The ELMO Domain Containing (ELMOD) Proteins: Phylogenetic, Structural, and Functional Characterization Open Access

East, Michael (2014)

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The ELMO domain containing proteins (ELMODs) are a novel group of GTPase activating proteins (GAPs) that act on members of the Arf family of regulatory GTPases, though they lack the previously characterized, conserved Arf GAP domain. Very little is known about the functions of the ELMODs but the human family members have been implicated in non-syndromic familial deafness or idiopathic pulmonary fibrosis. The ELMODs belong to the ELMO family of proteins which includes three ELMODs and three ELMOs in humans and is defined by the presence of the ELMO domain in each of its members. The ELMOs function as key regulators of cell migration and actin dynamics as unconventional guanine nucleotide exchange factors for the Rho family GTPase Rac1. Unlike the ELMODs, the ELMOs lack any detectable GAP activity for Arf family GTPases. In this dissertation, I show that ELMODs and ELMOs are two phylogenetically distinct groups of proteins despite being members of the same gene family. The ELMODs represent the more ancient form of the protein and were likely present in the last eukaryotic common ancestor, suggesting some ancient function(s) of the ELMODs important or even essential for eukaryotic life. I mapped the GAP motif of the ELMODs to a region within the ELMO domain and provided initial characterization of this motif by identifying an arginine residue essential for efficient GAP activity. I also provide the first functional information for the ELMODs and present models for the cellular roles of ELMOD1 and ELMOD3 as a regulator of Arf signaling at the Golgi and as an activator of RhoA signaling at the trailing edge of migrating cells, respectively. However, additional cellular localizations of each of the ELMODs and putative binding partners identified in this dissertation are suggestive of other, novel roles for the ELMODs in cells. Together these data represent the majority of the available information on the ELMODs and provide the foundation for our later studies to provide a better understanding of the biology of the ELMODs and their roles in cell biology and human diseases.

Table of Contents

General Introduction 1

Figure 1: The GTPase cycle 3

Figure 2: Domain architecture of the ELMO family of proteins 5

ELMO domains: evolutionary and functional characterization of a novel GTPase activating protein (GAP) domain for Arf family GTPases 14

Summary 16

Introduction 17

Experimental Procedures 20

Results 24

Figure 1: The six human ELMO domain-containing family members are equally divided into three ELMO and three ELMOD proteins 26

Figure 2: ELMOs cluster into a distinct phylogenetic sub-family 29

Figure 3: The ELMO sub-family is further divided into three distinct phylogenetic clades 30

Figure 4: Sequence alignment of the ELMODs reveals a highly conserved motif that includes a central arginine residue 32

Figure 5: The putative catalytic arginine and some of the other residues within the putative GAP domain of the ELMODs (top) are not conserved in members of the ELMO sub-family (bottom) 33

Figure 6: Mutation of the putative catalytic arginine residue reduces GAP activity of ELMOD1-myc/his and ELMOD2-myc/his in in vitro and cell-based assays 36

Figure 7: Over-expression of ELMOD1-HA alters Golgi morphology but this phenotype is absent for ELMOD1(R174K)-HA 40

Figure 8: Cellular localization of endogenous ELMOD1 and exogenous ELMOD1-HA or ELMOD2-HA 45

Discussion 47

References 55

Acknowledgments 61

Footnotes 61

Table S1: List of taxa and accession numbers for ELMO domain containing proteins used in our analyses 62

ELMOD3 is a novel regulator of the actin cytoskeleton through a RhoA dependent mechanism 65

Abstract 66

Introduction 66

Materials and Methods 69

Results 71

Figure 1: ELMOD3 migrates at a higher than expected apparent molecular weight on SDS gels 72

Figure 2: Endogenous ELMOD3 localizes to the trailing edge of migrating mouse embryonic fibroblasts (MEFs) 74

Figure 3: Endogenous ELMOD3 has a punctate staining pattern with organization along actin fibers in non-polarized mouse embryonic fibroblasts (MEFs) 75

Figure 4: ELMOD3 protein levels are unchanged after inhibiting protein synthesis for 16 hours 77

Figure 5: Endogenous ELMOD3 associates with the actin cytoskeleton 79

Figure 6: GFP-ELMOD3 causes dynamic plasma membrane blebbing 80

Figure 7: ELMOD3-HA causes changes in the actin cytoskeleton in a Rho-dependent fashion 82

Figure 8: Expression of dominant negative RhoA (T19N) inhibits blebbing and changes ELMOD3-HA localization 84

Figure 9: ELMOD3-HA induced plasma membrane blebbing is dependent on the RhoA signaling pathway 85

Discussion 86

References 91

Putative binding partners of ELMODs suggest novel functions and mechanisms 96

Introduction 97

Materials and Methods 99

Results and Discussion 103

Figure 1: Schematic of the SILAC co-IP methodology 105

Figure 2: Bovine testis enhances GST-ELMOD3 GAP activity 111

Figure 3: GST-ELMOD3 binds phosphoinositides 113

Figure 4: Staining of ELMOD3-HA and of PI(4,5)P2 show extensive overlap at the cell periphery 115

Figure 5: PI(4,5)P2 and PI(3,4,5)P3 have an asymmetrical distribution at the plasma membrane in migrating cells 117

References 118

Table 1: Putative binding partners of ELMOD1-HA 124

Table 2: Putative binding partners of ELMOD2-HA 126

Table 3: Putative binding partners of ELMOD3-HA 128

Discussion 130

ELMODs as GAPs 131

ELMOs and the ELMO domain 133

Figure 1: Model for the function of the ELMO domain in ELMOs and ELMODs 135

Models for the cellular functions of ELMODs 138

Figure 2: Schematic of confirmed and suspected subcellular localization of the ELMODs throughout the cell 139

Figure 3: The model for the function of ELMOD3 closely parallels its paralog ELMO1 146

General comments regarding models for the functions of ELMODs 151

Clinical aspects of the ELMODs 152

Concluding remarks 153

References 154

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