K645, K646, R648 and R650 in coiled-coil domain are essential for CIN85-phosphatidic acid interaction and membrane association
It was previously identified that the interaction of CIN85 with phosphatidic acid required the C-terminal coiled-coil domain [22]. In fact, coiled-coil domain alone was sufficient to bind phosphatidic acid (Figure 1A). In total 70 amino acid residues of coiled-coil domain there are 11 lysine and 5 arginine residues (Figure 1B). The enriched basic amino acid composition enables the coiled-coil domain to be positively charged in a neutral pH environment and interact with negatively charged proteins or lipids.These 16 basic amino acids naturally form 5 clusters, which were divided into 4 groups for generating mutations: Group C includes K596, K598, K613, R617 and R620; Group B K627, K631, R632 and K635; Group A K645, K646, R648 and R650; Group D K659, K660 and K665 (Figure 1B). They were mutated into alanine, a neutral amino acid, to verify which of these basic amino acids are involved in CIN85-phosphatidic acid interaction (Figure 1B). The mutation of all 16 basic amino acids (CIN85-16m) completely blocked CIN85-phosphatidic acid interaction (Figure 1C). It also inhibited CIN85 oligomerization or the association of CIN85 with membrane vesicles (Figure 1D and E). Essentially, mutation of these 16 basic amino acids exhibited the same effect as deletion of the coiled-coil domain (Figure 1).Mutations of the basic amino acids in individual groups revealed that the basic amino acids in Group A (K645, K646, R648 and R650) were the most important for coiled-coil domain (Figure 1C, D and E). Mutation of these 4 basic amino acids (CIN85-A4m) exhibited the same inhibitory effect as mutation of all 16 basic amino acids. Mutations in other groups (CIN85-C5m, B4m and D3m) did not affect or only partially inhibited the interaction of CIN85 with phosphatidic acid (Figure 1C).
The association of CIN85 with membrane vesicles was most likely mediated by its interaction with phosphatidic acid (Figure 1C and D). Mutations that inhibited CIN85-phosphatidic acid interaction also disrupted CIN85-membrane vesicle association (Figure 1C and D). In fact, CIN85 only associates with liposome containing phosphatidic acid (10%) and phosphatidyl choline (90%), but not liposome of phosphatidyl choline alone [22]. The association with membrane might also facilitate the alignment of CIN85 molecules on membrane surface, enabling them to oligomerize (Figure 1E).Further dissection of the basic amino acids in Group A revealed that the presence of positive charges in this group was more important than the position of these positive charges. Mutations of 2 basic amino acids (K645/646A), (K646/R648A) or (R648/650A) did not affect phosphatidic acid binding, membrane association or oligomerization (Figure 2). Mutations of 3 basic amino acids had mixed effect on the coiled-coil domain. Mutation of K646, R648 and R650 did not affect phosphatidic acid binding, membrane association or oligomerization, whereas mutation of K645, K646 and R648 inhibited these functions (Figure 2B-D). In addition, mutation of either one of these four basic amino acids exhibited no inhibitory effect (results not shown). Overall, there was no single basic amino acid in K645, K646, R648 and R650 that was more prominent than the others.
K645, K646, R648 and R650 are required for the recruitment of CIN85 by EGFR endocytic complex
EGFR endocytosis requires the coordinated interaction among the receptor complex, endocytic machinery and membrane. As an adaptor protein, CIN85 is recruited by c-Cbl to EGFR-Cbl complex [3]. The interaction of CIN85 with c-Cbl is mediated by SH3 domains in CIN85 [22, 23]. No direct interaction between c-Cbl and the coiled-coil domain of CIN85 has been identified [22]. Although the coiled-coil domain did not interact with c-Cbl, deletion of this domain led to the impairment of CIN85-Cbl interaction (Figure 3A).Because the basic amino acids in coiled-coil domain were involved in CIN85-lipid interaction (Figure 1), their role in CIN85-Cbl interaction was investigated. As shown in Figure 3A, mutations of the basic amino acids had strong inhibitory effect on CIN85-Cbl interaction. Except mutation in Group B (CIN85-B4m), which did not affect CIN85-Cbl interaction, all the other mutations (CIN85-16m, A4m, C5m and D3m) inhibited CIN85-Cbl interaction (Figure 3A). For most CIN85 mutants, the effect on CIN85-Cbl interaction was parallel to that on CIN85-phosphatidic acid interaction. CIN85-16m, A4m and D3m affected both phosphatidic and c-Cbl binding, while CIN85-B4m inhibited neither of them (Figure 1C and 3A). CIN85-C5m was the only exception that it inhibited the binding to c-Cbl, but not to phosphatidic acid (Figures 1C and 3A).
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 [3]. 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
In COS7 cells expressing CIN85 null-function mutant (CIN85-ΔCC or CIN85-A4m), EGF-stimulated EGFR degradation was decreased, whereas in cells expressing neutral mutant (CIN85-B4m) or wild type CIN85 the ligand-stimulated receptor degradation was not affected (Figure 3D and E). However, no difference in EGFR internalization was observed in COS7 cells expressing eGFP, CIN85 or CIN85 mutant (neutral or null-function) (Figure 4A). Thus, the expression of CIN85 null-function mutant did not inhibit the receptor internalization per se (Figure 4A). It only reduced the degradation of the internalized EGFR (Figure 3D and E). The inhibition of EGFR degradation but not EGFR internalization suggested that these CIN85 null-function mutants interfere with EGFR sorting/degradation 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 [25]. Vps (vacuolar protein sorting) is a group of ESCRT components that are recruited to endosomal membrane by adaptor proteins [26]. It is possible that CIN85 on endosomal membrane interacts with these ESCRT components to facilitate ESCRT assembly.
In our previous study, CIN85 was found to be co-localized with Vps4, Vps4m and CHMP4B (Vps32B) [22]. Moreover, coiled-coil domain deletion (CIN85-ΔCC) did not affect their cellular co-localization (Figure 5D-F). There were also protein interactions between CIN85 and Vps4, Vps4m and CHMP4B (Vps32B) (Figure 5A and B). Vps4 is a cytoplasmic ATPase that is recruited to ESCRT-III to regulate ESCRT dissociation [27]. It weakly interacted with CIN85 (Figure 5B) [22]. However, its ATPase-negative mutant, Vps4m that constitutively associates with endosomal membrane [28], dramatically increased its interaction with CIN85 (Figure 5B). For both Vps4 and Vps4m, their interaction with CIN85 or cellular co-localization was independent from the coiled-coil domain (Figure 5B,C,E and F). In addition, Vps4m appeared to be associated with intracellular membrane vesicles, whereas the wild type Vps4 was mostly cytoplasmic protein (Figure 6B).
There was only weak interaction between CIN85 and CHMP4B (Figure 5A and C), although they were co-localized in cells (Figure 5D) [22]. 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 [25]. 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.