Dopamine regulates GEF-H1 activity in striatum by phosphorylation with PKA
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1
Nagoya University Graduate School of Medicine, Department of Cell Pharmacology, Japan
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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
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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