Abstract
By transporting serotonin (5-HT) into neurons and other cells, serotonin transporter (SERT) modulates the action of 5-HT at cell surface receptors. SERT itself is modulated by several processes, including the cGMP signaling pathway. Activation of SERT by cGMP requires the cGMP-dependent protein kinase (PKG). Here we show that in HeLa cells lacking endogenous PKG, expression of PKGIα or PKGIβ was required for 8-bromoguanosine-3',5'-cyclic monophosphate (8-Br-cGMP) to stimulate SERT phosphorylation and 5-HT influx. Catalytically inactive PKG mutants and wild-type PKGII did not support this stimulation. However, a mutant PKGII (G2A) that was not myristoylated substituted for functional PKGI, suggesting that myristoylation and subsequent membrane association blocked productive interaction with SERT. PKG also influenced SERT expression and localization. PKGI isoforms increased total and cell surface SERT levels, and PKGII decreased cell surface SERT without altering total expression. Remarkably, these changes did not require 8-Br-cGMP or functional kinase activity and were also observed with a SERT mutant resistant to activation by PKG. Both PKGIα and PKGIβ formed detergent-stable complexes with SERT, and this association did not require catalytic activity. The nonmyristoylated PKGII G2A mutant stimulated SERT expression similar to PKGI isoforms. These results suggest multiple mechanisms by which PKG can modulate SERT and demonstrate that the functional difference between PKG isoforms results from myristoylation of PKGII.
Highlights
Cells co-expressing PKG and vasodilator-stimulated phosphoprotein (VASP) were treated with 8-Br-cGMP, and PKG-dependent VASP phosphorylation was detected by immunoblotting with a monoclonal antibody (16C2) specific for phosphorylated VASP [31]. 16C2 immunoreactivity indicates VASP phosphorylation at serines 157 and 239, two PKG-preferred phosphorylation sites in VASP [32]
The results presented here establish the basis of isoform selectivity in the PKG-dependent phosphorylation and activation of serotonin transporter (SERT)
These studies extend previous observations of SERT co-localization and association with PKGI␣ [27] by demonstrating that both PKGI isoforms associate with and activate SERT, that myristoylation of PKGII leads to its inability stimulate SERT activity, and that PKGI and PKGII have opposite effects on SERT surface expression and activity
Summary
32P Incorporation—Approximately 4 ϫ 105 cells co-expressing FLAG-tagged SERT and the indicated PKG isoforms or their inactive mutants were incubated with 1.0 mCi/ml carrier-free 32Pi for 60 min prior to the addition of PKG activators or inhibitors. 8-Br-cGMP did not stimulate SERT activity in cells co-expressing inactive PKG mutants PKGI␣ K390A, PKGI K405A, or PKGII K482A, JANUARY 28, 2011
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