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Hisaaki Shinohara^1,, Tomoharu Yasuda^1, and Yuji Yamanashi^1,2,*

¹ Department of Cell Regulation, Medical Research Institute, and ² School of Biomedical Science, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan

	Abstract

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Dok-1 is a common substrate of many protein tyrosine kinases (PTKs). It recruits rasGAP and other SH2-containing proteins and negatively regulates Ras-Erk signalling downstream of PTKs. However, the mechanisms of its inhibitory effect are yet unclear. Here, a series of C-terminal deletion mutants of Dok-1 delineated the core domain for the inhibition of Erk from 334 to 346 amino acid, which contains two SH2-binding motifs having Tyr-336 or Tyr-340. The Dok-1 mutants having tyrosine-to-phenylalanine (YF) substitution(s) at Tyr-336 and/or Tyr-340 lost their inhibitory effect on Ras and Erk downstream of Src-like PTK, Lyn or Fyn, whereas the rasGAP-binding of each mutant remained intact. However, the Dok-1 mutant having YF substitutions at the rasGAP-binding sites (Tyr-295 and Tyr-361) also showed incapability of Ras and Erk inhibition. Moreover, the Dok-1 mutant having YF substitutions at Tyr-336 and Tyr-340 showed an impaired inhibitory effect on v-Abl-induced transformation of NIH-3T3 cells. These results demonstrate that Tyr-336 and Tyr-340 of Dok-1 are dispensable for rasGAP-binding but essential for inhibition of Ras-Erk signalling and cellular transformation downstream of PTKs. Thus, Dok-1 probably recruits as yet unidentified molecule(s), which, in concert with rasGAP, negatively regulate Ras-Erk signalling.

	Introduction

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Dok-1, originally designated as Dok or p62^dok, was first identified as a major substrate of p210^bcr-abl, a causative factor of chronic myelogenous leukaemia (CML), and v-Abl protein tyrosine kinases (PTKs), which causes pro-B cell leukaemia (Carpino et al. 1997; Yamanashi & Baltimore 1997). Dok-1 has N-terminal pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains followed by C-terminal SH2-binding motifs, and each of these motifs contains a tyrosine residue to be phosphorylated. PH domain is generally involved in phosphoinositide binding and is important for Dok-1 to be proximal to the cellular membrane where many PTKs and Ras are localized to play their roles (Zhao et al. 2001). Consistently, a Dok-1 mutant lacking the PH domain was hardly tyrosine phosphorylated upon PDGF-stimulation of cells (Zhao et al. 2001). The PTB domain of Dok-1 preferentially binds to a peptide of the form Y/MxxNxLpY in vitro, in which pY represents a phosphorylated tyrosine. Interestingly, loss of the PTB function resulted in reduced levels of Dok-1 tyrosine phosphorylation by Src (Songyang et al. 2001). Multiple tyrosine residues in various SH2-binding motifs of Dok-1 are rapidly phosphorylated in a wide range of signalling situations (Berg et al. 1999; Carpino et al. 1997; Nelms et al. 1998; Noguchi et al. 1999; Tamir et al. 2000; Yamanashi et al. 2000; Zhao et al. 2001). When tyrosine-phosphorylated, Dok-1 works as an adaptor protein and recruits a variety of SH2-containing molecules such as p120 rasGAP (rasGAP hereafter), Nck, and Csk (Carpino et al. 1997; Noguchi et al. 1999; Shah & Shokat 2002; Yamanashi & Baltimore 1997).

Among the mammalian Dok family, Dok-1, Dok-2, and Dok-3 are preferentially expressed in haematopoietic cells; however, Dok-3 appears relatively distant and it does not bind to rasGAP unlike Dok-1 and Dok-2 (Cong et al. 1999; Lemay et al. 2000). The more distant Dok family members Dok-4 and Dok-5 are virtually absent in haematopoietic cells. Experiments with mice lacking Dok-1 demonstrated an indispensable role in the negative regulation of Ras and Erk downstream of PTKs in various cell types (Di Cristofano et al. 2001; Yamanashi et al. 2000; Zhao et al. 2001). In addition, over-expression of Dok-1 or Dok-2 in cultured cells also inhibited Ras-Erk signalling downstream of PTKs (Jones & Dumont 1999; Nelms et al. 1998; Wick et al. 2001; Yoshida et al. 2000). Because rasGAP is a potent negative regulator of Ras, its binding to Dok-1 and Dok-2 appears to be important for the negative regulation of the Ras-Erk pathway. However, the biological significance of rasGAP-binding in the function of the Dok family proteins is still controversial. A Dok-1 mutant lacking six SH2-binding motifs, including the rasGAP-binding sites, lost its inhibitory effect on Erk downstream of Ret PTK (Murakami et al. 2002). On the other hand, a couple of Dok-1 mutants lacking rasGAP-binding activity remained intact in the Erk inhibition upon PDGF- or insulin-stimulation of cells (Zhao et al. 2001; Wick et al. 2001). These findings, at least, suggest that Dok-1 recruits as yet unidentified effector(s) to inhibit Ras-Erk signalling. Here, we show evidence that Dok-1 requires another pathway in addition to rasGAP in negative regulation of Ras and Erk downstream of Lyn or Fyn.

