D-Cbl Binding to Drk Leads to Dose-Dependent Down-Regulation of EGFR Signaling and Increases Receptor-Ligand Endocytosis

Proper control of Epidermal Growth Factor Receptor (EGFR) signaling is critical for normal development and regulated cell behaviors. Abnormal EGFR signaling is associated with tumorigenic process of various cancers. Complicated feedback networks control EGFR signaling through ligand production, and internalization-mediated destruction of ligand-receptor complexes. Previously, we found that two isoforms of D-Cbl, D-CblS and D-CblL, regulate EGFR signaling through distinct mechanisms. While D-CblL plays a crucial role in dose-dependent down-regulation of EGFR signaling, D-CblS acts in normal restriction of EGFR signaling and does not display dosage effect. Here, we determined the underlying molecular mechanism, and found that Drk facilitates the dose-dependent regulation of EGFR signaling through binding to the proline-rich motif of D-CblL, PR. Furthermore, the RING finger domain of D-CblL is essential for promoting endocytosis of the ligand-receptor complex. Interestingly, a fusion protein of the two essential domains of D-CblL, RING- PR, is sufficient to down-regulate EGFR signal in a dose-dependent manner by promoting internalization of the ligand, Gurken. Besides, RING-SH2Drk, a fusion protein of the RING finger domain of D-Cbl and the SH2 domain of Drk, also effectively down-regulates EGFR signaling in Drosophila follicle cells, and suppresses the effects of constitutively activated EGFR. The RING-SH2Drk suppresses EGFR signaling by promoting the endosomal trafficking of ligand-receptor complexes, suggesting that Drk plays a negative role in EGFR signaling by enhancing receptor endocytosis through cooperating with the RING domain of D-Cbl. Interfering the recruitment of signal transducer, Drk, to the receptor by the RING-SH2Drk might further reduces EGFR signaling. The fusion proteins we developed may provide alternative strategies for therapy of cancers caused by hyper-activation of EGFR signaling.


Introduction
Ubiquitination occurs via sequential activation and conjugation of ubiquitin to target proteins by ubiquitin activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligase (E3) [1]. Aside from protein degradation, ubiquitination represents a crucial signal for the endocytosis of signaling molecules such as EGFR. The attenuation of EGFR signaling by endocytosis serves to properly control cell growth, differentiation, and normal developmental processes [2,3,4,5]. Consistent with an intimate role in signaling regulation, as well as in other cellular processes, emerging evidence has shown that derailed endocytosis disrupts developmental processes and leads to cancer formation [6,7].
A critical E3 ubiqutin ligase mediating the ubiquitiationdependent receptor endocytosis is the proto-oncogene Casitas Blineage lymphoma (Cbl), which was first identified as the cellular homolog of v-cbl, which induces pre-B-cell lymphomas and myeloid tumors [8,9]. Cbl is involved in multiple signaling pathways, and plays a negative role in EGFR signaling that is conserved among many species [10]. In Drosophila, D-Cbl negatively regulates EGFR signaling in dorsoventral patterning during oogenesis [11], in eye development [12,13,14], and in border cell migration [15]. The Cbl recognizes receptor tyrosine kinase (RTK, such as EGFR) and non-receptor tyrosine kinases through a phosphotyrosine-binding (PTB) domain [10,16]. Internalizing EGFR requires c-Cbl and Lys63-linked polyubiquitin chain modification mediated by c-Cbl is essential for sorting activated receptors to the lysosomal degradation compartment [17,18,19]. The RING finger domain in Cbl is highly conserved during evolution, particularly for critical amino acids related to E3 ligase activity [16,20]. The C-terminal proline-rich (PR) domain for protein-protein interaction and the ubiquitin association (UBA) domain are found only in some members of the family [21]. In Drosophila, two major isoforms, D-CblL and D-CblS are generated from the single D-Cbl gene [22]; the shorter isoform D-CblS lacks the proline-rich and UBA domains.
Studies in mammals have shown that the endocytosis of EGFR involves multiple pathways, depending on EGF concentration and exhibits cell-based specificities [2,23,24]. Therefore, redundancy of multiple pathway of endocytosis made it difficult to dissect which molecule the process requires. The Drosophila eggshell patterning has served as a sensitive and simple system to read out the levels of EGFR signaling levels [25,26], thus representing an ideal model of mechanistic studies. The advantage of this in vivo system is that it provides physiological conditions with a gradient of ligand concentration to induce different levels of EGFR activation that is reflecting through the D/V patterning of eggshell and embryo. The Gurken, a TGF-a homolog, is produced by the oocyte and activates EGFR in follicle cells to specify the dorsal cell fates, followed by attenuation of EGFR signaling via negative regulators, such as sprouty, and kekkon, which together determine the area of the follicle epithelium where the dorsal appendages (DAs) form [27,28]. Importantly, examining the Gurken distribution in loss of D-Cbl and D-CblL over-expression conditions has revealed that D-CblL promotes endocytosis of ligand-receptor to control the amount of available ligand. We demonstrated that D-CblL facilitates the activated receptor to traffic through the endocytic pathway for terminating signaling at lysosomal degradation compartment [29]. Therefore, over-expression of D-CblL at different levels resulted in different degrees of ventralization, corresponding to phenotypes resulting from different severities of gurken mutant alleles [30]. This dose-dependent, negative effect on EGFR signaling is specific to D-CblL and is not produced by overexpression of D-CblS.
To understand how D-CblL controls EGFR signaling at the molecular level, this study investigates which molecular interaction with D-CblL is sufficient to facilitate the endocytosis of the ligand-EGFR complex in Drosophila egg chambers. This work first demonstrates that the factor Downstream of receptor kinase (Drk) plays a major role in D-CblL mediated down-regulation of EGFR signaling in a dose-dependent fashion. In addition, E3 ligase activity is required for D-CblL activity, because over-expression of D70Z-D-CblL, an E3 defective mutant, blocked ligand-receptor internalization and produced a dominant-negative effect. We generated the RING-SH2 Drk chimeric protein, containing two functional domains of D-Cbl and Drk, and found that this chimeric protein not only attenuated EGFR signaling, but also down-regulated constitutively activated EGFR, l-top. We further demonstrated that RING-SH2 Drk suppresses EGFR signaling by enhancing the endosomal trafficking of the ligand-receptor complex and interfering with the recruitment of the endogenous Drk.

