GTP binding controls complex formation by the human ROCO protein MASL1

Sybille Dihanich,Adamantios Mamais,Rina Bandopadhyay,Claudia Manzoni,Patrick A Lewis,Laura Civiero,Elisa Greggio

doi:10.1111/febs.12593

Abstract

The human ROCO proteins are a family of multi-domain proteins sharing a conserved ROC-COR supra-domain. The family has four members: leucine-rich repeat kinase 1 (LRRK1), leucine-rich repeat kinase 2 (LRRK2), death-associated protein kinase 1 (DAPK1) and malignant fibrous histiocytoma amplified sequences with leucine-rich tandem repeats 1 (MASL1). Previous studies of LRRK1/2 and DAPK1 have shown that the ROC (Ras of complex proteins) domain can bind and hydrolyse GTP, but the cellular consequences of this activity are still unclear. Here, the first biochemical characterization of MASL1 and the impact of GTP binding on MASL1 complex formation are reported. The results demonstrate that MASL1, similar to other ROCO proteins, can bind guanosine nucleotides via its ROC domain. Furthermore, MASL1 exists in two distinct cellular complexes associated with heat shock protein 60, and the formation of a low molecular weight pool of MASL1 is modulated by GTP binding. Finally, loss of GTP enhances MASL1 toxicity in cells. Taken together, these data point to a central role for the ROC/GTPase domain of MASL1 in the regulation of its cellular function.Structured digital abstractHSP60 and MASL1 physically interact by molecular sieving (View interaction)MASL1 physically interacts with HSP70 and HSP60 by anti tag coimmunoprecipitation (View interaction)MASL1 physically interacts with HSP60 by anti tag coimmunoprecipitation (View interaction)

Highlights

Malignant fibrous histiocytoma amplified sequences with leucine-rich tandem repeats 1 (MASL1) is the smallest member of the human ROCO protein family, members of which are characterized by a conserved supra-domain containing a Ras of complex proteins (ROC) domain and a C-terminal of ROC (COR)domain [1]
To investigate the function of the ROC domain of MASL1, an artificial mutation (K442A), designed to disrupt nucleotide binding, was introduced into the open reading of hemagglutinin (HA) tagged MASL1. This mutation was chosen based on homology to small GTPases and leucine-rich repeat kinase 2 (LRRK2), where it has previously been shown to impact upon guanosine triphosphate (GTP) binding properties [14]
This study provides an insight into the biology of MASL1, the smallest member of the human ROCO protein family

Summary

Introduction

Malignant fibrous histiocytoma amplified sequences with leucine-rich tandem repeats 1 (MASL1) is the smallest member of the human ROCO protein family, members of which are characterized by a conserved supra-domain containing a Ras of complex proteins (ROC) domain and a C-terminal of ROC (COR)domain [1]. Malignant fibrous histiocytoma amplified sequences with leucine-rich tandem repeats 1 (MASL1) is the smallest member of the human ROCO protein family, members of which are characterized by a conserved supra-domain containing a Ras of complex proteins (ROC) domain and a C-terminal of ROC (COR). In addition to MASL1, this protein family includes the leucine-rich repeat kinases 1 and 2 (LRRK1/2) and death-associated protein kinase 1 (DAPK1) (Fig. 1) [2]. Abbreviations BN, blue native; COR, C-terminal of ROC; DAPK1, death-associated protein kinase 1; FACS, fluorescence-activated cell sorting; HA, hemagglutinin; HMW, high molecular weight; HSP, heat shock protein; LMW, low molecular weight; LRRK1, leucine-rich repeat kinase 1; LRRK2, leucine-rich repeat kinase 2; MASL1, malignant fibrous histiocytoma amplified sequences with leucine-rich tandem repeats 1; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ROC, Ras of complex proteins; SEC, size exclusion chromatography. It is clear that understanding the cellular and molecular functions of these proteins is an important challenge in several areas of biomedical research

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