Dok5 is substrate of TrkB and TrkC receptors and involved inneurotrophin induced MAPK activation
Lei Shi a,1, Jiping Yue a,1, Yuangang You a, Bin Yin a, Yanhua Gong a, Caimin Xu a,Boqin Qiang a, Jiangang Yuan a, Yongjian Liu b,⁎, Xiaozhong Peng a,⁎
a The National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences andPeking Union Medical College, Chinese National Human Genome Center, Beijing 100005, China
b Department of Neurology, University of Pittsburgh School of Medicine, PA 15213, USA
Received 4 February 2006; received in revised form 3 March 2006; accepted 10 March 2006Available online 2 May 2006
The neurotrophin family growth factors, including nervegrowth factor (NGF), brain-derived neurotrophic factors (BDNF),neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5), haveimportant functions in nervous system. They play multiple rolesin vertebrate neural development, differentiation, survival, synap-tic plasticity, myelination and many other processes [1,2]. Theirhigh affinity receptors, tropomyosin-related kinase (Trk) familyreceptors, are cell membrane receptor tyrosine kinase (RTK). Thisreceptor family has three structural conserved members: TrkA,TrkB and TrkC. Each neurotrophin binds to different receptors:NGF binds to TrkA, BDNF binds to TrkB, and NT-3 binds to
TrkC. Also NT-3 can bind to TrkA and NT-4 can bind to TrkB[1,2]. Stimulation of neurotrophins can result in dimerization andactivation of Trk receptors. Then the activated receptors phos-phorylate several tyrosines of its intracellular domain (ICD). Thephosphorylated tyrosines may serve as the docking site for signalmolecules and mediate the signal transduction. Many signalmolecules such as Src homology 2 domain containing transform-ing protein (Shc), FGF receptor substrate 2 (FRS2), growth factorreceptor bound protein 2 (Grb2), phospholipase Cγ(PLC-γ) havebeen proved to be substrates of Trk receptors [3–5]. They can bephosphorylated by Trk receptors and cascade the signal todownstream.
Downstream of tyrosine kinase/Docking proteins (Dok) areadaptor proteins that can act as substrate of multiple tyrosinekinase including both receptor tyrosine kinases and non-receptortyrosine kinase [6–10]. The first member of Dok family, Dok1/p62Dok, was identified by Carpino et al. [11] and Yamanashi and
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Baldimore [12]. Subsequently other members were identified,and now there are six members in this family: Dok1/p62Dok,Dok2/DokR/p56Dok, Dok3/DokL, Dok4, Dok5, and Dok6/Dok5 like [13–17]. Dok proteins have classic feature of adaptorproteins: N-terminal pleckstrin homology (PH) domain, centralphosphotyrosine binding (PTB) domain and C-terminal region.
According to different expression patterns, Dok family pro-teins are divided into two subgroups. The first subgroup, Dok1–3 are predominantly expressed in hematopoietic tissues and actas negative regulator in many signal pathways. Dok1/2 inhibitMAPK activation through their association with RasGTPase-activating protein (RasGAP) [18,19]. Van Slyke et al. showedDok2 attenuated EGFR signaling by recruiting c-Src and Csk,providing an alternative regulatory way [20]. Dok3, as thenegative regulator of the immunoreceptor signaling in B cellsand macrophages, exerts its function by the formation of Dok3-SHIP1 complex [15,21]. Recently, studies from double or singleknockout mice of Dok1 and/or Dok2 provided solid evidencesin vivo for their negative roles in signal transduction and sup-pressant functions in leukemogenesis [22–24].
Another subgroup, Dok4–6 are expressed in non-hemato-poietic tissues. Dok4 is widely expressed, especially in tissuesof epithelial origin, and strongly inhibit Ret or Fyn signaling[25]. Itoh et al. found Dok4 localized in mitochondrial and
Fig. 1. Dok5 interacts with TrkB and TrkC ICD in yeast two-hybrid system. (A) Schemwith TrkB and TrkC ICD, not with TrkA ICD in yeast two-hybrid system. Yeast (SFvector, then cultured on SD/-Trp/-Leu plates for 4 days at 30 °C. Colony liftβ-galadomain, not PH domain or C-terminal region, binds to TrkB and TrkC ICD in yeas
played a role in tumor necrosis factor-alpha (TNF-alpha) me-diated reactive oxygen species (ROS) production [26]. Dok6,the newest member, is mainly expressed in nervous system andpromotes Ret mediated neurite outgrowth in N2A-α1 cells [17].And for Dok5, Northern blot and in situ hybrid results showedthat Dok5 was mainly expressed in nervous system, especiallyin neural tube, dorsal root ganglia (DRG) and cranial ganglia.Studies also showed Dok5 was involved in insulin and GDNFsignal pathway and it mediated GDNF induced neuriteoutgrowth in PC12 cells [16,27]. These results suggest Dok5has very important function in neural development. But asrecently identified member, its function remains mostly unclear.
