The ELMO Domain Containing (ELMOD) Proteins: Phylogenetic, Structural, and Functional Characterization Open Access
East, Michael (2014)
Abstract
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
About this Dissertation
| School | |
|---|---|
| Department | |
| Subfield / Discipline | |
| Degree | |
| Submission | |
| Language |
|
| Research Field | |
| Keyword | |
| Committee Chair / Thesis Advisor | |
| Committee Members |
Primary PDF
| Thumbnail | Title | Date Uploaded | Actions |
|---|---|---|---|
|
|
The ELMO Domain Containing (ELMOD) Proteins: Phylogenetic, Structural, and Functional Characterization () | 2018-08-28 15:10:22 -0400 |
|
Supplemental Files
| Thumbnail | Title | Date Uploaded | Actions |
|---|