGF-induced cell migration. Monolayer of PDGFRbeta-null MEFs stably reconstituted with WT or Y1021F mutant PDGFRbeta were plated in 6-well plates, serum-starved for 48 h, and subjected to scratch wounds with a plastic pipette tip (marked with a large black line). PDGF BB (20 ng/ml) was added, and the cells were photographed every 3 h for 12 h.

 
Cbl Deficiency Promotes the PLC{gamma}1-mediated Cell Migration Downstream of PDGFR—To assess whether Cbl deficiency promotes the PLC{gamma}1-mediated biological responses, we compared Cbl+/+ and Cbl-/- MEFs for PDGF-induced cell migration using a modified Boyden chamber assay. Indeed, the PDGF BB dose-dependent cell migration was significantly (p < 0.05) higher in Cbl-/- cells compared with that in Cbl+/+ cells (Fig. 7A). To further assess whether the enhanced cell migration in Cbl-/- MEFs required PLC{gamma}1 activity, we used chemical inhibitors of PLC. The specific PLC inhibitor U73122 [GenBank] (54) inhibited the PLC{gamma}1 phosphorylation (≥ 90% at 1-3 µM) as well as the phosphorylation of its downstream target MAPK; the inactive analog U73343 [GenBank] or Me2SO had no effect (Fig. 7B). U73122 [GenBank] but not U73343 [GenBank] blocked the PDGF-induced migration of MEFs (Fig. 7C), suggesting that a lack of Cbl results in hyperactivation of PLC{gamma}1 by PDGFR, which in turn leads to enhanced downstream biological responses.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we establish that endogenous Cbl is critical for ligand-dependent ubiquitinylation and degradation of PDGFRbeta. Importantly, our studies identify the Cbl TKB domain-binding site on PDGFRbeta, which overlaps with the known PLC{gamma}1 SH2 domain-binding site, thereby revealing a novel mechanism of negative regulation of a RTK by Cbl via competition for recruitment sites of a positive mediator of downstream signaling. Given the recent studies in lymphocytes that Cbl TKB domain-binding site on B-cell linker protein (BLNK) also corresponds to a PLC{gamma}1 recruitment site (55), our analyses of a RTK (PDGFR) suggests that Cbl-mediated inhibition of PLC{gamma}1 may provide a prevalent mechanism of reciprocal biological regulation of tyrosine kinase signaling.

Most of the analyses on Cbl-mediated ubiquitin modification and its role in lysosomal targeting of activated RTKs, including those of PDGFRs, have solely relied on the use of ectopically overexpressed Cbl proteins, thereby raising questions about the significance of endogenous Cbl in RTK regulation. Thus, our analyses of endogenous PDGFRbeta on two distinct MEF lines with or without Cbl expression, together with Cbl knock-down in a human cell line expressing ectopic PDGFRbeta, provide the first direct evidence that Cbl indeed is the primary determinant of ligand-induced ubiquitinylation, lysosomal targeting, and degradation of PDGFR. These results further extend prior studies of human EGFR transfected into MEFs (24) and endogenous CSF-1 receptor in Cbl-null macrophages (25) and together provide a firm basis for a crucial regulatory role of Cbl in controlling activated RTK down-regulation via ubiquitin-dependent lysosomal targeting.

Prior studies of PDGFRs have demonstrated that an intact Cbl TKB domain is crucial for negative regulation. Yet, the Cbl TKB domain-binding site(s) on PDGFRs have not been identified. Previous work by us and others (29, 56, 57) has delineated a consensus Cbl TKB domain-binding motif (D/N)XpYXXX{phi} that is found in a number of known Cbl targets including EGFR, ZAP-70, Syk, and Sprouty 2 (29, 56, 58-60). A close match of this motif corresponds to sequences surrounding Y1021 on human PDGFRbeta (DNDYIIPL), and our analyses (Fig. 2 and supplemental Fig. S6) demonstrate that this motif indeed mediates Cbl TKB domain binding. The Y1021F mutation reduces the PDGFRbeta ubiquitinylation and lysosomal traffic (Fig. 4, B and D), indicating that the ability of Cbl to interact with this motif is critical for Cbl-mediated regulation of PDGFR. Previous analysis of the PDGFRbeta Y1009F/Y1021F mutant in porcine aortic endothelial cells also showed diminution of PDGF-induced ubiquitinylation (61).