In this study, we identified Dok5 as novel substrate of TrkB/C receptors. We proved Dok5 could selectively bind to NPQYmotif of TrkB and TrkC receptors, not that of TrkA receptors ina kinase-activity-dependent manner. Co-localization of Dok5and TrkB or TrkC receptors in differentiated PC12 cells provideanother evidence for their interaction. In competition assay, weshowed that Dok5 competed with N-shc for Trk receptor bin-ding. Furthermore, we demonstrated that Dok5 was involved inthe activation of MAPK signal pathway upon neurotrophinstimulation. Taken together, we proved that Dok5 could act assubstrate of Trk receptors and was involved in neurotrophininduced MAPK activation. These results indicate that Dok5
atic diagram shows the structures of Dok5 and Trk receptors. (B) Dok5 interactsY526) were transformed with Trk ICD in pAS2-1 vector and Dok5 in pACT2ctosidase assays were performed according to the user manual. (C) Dok5 PTBt two-hybrid assay.
1997L. Shi et al. / Cellular Signalling 18 (2006) 1995–2003
plays an important role in neurotrophin signal pathway, and alsoprovide new clues for Dok5 function in neuronal developmentand differentiation.
2. Materials and methods
2.1. Reagents and cell culture
Human recombinant NGF, BDNF and NT-3 were purchased from PeprotechInc. The anti-V5 polyclonal antibody was purchased from Novus. The anti-Flagmonoclonal antibody was from Sigma. The anti-Myc monoclonal antibody(9E10) was purchased from Convance. The anti-Erk and anti-phosphop44/42ErkMAP kinase antibodywere purchased from Cell signaling. The anti-pTyr (PY99)monoclonal antibody was product of Santa Cruz Biotechnology.
Human Embryonic Kidney (HEK)293 cells were cultured in DMEMmedium with 10% fetal bovine serum (Hyclone). PC12 cells were maintained inDMEM medium with 10% horse serum and 5% fetal bovine serum. For trans-fection, cells were split into 6-well plate and cultured to 90% confluency. Thenthe transfection was done with Lipofectamine2000 (Invitrogen) according to theuser manual.
2.2. Plasmid construction
The human TrkA full length cDNAwas a kindly gift from Dr. Lloyd Greene.The human TrkB and TrkC full length cDNAwere amplified from human fetalbrain cDNA library. For yeast two-hybrid assay, the Trk receptors intracellulardomains (ICD) were subcloned into pAS2-1 vector. Dok5 full length cDNA andthe sequence coding for PH domain, PTB domain and C-terminal region weresubcloned into pACT2 vectors, respectively. All the mutant TrkB and TrkCreceptors, including TrkBM1(K572A), TrkBM2(Y516F), TrkBM3(Y702A),TrkBM4(Y706D), TrkBM5(Y707E), TrkBM6(Y817F), TrkCM1(K572A),TrkCM2(Y516F), TrkCM3(Y705A), TrkCM4(Y709D), TrkCM5(Y710E) andTrkCM6(Y820F) were generated by using QuikChange Site-Directed Mutagen-
Fig. 2. Dok5 PTB domain binds to TrkB and TrkC receptors in vitro. (A) Dok5 PTB dwere transfected with pcDNA3.1V5-Trk and treated with or without neurotrophin (104B beads at 4 °C for 2 h. The bound proteins were then analyzed by Western blottinganti-pTyr antibody to detect the expression and phosphorylation level of Trk recCoomassie Brilliant Blue staining (bottom panel). (B) Alignment of amino acids sequblue, ≥50%. (For interpretation of the references to colour in this figure legend, the
esis Kit (Stratagene) [3,4,28,29]. For prokaryotic expression, the sequencecoding for Dok5 PTB domain was subcloned into pGEX6P-1 vector. For eu-karyotic expression, wild type and mutant Trk receptors were subcloned intoexpression vector pcDNA3.1V5. Dok5 full length cDNA and Dok5ΔC-terminalregion(Dok5ΔC) were subcloned into expression vector pcDNA3.1Flag vector.For image analysis, TrkB and TrkC full length cDNA was subcloned intopmRFP-N1 vector and Dok5 cDNAwere subcloned into pEGFP-N1 vector.