Drk plays a major role in D-CblL mediated downregulation of EGFR signaling
In this study, we set out to elucidate the molecular mechanism by which D-CblL promotes the endocytosis of the ligand-receptor complex. Since this effect of D-CblL was not observed for D-CblS even when expressed at a similar level [30], we suspected that D-CblL may mediate the internalization by its extra C-terminus that is distinct from D-CblS. In mammals, Grb2 (Growth factor receptor binding protein 2), Eps15 and the CIN85-Endophilin complex are involved in Cbl-mediated down-regulation of EGFR signaling [31,32,33]. We then tested for their involvement in D-CblL mediated down-regulation of EGFR signaling in Drosophila oogenesis by a sensitive genetic assay. While Drosophila Eps15, endophilin A and endophilin B had no or minor effects (Table S1), the Drosophila Grb2 homolog drk exhibits a strong link to D-CblL activity described below.
Mammalian studies demonstrated that c-Cbl is recruited to EGFR through directly binding the Y1045 residue of EGFR or indirectly interacting with Grb2 [34,35,36,37]. The Tyr1068/ 1086 of EGFR is the direct docking site for the SH2 domain of Grb2 [38]. The SH3 domain of Grb2 binds to the proline-rich region of D-CblL, which is absent in D-CblS. We used the drk EOA mutant, which loses binding to EGFR caused by mutation in the SH2 domain [39], for a genetic interaction assay. The ventralized effect by EQ1-Gal4-driven over-expression of D-CblL in the follicle cells was significantly reduced in the heterozygous drk EOA mutant background (Table 1 and Figure 1A-D). EQ1-Gal4 is mainly expressed in the follicle cells [30]. We reasoned that if Drk was required for the effect of D-CblL over-expression, the interruption of interaction between Drk and D-CblL would block the D-CblL over-expression effects. To address this issue, we found one consensus sequence PPLPPR of the Grb2/Drk binding motif on D-CblL (named PR), and generated a mutant in this motif by replacing the first and fifth prolines with alanines (mPR) ( Figure 1E). The results from the yeast two hybrid system showed that the wild-type PR, but not the mPR, interacted with full-length Drk ( Figure S2). Consistently, in the anti-D-Cbl immunoprecipitation assay, much less Drk was pulled down in the D-CblL-mPR complex than in the wild-type D-CblL complex, even though D-CblL-mPR was expressed at a higher level compared to that of D-CblL ( Figure 1F). We then over-expressed D-CblL-mPR or D-CblL in follicle cells using EQ1-Gal4. At comparable levels ( Figure  S1A), the over-expression effects of D-CblL-mPR on EGFR signaling were much weaker than that of D-CblL. Furthermore, the effect of D-CblL-mPR over-expression was not suppressed in the heterozygous drk EOA mutant background, suggesting that PR of D-CblL might be a critical binding domain for Drk (Table 1).
We further analyzed the function of the D-CblL-mPR mutant using a constitutive expression promoter, HS83 [40], which was also used in rescue assays by D-CblL and D-CblS. Two transgenic lines, hs83-D-cblL-mPR-7 and hs83-D-cblL-mPR-4, rescued the lethality of the cbl F165 null mutant and 75%,80% of the expected animals developed to adults ( Table 2). The rescue ability of the D-CblL-mPR mutant was comparable to that of D-CblS (90,100%). Moreover, unlike the wild-type D-CblL, cbl mutant flies rescued by D-CblL-mPR showed no pattern defect in wing or the eggs they laid (data not shown), although the expression level of hs83-D-cblL-mPR was similar to that of hs83-D-cblL ( Figure S1B). These data indicate that the interaction between D-CblL and Drk underlies the functional difference between the D-CblS and D-CblL, and provide the basis for the dose-dependent, negative effects of D-CblL on EGFR signaling. However, this interaction is not essential for D-CblL function in terms of the normal restriction of EGFR signaling.

