The basic amino acids in the coiled-coil domain of CIN85 regulate its interaction with c-Cbl and phosphatidic acid during epidermal growth factor receptor (EGFR) endocytosis
© Zheng et al.; licensee BioMed Central Ltd. 2014
Received: 14 April 2014
Accepted: 3 July 2014
Published: 8 July 2014
During EGFR internalization CIN85 bridges EGFR-Cbl complex, endocytic machinery and fusible membrane through the interactions of CIN85 with c-Cbl, endophilins and phosphatidic acid. These protein-protein and protein-lipid interactions are mediated or regulated by the positively charged C-terminal coiled-coil domain of CIN85. However, the details of CIN85-lipid interaction remain unknown. The present study suggested a possible electric interaction between the negative charge of phosphatidic acid and the positive charge of basic amino acids in coiled-coil domain.
Mutations of the basic amino acids in the coiled-coil domain, especially K645, K646, R648 and R650, into neutral amino acid alanine completely blocked the interaction of CIN85 with c-Cbl or phosphatidic acid. However, they did not affect CIN85-endophilin interaction. In addition, CIN85 was found to associate with the internalized EGFR endosomes. It interacted with several ESCRT (Endosomal Sorting Complex Required for Transport) component proteins for ESCRT assembly on endosomal membrane. Mutations in the coiled-coil domain (deletion of the coiled-coil domain or point mutations of the basic amino acids) dissociated CIN85 from endosomes. These mutants bound the ESCRT components in cytoplasm to prevent them from assembly on endosomal membrane and inhibited EGFR sorting for degradation.
As an adaptor protein, CIN85 interacts with variety of partners through several domains. The positive charges of basic amino acids in the coiled-coil domain are not only involved in the interaction with phosphatidic acid, but also regulate the interaction of CIN85 with c-Cbl. CIN85 also interacts with ESCRT components for protein sorting in endosomes. These CIN85-protein and CIN85-lipid interactions enable CIN85 to link EGFR-Cbl endocytic complex with fusible membrane during EGFR endocytosis and subsequently to facilitate ESCRT formation on endosomal membrane for EGFR sorting and degradation.
KeywordsCIN85 EGFR endocytosis Phosphatidic acid Coiled-coil ESCRT
CIN85 is identified as a Cbl-interacting protein of 85 kDa and belongs to adaptor/scaffold proteins [1, 2]. It consists of three Src homology (SH3) domains, a proline-rich region and a putative α-helical coiled-coil domain at the extreme C-terminal end . As an adaptor protein, CIN85 is involved in Cbl-dependent EGFR internalization, intracellular receptor trafficking, sorting and degradation [3–8]. However, the detailed mechanisms by which CIN85 mediates EGFR endocytic process are not fully defined.
The activation of EGFR by EGF leads to the binding and ubiquitylation of the receptor by c-Cbl, which then recruits CIN85 to EGFR-Cbl complex to initiate the receptor endocytosis [3, 9–11]. CIN85, on the other hand, constitutively binds to endophilins, the regulatory proteins that control endocytosis through clathrin-coated pits [3, 4, 12, 13]. Thus, CIN85 tethers EGFR-Cbl complex to the endocytic machinery in an EGF-dependent manner . After internalization, c-Cbl further catalyzes the receptor multi-ubiquitylation and CIN85 mono-ubiquitylation in endosomes [6, 14]. The ubiquitylated EGFR is then sorted into multivesicular bodies and fused into lysosomes for degradation [15–17].
EGFR internalization and degradation is enhanced by overexpression of either phospholipase D1 or D2, and inhibited by expression of inactive phospholipase D mutants or by treatment of cells with primary alcohols . The function of phospholipase D in facilitating membrane endocytosis has largely been attributed to its ability to generate phosphatidic acid, a fusogenic lipid that lowers the activation energy required for inward curving of membrane [19, 20]. Upon EGF stimulation, phospholipase D is activated in plasma membrane and the production of phosphatidic acid, which enhances membrane fusibility for endocytosis, is increased [19, 21].