2.3. Yeast two-hybrid assay
For yeast two-hybrid assay, Trk receptor intracellular domains were fused tothe GAL4 DNA binding domain in pAS2-1 vector. Wild type Dok5, or Dok5 PHdomain, PTB domain and C-terminal region were fused to GAL4 activationdomain in pACT2 vectors, respectively. The co-transformation was performedin yeast strain SFY526 by using TE/LiAc method according to the user manualof MATCHMAKER yeast two-hybrid system 2 (Clontech). Transformed yeastwere selected on SD dropout medium/-Leu/-Trp plates at 30 °C for 4 days. Thencolony liftβ-galactosidase assays were performed.
2.4. GST pull-down assay
pGEX6P-Dok5-PTB plasmid was transformed into BL21 Escherichia colistrain. Then the overnight cultures were diluted 1:500 in 200 ml LB medium andcultured at 37 °C until OD600 to 0.6. The induction was performed by shaking at30 °C for 4 h with 0.8 mM isopfopyl-β-D-thiogalactopyranoside. GST-Dok5-PTB fusion proteins were immobilized onto Sepharose 4B beads according to theprotocol (Amersham Biosciences). The HEK293 cells were transient transfectedwith pcDNA3.1V5-Trk plasmids. 24 h later, the cell were treated with or withoutneurotrophin for 10 min and lysed. The cell lysates were centrifuged at 12,000×gfor 15 min and the supernatants were incubated with the same amount of GST-Dok5-PTB Sepharose 4B beads at 4 °C for 2 h. The beads were washed with 1×PBS for 3 times and then resuspended in 1× SDS loading buffer and boiled for5 min. Protein samples were analyzed byWestern blotting or Coomassie BrilliantBlue staining.
omain binds to TrkB and TrkC receptors in GST pull-down assay. HEK293 cells0 ng/ml) for 10 min. The lysates were incubated with Dok5-PTB-GST Sepharosewith anti-V5 antibody (upper panel). Cell lysates were examined by anti-V5 or
eptors (second and third panel). Dok5-PTB-GST proteins were examined byence around NPQY motif of Trk family receptors. Homology level: red, 100%;reader is referred to the web version of this article.)
Fig.3.D
ok5bind
stoNPQYmotifof
TrkBandTrkCreceptorsby
akinase-activity
-dependentmanner.(A
)Schem
aticdiagramshow
stheim
portantaminoacidsof
TrkBandTrkC.(B)M
utationanalysisof
thekeyam
ino
acidsof
Trk
receptor
forDok
5bind
ing.Yeasttwo-hybrid
assaywas
performed
with
pACT2-Dok
5-PT
BandTrkBor
TrkCICDmutantsin
pAS2-1vector,including
BM1(K572A
),BM2(Y516F
),BM3(Y702A
),BM4
(Y706D
),BM5(Y707E
),BM6(Y817F
),or
CM1(K572A
),CM2(Y516F
),CM3(Y705A
),CM4(Y709D
),CM5(Y710E
),CM6(Y820F
).(C)Dok
5bind
sto
NPQYmotifof
TrkBandTrkCreceptorsby
akinase-activity
-dependentm
annerin
vivo.C
o-IP
experimentsweredone
inHEK293cells
co-transfected
with
Dok5andwild
type
ormutantreceptors.B
eforeharvest,thecells
weretreatedwith
100ng/m
lBDNFor
NT-3for10
min.
Protein
samples
werethen
detected
byWestern
blottin
gwith
anti-V5,
anti-Flagor
anti-pT
yrantib
ody.(D
)Dok
5isph
osph
orylated
byTrkBor
TrkCreceptorsafterstim
ulation.
WT,
wild
type.
1998 L. Shi et al. / Cellular Signalling 18 (2006) 1995–2003
1999L. Shi et al. / Cellular Signalling 18 (2006) 1995–2003
2.5. Co-immunoprecipitation
HEK293 cells were plated into 6-well plate and cultured to 90% confluency.The cells were then transient co-transfected with pcDNA3.1V5-Trk andpcDNA3.1Flag-Dok5, pcDNA3.1V5-TrkM1 and pcDNA3.1Flag-Dok5,pcDNA3.1V5-TrkM2 and pcDNA3.1Flag-Dok5, or pcDNA3.1V5-TrkM6 andpcDNA3.1Flag-Dok5. 24 h after transfection, stimulate the cells with 100 ng/mlneurotrophin (PeproTech) for 10 min. Then cell were lysed in 750 μl lysis buffer(50 mM Tris–HCl pH 7.5, 1 mM EDTA, 120 mM NaCl, 0.5% NP-40, 10%glycerol, 1 mM Na3VO4, 25 mM NaF) containing protease inhibitors. Afterincubation on ice for 30 min, detergent-insoluble materials were removed bycentrifugation at 4 °C at 12,000×g for 15 min. The 2-μg anti-Flag monoclonalantibody (Sigma) was added to the lysates and incubated at 4 °C rotating for 3 hfollowed by addition of 30 μl protein G-agarose (Roche). The incubation wascontinued for 3 h. The mixture was centrifuged at 12,000×g for 1 min, and thepellet was washed 3 times with 1 ml washing buffer (20 mM Tris–HCl pH 7.4,1 mM EDTA, pH 8.0, 200 mM NaCl, 0.5% NP-40, 100 μM Na3VO4). Thesamples were analyzed by Western blotting with anti-V5 antibody (Invitrogen),anti-Flag antibody (Sigma) or anti-pTyr antibody (PY99, Santa Cruz).