Figure 6
View larger version (40K):
[in this window]
[in a new window]

  		FIGURE 6.
Cbl-deficient cells show increased recruitment of PLC{gamma}1 to PDGFRbeta. A, Cbl-/- MEFs or their HA-Cbl-reconstituted derivatives (Cbl-/-/HA-Cbl) were serum-starved for 48 h and stimulated with 20 ng/ml PDGF BB for 10 min prior to lysis. 1-mg aliquots of protein lysate were subjected to anti-PLC{gamma}1 IP followed by anti-PDGFRbeta (top panel), anti-PLC{gamma}1 antibody (middle panel), and anti-Tyr(P) IB (bottom panel). B, enhanced PDGF-induced PLC{gamma}1 phosphorylation in Cbl-deficient cells. MEFs (Cbl-/-, Cbl+/+, and Cbl-/-/HA-Cbl) were serum-starved for 48 h and stimulated with 20 ng/ml PDGF BB for the indicated time points. 100-µg aliquots of cell lysate protein were subjected to anti-PLC{gamma}1 IB and reprobed with anti-phospho-PLC{gamma}1 (Tyr783) antibody. C and D, lentiviral infection was used to introduce Cbl shRNA or scrambled control shRNA into MCF-7-PDGFR-beta-GFP cells. 50-µg aliquots of lysate protein were subjected to anti-Cbl (upper part of filter) and anti-beta-actin (lower part of filter; loading control) IB. Cbl-/- MEFs lysates (50 µg) were served as a control (C). MCF-7-PDGFRbeta-GFP cells with indicated shRNAs were serum-starved for 72 h and stimulated with 20 ng/ml PDGF BB for the indicated time points prior to lysis. 100-µg aliquots of lysate protein were subjected to anti-PLC{gamma}1 followed by anti-phospho-PLC{gamma}1 IB (D).

 
Although the previously identified Cbl TKB domain-binding motifs on other Cbl targets are apparently Cbl-selective, Tyr1021 on PDGFRbeta is a known binding site for PLC{gamma}1 SH2 domains (42, 43, 50). Potential interaction of Cbl, a negative regulator, and PLC{gamma}1, a positive effector, with the same phosphotyrosine-containing motif predicted that (1), unlike other Cbl-regulated PTKs such as ZAP70 or Syk (29, 58), Tyr -> Phe mutation of Tyr1021 may not render PDGFR hyperactive because of concurrent loss of interaction with PLC{gamma}1, and (2) Cbl binding to a PLC{gamma}1-binding motif on PDGFRbeta could provide an additional mechanism for negative regulation of receptor signaling distinct from Cbl-mediated PDGFR ubiquitinylation and lysosomal sorting. Indeed, Y1021F PDGFRbeta, instead of being hyperactive, was defective in PDGF-induced cell migration, a function linked to PLC{gamma}1 activation (36, 41). Our results in this regard are consistent with published observations that PDGFRbeta Y1021F mutant is impaired in PDGF-induced migration in canine kidney epithelial cells (36), and a triple mutant (Tyr -> Phe mutation of phosphatidylinositol 3-kinase binding sites at Tyr739 and Tyr750 together with Tyr -> Ile mutation of Tyr1020; equivalent to human Tyr1021) was impaired in PDGF-induced cell proliferation and migration in mesangial cells (62). Strong evidence for functionally antagonistic effects of Cbl and PLC{gamma}1 binding to the Tyr1021 motif is provided by our analyses using Cbl-deficient cell lines and their parental and reconstituted controls, which demonstrated that lack of Cbl indeed promotes prolonged phosphorylation of PDGFRbeta (including that on Tyr1021), prolonged PLC{gamma}1 activation, and enhanced cell migration (Figs. 6, B and D, and 7A); the latter in our cell system was critically dependent on PLC{gamma}1 activity as seen with the use of inhibitors (Fig. 7C).

