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Exchange factor EFA6R requires C-terminal targeting to the plasma membrane to promote cytoskeletal rearrangement through the activation of ARF6

V. Kanamarlapudi, Venkat Kanamarlapudi Orcid Logo

Journal of Biological Chemistry, Volume: 289, Issue: 48, Pages: 33378 - 33390

Swansea University Author: Venkat Kanamarlapudi Orcid Logo

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DOI (Published version): 10.1074/jbc.M113.534156

Abstract

ADP-ribosylation factor 6 (ARF6) small GTPase regulates membrane trafficking and cytoskeleton rearrangements at the plasma membrane (PM) by cycling between the GTP-bound active and GDP-bound inactive conformations. Guanine nucleotide exchange factors (GEFs) activate ARF6. The exchange factor for ARF...

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Published in: Journal of Biological Chemistry
Published: 2014
URI: https://cronfa.swan.ac.uk/Record/cronfa18668
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spelling 2019-10-11T18:36:23.6561182 v2 18668 2014-10-09 Exchange factor EFA6R requires C-terminal targeting to the plasma membrane to promote cytoskeletal rearrangement through the activation of ARF6 63741801137148abfa4c00cd547dcdfa 0000-0002-8739-1483 Venkat Kanamarlapudi Venkat Kanamarlapudi true false 2014-10-09 BMS ADP-ribosylation factor 6 (ARF6) small GTPase regulates membrane trafficking and cytoskeleton rearrangements at the plasma membrane (PM) by cycling between the GTP-bound active and GDP-bound inactive conformations. Guanine nucleotide exchange factors (GEFs) activate ARF6. The exchange factor for ARF6 (EFA6) R has been identified as a biomarker for ovarian cancer. EFA6R shares the catalytic Sec7, pleckstrin homology (PH), and coiled coil (CC) domains of the other EFA6 family GEFs. Here we report the functional characterization of EFA6R. Endogenous EFA6R was present in the plasma membrane fraction. The exogenously expressed FLAG- and GFP-tagged EFA6R were targeted to the PM. In vitro, GFP-EFA6R associated weakly but preferentially with phosphatidylinositol 4,5-bisphosphate (PIP2) through the PH domain. EFA6R required both its PH and CC domains localized at the C terminus to target the PM. Consistent with this, EFA6R lacking the CC domain (EFA6R deltaCC) was released from the PM into the cytosol upon PIP2 depletion, whereas EFA6R release from the PM required both PIP2 depletion and actin destabilization. These results suggest that the dual targeting via the PH and CC domains is important for the PM localization of EFA6R. EFA6R specifically catalyzed the GTP loading of ARF6 in mammalian cells. Moreover, EFA6R regulated ARF6 localization and thereby actin stress fiber loss. The GEF activity of EFA6R was dependent on the presence of the Sec7 domain. The PH and CC domains were also required for the in vivo GEF activity of EFA6R but could be functionally replaced by the CAAX motif of K-Ras, suggesting a role for these domains in the membrane targeting of EFA6R. Journal Article Journal of Biological Chemistry 289 48 33378 33390 EFA6R, ARF6, GEF, actin, PIP2, PH, CC, plasma membrane, cytoskeleton, ovarian cancer 8 10 2014 2014-10-08 10.1074/jbc.M113.534156 COLLEGE NANME Biomedical Sciences COLLEGE CODE BMS Swansea University 2019-10-11T18:36:23.6561182 2014-10-09T12:56:51.5371544 Faculty of Medicine, Health and Life Sciences Swansea University Medical School - Medicine V. Kanamarlapudi 1 Venkat Kanamarlapudi 0000-0002-8739-1483 2 0018668-16122014134103.pdf EFA6R__JBC14.pdf 2014-12-16T13:41:03.8030000 Output 3753587 application/pdf Version of Record true 2014-12-16T13:41:03.0000000 Distributed under the terms of a Creative Commons Attribution Non-Commercial (CC-BY-3.0) false
title Exchange factor EFA6R requires C-terminal targeting to the plasma membrane to promote cytoskeletal rearrangement through the activation of ARF6