2.6. Fluorescence microscopy
pmRFPN1-TrkB or pmRFPN1-TrkC and pEGFPN1-Dok5 were co-trans-fected into PC12 cells. Twenty-four hours later, PC12 cells were split into 24-wellplate with polylysine coated cover slips and cultured in low serum medium (1%equine serum) with 50 ng/ml BDNF or NT-3 for 48 h. Cells then were fixed with4% PFA for 30 min at RTand washed with PBS for 3 times. The cover slips weremounted and co-localization was analyzed under a confocal imaging system.
2.7. MAPK signal pathway detection
HEK293 cells were co-transfected with pcDNA3.1V5-Trk and pcDNA3.1Flag, pcDNA3.1V5-Trk and pcDNA3.1Flag-Dok5, or pcDNA3.1V5-Trk and
Fig. 4. Dok5 co-localizes with TrkB or TrkC in differentiated PC12 cells. PC12 cells wNT-3 for 2 days.
pcDNA3.1Flag-Dok5ΔC. The cells were treated with 100 ng/ml BDNF or NT-3and harvested at 0 min, 5 min, 15 min, 30 min and 60 min. The lysates wereexamined byWestern blotting with anti-Erk1 (k-23, Santa Cruz), anti-pErk (E-4,Santa Cruz), anti-Flag antibody (Sigma) or anti-V5 antibody (Invitrogen).
3. Results
3.1. Dok5 PTB domain binds to TrkB and TrkC ICD in yeasttwo-hybrid system
Bioinformatics analysis and our previous structural studiesshowed that Dok5 has a conserved PTB domain [30], whichmakeit possible for Dok5 to be the potential substrate of multipletyrosine kinases. In consideration of Dok5 neural specificexpression, we try to identify new tyrosine kinase expressed innerve system that coupled with Dok5. Trk receptors, which arevery important RTKs during neural development and differenti-ation, could be good candidates. To verify this possibility, we useyeast two-hybrid system to examinewhether there are interactionsbetween Dok5 and Trk receptors. There was evidence showingthat the dimerization of the Gal4 BD domain could activate TrkICD [5], which enable yeast two-hybrid assay to be used to detectthe interaction between substrate and receptors. So we performedyeast two-hybrid with Trk ICD in pAS2-1 vector and Dok5 inpACT2 vector. Interestingly, the result showed that there wereinteractions between Dok5 and TrkB or TrkC ICD, while therewas no obvious interaction between Dok5 and TrkA ICD(Fig. 1B). This implies that Dok5 has binding selectivity among
ere transfected with the constructs as indicated and induce by 50 ng/ml BDNF or
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three members of Trk family receptors. In order to determine theinteraction domain of Dok5, we further subcloned Dok5 PHdomain, PTB domain and C-terminal region into pACT2 vector,respectively, and performed yeast two-hybrid assay again. Asshown in Fig. 1C, Dok5 PTB domain is key domain responsiblefor the interaction with TrkB and TrkC ICD, while neither PHdomain nor C-terminal region can interact with the receptors.
3.2. Dok5 binds to TrkB and TrkC receptor in GST pull-down
To confirm the results of yeast two-hybrid assay, we used GSTpull-down assay to examinewhether there are interaction betweenDok5 and Trk receptors in vitro. The result proved that Dok5 PTBdomain can bind to the activated TrkB and TrkC receptors (Fig.2A). Upon neurotrophin stimulation, Trk receptors becamehighly phosphorylated comparing with those without stimulation.And only activated TrkB or TrkC receptors can be pulled down byGST-Dok5-PTB fusion protein. However, GST-Dok5-PTB
Fig. 5. Dok5 and N-shc compete for TrkB (A) or TrkC (B) receptors binding.HEK293 cells were transfected with Trk, Dok5 and N-shc and then treated with100 ng/ml BDNF or NT-3 for 10 min. Lysates were immunoprecipitated withanti-Flag antibody and the precipitated proteins were detected with anti-V5 andanti-Flag antibody. Cell lysates were detected with anti-V5 and anti-Mycantibody to examine the expression of Trk and N-shc.
fusing protein could not bind to TrkA receptors no matterwhether TrkA was activated or not (Fig. 2A). The consistentresults from yeast two-hybrid and pull-down assay indicated thatDok5 selectively bind to TrkB and TrkC, but not TrkA.