Our findings that Tyr1021 on PDGFRbeta is a shared binding site for Cbl and PLC{gamma}1 may help explain, in part, the variability in the functional effects of Y1021F mutation when examined in different cell systems. For example, Y1021F mutant when expressed in canine kidney epithelial cells was shown to be defective in PLC{gamma}1 activation and cell migration (36), whereas when expressed in porcine aortic endothelial cells, it did not affect cell migration in response to PDGF (63). The balance between the levels of Cbl and PLC{gamma}1 and/or their relative recruitment to PDGFR could determine whether phosphorylation of Tyr1021 functions as a positive or a negative regulatory mechanism. In addition, the effect of Tyr1021 mutation could be influenced by how important a role other signaling pathways, such as the phosphatidylinositol 3-kinase activation, play in the cell migration response (63).


Figure 7
View larger version (20K):
[in this window]
[in a new window]

  		FIGURE 7.
Enhanced PDGF-induced chemotaxis in Cbl-deficient MEFs is PLC-dependent. A, serum-starved Cbl-/- or Cbl+/+ cells were allowed to attach to the top surface of Boyden chamber filters for 4 h and allowed to migrate toward medium containing the indicated concentrations of PDGF beta for 4 h. The migrated cells were counted under light microscopy. The data are presented as the means plus one S.D. of six replicates, and the asterisks mark statistically significant difference (p < 0.005). Similar results observed in both HG and DB MEFs. B, the indicated serum-starved MEFs were treated with various concentration of PLC inhibitor U73122, its inactive analog U73343, or Me2SO (vehicle control) for 10 min, followed by a wash with starvation medium. The cells were stimulated with PDGF BB (20 ng/ml) for 10 min prior to lysis. 50-µg aliquots of lysate protein were subjected to anti-phospho-p44/42 MAPK, anti-phospho-PLC{gamma}1 (Tyr783), and anti-PLC{gamma}1 IB. C, serum-starved MEFs (Cbl-/-, Cbl-/-/HA-Cbl, and Cbl+/+) were incubated with PLC inhibitor U73122 or U73343 (1 µM) for 10 min, washed with starvation medium, and trypsinized. The cells were analyzed for PDGF-induced chemotaxis as in A.

 
The ability of Cbl to bind to PLC{gamma}1-binding site on PDGFRbeta provides a biochemical basis for how Cbl could selectively dampen PLC{gamma}1-mediated signaling output in cells far beyond its general negative regulatory effect as a result of targeting receptors to lysosomal degradation. Somatic knock-out of Cbl in a chicken B cell line was shown to dramatically increase the PLC{gamma}2-dependent calcium flux and apoptosis (64). Biochemical analyses in this system suggested that the Cbl TKB domain can directly bind to phosphorylated BLNK, an essential adaptor to recruit PLC{gamma}1 to the B cell receptor, and that Cbl competes for PLC{gamma}1 binding (65). Because PLC{gamma} binds to multiple phosphotyrosine motifs on BLNK and the Cbl TKB domain-binding site on BLNK has not been directly identified, it is not known whether a shared binding site on BLNK may also account for Cbl-PLC{gamma} competition in lymphocytes. Consistent with this possibility, the PLC{gamma}-binding sites on BLNK (65) correspond to potential Cbl TKB domain-binding motifs, with one motif (DDSY115) a close match. Cbl was also found to attenuate PLC{gamma}1 signaling downstream of EGFR, and it was suggested that Cbl may compete with PLC{gamma}1 activation; however, no evidence for a shared binding site or another mechanism for direct competition was provided in this study (66). It is more likely that reduction of EGFR-mediated PLC{gamma}1 activation by Cbl is a consequence of general negative regulation of EGFR rather than the competitive binding mechanisms deduced from our study.