	Results

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Previous studies demonstrated that multiple tyrosine residues at the C-terminal half of Dok-1 are phosphorylated downstream of PTKs and the C-terminal region was required for the inhibition of Erk, probably due to phosphotyrosine-based interactions with SH2 domains of downstream effector protein(s) (Shah & Shokat 2002; Tamir et al. 2000). Because the Dok-1 amino acids from 336 to 363, having three tyrosines in total, at the C-terminal half are critical for the inhibition of Src-mediated cellular transformation (Songyang et al. 2001), we focused on the roles of Tyr-336, Tyr-340, and Tyr-361 for the inhibition of the Ras-Erk pathway (Fig. 1). When ectopically expressed in 293T cells, the C-terminal deletion mutants (Dok368, Dok346, Dok333, Dok277; Fig. 1) were tyrosine phosphorylated by Lyn or Fyn at levels comparable to wild-type Dok-1 (Fig. 2). As previously reported, over-expression of Dok-1 inhibited Erk activation induced by Lyn or Fyn in 293T cells (Fig. 2). However, this inhibitory effect was diminished in cells expressing Dok333 or Dok277, but not in cells expressing Dok368 or Dok346, indicating that the amino acids from 334 to 346 of Dok-1 are essential for the inhibition of Erk.

View larger version (23K): [in this window] [in a new window]   Figure 1  Schematic illustration of mouse Dok-1 and its mutants. The positions of tyrosine residues at the C-terminal half are indicated (Y) by amino acid numbers (top), and the total amino acid length for each protein is also indicated (right). WT, PH, and PTB represent wild-type, pleckstrin homology domain, and phosphotyrosine binding domain, respectively, and the positions of YF substitutions are indicated (F). Tyrosine residues responsible for the rasGAP binding are indicated by arrows (top).

View larger version (67K): [in this window] [in a new window]   Figure 2  Effect of C-terminal deletions in Dok-1 on the inhibition of Erk. 293T cells were transfected with expression plasmids for indicated proteins (top) and HA-Erk2. The whole cell lysates were subjected to immunoblotting (IB) with antibodies to activated Erk (pErk), HA-epitope tag (HA), Dok-1, or phosphotyrosine (pTyr). The positions and sizes (kDa) of molecular weight markers are indicated (left). Arrowheads indicate the positions of HA-Erk2, Dok-1 (WT), or Dok-1 mutants.

View larger version (51K): [in this window] [in a new window]   Figure 3  Effect of YF substitutions in Dok-1 on the inhibition of Erk. 293T cells were transfected with expression plasmids for indicated proteins (top) and HA-Erk2. The whole cell lysates were subjected to immunoblotting (IB) with antibodies to activated Erk (pErk), HA-epitope tag (HA), Dok-1, or phosphotyrosine (pTyr). The positions and sizes (kDa) of molecular weight markers are indicated (left). Arrowheads indicate the positions of HA-Erk2, Dok-1, or Dok-1 mutants.

View larger version (28K): [in this window] [in a new window]   Figure 4  Effect of YF substitutions in Dok-1 on interaction with rasGAP. 293T cells were transfected with expression plasmids for indicated proteins (top). The anti-Dok-1 immunoprecipitates (IP) or whole cell lysates (WCL) were subjected to immunoblotting (IB) with antibodies to rasGAP, Dok-1, or phosphotyrosine (pTyr). Arrowheads indicate the positions of rasGAP, Dok-1, or Dok-1 mutants.

View larger version (18K): [in this window] [in a new window]   Figure 5  Effect of YF substitutions in Dok-1 on the inhibition of Ras. 293T cells were transfected with expression plasmids for indicated proteins (top). The whole cell lysates were subjected to RBD pull-down assay for visualization of activated Ras, which binds GST-Raf RBD, by immunoblotting (IB) with anti-H-Ras antibody. The amount of GST-Raf RBD applied for each pull-down assay was monitored by immunoblotting (IB) with anti-GST antibody. Arrowheads indicate the positions of activated Ras or GST-Raf RBD.