3.3. Dok5 binds to NPQY motif of TrkB and TrkC in a kinase-activity-dependent manner
We have proved that Dok5 interacted with the Trk receptorsthrough its PTB domain. As known, PTB domain can recognizethe NPQY motif by two manners: dependent or independent onphosphorylation [31,32]. To determine by which tyrosine andby which manner that Dok5 binds to Trk receptors, we con-structed serial mutant Trk receptors for yeast two-hybrid assayand Co-IP experiment. As showed in Fig. 3A, Trk receptorshave 5 tyrosine residues in their ICD that can be phosphorylatedupon activation. For TrkB, they are Tyr516, Tyr702, Tyr706,Tyr707 and Tyr817. Each tyrosine mutation would disrupt theinteraction between the receptor and the substrate binding at thissite. The juxtamembrane Tyr516 lies in the conserved NPQYmotif, which is typical binding motif for PTB domain. Manyadaptor proteins containing PTB domain, such as Shc and Frs2,bind to Trk receptors at this site. And we supposed that thistyrosine is also the binding site for Dok5. Another key aminoresidue, Lys572, is essential for tyrosine kinase activity of thereceptor. The mutation of this lysine would produce kinaseinactive receptor.
As expected, the yeast two-hybrid results proved that NPQYmotif is the binding site for Dok5, because the mutant receptorY516F lost the interaction with Dok5. Other tyrosine mutationshave no obvious influence on the interaction. And the kinaseinactive TrkB (K572A) had no interaction with Dok5 either,which implied that the kinase activity is essential for the inter-action (Fig. 3B). Therewas similar result betweenDok5 andTrkC.
By Co-IP experiment, we further proved that Dok5 bind toNPQY motif of TrkB depending on phosphorylation. The resultshowed that wild type TrkB receptors could be immunopreci-pitated by Dok5 upon BDNF stimulation; while the mutantreceptor, TrkBM1 (K572A) or TrkBM2 (Y516F), could not beco-immunoprecipitated by Dok5 with the stimulation. Theunrelated tyrosine mutation, TrkBM6 (Y817F), did not affect theinteraction. Similar results were obtained between Dok5 andTrkC with stimulation of NT-3 (Fig. 3C). Moreover, as sub-strates of TrkB and TrkC, Dok5 phosphorylation level wasgreatly enhanced upon neurotrophin stimulation (Fig. 3D).These results strongly suggested that Dok5 binds to NPQYmotifof TrkB/C receptors in a kinase-activity-dependent manner.
3.4. Dok5 co-localizes with TrkB and TrkC receptors indifferentiated PC12 cells
PC12 cell line has been widely used as a good cell model forresearches on neurotrophin and Trk receptors. With induction ofNGF, PC12 cells would manifest characteristics of neuronal cells.So we choose PC12 cells for Dok5 and Trk receptors localizationstudy. We constructed EGFPN1-Dok5 and mRFPN1-TrkB/Cclones and co-transfected them into PC12 cells. The cells were
Fig. 6. Dok5 mediate the activation of MAPK pathway upon neurotrophin stimulation in HEK293 cells. HEK293 cells were transfected with the constructs asindicated. The cells were treated with BDNF (A) or NT-3 (B) and harvested at 0 min, 5 min, 15 min, 30 min and 60 min. Cell lysates were examined by Westernblotting with antibody indicated.
2001L. Shi et al. / Cellular Signalling 18 (2006) 1995–2003
inducedwith 50 ng/ml BDNForNT-3 in low concentration serummedium (1% equine serum) for 2 days and then analyzed byconfocalmicroscopy. Obviously, Dok5 co-localize with TrkB andTrkC receptors in differentiated PC12 cells (Fig. 4). The co-localization of Dok5 and Trk receptors provided another evidencefor their interaction.