In our previous study, it is identified that CIN85 binds to phosphatidic acid . This ability enables CIN85 to target phosphatidic acid-enriched membrane. By interacting with EGFR-Cbl complex, endophilins and phosphatidic acid, CIN85 acts as a mediator to bring the receptor, endocytic machinery and fusible membrane together. It is further identified that deletion of the coiled-coil domain blocks CIN85-phosphatidic acid interaction, weakens CIN85-Cbl interaction, dissociates CIN85 from membrane and reduces EGFR degradation .
The coiled-coil domain is well-known for its ability to form oligomers in coiled-coil domain-containing proteins, including CIN85 [22–24]. By adding chemical cross-linking reagents, CIN85 is found to form oligomers through its coiled-coil domain [22, 23]. Of 70 amino acid residues in the coiled-coil domain, 16 residues are basic amino acids (K or R) . The basic amino acid frequency (23%) is much higher than that in an average protein. Because phosphatidic acid is a negatively charged lipid, the CIN85-phosphatidic acid interaction could be mediated by electric interaction between the negative charge of the lipid and the positive charge of basic amino acids in coiled-coil domain.
In the present study, we found that the basic amino acids in the coiled-coil domain were essential for CIN85-phosphatidic acid interaction. Of 16 K and R residues in the coiled-coil domain, K645, K646, R648 and R650 were the most important. The alanine mutation of these 4 basic amino acids inhibited CIN85-phosphatidic acid interaction, impaired the recruitment of CIN85 by c-Cbl, dissociated CIN85 from EGFR endosomes and reduced EGFR degradation.
K645, K646, R648 and R650 in coiled-coil domain are essential for CIN85-phosphatidic acid interaction and membrane association
K645, K646, R648 and R650 are required for the recruitment of CIN85 by EGFR endocytic complex
Through the constitutive CIN85-endophilin interaction, CIN85 links EGFR-Cbl complex with endocytic machinery of clathrin-coated pits [3, 13]. Because CIN85-endophilin interaction is mediated by the C-terminal part of CIN85, deletion of coiled-coil domain might disrupt the endophilin interacting region in CIN85 C-terminus . That was the case as deletion of the coiled-coil domain disrupted CIN85-endophilin interaction (Figure 3B). The less disruptive point mutations of the basic amino acids in coiled-coil domain revealed that CIN85-endophilin interaction was independent from the positive charge in coiled-coil domain. There was no difference for endophilin to interact with CIN85, CIN85-A4m, CIN85-B4m or other mutants (Figure 3B).Since CIN85 is recruited by c-Cbl in EGFR-Cbl complex, the interaction between CIN85 and c-Cbl should not affect EGFR-Cbl complex. As shown in Figure 3C, no matter whether the mutants could interact with c-Cbl (CIN85-B4m) or not (CIN85-ΔCC or CIN85-A4m), the presence of CIN85 mutants had no effect on EGFR-Cbl interaction.
Mutation of K645, K646, R648 and R650 dissociates CIN85 from EGFR endosomal membrane and affects the protein sorting process
After EGFR internalization, CIN85 or the neutral mutant CIN85-B4m was found to be associated with EGFR endosomes (Figure 4A). Both of them exhibited similar endosome binding (Figure 4B). In contrast, CIN85 null-function mutants (CIN85-ΔCC, CIN85-16m or CIN85-A4m) did not associated with EGFR endosomes (Figure 4A and B). The internalized EGFR is sorted by the assembly of ESCRT (Endosomal Sorting Complex Required for Transport) on endosomal membrane . Vps (vacuolar protein sorting) is a group of ESCRT components that are recruited to endosomal membrane by adaptor proteins . It is possible that CIN85 on endosomal membrane interacts with these ESCRT components to facilitate ESCRT assembly.