The competition between Cbl and PLC{gamma}1 can be considered an example of an increasing numbers of regulatory interactions that appear to fine tune the activity of Cbl as a negative regulator of RTK signaling. The interaction of Cbl TKB domain with Sprouty 2, itself a negative regulator of RTK signaling, can allow bimodal regulation with two negative regulators counteracting each other's action at certain times while allowing Cbl action at other times (67). Additional studies point to a potential reciprocal regulation between Cbl and c-Src in which Cbl-mediated ubiquitinylation facilitates Src degradation, whereas Src-dependent phosphorylation of Cbl targets it for auto-ubiquitinylation and degradation (68). Finally, activated Cdc42 appears to inhibit the EGFR degradation by sequestering Cbl in a Cbl-Cool-1/beta-Pix-CDC42 ternary complex (69). Thus, competition between Cbl and PLC{gamma} binding to RTKs or adaptor proteins may provide an analogous regulatory mechanism to more precisely regulate RTK signaling via specific pathways and control Cbl action temporally and/or spatially in an activated cell. It is notable, however, that in some cell types PLC{gamma} itself appears to be a target of Cbl-mediated negative regulation. For example, Cbl-b-mediated ubiquitinylation of PLC{gamma}1 in T lymphocytes reduced its tyrosine phosphorylation and activation apparently in a proteolysis-independent manner (70). In another study, up-regulation in the levels of Cbl-b and other E3s in anergic T lymphocytes led to a ubiquitin-dependent loss of PLC{gamma}1 protein, apparently because of lysosomal degradation (71).

In summary, the results presented here demonstrate the essential role of endogenous Cbl in ligand-induced ubiquitinylation and lysosomal targeting of PDGFRbeta. We identify the Cbl TKB domain-docking site on PDGFRbeta to be coincident with a known PLC{gamma}1-binding site and demonstrate that loss of endogenous Cbl exaggerates the PLC{gamma}1-mediated biochemical and cell biological responses. Our studies illustrate a biochemical mechanism of competitive binding of a negative regulator (Cbl) and a positive effector (PLC{gamma}1) in fine-tuning signaling downstream of a prototypical RTK, PDGFRbeta. Future studies using mutagenesis of Cbl, PDGFRbeta, and PLC{gamma}1 in the context of WT and Cbl-null cells should help assess the relative role of the competitive interaction of Cbl versus PLC{gamma}1 in regulating RTK signaling.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants CA 99900, CA99163, CA 87986, and CA76118 (to H. B.) and CA94143, CA96844, and CA81076 (to V. B.); Department of Defense Breast Cancer Research Grants DAMD17-02-1-0303 (to H. B.) and DAMD17-02-1-0508 (to V. B.); Center for Cancer Nanotechnology Excellence Grant NCI 1U54 CA119341-01 (to H. B. and V. B.); the Jean Ruggles-Romoser Chair of Cancer Research (to H. B.); and the Duckworth Family Chair of Breast Cancer Research (to V. B.). 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

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1-S3 and supplemental Figs. S1-S6. Back

1 To whom correspondence should be addressed: ENH Research Institute, 1001 University Place, Evanston, IL 60201. Tel.: 224-364-7401/7424; Fax: 224-364-7402; E-mail: h-band{at}northwestern.edu.