3.5. Dok5 competes with N-shc for interaction with the samesite of Trk receptor
As well known, Shc can bind to the NPQY motif of Trkreceptors through its PTB domain. In this study, we showed thatDok5 PTB domain also bind to the same site of Trk receptor. Soit is possible that Dok5 competes with Shc for Trk receptorbinding. Shc adaptor protein family comprises three members;ShcA, B and C. Among them, ShcC/N-shc is exclusively ex-pressed in brain and also interact with Trk receptors [33–35].We subcloned N-shc into pcDNA4Myc vector and performed acompetition experiment. In competition assay, pcDNA3.1V5-Trk, pcDNA3.1Flag-Dok5 and pcDNA4Myc-N-shc were co-transfected into HEK293 cells. The plasmid amounts of Trk andDok5 were kept equal, with the plasmid amount of N-shcincreasing. As indicated in Fig. 5, with the increasing expresionof N-shc, the amount of Trk receptors that interacted with Dok5reduced gradually (Fig. 5). When there was a huge amount of N-shc, almost no Trk receptors were co-immunoprecipitated by
Dok5. This result provided a good competitive model for Dok5and N-shc interacting with Trk receptor.
3.6. Dok5 is involved in the activation of MAPK signalpathway induced by BDNF and NT-3
To make clear the function of Dok5 in neurotrophin sig-naling, we detected its effects on MAPK signal pathway. We co-transfected Dok5 and Trk into HEK293 cells, another group theempty vector and Trk were used as control. Twenty-four hoursafter transfection, cells were treated with BDNF or NT-3 andthen harvest cells at indicated time and detected MAPK path-way. Obviously, the existence of Dok5 enhanced the phosphor-ylation level of Erk1/2 compared with the control group.Meanwhile Dok5 also prolonged the activation time of MAPKpathway (Fig. 6). Previous studies showed that the C-terminalregion of Dok protein was essential for signal transduction.Dok1/2 C-terminal region have multiple tyrosine residues andPXXP motifs that can serve as docking site for SH2/SH3 con-taining signal proteins [13,36]. The deletion of C-terminal re-gion would disrupt their functions. So we were motivated toconstruct Dok5ΔC mutant and detect MAPK pathway again.As shown in Fig. 6, Dok5ΔC could inhibit MAPK pathway byreducing the strength and shorten the activation time. Thus, theresult demonstrated that Dok5 participated in MAPK pathwayactivation induced by BDNF and NT-3.
2002 L. Shi et al. / Cellular Signalling 18 (2006) 1995–2003
4. Discussion
In this study, by using yeast two-hybrid and pull-down assay,we identified Dok5 as substrate of TrkB and TrkC receptors.There was no obvious interaction between Dok5 and TrkA. Asmembers of same family, TrkA, B and C share high homology instructure and they all have juxtamembrane NPQYmotif for PTBdomain binding. By aligning the amino acid sequence aroundNPQY motif, we found there were several amino acid residuedeletions in TrkA compared with TrkB and TrkC receptors(Fig. 2B). These amino residues at the N-terminal or C-terminalof NPQYmotif might provide the recognition specificity for thesubstrates. Interestingly, study from Shi et al. illustrated thestructural basis of recognition between Dok1 PTB domain andc-Ret, and they also showed that Dok1 PTB domain did notrecognize phosphopeptide including NPQY motif from TrkA[37]. The structural information fromDok1may provide the cluefor Dok5 specific recognition among Trk receptors. Further-more, Dok5 selectively binding to TrkB and TrkC may suggestits distinct function in BDNF and NT-3 signal pathway. Thoughevidences showed that there might be functional redundancyamong Trk signal pathways, studies also proved TrkA/B/C hadnonoverlapping functions in neural development. The signifi-cant loss of neurons in BDNF/TrkB and NT-3/TrkC knockoutmouse demonstrated their indispensable functions duringsensory and motor neurons development [38]. The overlappedexpression patterns of Dok5 and TrkB/C receptors and theirinteraction imply that Dok5 may have important functions insensory and motor neurons development and survival.
Moreover, we proved that Dok5 binds to TrkB and TrkCreceptors NPQY motif in a kinase-activity-dependent mannerand Dok5 is phosphorylated by Trk receptors upon neurotro-phin stimulation. Co-localization of Dok5 and TrkB or TrkCreceptors in differentiated PC12 cells provides another evidencefor their physical interaction. The identification of Dok5 assubstrate of TrkB/C receptors will enrich the signal network ofneurotrophin pathway.
Besides Dok5, many other adaptor proteins containing PTBdomain have been reported to bind to the NPQY motif of Trkreceptors, for example Shc, IRS and FRS2 [4,39]. It is hard toimagine that so many kinds of adaptor proteins bind to thereceptor at the same site and at the same time. The mostpossibility is that they compete with each other for receptorbinding. And our result established a competitive modelbetween Dok5 and N-shc for Trk binding. This might be agood explanation for several adaptor proteins binding to thereceptors at the same site to mediate different downstreamsignal pathway. But actual situation in vivo may be much morecomplicated. The different expression tissues and cell types ofthese adaptor proteins, and their different binding affinity withthe receptor and many other factors should be considered.