There was only weak interaction between CIN85 and CHMP4B (Figure 5A and C), although they were co-localized in cells (Figure 5D) . In comparison to the interactions with other ESCRT proteins [Vps4m, CHMP2A (Vps2A) and CHMP4C (Vps32C)], CIN85-CHMP4B interaction was negligible (Figure 5C). Little amount CHMP4B was co-precipitated with either CIN85 or CIN85-ΔCC (Figure 5A). On the other hand, CHMP2A (Vps2A) and CHMP4C (Vps32C), two core members in ESCRT-III that are assembled in sequential manner on endosomal membrane, interacted with CIN85 strongly (Figure 5C).
In ESCRT complex not all the components are in similar position to contact with CIN85 . The domains of CIN85 involved in the interaction varied with different Vps proteins. The interaction of CIN85 with CHMP2A was mediated by the C-terminus containing the proline-rich region, as CHMP2A was colocalized with CIN85 C-terminus, but not N-terminal SH3 domains (Figure 6C). In contrast, the interaction between CIN85 and Vps4 was most likely mediated by the N-terminal SH3 domains (Figure 6D). Vps4m was colocalized with N-terminal SH3 domains, but not C-terminus containing the proline-rich region (Figure 6D). Whatever the differences were in the interactions between CIN85 and Vps proteins, they were all independent from the coiled-coil domain (Figure 5C). As CIN85 with mutations in coiled-coil domain (CIN85-ΔCC, CIN85-16m or CIN85-A4m) dissociated from EGFR endosomes, their interaction with Vps proteins in cytoplasm prevented the Vps proteins to interact with EGFR-Cbl-CIN85 complex on endosomal membrane. Thus, the presence of exogenous CIN85 mutants interfered with EGFR sorting process in endosomes and less EGFR was sorted to lysosome for degradation.
The recruitment of CIN85 by c-Cbl in EGFR-Cbl complex has been indicated as the initial step for EGFR internalization . The interaction of CIN85 with c-Cbl is mediated by the SH3 domains in CIN85 and a novel proline-arginine motif present in the distal carboxyl-terminus of Cbl [3, 22, 23, 29]. Interestingly, c-Cbl can bind to wild type CIN85 or CIN85 fragment containing only the SH3 domains (CIN85-SH3), but cannot bind to CIN85 with coiled-coil domain deletion (CIN85-ΔCC) [3, 22]. The SH3 domains in CIN85-ΔCC appear to be blocked for c-Cbl interaction, possibly due to the conformational change in CIN85 lacking the coiled-coil domain . However, the detailed mechanism by which the deletion of coiled-coil domain affects CIN85-Cbl interaction is still not clear. It is possible that without coiled-coil domain the proline-rich region in CIN85 may engage in auto-interaction with the SH3 domains to prevent them to bind c-Cbl . The difference of CIN85-SH3 and CIN85-ΔCC in c-Cbl binding leads to different effects on EGFR internalization. CIN85-SH3 has a dominant negative effect on EGF stimulated EGFR internalization , whereas CIN85-ΔCC exhibits no inhibitory effect on EGFR internalization (Figure 4). The interaction of CIN85-SH3 with c-Cbl competes off the binding of functional CIN85 to EGFR-Cbl complex. Due to the lack of C-terminal endophilin-interacting domains, CIN85-SH3 cannot tether EGFR-Cbl complex with endocytic machinery, blocking endophilin-mediated EGFR endocytosis. CIN85-ΔCC, on the other hand, cannot interact with c-Cbl and is a bystander to EGFR-Cbl complex. The endogenous CIN85 can still be recruited by c-Cbl to form EGFR-Cbl-CIN85 complex, which then recruits endocytic proteins for receptor internalization. Thus, binding of CIN85 mutant to c-Cbl is essential for the dominant negative inhibition of EGFR internalization.
