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

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Figure 1

K645, K646, R648 and R650 in coiled-coil domain are essential for CIN85-phosphatidic acid interaction and membrane association. (A) Interaction of the coiled-coil domain with phosphatidic acid. The coiled-coil domain (CC) and the C-terminal fragment (PRC) were eGFP-tagged, expressed and purified from HEK293 cells to blot PIP Strip^TM membrane. The phospholipids on the PIP Strip^TM membrane are: 1, lysophosphatidic acid; 2, lysophosphatidylcholine; 3, phosphatidylinositol; 4, PtdIns(3)P; 5, PtdIns(4)P; 6, PtdIns(5)P; 7, phosphatidylethanolamine; 8, phosphatidylcholine; 9, sphingosine 1-phosphate; 10, PtdIns(3,4)P; 11, PtdIns(3,5)P; 12, PtdIns(4,5)P; 13, PtdIns(3,4,5)P; 14, phosphatidic acid; 15, phosphatidylserine; 16, blank. (B) The point mutations of the basic amino acids in the coiled-coil domain and other truncation mutations. The 16 basic amino acids in coiled-coil domain were divided into 4 groups (C5, B4, A4 and D3) and were mutated to alanine (A). (C) Interaction of CIN85 mutants with phosphatidic acid. Phosphatidic acid (PA) was dotted on nitrocellulose membrane at 1 and 10 nmole. The Western blot showed the relative protein level of each mutant used to blot the phosphatidic acid membrane. The mutants were Flag-tagged. (D) The association of CIN85 mutants with intracellular membrane vesicles. M, the membrane fraction of ultracentrifugation; C, the cytosolic fraction of ultracentrifugation. (E) Chemical cross-link of CIN85 mutants by dimethylpimelimidate (DMP). +DMP, cell lysate was treated with DMP; −DMP, control cell lysate. The numbers indicate the molecular weight markers.

Figure 2

The net positive charges in group A (K645, K646, R648 and R650) are more important than their positions. (A) The construction of point mutations in group A. (B) Interaction of the mutants with phosphatidic acid. (C) The association of the mutants with intracellular membrane vesicles. (D) Chemical cross-link of CIN85 mutants by dimethylpimelimidate (DMP). The labels in this figure are the same as in Figure 1.

Figure 3

The mutation of K645, K646, R648 and R650 disrupts CIN85-Cbl interaction and inhibits EGFR degradation. (A) Interaction of CIN85 mutants with c-Cbl. HA-tagged c-Cbl and Flag-tagged CIN85 mutants were co-expressed in HEK293 cells and HA-Cbl was immunoprecipitated by anti-HA antibody (IP: α-HA-Cbl). CIN85 mutants were detected by immunoblot with anti-Flag antibody (IB). (B) Interaction of CIN85 mutants with endophilins. CIN85 mutants were expressed in COS7 cells and endogenous endophilins were immunoprecipitated by anti-endophilin antibody. (C) Interaction of EGFR and c-Cbl in the presence of CIN85 mutants. COS7 cells co-expressing CIN85 mutant and HA-Cbl were serum starved and stimulated with 25 ng/ml EGF for 30 minutes. HA-Cbl was immunoprecipitated by anti-HA antibody. CIN85 mutants and EGFR were detected by immunoblot with anti-Flag antibody and anti-EGFR antibody respectively. (D) Inhibition of EGFR downregulation by CIN85-ΔCC or CIN85-A4m. COS7 cells transfected with CIN85, CIN85-ΔCC, CIN85-A4m, CIN85-B4m or blank vector were serum starved and stimulated with 25 ng/ml EGF for indicated times (0, 15, 30, 60, 120, 180 min). Cells were harvested and EGFR was detected by Western blot. Tubulin was used as loading control. (E) Image gray statistical plot of EGFR downregulation in Figure 3D. Each gray level of EGFR electrophoretic band was normalized against relative EGFR level at 0 min. The final data were averaged from two independent western blot assays.