Unlike Dok1 and Dok2 as negative regulator in MAPKpathways [22,40,41], Dok5 mediated GDNF-induced MAPKactivation [16]. Here we also showed that overexpression ofDok5 could augment MAPK activation induced by BDNF orNT-3; while the deletion of Dok5 C-terminal region disruptedthis effect. This indicates that Dok5 C-terminal region is res-
ponsible for cascading the signal to downstream. However, tillnow downstream signal molecules that associate with Dok5 C-terminal region have not been identified yet. Compared withDok1/2, Dok5 has a much shorter C-terminal region. Many SH2/SH3 containing proteins that associated with Dok1/2, includingRasGAP and NCK, showed no interaction with Dok5 [16,27].Crowder et al. tried to find downstream signal proteins coupledwith Dok4/5/6 subgroup by bioinformatics database search, butno functional known proteins were obtained [17]. So the studiesof identifying the downstream partners of Dok5 become veryimportant for explaining its function in signal transduction.
In summary, we identified Dok5 as the new substrate of TrkBand TrkC receptors and it was involved in MAPK activationinduced by BDNF and NT-3. Dok5 bind to the NPQY motif ofTrk receptors in a kinase-activity-dependent manner through itsPTB domain. These results enrich the neurotrophin signalingnetwork and supply important information about Dok5 functionin nervous system. Our studies as well as previous researcheshave showed that Dok5 was involved in TrkB/C, c-Ret, andinsulin signal transduction, however the detailed regulatorymechanism and comprehensive functions of Dok5 in thesepathways are yet unknown, more studies need to be done.
Acknowledgements
This work was supported by grants from the National Programfor the Key Basic Research Project (‘973’ Nos. 2001CB510206,2004CB518604 and 2005CB522507), and the National SciencesFoundation of China (Nos. 30421003 and 30430200).
References
[1] E.J. Huang, L.F. Reichardt, Annu. Rev. Neurosci. 24 (2001) 677.[2] E.J. Huang, L.F. Reichardt, Annu. Rev. Biochem. Allied Res. India 72
(2003) 609.[3] A. Obermeier, R. Lammers, K.H. Wiesmuller, G. Jung, J. Schlessinger, A.
Ullrich, J. Biol. Chem. 268 (1993) 22963.[4] S.O. Meakin, J.I. MacDonald, E.A. Gryz, C.J. Kubu, J.M. Verdi, J. Biol.
Chem. 275 (2000) 18225.[6] N. Jones, S.H. Chen, C. Sturk, Z. Master, J. Tran, R.S. Kerbel, D.J.
Dumont, Mol. Cell Biol. 23 (2003) 2658.[7] T.B. vanDijk, E. vanDenAkker, M.P. Amelsvoort, H.Mano, B. Lowenberg,
M. von Lindern, Blood 96 (2000) 3406.[8] X. Liang, D. Wisniewski, A. Strife, Shivakrupa, B. Clarkson, M.D. Resh,
J. Biol. Chem. 277 (2002) 13732.[9] M.J. Wick, L.Q. Dong, D. Hu, P. Langlais, F. Liu, J. Biol. Chem. 276
(2001) 42843.[10] N. Jones, D.J. Dumont, Curr. Biol. 9 (1999) 1057.[11] N. Carpino, D. Wisniewski, A. Strife, D. Marshak, R. Kobayashi, B.
Stillman, B. Clarkson, Cell 88 (1997) 197.[12] Y. Yamanashi, D. Baltimore, Cell 88 (1997) 205.[13] A. Di Cristofano, N. Carpino, N. Dunant, G. Friedland, R. Kobayashi, A.
Strife, D. Wisniewski, B. Clarkson, P.P. Pandolfi, M.D. Resh, J. Biol.Chem. 273 (1998) 4827.
[14] N. Jones, D.J. Dumont, Oncogene 17 (1998) 1097.[15] S. Lemay,D.Davidson, S. Latour,A.Veillette,Mol. Cell Biol. 20 (2000) 2743.[16] J. Grimm, M. Sachs, S. Britsch, S. Di Cesare, T. Schwarz-Romond, K.