The E3 ligase activity of D-CblL is essential for its negative role in EGFR signaling
A screen for D-Cbl loss-of-function alleles has identified mutations in the RING domain [13]. Indeed, mouse fibroblasts that express D70Z-Cbl, an E3 defective mutant with a deletion of 17 amino acids prior to the RING finger domain, show increased EGFR activation upon ligand stimulation [41,42]. In addition, the D-v-cbl and D-cblS-onco (D-CblS-D70Z) act as dominant negative mutants in the Drosophila eye and wing, which is presumably resulted from competing with wild-type D-Cbl for binding to the EGFR [43]. However, one proposed inhibitory function of Cbl in EGFR signaling is acting as an competitor with Sos (Son of sevenless) by binding to Grb2, thereby blocking signaling through the Ras-MAPK pathway [21]. We decided to test whether the E3 activity is essential for D-CblL function in the dose-dependent EGFR regulation. First we tested the involvement of ubiquitination in D-CblL-mediated regulation using the E2-conjugase mutant, eff 8 , which has been shown to be involved in D-v-cbl function [43,44,45]. The effect of D-CblL over-expression was reduced in the eff 8 heterozygous mutant (Table S2), indicating the attribution of ubiquitination in D-CblL effects. A deletion mutant similar to D-CblS-D70Z, which should be E3 defective, was generated for D-CblL, and notably dominant negative effects on EGFR signaling were observed upon its over-expression in the wing, eye and follicle cells ( Figure 2). Significantly, the dominant effect of D-CblL-D70Z could be suppressed in drk EOA heterozygous mutant background ( Figure 2I), indicating that the interaction between Drk and D-CblL is required for the dose-dependent effect of D-CblL on EGFR signaling. Furthermore, the HS83-D-CblL-D70Z could not rescue the cbl F165 null mutant (data not shown), reinforcing the dependence of the negative role of D-CblL in EGFR signaling depends on the E3 activity.
We had previously shown that there is an increase of the endocytic Gurken (HRP-Gurken) in follicle cells after D-CblL over-expression, implying that D-CblL promotes the endocytosis of the Gurken-EGFR complex [29]. To clarify the requirement of ubiquitination in D-CblL-mediated ligand-receptor endocytosis, the distribution of HRP-Gurken in follicle cells was examined. The HRP-Gurken signal was abolished in follicle cells with D-CblL-D70Z over-expression ( Figure 3C, C9 and C0), whereas the signal was clearly detected in wild type or D-CblL expressing follicle cells (Figure 3 A and B). Taken together, our results showed that the E3 activity of D-CblL plays an essential role in promoting ligand-receptor endocytosis.

