Actin Cytoskeletal Reorganization Function of JRAB/MICAL-L2 Is Fine-tuned by Intramolecular Interaction between First LIM Zinc Finger and C-terminal Coiled-coil Domains

Kazuhisa Miyake,Issei Imoto,Yoko Tomida,Yuko Tsuchiya,Daisuke Higo,Jiro Kasahara,Kenji Mizuguchi,Shio Watanabe,Ikuko Sagawa,Ayuko Sakane,Takuya Sasaki

doi:10.1038/s41598-019-49232-8

Abstract

JRAB/MICAL-L2 is an effector protein of Rab13, a member of the Rab family of small GTPase. JRAB/MICAL-L2 consists of a calponin homology domain, a LIM domain, and a coiled-coil domain. JRAB/MICAL-L2 engages in intramolecular interaction between the N-terminal LIM domain and the C-terminal coiled-coil domain, and changes its conformation from closed to open under the effect of Rab13. Open-form JRAB/MICAL-L2 induces the formation of peripheral ruffles via an interaction between its calponin homology domain and filamin. Here, we report that the LIM domain, independent of the C-terminus, is also necessary for the function of open-form JRAB/MICAL-L2. In mechanistic terms, two zinc finger domains within the LIM domain bind the first and second molecules of actin at the minus end, potentially inhibiting the depolymerization of actin filaments (F-actin). The first zinc finger domain also contributes to the intramolecular interaction of JRAB/MICAL-L2. Moreover, the residues of the first zinc finger domain that are responsible for the intramolecular interaction are also involved in the association with F-actin. Together, our findings show that the function of open-form JRAB/MICAL-L2 mediated by the LIM domain is fine-tuned by the intramolecular interaction between the first zinc finger domain and the C-terminal domain.

Highlights

In our previous studies, junctional Rab13-binding protein (JRAB)/molecule interacting with CasL-like[2] (MICAL-L2) is identified as an effector protein of Rab[131], a member of the Rab family of small GTPases (Rab), which contributes to the regulation of membrane trafficking[2,3,4]
By a combination of bioinformatic and biochemical analyses, we generated a structural model that suggested that the hydrophobic or negatively charged region of the C-terminal part of JRAB/MICAL-L2 is involved in the interaction with the deduced structure of the N-terminal LIM domain and Rab[138]
We provided structural models for JRAB-C-Rab[13] and JRAB-C-JRAB-LIM using a combination of biochemical analysis and bioinformatics[8]

Summary

Introduction

Junctional Rab13-binding protein (JRAB)/molecule interacting with CasL-like[2] (MICAL-L2) is identified as an effector protein of Rab[131], a member of the Rab family of small GTPases (Rab), which contributes to the regulation of membrane trafficking[2,3,4]. JRAB/MICAL-L2 adopts a closed form via the intramolecular interaction between the N-terminal LIM domain and the C-terminal CC domain. Rab[13] competes with the LIM domain for a part of JRAB/MICAL-L2 C-terminus, leading to the conformational change of JRAB/MICAL-L2 from closed to open form. Closed-form JRAB/MICAL-L2 forms thick F-actin bundles along the leading edge, followed by generation of traction force that pulls the cell population[7,8]. To understand how JRAB/MICAL-L2 performs its various functions, it is necessary to establish a linkage between its phenotypes and conformations To this end, we focused on the LIM domain of JRAB/MICAL-L2, for the following reasons. We concluded that the function of open-form JRAB/MICAL-L2 mediated by the LIM domain is fine-tuned by the intramolecular interaction between the first zinc finger domain and the C-terminal domain

Methods

Results

Conclusion