2 The abbreviations used are: RTK, receptor tyrosine kinase; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; PLC, phospholipase C; E3, ubiquitin-protein isopeptide ligase; TKB, tyrosine kinase-binding; HA, hemagglutinin; MAPK, mitogen-activated protein kinase; MEF, murine embryonic fibroblast; GFP, green fluorescent protein; shRNA, small hairpin RNA; IP, immunoprecipitation; IB, immunoblotting; GST, glutathione S-transferase; WT, wild type. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Philippe Soriano for PDGFRbeta-null cells; Drs. Karl Heldin, Lena Claesson-Welch, and Andrius Kazlauskas for PDGFR plasmids and cells lines; Drs. Sri Raja and Mark Rainey for comments on the manuscript; and other Band laboratory members for suggestions and discussion.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Betsholtz, C., Karlsson, L., and Lindahl, P. (2001) Bioessays 23, 494-507[CrossRef][Medline] [Order article via Infotrieve]
  2. Claesson-Welsh, L. (1996) Int. J. Biochem. Cell Biol. 28, 373-385[CrossRef][Medline] [Order article via Infotrieve]
  3. Hoch, R. V., and Soriano, P. (2003) Development 130, 4769-4784[Abstract/Free Full Text]
  4. Ronnstrand, L., and Heldin, C. H. (2001) Int. J. Cancer 91, 757-762[CrossRef][Medline] [Order article via Infotrieve]
  5. Rosenkranz, S., and Kazlauskas, A. (1999) Growth Factors 16, 201-216[Medline] [Order article via Infotrieve]
  6. Yu, J., Ustach, C., and Kim, H. R. (2003) J. Biochem. Mol. Biol. 36, 49-59[Medline] [Order article via Infotrieve]
  7. Ding, H., Wu, X., Bostrom, H., Kim, I., Wong, N., Tsoi, B., O'Rourke, M., Koh, G. Y., Soriano, P., Betsholtz, C., Hart, T. C., Marazita, M. L., Field, L. L., Tam, P. P., and Nagy, A. (2004) Nat. Genet. 36, 1111-1116[CrossRef][Medline] [Order article via Infotrieve]
  8. Betsholtz, C., Lindblom, P., Bjarnegard, M., Enge, M., Gerhardt, H., and Lindahl, P. (2004) Curr. Opin. Nephrol. Hypertens. 13, 45-52[CrossRef][Medline] [Order article via Infotrieve]
  9. Schmahl, J., Raymond, C. S., and Soriano, P. (2007) Nat. Genet. 39, 52-60[CrossRef][Medline] [Order article via Infotrieve]
  10. Plate, K. H., Breier, G., Farrell, C. L., and Risau, W. (1992) Lab. Investig. 67, 529-534[Medline] [Order article via Infotrieve]
  11. Westermark, B., Heldin, C. H., and Nister, M. (1995) Glia 15, 257-263[CrossRef][Medline] [Order article via Infotrieve]
  12. Golub, T. R., Barker, G. F., Lovett, M., and Gilliland, D. G. (1994) Cell 77, 307-316[CrossRef][Medline] [Order article via Infotrieve]
  13. Uren, A., Merchant, M. S., Sun, C. J., Vitolo, M. I., Sun, Y., Tsokos, M., Illei, P. B., Ladanyi, M., Passaniti, A., Mackall, C., and Toretsky, J. A. (2003) Oncogene 22, 2334-2342[CrossRef][Medline] [Order article via Infotrieve]
  14. Kitadai, Y., Sasaki, T., Kuwai, T., Nakamura, T., Bucana, C. D., Hamilton, S. R., and Fidler, I. J. (2006) Int. J. Cancer 119, 2567-2574[CrossRef][Medline] [Order article via Infotrieve]
  15. Cho, S., Kitadai, Y., Yoshida, S., Tanaka, S., Yoshihara, M., Yoshida, K., and Chayama, K. (2006) Int. J. Oncol. 28, 1361-1367[Medline] [Order article via Infotrieve]