The D-CblL fusion proteins down-regulate EGFR signaling
Based on the functional implications of the D-CblL's interaction with Drk and E3 ligase activity, we therefore aimed to test next whether these two functional domains are sufficient to effectively down-regulate EGFR. A fusion protein containing the RING finger domain and the PR motif of D-CblL was generated (Figure 4 A). We expected that the fusion protein could interact with EGFR through binding to Drk. In line with this notion, we also generated a chimeric protein that contained the RING finger domain of D-CblL and the SH2 domain of Drk (Figure 4 A). This RING-SH2 Drk chimera should be able to bind to the activated EGFR on pY1068 or pY1086 that can be recognized by the SH2 domain of Drk.
To test the effects of these fusion proteins on EGFR signaling, ectopic expression of UAS-Flag-RING-SH2 Drk and of UAS-RING-PR were induced in follicle cells by GR1-Gal4 at 29uC and 32uC, respectively ( Figure S1). At 25uC, only low levels of D-Cbl fusion proteins were expressed, resulting in a slight defect on the eggshell morphology (data not shown). High-level expression of RING-SH2 Drk at 29uC resulted in significantly reduced EGFR signaling, and about half of the eggshells showed an intermediate ventralization phenotype indicated by the fusion of two dorsal appendages (Table 3 and Figure 1C). The effects were correlated with expression levels of the fusion protein ( Figure S1). Even though expression of RING-PR had a weaker effect than that caused by expression of RING-SH2 Drk , high level expression of RING-PR, induced by hsGal4 ( Figure S1D), also effectively downregulated EGFR signaling and caused about 1/3 of the eggshells to display the intermediate ventralization phenotype (Table 3, Figure 1C). Consistent with our previous observations [29], Gurken distribution outside of the follicle cells was also reduced when RING-SH2 Drk or RING-PR were over-expressed in follicle cells, similar to the effects caused by D-CblL over-expression ( Figure 4). These results showed that these small fusion proteins, as well as full length D-CblL, could down-regulate EGFR signaling in a dose-dependent manner. Furthermore, RING-SH2 Drk and RING-PR can promote the internalization of ligand-receptor complexes and lead to a reduction of extracellular Gurken distribution.

RING-SH2 Drk down-regulates EGFR signaling through endosomal sorting and competition with Drk
We previously demonstrated that D-CblL promotes the internalization of the Grk/EGFR complex via the Rab5/Rab7 endocytic pathway [29]. To determine the route in endosomal trafficking of RING-SH2 Drk -mediated endocytosis, we assayed the HRP-Grk/EGFR complex using the anti-HRP antibody in follicle cells expressing the RING-SH2 Drk chimera protein. More HRP-Grk signals were co-localized with Rab5-GFP and Rab7-GFP in follicle cells expressing either D-CblL ( Figure 5E Because the SH2 domain of RING-SH2 Drk was derived from Drk, we assumed that the docking site for RING-SH2 Drk on EGFR is the same as that for Drk. Therefore, this chimeric protein might compete with endogenous Drk for binding to EGFR. This possibility was tested by immunoprecipitation using anti-EGFR antibodies to determine the amount of Drk in the receptor complex. 40% of Drk in the EGFR complex was reduced in egg chambers expressing RING-SH2 Drk or full length D-CblL, compared to the wild-type egg chambers ( Figure 6). This result indicates that RING-SH2 Drk interferes with the interaction between endogenous Drk and EGFR, which may lead to reduced signal transduction. Taken together, the chimeric protein RING-SH2 Drk may down-regulate EGFR signaling through promoting the endosomal trafficking of the EGFR complex and reducing the recruitment of Drk/Sos in signal transduction.

Discussion
To dissect the molecular machinery for EGFR endocytosis, we studied the mechanism by which D-CblL promotes EGFR