View larger version (18K): [in this window] [in a new window]   Figure 6  Effect of YF substitutions in Dok-1 on v-Abl-induced cellular transformation. NIH-3T3 cells were transfected with expression plasmids for indicated proteins (bottom). The focus-forming ability of each combination of plasmids was determined by enumerating cellular foci. Mean values and SDs are calculated from triplicate experiments.

	Discussion

Top Abstract Introduction Results Discussion Experimental procedures References

We previously demonstrated that the Dok-1 amino acids from 336 to 363 are critical for binding with rasGAP and inhibition of cellular transformation upon Src-mediated stimulation (Songyang et al. 2001). Here we performed detailed structure-function analyses and found that Dok-1 mutants having YF substitution(s) at Tyr-336 and/or Tyr-340 retained binding capability to rasGAP, but lost the inhibitory effects on Erk activation downstream of Lyn or Fyn. Because these mutants can recruit rasGAP when tyrosine phosphorylated, they may have lost a pathway to inhibit Erk independently of Ras. However, to our surprise, none of the mutants showed an inhibitory effect on Ras activation, although they recruited rasGAP to the same extent as wild-type Dok-1 (Figs 4 and 5). In addition, DokY295/361F mutant lacking the rasGAP-binding sites failed to suppress Ras upon LynYF stimulation. These results indicate that rasGAP-binding does not suffice the inhibitory function of Dok-1. Therefore, Dok-1 requires at least another pathway in addition to rasGAP association for the negative regulation of Ras-Erk signalling. Because Tyr-336 and Tyr-340 are necessary for the regulation, Dok-1 probably recruits novel effector protein(s) having the SH2 domain that would bind the peptides containing those tyrosine residues upon their phosphorylation.

Tyr-336 and Tyr-340 of Dok-1 are in the SH2-binding motifs of the form pYWDL for the SHP-1 SH2 and the form pYGHV for the SH2 of Src family PTK. Although SHP-1 and Lyn bound Dok-1 as reported (Berg et al. 1999; van Dijk et al. 2000), Tyr-336 and Tyr-340 were dispensable for these associations (data not shown). Other SH2-containing proteins, Nck and Csk, were also reported to associate Dok-1 downstream of PTKs. However, the SH2 domains of Nck and Csk target the SH2-binding motifs having Tyr-361 and Tyr-450, respectively (Noguchi et al. 1999; Shah & Shokat 2002). Thus, it is likely that there is other key molecule(s) to bind Dok-1 via Tyr-336 and/or Tyr-340 and block the Ras-Erk pathway, in concert with rasGAP, downstream of PTK. However, we cannot completely rule out the possibility that YF substitution(s) at Tyr-336 and Tyr-340 may perturb the overall structural integrity of Dok-1.

In summary, we have found that Tyr-336 and Tyr-340 of Dok-1 are indispensable for the inhibition of the Ras-Erk pathway irrespective of rasGAP-binding upon Lyn- or Fyn-mediated stimulation of cells. Therefore, we propose that Dok-1 requires another pathway in addition to rasGAP in the negative regulation of Ras-Erk signalling downstream of PTKs. That DokY336/340F showed impaired inhibition of v-Abl-mediated transformation of NIH-3T3 cells further supports this proposal. Identification of protein(s) that bind Dok-1 via Tyr-336 and/or Tyr-340 upon PTK-mediated stimulation of cells may provide a clue to understand how Dok-1 regulates the Ras-Erk pathway, which plays a pivotal role in controlling cellular functions and fates in many signalling situations.

	Experimental procedures

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Cell culture and transfection

293T cells and NIH-3T3 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% foetal calf serum (FCS), penicillin, and streptomycin. Transient transfection of cells with expression plasmids was performed with Fugene 6 reagents (Roche Diagnostics), following the manufacturer's instructions.

Plasmid constructions

The coding region of the wild-type murine Dok-1 cDNA was cloned into the pcDNA3.1 expression vector (Invitrogen). cDNAs encoding C-terminal deletion mutants of Dok-1 were generated as described (Songyang et al. 2001). The Dok368, 346, 333 and 277 plasmids encode amino acids 1-368, 1-346, 1-333 and 1-277 of Dok-1, respectively. The cDNAs encoding DokY336F, DokY340F, DokY336/340F and DokY295/361F were generated by site-directed mutagenesis using PCR with the wild-type Dok-1 cDNA and the KODplus polymerase (TOYOBO); the cDNA encoding DokY361F, a kind gift from K. M. Shokat (Shah & Shokat 2002), was used as a template for the PCR to generate the DokY295/361F cDNA. These cDNAs were cloned into the pcDNA3.1 expression vector. All plasmid constructs were confirmed by DNA sequencing. The expression plasmids pME-Lyn, pME-LynY508F, pME-Fyn and pGD-v-Abl have been described (Fusaki et al. 1994; Takeuchi et al. 1993; Yamanashi & Baltimore 1997). The expression plasmid for HA epitope-tagged Erk2 was a kind gift from Y. Goto (Wakioka et al. 2001).

