Dopamine regulates GEF-H1 activity in striatum by phosphorylation with PKA

Abstract

Event Abstract Back to Event Dopamine regulates GEF-H1 activity in striatum by phosphorylation with PKA Xinjian Zhang1, Keisuke Kuroda1, Hiroyuki Takenaka1, Reon Kondo1, Kaishu Oda1, Tomoki Nishioka1, Shinichi Nakamuta1, Taku Nagai2, Kiyofumi Yamada2 and Kozo Kaibuchi1* 1 Nagoya University Graduate School of Medicine, Department of Cell Pharmacology, Japan 2 Nagoya University Graduate School of Medicine, Department of Neuropsychopharmacology and Hospital Pharmacy, Japan In the brain, dopamine is a neurotransmitter involved in motor function, motivation, working memory, and reward. The functions of dopamine are mediated by Protein kinase A (PKA) and its downstream signaling cascades in dopaminergic neurons. PKA regulates a lot of neural functions including the structural and functional plasticity via phosphorylation of its substrates. However, PKA substrates that regulate plasticity are still unknown. Here, we performed phosphoproteomic analysis to search for the substrates for PKA and their function. We treated mouse striatum slices with a D1 agonist (SKF81297) or cAMP inducer forskolin, and then analyzed phosphoproteins pulled down with GST-14-3-3z by LC-MS/MS. More than 100 candidate substrates of PKA were identified. It is known that Rho family GTPases regulate actin cytoskeleton, whose dynamics have been implicated as the structural basis of synaptic plasticity in neurons. Rho family GTPases are activated by GEFs and inactivated by GAPs. Thereby, among candidate substrates, we chose and focused on GEF-H1, a Rho guanine nucleotide exchange factor, which was previously shown to be phosphorylated by PKA at Ser 885 in vitro. To confirm that PKA phosphorylates GEF-H1 S885 in striatum, we performed a western blot analysis using anti-phospho-GEF-H1 S885 antibody. Phosphorylation level of GEF-H1 S855 was increased in forskolin-stimulated striatum slices compared with control. This result indicates PKA phosphorylates GEF-H1 at Ser 885 in striatum. To investigate whether the activity of GEF-H1 is regulated by PKA, we performed an affinity precipitation assay using nucleotide-free RhoA (RhoA G17A)-coated beads. Since active GEF-H1 binds to inactive RhoA specifically, this method made us examine whether GEF-H1 is activated in striatum slices. Precipitated GEF-H1 was decreased in forskolin-stimulated striatum slices. These results suggest that dopamine can decrease RhoA activity through phosphorylation of GEF-H1 in striatum. Keywords: Dopamine, Phosphorylation, Striatum, in vitro, neurotransmitter Conference: 14th Meeting of the Asian-Pacific Society for Neurochemistry, Kuala Lumpur, Malaysia, 27 Aug - 30 Aug, 2016. Presentation Type: O01: Postgraduate Travel Awardees Oral Session 1 Topic: 14th Meeting of the Asian-Pacific Society for Neurochemistry Citation: Zhang X, Kuroda K, Takenaka H, Kondo R, Oda K, Nishioka T, Nakamuta S, Nagai T, Yamada K and Kaibuchi K (2016). Dopamine regulates GEF-H1 activity in striatum by phosphorylation with PKA. Conference Abstract: 14th Meeting of the Asian-Pacific Society for Neurochemistry. doi: 10.3389/conf.fncel.2016.36.00079 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 Aug 2016; Published Online: 11 Aug 2016. * Correspondence: Prof. Kozo Kaibuchi, Nagoya University Graduate School of Medicine, Department of Cell Pharmacology, Nagoya, Aichi, Japan, kaibuchi@med.nagoya-u.ac.jp Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Xinjian Zhang Keisuke Kuroda Hiroyuki Takenaka Reon Kondo Kaishu Oda Tomoki Nishioka Shinichi Nakamuta Taku Nagai Kiyofumi Yamada Kozo Kaibuchi Google Xinjian Zhang Keisuke Kuroda Hiroyuki Takenaka Reon Kondo Kaishu Oda Tomoki Nishioka Shinichi Nakamuta Taku Nagai Kiyofumi Yamada Kozo Kaibuchi Google Scholar Xinjian Zhang Keisuke Kuroda Hiroyuki Takenaka Reon Kondo Kaishu Oda Tomoki Nishioka Shinichi Nakamuta Taku Nagai Kiyofumi Yamada Kozo Kaibuchi PubMed Xinjian Zhang Keisuke Kuroda Hiroyuki Takenaka Reon Kondo Kaishu Oda Tomoki Nishioka Shinichi Nakamuta Taku Nagai Kiyofumi Yamada Kozo Kaibuchi Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.