Alitalo, W. Birchmeier, J. Cell Biol. 154 (2001) 345.[17] R.J. Crowder, H. Enomoto, M. Yang, E.M. Johnson Jr., J. Milbrandt,
J. Biol. Chem. 279 (2004) 42072.
2003L. Shi et al. / Cellular Signalling 18 (2006) 1995–2003
[18] R. Gugasyan, C. Quilici, S.T. I., D. Grail, A.M. Verhagen, A. Roberts, T.Kitamura, A.R. Dunn, P. Lock, J. Cell Biol. 158 (2002) 115.
[19] I. Tamir, J.C. Stolpa, C.D. Helgason, K. Nakamura, P. Bruhns, M. Daeron,J.C. Cambier, Immunity 12 (2000) 347.
[20] P. Van Slyke, M.L. Coll, Z. Master, H. Kim, J. Filmus, D.J. Dumont, Mol.Cell Biol. 25 (2005) 3831.
[21] J.D. Robson, D. Davidson, A. Veillette, Mol. Cell Biol. 24 (2004) 2332.[22] T. Yasuda, M. Shirakata, A. Iwama, A. Ishii, Y. Ebihara, M. Osawa, K.
Honda, H. Shinohara, K. Sudo, K. Tsuji, H. Nakauchi, Y. Iwakura, H.Hirai, H. Oda, T. Yamamoto, Y. Yamanashi, J. Exp. Med. 200 (2004) 1681.
[23] M. Niki, A. Di Cristofano, M. Zhao, H. Honda, H. Hirai, L. Van Aelst, C.Cordon-Cardo, P.P. Pandolfi, J. Exp. Med. 200 (2004) 1689.
[24] A. Di Cristofano, M. Niki, M. Zhao, F.G. Karnell, B. Clarkson, W.S. Pear,L. Van Aelst, P.P. Pandolfi, J. Exp. Med. 194 (2001) 275.
[25] A. Bedirian, C. Baldwin, J. Abe, T. Takano, S. Lemay, J. Biol. Chem. 279(2004) 19335.
[26] S. Itoh, S. Lemay, M. Osawa, W. Che, Y. Duan, A. Tompkins, P.S.Brookes, S.S. Sheu, J.I. Abe, J. Biol. Chem. 280 (2005) 26383.
[27] D. Cai, S. Dhe-Paganon, P.A. Melendez, J. Lee, S.E. Shoelson, J. Biol.Chem. 278 (2003) 25323.
[28] H. Yamashita, S. Avraham, S. Jiang, I. Dikic, H. Avraham, J. Biol. Chem.274 (1999) 15059.
[29] R.M. Stephens, D.M. Loeb, T.D. Copeland, T. Pawson, L.A. Greene, D.R.Kaplan, Neuron. 12 (1994) 691.
[30] N. Shi, W. Zhou, K. Tang, Y. Gao, J. Jin, F. Gao, X. Peng, M. Bartlam, B.Qiang, J. Yuan, Z. Rao, Acta Crystallogr., D Biol. Crystallogr. 58 (Pt 12)(2002) 2170.
[31] B. Margolis, J. Lab. Clin. Med. 128 (1996) 235.[32] M.T. Uhlik, B. Temple, S. Bencharit, A.J. Kimple, D.P. Siderovski, G.L.
Johnson, J. Mol. Biol. 345 (2005) 1.[33] T. Nakamura, R. Sanokawa, Y. Sasaki, D. Ayusawa, M. Oishi, N. Mori,
Oncogene 13 (1996) 1111.[34] T. Nakamura, S. Muraoka, R. Sanokawa, N. Mori, J. Biol. Chem. 273
(1998) 6960.[35] H.Y. Liu, S.O. Meakin, J. Biol. Chem. 277 (2002) 26046.[36] F. Cong, B. Yuan, S.P. Goff, Mol. Cell Biol. 19 (1999) 8314.[37] N. Shi, S. Ye, M. Bartlam, M. Yang, J. Wu, Y. Liu, F. Sun, X. Han, X.
Peng, B. Qiang, J. Yuan, Z. Rao, J. Biol. Chem. 279 (2004) 4962.[38] R. Klein, FASEB J. 8 (1994) 738.[39] C. Miranda, A. Greco, C. Miele, M.A. Pierotti, E. Van Obberghen, J. Cell
Physiol. 186 (2001) 35.[40] H. Shinohara, T. Yasuda, Y. Yamanashi, Genes Cells 9 (2004) 601.[41] H. Shinohara, A. Inoue, N. Toyama-Sorimachi, Y. Nagai, T. Yasuda, H.
Suzuki, R. Horai, Y. Iwakura, T. Yamamoto, H. Karasuyama, K. Miyake,Y. Yamanashi, J. Exp. Med. 201 (2005) 333.
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