Drk plays two roles in EGFR signaling in Drosophila oogenesis
Mammalian studies have reported that c-Cbl regulates EGFR signaling through its interacting molecules, such as Grb2, CIN85-Endophilin complex and Eps15 [21]. This study investigated the role of these molecules in D-CblL mediated regulation, and found that elimination of Drk interaction resulted in significant reduction of the effect of D-CblL over-expression (Table 1). In consideration of our data and results from previous studies, we conclude that the Grb2/Drk has dual roles in EGFR signaling both in mammals and Drosophila. Acting as a signaling transducer, Grb2 binds to the proline rich motif of RasGEF/Sos through the SH3 domain, leading to Ras activation and activation of MAPK cascade [46]. Similarly, loss of function of a drk mutant in Drosophila caused a ventralization of the egg, a phenotype representing hypoactivation of EGFR signaling [47]. In contrast, a recombinant SH2 domain of Grb2 inhibited EGFR endocytosis, indicating the requirement of Grb2 in EGFR endocytosis [48]. Here, we demonstrated that interaction between D-CblL and Drk is crucial for promoting EGFR endocytosis by D-CblL. Our protein interaction data in this study ( Figure 1F and Figure S2) agree with previous finding that Drk/Grb2 interacts with the prolinerich motif of Cbl [49,50]. In cell culture studies, the indirect binding of EGFR to Cbl through Grb2 is necessary for receptor internalization, whereas endosomal sorting requires direct binding to Cbl [31,35]. Our findings showed that the chimeric protein is sufficient to down-regulate EGFR signaling and suppresses the effect of l-top over-expression in Drosophila ovaries (Table 3 and 4). Expression of RING-SH2 Drk facilitated the endosomal trafficking of ligand-receptor complex through Rab5 (early) and Rab7 (late) endosomes, and MVBs ( Figure 5). This result argues that recruiting the RING domain of Cbl to EGFR by the SH2 domain of Drk can promote the trafficking of EGFR to degradation compartments, such as MVBs. Furthermore, expression of RING-SH2 Drk led to reduced Drk binding on EGFR ( Figure 6). This observation suggests that RING-SH2 Drk chimeric protein not only promotes the trafficking of EGFR to lysosomal degradation pathway, but also competes away the endogenous Drk, which may also contribute to reducing EGFR signaling. Interestingly, over-expression of D-CblL also reduced the binding of Drk to EGFR, suggesting that D-CblL might sequester Drk from binding to the receptor for signaling.
Importantly, the lethal effect of D-CblL was significantly reduced when the Drk binding motif was mutated in D-CblL (Table 2). This indicates that D-CblL efficiently down-regulates EGFR signaling through Drk. Large amounts of D-CblL in the cell may lead to comprised EGFR signaling levels that are too low for survival. However, even when we eliminated the interaction with Drk in the D-CblL-mPR mutant, this protein could still down-regulate l-top (Table S3). This result further demonstrated that D-CblL down-regulates EGFR through multiple mechanisms, and other D-CblL interacting molecules besides Drk might play important roles in down-regulating EGFR signaling even when Drk is absent.

The role of the RING finger domain in Cbl-mediated down-regulation of EGFR signaling
Our previous study demonstrated that D-cbl is required for down-regulation of EGFR signaling during DV patterning of the eggshell and embryo. Here we further showed that the RING finger domain of D-CblL is essential for its negative effect on EGFR, since the D-CblL mutant protein lacking this domain (D70Z-D-CblL) exhibited a dominant negative effect (Figure 2). In addition, the D70Z-D-CblL mutant also failed to rescue cbl F165 mutant, indicating that RING finger domain activity plays a major role in D-CblL function. These data are consistent with results from studies on the D-Cbl loss-of-function alleles by Wang et al. [13] and from research reports in mammals [42,51,52]. Ubiquitination has been considered as a signal to mediate EGFR endocytosis at two critical steps: receptor internalization and endosormal sorting [17,31,35,53,54]. D-CblL promotes EGFR endocytosis in a dose-dependent manner, but the endocytosis of the ligand-receptor complex was significantly reduced in D70Z-D-CblL over-expressing follicle cells. Interestingly, a cell culture system that expressed an EGFR mutant with a reduced ubiquitination level (to only 1%) still displayed normal internalization [53]. Therefore, one possibility is that the endocytic signal is not ubiquitination of EGFR itself [53], and that other molecules might be involved and ubiquitinated by Cbl. Cbl can directly bind the Y1045 residue of EGFR through its SH2 domain. However, the binding-deficient Y1045F EGFR mutant is internalized almost as efficiently as the wild-type EGFR, indicating that direct binding of Cbl to EGFR is not necessary for EGFR endocytosis [55]. Indeed, the chimeric protein (RING-SH2 Drk ) we generated promoted ligand-receptors endocytosis as well (Figure 4 and Figure 5). Therefore, we conclude that direct interaction between Cbl and EGFR may not be necessary for ligand-receptor endocytosis in vertebrate and invertebrate cells. Considering that Cbl acts as an adaptor in many signaling pathways [21], we were surprised to find that the RING finger domain, while recruited specifically to EGFR, is sufficient to down-regulate EGFR signaling. This finding implies the possibility of using the RING finger domain as a therapeutic tool in human diseases treatment.