  16. Lev, D. C., Kim, S. J., Onn, A., Stone, V., Nam, D. H., Yazici, S., Fidler, I. J., and Price, J. E. (2005) Clin. Cancer Res. 11, 306-314[Abstract/Free Full Text]
  17. Carvalho, I., Milanezi, F., Martins, A., Reis, R. M., and Schmitt, F. (2005) Breast Cancer Res. 7, R788-795[CrossRef][Medline] [Order article via Infotrieve]
  18. Bonita, D. P., Miyake, S., Lupher, M. L., Jr., Langdon, W. Y., and Band, H. (1997) Mol. Cell. Biol. 17, 4597-4610[Abstract]
  19. Miyake, S., Lupher, M. L., Jr., Druker, B., and Band, H. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7927-7932[Abstract/Free Full Text]
  20. Miyake, S., Mullane-Robinson, K. P., Lill, N. L., Douillard, P., and Band, H. (1999) J. Biol. Chem. 274, 16619-16628[Abstract/Free Full Text]
  21. Levkowitz, G., Oved, S., Klapper, L. N., Harari, D., Lavi, S., Sela, M., and Yarden, Y. (2000) J. Biol. Chem. 275, 35532-35539[Abstract/Free Full Text]
  22. Waterman, H., Levkowitz, G., Alroy, I., and Yarden, Y. (1999) J. Biol. Chem. 274, 22151-22154[Abstract/Free Full Text]
  23. Soubeyran, P., Kowanetz, K., Szymkiewicz, I., Langdon, W. Y., and Dikic, I. (2002) Nature 416, 183-187[CrossRef][Medline] [Order article via Infotrieve]
  24. Duan, L., Miura, Y., Dimri, M., Majumder, B., Dodge, I. L., Reddi, A. L., Ghosh, A., Fernandes, N., Zhou, P., Mullane-Robinson, K., Rao, N., Donoghue, S., Rogers, R. A., Bowtell, D., Naramura, M., Gu, H., Band, V., and Band, H. (2003) J. Biol. Chem. 278, 28950-28960[Abstract/Free Full Text]
  25. Lee, P. S., Wang, Y., Dominguez, M. G., Yeung, Y. G., Murphy, M. A., Bowtell, D. D., and Stanley, E. R. (1999) EMBO J. 18, 3616-3628[CrossRef][Medline] [Order article via Infotrieve]
  26. Wang, Y., Yeung, Y. G., and Stanley, E. R. (1999) J. Cell. Biochem. 72, 119-134[CrossRef][Medline] [Order article via Infotrieve]
  27. Rohde, C. M., Schrum, J., and Lee, A. W. (2004) J. Biol. Chem. 279, 43448-43461[Abstract/Free Full Text]
  28. Zeng, S., Xu, Z., Lipkowitz, S., and Longley, J. B. (2005) Blood 105, 226-232[Abstract/Free Full Text]
  29. Lupher, M. L., Jr., Songyang, Z., Shoelson, S. E., Cantley, L. C., and Band, H. (1997) J. Biol. Chem. 272, 33140-33144[Abstract/Free Full Text]
  30. Babst, M. (2005) Traffic 6, 2-9[CrossRef][Medline] [Order article via Infotrieve]
  31. Hurley, J. H., and Emr, S. D. (2006) Annu. Rev. Biophys Biomol. Struct. 35, 277-298[CrossRef][Medline] [Order article via Infotrieve]
  32. Slagsvold, T., Pattni, K., Malerod, L., and Stenmark, H. (2006) Trends Cell Biol. 16, 317-326[CrossRef][Medline] [Order article via Infotrieve]
  33. Sorkin, A., Westermark, B., Heldin, C. H., and Claesson-Welsh, L. (1991) J. Cell Biol. 112, 469-478[Abstract/Free Full Text]
  34. Sorkin, A., Eriksson, A., Heldin, C. H., Westermark, B., and Claesson-Welsh, L. (1993) J. Cell. Physiol. 156, 373-382[CrossRef][Medline] [Order article via Infotrieve]
  35. Rhee, S. G. (2001) Annu. Rev. Biochem. 70, 281-312[CrossRef][Medline] [Order article via Infotrieve]
  36. Kundra, V., Escobedo, J. A., Kazlauskas, A., Kim, H. K., Rhee, S. G., Williams, L. T., and Zetter, B. R. (1994) Nature 367, 474-476[CrossRef][Medline] [Order article via Infotrieve]