The point mutations in the coiled-coil domain minimize the disruptive effect of protein truncation . In fact, the point mutation of the basic amino acids in the coiled-coil domain (CIN85-16m) is sufficient to disrupt CIN85-Cbl interaction (Figure 3A). It not only inhibits CIN85-Cbl interaction, but also blocks CIN85-phosphatidic acid interaction (Figures 1 and 3). The results from the mutations of basic amino acids in coiled-coil domain support the electric charge interaction between phosphatidic acid and CIN85 (Figure 1). K645, K646, R648 and R650 out of 16 basic amino acids in coiled-coil domain are the most important in phosphatidic acid binding, membrane association and c-Cbl interaction (Figure 1 and 3). Mutation of these four basic amino acids (CIN85-A4m) is sufficient to disrupt every CIN85 interactions except CIN85-endophilin interaction (Figure 3B).
The interactions with c-Cbl and phosphatidic acid enable CIN85 to couple EGFR endocytic complex with fusible membrane. The results from study of phospholipase D activation and EGFR endocytosis support this kind of association, since EGF stimulation induces the activation of phospholipase D as well as EGFR internalization . Phosphatidic acid is generated by phospholipase D in response to EGF or other growth factors [19–21]. The activation of phospholipase D increases phosphatidic acid concentration in plasma membrane, while the stimulation of EGFR triggers the c-Cbl binding to initiate the endocytic complex formation . By bridging these two events, CIN85 facilitates the endocytic complex formation at membrane area enriched with fusogenic lipid.
Reports have shown that knockdown of CIN85 by RNA interference has no significant effect on EGFR internalization, but rather delays the degradation of internalized EGFR [8, 11]. It is further identified that the internalized EGFR is recycled rather than degraded in CIN85-depleted cells . Depletion of CIN85 shifts the trafficking of internalized EGFR from the degradative to the recycling pathway. It seems to be that CIN85 is required for the sorting of the internalized EGFR. And CIN85 independent endocytic pathways are involved in EGFR internalization in CIN85-depleted cells.
During EGFR endocytosis, CIN85 tethers EGFR, phosphatidic acid and clathrin-coated pits to facilitate the receptor internalization. The interaction of CIN85 with EGFR-Cbl complex and phosphatidic acid targeted the internalized proteins to membrane areas enriched with fusogenic lipid. The coiled-coil domain, especially the basic amino acids in the domain, is the key regulatory element for the interactions of CIN85 with EGFR-Cbl as well as phosphatidic acid. After endocytosis, CIN85 in association with EGFR-Cbl complex on endosomal membrane recruits several ESCRT components to endosome for ESCRT assembly. Then the ESCRT complex on endosomal membrane induces the formation of multi-vesicular bodies to sort the internalized receptor for degradation or recycling.
All results of this research were based on cultured HEK293 or COS7 cells lines. Neither human (human subjects, human material or human data) nor animals (vertebrates or any regulated invertebrates) were used in this experimental research.
Anti-EGFR, anti-Cbl and anti-endophilins antibodies were purchased from Santa Cruz. Anti-CIN85 monoclonal antibody was from Upstate. Anti-flag monoclonal antibody, phosphatidic acid, DMP (dimethylpimelimidate) and horse radish peroxidase-conjuated secondary antibodies were from Sigma. EGF, Alexa Fluor 647 labeled EGF, PIP Strips™ membrane and fluorescence labeled secondary antibodies (Alexa Fluor 488/546 conjugates) for immunofluorescence were the products of Life Technologies. The leica laser scanning confocal microsystem, including the leica TCS SP2 confocal microscope, Leica confocal scanner, and Leica confocal acquisition software were used with the HCX PL APO 1bd. BL 63.0 X/1.4 oil objective at 1.4 numerical aperture at a working temperature of 22°C.
The truncation and point mutations of CIN85 were constructed by polymerase chain reaction amplification and the mutants were tagged with Flag-tag or eGFP at the N-terminus of the fusion protein. CIN85-ΔCC was the deletion of amino acid 601 to 665. CIN85-PRC was the fragment of amino acid 334 to 665. CIN85-CC was the coiled-coil domain of amino acid 594 to 665. CIN85-SH3 was the N-terminus of amino acid 1 to 331. The 16 basic amino acids in CIN85 from amino acid 596 to 665 (K596, K598, K613, R617, R620, K627, K631, R632, K635, K645, K646, R648, R650, K659, K660 and K665) were divided into several groups and mutated to alanine residues.
