Identification of an FHL1 Protein Complex Containing Gamma-Actin and Non-Muscle Myosin IIB by Analysis of Protein-Protein Interactions

Lili Wang,Lianyong Li,Zhengwei Yuan,Lijun Zhang,Kailai Sun,Yi Zhang,Di Wu,Jianing Miao,Zhaohong Peng,Leonard Eisenberg

doi:10.1371/journal.pone.0079551

Abstract

FHL1 is multifunctional and serves as a modular protein binding interface to mediate protein-protein interactions. In skeletal muscle, FHL1 is involved in sarcomere assembly, differentiation, growth, and biomechanical stress. Muscle abnormalities may play a major role in congenital clubfoot (CCF) deformity during fetal development. Thus, identifying the interactions of FHL1 could provide important new insights into its functional role in both skeletal muscle development and CCF pathogenesis. Using proteins derived from rat L6GNR4 myoblastocytes, we detected FHL1 interacting proteins by immunoprecipitation. Samples were analyzed by liquid chromatography mass spectrometry (LC-MS). Dynamic gene expression of FHL1 was studied. Additionally, the expression of the possible interacting proteins gamma-actin and non-muscle myosin IIB, which were isolated from the lower limbs of E14, E15, E17, E18, E20 rat embryos or from adult skeletal muscle was analyzed. Potential interacting proteins isolated from E17 lower limbs were verified by immunoprecipitation, and co-localization in adult gastrocnemius muscle was visualized by fluorescence microscopy. FHL1 expression was associated with skeletal muscle differentiation. E17 was found to be the critical time-point for skeletal muscle differentiation in the lower limbs of rat embryos. We also identified gamma-actin and non-muscle myosin IIB as potential binding partners of FHL1, and both were expressed in adult skeletal muscle. We then demonstrated that FHL1 exists as part of a complex, which binds gamma-actin and non-muscle myosin IIB.

Highlights

The Fhl1 gene is located on chromosome Xq36, and encodes four-and-a-half LIM protein-1 (FHL1) and its spliced isoforms, SLIMMER and FHL1C [1]
Mice lacking Fhl1 were protected from the onset of hypertrophic cardiomyopathy, which is normally induced by biomechanical stress, whereas transgenic expression of Fhl1 in mice promoted skeletal muscle hypertrophy [13,16,17,18,19,20]
Twentyseven mutations have been identified in the FHL1 gene that contribute to the development of six different myopathies, each of which present a combination of various protein aggregates, joint contractures, muscle atrophy/hypertrophy, and cardiovascular diseases [4]

Summary

Introduction

The Fhl gene is located on chromosome Xq36, and encodes four-and-a-half LIM protein-1 (FHL1) and its spliced isoforms, SLIMMER and FHL1C [1]. Twentyseven mutations have been identified in the FHL1 gene that contribute to the development of six different myopathies, each of which present a combination of various protein aggregates, joint contractures, muscle atrophy/hypertrophy, and cardiovascular diseases [4]. These observations suggest that FHL1 plays an important role in muscle growth and development

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Discussion

Conclusion