  37. Firth, J. D., Putnins, E. E., Larjava, H., and Uitto, V. J. (2001) Wound Repair Regen. 9, 86-94[CrossRef][Medline] [Order article via Infotrieve]
  38. Song, H. J., and Poo, M. M. (1999) Curr. Opin. Neurobiol. 9, 355-363[CrossRef][Medline] [Order article via Infotrieve]
  39. Yu, H., Fukami, K., Itoh, T., and Takenawa, T. (1998) Exp. Cell Res. 243, 113-122[CrossRef][Medline] [Order article via Infotrieve]
  40. Amyere, M., Payrastre, B., Krause, U., Van Der Smissen, P., Veithen, A., and Courtoy, P. J. (2000) Mol. Biol. Cell 11, 3453-3467[Abstract/Free Full Text]
  41. Yamamoto, M., Hilgemann, D. H., Feng, S., Bito, H., Ishihara, H., Shibasaki, Y., and Yin, H. L. (2001) J. Cell Biol. 152, 867-876[Abstract/Free Full Text]
  42. Ronnstrand, L., Mori, S., Arridsson, A. K., Eriksson, A., Wernstedt, C., Hellman, U., Claesson-Welsh, L., and Heldin, C. H. (1992) EMBO J. 11, 3911-3919[Medline] [Order article via Infotrieve]
  43. Valius, M., Bazenet, C., and Kazlauskas, A. (1993) Mol. Cell. Biol. 13, 133-143[Abstract/Free Full Text]
  44. Valius, M., and Kazlauskas, A. (1993) Cell 73, 321-334[CrossRef][Medline] [Order article via Infotrieve]
  45. Druker, B. J., Mamon, H. J., and Roberts, T. M. (1989) N. Engl. J. Med. 321, 1383-1391[Medline] [Order article via Infotrieve]
  46. Wilson, I. A., Niman, H. L., Houghten, R. A., Cherenson, A. R., Connolly, M. L., and Lerner, R. A. (1984) Cell 37, 767-778[CrossRef][Medline] [Order article via Infotrieve]
  47. Murphy, M. A., Schnall, R. G., Venter, D. J., Barnett, L., Bertoncello, I., Thien, C. B., Langdon, W. Y., and Bowtell, D. D. (1998) Mol. Cell. Biol. 18, 4872-4882[Abstract/Free Full Text]
  48. Naramura, M., Kole, H. K., Hu, R. J., and Gu, H. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 15547-15552[Abstract/Free Full Text]
  49. Claesson-Welsh, L., Eriksson, A., Moren, A., Severinsson, L., Ek, B., Ostman, A., Betsholtz, C., and Heldin, C. H. (1988) Mol. Cell. Biol. 8, 3476-3486[Abstract/Free Full Text]
  50. Finerty, P. J., Jr., Mittermaier, A. K., Muhandiram, R., Kay, L. E., and Forman-Kay, J. D. (2005) Biochemistry 44, 694-703[CrossRef][Medline] [Order article via Infotrieve]
  51. Pascal, S. M., Singer, A. U., Gish, G., Yamazaki, T., Shoelson, S. E., Pawson, T., Kay, L. E., and Forman-Kay, J. D. (1994) Cell 77, 461-472[CrossRef][Medline] [Order article via Infotrieve]
  52. Mellman, I. (1996) Annu. Rev. Cell Dev. Biol. 12, 575-625[CrossRef][Medline] [Order article via Infotrieve]
  53. Hamilton, T. G., Klinghoffer, R. A., Corrin, P. D., and Soriano, P. (2003) Mol. Cell. Biol. 23, 4013-4025[Abstract/Free Full Text]
  54. Smallridge, R. C., Kiang, J. G., Gist, I. D., Fein, H. G., and Galloway, R. J. (1992) Endocrinology 131, 1883-1888[Abstract/Free Full Text]
  55. Ishiai, M., Sugawara, H., Kurosaki, M., and Kurosaki, T. (1999) J. Immunol. 163, 1746-1749[Abstract/Free Full Text]
  56. Peschard, P., Ishiyama, N., Lin, T., Lipkowitz, S., and Park, M. (2004) J. Biol. Chem. 279, 29565-29571[Abstract/Free Full Text]
  57. Hu, J., and Hubbard, S. R. (2005) J. Biol. Chem. 280, 18943-18949[Abstract/Free Full Text]