spellingShingle Exchange factor EFA6R requires C-terminal targeting to the plasma membrane to promote cytoskeletal rearrangement through the activation of ARF6
Venkat Kanamarlapudi
title_short Exchange factor EFA6R requires C-terminal targeting to the plasma membrane to promote cytoskeletal rearrangement through the activation of ARF6
title_full Exchange factor EFA6R requires C-terminal targeting to the plasma membrane to promote cytoskeletal rearrangement through the activation of ARF6
title_fullStr Exchange factor EFA6R requires C-terminal targeting to the plasma membrane to promote cytoskeletal rearrangement through the activation of ARF6
title_full_unstemmed Exchange factor EFA6R requires C-terminal targeting to the plasma membrane to promote cytoskeletal rearrangement through the activation of ARF6
title_sort Exchange factor EFA6R requires C-terminal targeting to the plasma membrane to promote cytoskeletal rearrangement through the activation of ARF6
author_id_str_mv 63741801137148abfa4c00cd547dcdfa
author_id_fullname_str_mv 63741801137148abfa4c00cd547dcdfa_***_Venkat Kanamarlapudi
author Venkat Kanamarlapudi
author2 V. Kanamarlapudi
Venkat Kanamarlapudi
format Journal article
container_title Journal of Biological Chemistry
container_volume 289
container_issue 48
container_start_page 33378
publishDate 2014
institution Swansea University
doi_str_mv 10.1074/jbc.M113.534156
college_str Faculty of Medicine, Health and Life Sciences
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hierarchy_top_id facultyofmedicinehealthandlifesciences
hierarchy_top_title Faculty of Medicine, Health and Life Sciences
hierarchy_parent_id facultyofmedicinehealthandlifesciences
hierarchy_parent_title Faculty of Medicine, Health and Life Sciences
department_str Swansea University Medical School - Medicine{{{_:::_}}}Faculty of Medicine, Health and Life Sciences{{{_:::_}}}Swansea University Medical School - Medicine
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description ADP-ribosylation factor 6 (ARF6) small GTPase regulates membrane trafficking and cytoskeleton rearrangements at the plasma membrane (PM) by cycling between the GTP-bound active and GDP-bound inactive conformations. Guanine nucleotide exchange factors (GEFs) activate ARF6. The exchange factor for ARF6 (EFA6) R has been identified as a biomarker for ovarian cancer. EFA6R shares the catalytic Sec7, pleckstrin homology (PH), and coiled coil (CC) domains of the other EFA6 family GEFs. Here we report the functional characterization of EFA6R. Endogenous EFA6R was present in the plasma membrane fraction. The exogenously expressed FLAG- and GFP-tagged EFA6R were targeted to the PM. In vitro, GFP-EFA6R associated weakly but preferentially with phosphatidylinositol 4,5-bisphosphate (PIP2) through the PH domain. EFA6R required both its PH and CC domains localized at the C terminus to target the PM. Consistent with this, EFA6R lacking the CC domain (EFA6R deltaCC) was released from the PM into the cytosol upon PIP2 depletion, whereas EFA6R release from the PM required both PIP2 depletion and actin destabilization. These results suggest that the dual targeting via the PH and CC domains is important for the PM localization of EFA6R. EFA6R specifically catalyzed the GTP loading of ARF6 in mammalian cells. Moreover, EFA6R regulated ARF6 localization and thereby actin stress fiber loss. The GEF activity of EFA6R was dependent on the presence of the Sec7 domain. The PH and CC domains were also required for the in vivo GEF activity of EFA6R but could be functionally replaced by the CAAX motif of K-Ras, suggesting a role for these domains in the membrane targeting of EFA6R.
published_date 2014-10-08T03:21:54Z
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