Protein expression, immunofluorescence staining, immunoprecipitation and western blot
CIN85 mutant plasmid was transiently transfected into HEK293 or COS7 cells and experiments were performed in 48 hours. For immunofluorescence staining, cells were fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS). EGFP fusion proteins and Fluor-labeled EGF were directly visualized by confocal microscope. Other proteins were stained with appropriate primary antibodies and Fluor-labeled secondary antibodies. For immunoprecipitation, two plasmids in 1:3 ratio (primary immunoprecipitated protein/co-precipitated protein) were co-transfected into cells and the cells were lysed in a buffer containing 1% Triton X-100, 10% glycerol, 20 mM Hepes, pH7.4, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF and 2 μl/ml protease inhibitor cocktails 1 and 2. The immunoprecipitation and western blots were conducted as described previously .
Cell fractionation for intracellular membrane vesicles
Cells were homogenized in isotonic buffer containing 20 mM Hepes, pH 7.4, 0.25 M sucrose, 1 mM EDTA, 2.5 mM dithiothreitol, 1 mM PMSF and 2 μl/ml protease inhibitor cocktails 1 and 2. The homogenized cell lysate was centrifuged at 12,000 × g for 15 min. The pellet was discarded and the supernatant was then ultracentrifuged at 100,000 × g for 90 minutes to separate intracellular membrane vesicles and cytoplasm.
Phosphatidic acid association
The eGFP-tagged CIN85-PRC or CIN85-CC was expressed in HEK293 cells and purified by immunoprecipitation. The immunopurified fusion protein was incubated with PIP StripsTM following the manufacturer’s instruction and detected by immunoblot with anti-eGFP antibody. The phosphatidic acid membrane was prepared by dotting nitrocellulose membrane with phosphatidic acid in chloroform solution. CIN85 mutants expressed in HEK293 cell were prepared by homogenizing the cell in buffer containing 20 mM Hepes, pH7.4, 150 mM NaCl, 2 mM MgCl2, 15 mM KCl, 1 mM EDTA, 1 mM PMSF and 2 μl/ml protease inhibitor cocktails 1 and 2. The mixture was then centrifuged at 12,000 × g for 15 min and the supernatant was used to blot the phosphatidic acid membrane. The proteins were detected by anti-Flag antibody.
Downregulation and internalization of EGFR
COS7 cells were transfected with CIN85 mutant plasmid, serum-starved and stimulated with 25 ng/ml EGF. The cells were harvested at various time points and EGFR protein was detected by Western blot. The analysis of EGFR internalization followed the protocol developed by Doyotte et al.  and Raiborg et al. . Briefly, COS7 cells were transfected with CIN85 mutants. After 72 hours, the cells were serum-starved overnight and incubated with Alexa Fluor-labeled EGF (200 ng/ml in Eagle’s minimal essential medium supplemented with 20 mM Hepes and 2 mg/ml bovine serum albumin) on ice for 30 minutes. Cells were then washed with pre-warmed binding buffer (37°C) three times and incubated in binding buffer for indicated length of time before fixation with 4% PFA.
Chemical cross-link by dimethylpimelimidate (DMP)
HEK293 cells expressing CIN85 mutants were lysed in 0.2 M triethanolamine (pH 8.0). 100 μM DMP was added to the lysate and the mixture was incubated for 1 hour at 4°C. The cross-linking reaction was terminated by adding 100 mM Tris–HCl (pH 7.5). The samples were subjected to SDS-PAGE for western blot.
Cbl-interacting protein of 85 kDa
Casitas B-lineage lymphoma proto-oncoprotein
Epidermal growth factor receptor
Endosomal sorting complex required for transport
Src homology domain
Coiled-coil domain deletion
Proline-rich C-terminal region
Vacuolar protein sorting
Charged multivesicular body protein.
This work was supported by the Ministry of Science and Technology of China [grant numbers 2010CB912102], and the China National Nature Sciences Foundation [grant numbers 31370818].
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