Immunoprecipitation and immunoblotting

293T cells transfected with pME-Lyn or pME-Fyn together with other plasmids were solubilized in TNN buffer (10 mM Tris [pH 7.5], 150 mM NaCl, 1.0 mM EGTA, 1.5 mM MgCl₂, 1.0% Nonidet P-40, 1 mM Na₃VO₄, 10 mM NaF, 10 µg/mL leupeptin, 10 µg/mL aprotinin, 10 µg/mL pepstain, 10 µg/mL antipain, 160 µg/mL benzamidine and 10 µg/mL soybean trypsin inhibitor). 293T cells transfected with pME-LynY508F together with other plasmids were solubilized in RIPA buffer (10 mM Tris [pH 7.4], 150 mM NaCl, 0.5 mM EDTA, 1.0% Nonidet P-40, 1 mM Na₃VO₄, 10 mM NaF, 10 µM sodium molybdate, 2 µg/mL aprotinin, 1 mM benzamidine, 5 µg/mL chymostatin, 5 µg/mL leupeptin, 1 µg/mL pepstatin and 0.2 mM PMSF). Cleared lysates were then sequentially incubated with anti-Dok-1 antibody (A-3; Santa Cruz Biotechnology) and protein G-Sepharose (Amersham Biosciences). The immune complexes were precipitated and the immunoprecipitates were washed four times with TNN or RIPA buffer. Proteins in whole cell lysates or immunoprecipitates were separated by SDS-polyacrylamide gel electrophoresis, transferred to PVDF membrane (Bio-Rad Laboratories), and incubated with appropriate antibodies for immunoblotting. The membranes were then incubated with horseradish peroxidase-labelled (Amersham Biosciences) or alkaline phosphatase-labelled secondary antibodies (Santa Cruz Biotechnology), and visualized by the ECL‘ system (Amersham Biosciences) or BCIP/NBT Color Development system (Promega), respectively. The following antibodies were used for immunoblotting: anti-Dok-1 (A-3), anti-phospho-Erk1/2 (Thr202/Tyr204), anti-rasGAP (B4F8) and anti-SHP-1 (C-19) from Santa Cruz Biotechnology; anti-phosphotyrosine (4G10) and anti-Lyn from Upstate Biotechnologies; anti-HA (12CA5) from Berkeley Antibody Company.

RBD pull-down assay

A bacterially expressed glutathione S-transferase fused with the Ras-binding domain of human cRaf-1 (amino acids 1-149), GST-Raf RBD, bound with glutathione-sepharose beads was prepared as described (Taylor & Shalloway 1996). 293T cells transfected with an appropriate set of expression plasmid(s) were solubilized in Mg²⁺-containing lysis buffer (Taylor & Shalloway 1996). The lysates were then incubated with 20 µg of the GST-Raf RBD beads for 60 min at 4 °C. After extensive washing, proteins bound with the beads were eluted with SDS-loading buffer (50 mM Tris [pH 6.8], 5 mM EDTA, 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue and 10% glycerol) and separated by SDS-polyacrylamide gel electrophoresis to be subjected to immunoblotting with anti-H-Ras (259) or anti-GST (B-14) antibodies (Santa Cruz Biotechnology).

Transformation assay

v-Abl-induced transformation of NIH-3T3 cells was evaluated by enumerating the foci generated in each assay. 2 x 10⁵ cells co-transfected with 1 µg of v-Abl along with 1 µg of expression plasmid for wild-type Dok-1 or its mutant were maintained in Dulbecco's modified Eagle's media containing 10% FCS, penicillin and streptomycin for 14 days as described (Fujimoto et al. 1996). Triplicate culture plates were examined in each assay.

	Acknowledgements

 
We thank Y. Goto (University Tokyo, Japan) and K. M. Shokat (University California, USA) for plasmids. We are also grateful to K. Matsumoto for technical assistance. This work was supported by Grants-in-Aid for Scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by grants from the Uehara Memorial, the Tokyo Biochemical Research, and the Yamanouchi Foundations.

	Footnotes

 
These authors contributed equally to this work.

Communicated by: Tadashi Yamamoto

^* Correspondence: E-mail: yamanashi.creg{at}mri.tmd.ac.jp

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Received: 11 March 2004
Accepted: 29 March 2004

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Shinohara, H. Articles by Yamanashi, Y. Search for Related Content PubMed PubMed Citation Articles by Shinohara, H. Articles by Yamanashi, Y.