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The egg collection was done at two temperatures, 29uC and 32uC, and expressions were driven by GR1-Gal4.

DNA constructs
To generate Flag-D-CblL, the D-CblL 328-3533 cDNA fragment was cloned into pUAST-Flag vector with BagII/KpnI sites. D-CblL-D70Z was generated by site-directed mutagenesis, which was designed according to the previously reported method [20]. To generate the D-CblL-mPR Drk mutant, we replaced the Pro 475 and Pro 479 with Ala using site-directed mutagenesis. The primers used include: Cbl-mut-2270-F 59-gtggtggctgctcccctgccagcccgaaagtcctcacc-39 and Cbl-mut-2270-R 59-ggtgaggactttcgggc tggcaggggagcagccaccac-3. To generate pUAST-Flag-RING, the RING finger domain of D-CblL was amplified by PCR using the following primers: RING-F-KpnI 59-aaggtaccggaccac ataaccgttacccaagag-39 and RING-R-EcoRI 59-aagaattctcagtgtcgtcttcttcc-39. The RING fragment was cloned into pUAST-Flag using KpnI and EcoRI sties and was in-frame fused to 39 of the Flag-tag. To generate pUAST-Flag-RING-SH2 Drk , the SH2 domain of Drk (amino acid 53 to 160) was amplified from the Drosophila ovarian cDNA library by PCR using the following primers: SH2-F-RI 59aagaattcaatagaaatgaagaatcacgactggtat-39 and SH2-R-XbaI 59aatctagacagcgcctg cacgag-39. The SH2 Drk fragment was cloned into pUAST-Flag-RING using EcoRI and XbaI sties and was inframe fused to 39 of the RING finger domain. To generate the pUAST-Flag-RING-PR, the following two poly-nucleotides PR-F 59-aattcacctcccctgccaccccgat-39 and PR-R 59-ctagatcggggtggcaggggaggtg-39 were synthesized in vitro, and they were ligated to the pUAST-Flag-RING. Therefore, the PR motif is in-frame fused with the RING domain.

Yeast two-hybrid analyses
The full-length Drk was amplified by PCR from the Drosophila ovarian cDNA library and cloned into the yeast expression plasmid pGBDT7 (Clontech) to fuse with the DNA-binding domain of GAL4 protein. The pGBKT7 vector was the negative control bait. The proline-rich domain of D-CblL was amplified by PCR and cloned into the yeast expression plasmid pGADT7 to fuse with the activation domain of GAL4 protein. The bait vector carrying the TRP1 gene and the prey vector carrying the LEU2 gene were for auxotrophic selection. The pCL1 plasmid carrying full length GAL4 protein was used as a positive control. The pGBDT7-Drk was co-transformed into an YH109 yeast strain (Clontech) either with pGADT7-mPR, or pGADT7-wild-type-PR or pGADT7 vector, and the transformants were grown on leucinetryptophan double selective plates.  *The eggshell pattern of Wt, V1, V2 and V3 were the same as that described in Table1. WtD indicated extra dorsal appendage appeared in the dorsa-lateral region; D1 indicated two dorsal appendages appeared in lateral region, and the dorsal midline area was expanded. D2 indicated the dorsalized eggshell with dorsal appendage around anterior of egg.

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The egg collection was done at 29uC, and expressions were driven by EQ1-Gal4. doi:10.1371/journal.pone.0017097.t004 Flag-RING-PR is about 14kD, and Flag-RING-SH2 Drk is about 25kD. (TIF) Figure S2 Drk interacts with the PR motif of D-CblL. (A) The interaction between Drk and D-CblL was analyzed by a yeast-two hybrid system. Yeasts are co-transformed with pGFBKT7-Drk-FL and pCL1 (as a positive control), pGADT7 vector (as a negative control), wild-type or mPR proline-rich domain. (B) On the G2 selection plate, the duplicated experiments show the successful transformation of each line. (C) On the G3 selection plate, yeast containing the PR mutant or the pGADT7 vector could not grow, whereas yeast containing the wild-type or pCL1 plasmid could grow. (TIF) Table S1 Over-expression of D-CblL has little genetic interaction with D-eps15 and D-endophilin B. (DOC)