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.

Xiudan Zheng,Kan Liao,Jing Zhang

doi:10.1186/1471-2091-15-13

Abstract

BackgroundDuring 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.ResultsMutations 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.ConclusionsAs 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.

Highlights

During Epidermal growth factor receptor (EGFR) internalization Casitas B-lineage lymphoma proto-oncoprotein (Cbl)-interacting protein of 85 kDa (CIN85) bridges EGFR-Cbl complex, endocytic machinery and fusible membrane through the interactions of Cbl-interacting protein of 85 kDa (CIN85) with c-Cbl, endophilins and phosphatidic acid
We found that the basic amino acids in the coiled-coil domain were essential for CIN85phosphatidic acid interaction
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 coiledcoil domain [22]

Summary

Introduction

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. 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 [1]. The ubiquitylated EGFR is sorted into multivesicular bodies and fused into lysosomes for degradation [15,16,17]

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