Figure 4

The mutation of K645, K646, R648 and R650 dissociates CIN85 from EGFR endosome. (A) EGF endocytosis analysis in eGFP/eGFP-CIN85 mutants transfected COS7 cells. COS7 cells expressing eGFP-tagged CIN85 mutant were incubated with 200 ng/ml Alexa Fluor 647 labeled EGF for 30 min on ice and then shifted to 37°C. At indicated time the cells were fixed and immunostained with anti-EGFR antibody. EGFP-tagged CIN85 mutant was visualized by eGFP fluorescence and the labeled EGF was visualized by the fluorescence of Alexa Fluor 647. A selected area is enlarged 6 times to reveal the details. The arrows point the white dots which are the colocalization of EGF (purple), EGFR (red) and eGFP-CIN85 mutant (green). The bar is 10 μm. (B) Plot of the percentage of EGF-EGFR endosomes with eGFP-CIN85 or mutant association. For each mutant, 10 cells were used to calculate white dots and total EGF-EGFR endosomes, and the percentage was normalized against the average percentage in eGFP-CIN85 expressing cell. Student’s t-test was performed. The P values between eGFP-CIN85 expressing cells and eGFP expressing cells or eGFP-CIN85 mutants expressing cells were ***9.34E-05 (10 min) and ***0.000272 (20 min) for eGFP; ***0.000187 (10 min) and **0.001979 (20 min) for eGFP-ΔCC; ***0.000252 (10 min) and **0.007448 (20 min) for eGFP-16 m; **0.001818 (10 min) and **0.002121 (20 min) for eGFP-A4m; 0.329283 (10 min) and 0.739651 (20 min) for eGFP-B4m.

Figure 5

Interactions of CIN85 with Vps proteins. (A) Interactions of CIN85 and CIN85-ΔCC with CHMP4B. Flag-tagged CIN85 and eGFP-tagged CHMP4B were co-expressed in HEK293 cells and Flag-CIN85 was immunoprecipitated by anti-Flag antibody. CHMP4B in immunoprecipitated sample was detected by Western blot. (B) Interactions of CIN85 and CIN85-ΔCC with Vps4 and Vps4m (ATPase negative mutant). Flag-tagged CIN85 and eGFP-tagged Vps4 were co-expressed in HEK293 cells and Flag-CIN85 was immunoprecipitated by anti-Flag antibody. Vps4 in immunoprecipitated sample was detected by Western blot. (C) Interactions of CIN85 and CIN85-ΔCC with different Vps proteins. The tagged CIN85 and Vps protein were co-expressed in HEK293 cells and CIN85 was immunoprecipitated. Vps proteins were detected by Western blot. (D) Co-localization of CIN85 and CIN85-ΔCC with CHMP4B. Flag-tagged CIN85 was co-expressed with eGFP-tagged CHMP4B in HEK293 cells. Cells were fixed with 4% PFA and observed by confocal microscopy. Τhe bar is 10 μm. (E, F) Co-localization of Flag-tagged CIN85 and CIN85-ΔCC with eGFP-tagged Vps4 and Vps4m.

Figure 6

Cellular colocalization of Vps4 and CHMP2A with CIN85 domains. Τhe bar is 10 μm. (A) Construction of fusion proteins of CIN85 fragment. CIN85 fragment was tagged with eGFP. (B) Intracellular localization of Vps4 and Vps4m (ATPase negative mutant). The proteins were tagged with DsRed and expressed in HEK293 cells. (C) Colocalization of CIN85 fragment with CHMP2A. EGFP-tagged CIN85 mutant and DsRed-tagged CHMP2A were co-expressed in HEK293 cells. The proteins were visualized by the tagged fluorescence protein. (D) Colocalization of CIN85 fragment with Vps4m.

Figure 7

Illustration of EGFR endocytosis process. The functions of CIN85 during EGFR internalization and sorting are depicted in this illustration. It is not a comprehensive model for EGFR endocytosis process, rather a model for understanding the functions of CIN85 in EGFR endocytosis process. PA, phosphatidic acid; endo-complex, endophilins and endocytic protein complex; MVB, multi-vesicular bodies.