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Department of Molecular Physiology and Biophysics and of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615
Guanosine
3',5'-cyclic monophosphate (cGMP)-binding, cGMP-specific
phosphodiesterase (PDE5) is abundant in vascular smooth muscle, and
this enzyme is a potent substrate for cGMP-dependent protein kinase
(PKG) in vitro. Binding of cGMP to the allosteric sites of PDE5 is
required for this phosphorylation to occur. Vascular smooth muscle
cells (VSMC) were used to determine if PDE5 is phosphorylated in intact
cells when cGMP is increased. With the use of anti-PDE5 antibodies, a
phosphorylated 93-kDa protein band was immunoprecipitated from early
passaged primary cultures of VSMC that had been preincubated with
32Pi
to label cellular ATP and then treated with atrial natriuretic factor
(ANF). In the absence of ANF, there was no detectable incorporation of
radiolabeled phosphate into this band. Phosphorylation of the 93-kDa
protein was augmented by pretreating cells with 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) to activate PKG
before addition of ANF. 8-BrcGMP, which interacts poorly with the
allosteric sites of PDE5, had no effect on PDE5 phosphorylation in the
absence of ANF. Phosphorylation of PDE5 in response to treatment of
cells with ANF was associated with a two- to fourfold increase in PDE activity in immunoprecipitates. Multiple-passaged VSMC, which are
deficient in PKG but retain PDE5, demonstrated no ANF-dependent increase in phosphorylation or catalytic activity of PDE5. However, incubation of immunoprecipitated PDE5 from these cells with purified PKG, cGMP, and a phosphorylation mixture containing
[
-32P]ATP resulted
in
32Pi
incorporation into PDE5 that was correlated with increased catalytic
activity. These studies are the first to demonstrate phosphorylation of
PDE5 in intact cells, thus suggesting a physiological role for this
enzyme in smooth muscle regulation.
cyclic nucleotides; protein phosphorylation; protein kinases; smooth muscle relaxation
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