  58. Lupher, M. L., Jr., Rao, N., Lill, N. L., Andoniou, C. E., Miyake, S., Clark, E. A., Druker, B., and Band, H. (1998) J. Biol. Chem. 273, 35273-35281[Abstract/Free Full Text]
  59. Rubin, C., Litvak, V., Medvedovsky, H., Zwang, Y., Lev, S., and Yarden, Y. (2003) Curr. Biol. 13, 297-307[CrossRef][Medline] [Order article via Infotrieve]
  60. Ohrt, T., Mancini, A., Tamura, T., and Niedenthal, R. (2004) Cell Signal. 16, 1291-1298[CrossRef][Medline] [Order article via Infotrieve]
  61. Mori, S., Heldin, C. H., and Claesson-Welsh, L. (1993) J. Biol. Chem. 268, 577-583[Abstract/Free Full Text]
  62. Tallquist, M. D., Klinghoffer, R. A., Heuchel, R., Mueting-Nelsen, P. F., Corrin, P. D., Heldin, C. H., Johnson, R. J., and Soriano, P. (2000) Genes Dev. 14, 3179-3190[Abstract/Free Full Text]
  63. Wennstrom, S., Siegbahn, A., Yokote, K., Arvidsson, A. K., Heldin, C. H., Mori, S., and Claesson-Welsh, L. (1994) Oncogene 9, 651-660[Medline] [Order article via Infotrieve]
  64. Yasuda, T., Tezuka, T., Maeda, A., Inazu, T., Yamanashi, Y., Gu, H., Kurosaki, T., and Yamamoto, T. (2002) J. Exp. Med. 196, 51-63[Abstract/Free Full Text]
  65. Ishiai, M., Kurosaki, M., Pappu, R., Okawa, K., Ronko, I., Fu, C., Shibata, M., Iwamatsu, A., Chan, A. C., and Kurosaki, T. (1999) Immunity 10, 117-125[CrossRef][Medline] [Order article via Infotrieve]
  66. Choi, J. H., Bae, S. S., Park, J. B., Ha, S. H., Song, H., Kim, J. H., Cocco, L., Ryu, S. H., and Suh, P. G. (2003) Mol Cells 15, 245-255[Medline] [Order article via Infotrieve]
  67. Hall, A. B., Jura, N., DaSilva, J., Jang, Y. J., Gong, D., and Bar-Sagi, D. (2003) Curr. Biol. 13, 308-314[CrossRef][Medline] [Order article via Infotrieve]
  68. Bao, J., Gur, G., and Yarden, Y. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 2438-2443[Abstract/Free Full Text]
  69. Schmidt, M. H., Husnjak, K., Szymkiewicz, I., Haglund, K., and Dikic, I. (2006) Oncogene 25, 3071-3078[CrossRef][Medline] [Order article via Infotrieve]
  70. Jeon, M. S., Atfield, A., Venuprasad, K., Krawczyk, C., Sarao, R., Elly, C., Yang, C., Arya, S., Bachmaier, K., Su, L., Bouchard, D., Jones, R., Gronski, M., Ohashi, P., Wada, T., Bloom, D., Fathman, C. G., Liu, Y. C., and Penninger, J. M. (2004) Immunity 21, 167-177[CrossRef][Medline] [Order article via Infotrieve]
  71. Heissmeyer, V., Macian, F., Im, S. H., Varma, R., Feske, S., Venuprasad, K., Gu, H., Liu, Y. C., Dustin, M. L., and Rao, A. (2004) Nat. Immunol. 5, 255-265[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
J. Y. Cha, S. Maddileti, N. Mitin, T. K. Harden, and C. J. Der
Aberrant Receptor Internalization and Enhanced FRS2-dependent Signaling Contribute to the Transforming Activity of the Fibroblast Growth Factor Receptor 2 IIIb C3 Isoform
J. Biol. Chem., March 6, 2009; 284(10): 6227 - 6240.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
282/40/29336    most recent
M701797200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Reddi, A. L.
Right arrow Articles by Band, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Reddi, A. L.
Right arrow Articles by Band, H.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement