Phosphodiesterase-8A binds to and regulates Raf-1 kinase

Abstract

V-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1) is a key activator of the ERK pathway and is a target for cross-regulation of this pathway by the cAMP signaling system. The cAMP-activated protein kinase, PKA, inhibits Raf-1 by phosphorylation on S259. Here, we show that the cAMP-degrading phosphodiesterase-8A (PDE8A) associates with Raf-1 to protect it from inhibitory phosphorylation by PKA, thereby enhancing Raf-1's ability to stimulate ERK signaling. PDE8A binds to Raf-1 with high (picomolar) affinity. Mapping of the interaction domain on PDE8A using peptide array technology identified amino acids 454-465 as the main binding site, which could be disrupted by mutation. A cell-permeable peptide corresponding to this region disrupted the PDE8A/Raf-1 interaction in cells, thereby reducing ERK activation and the cellular response to EGF. Overexpression of a catalytically inactive PDE8A in cells displayed a dominant negative phenotype on ERK activation. These effects were recapitulated at the organism level in genetically modified (PDE8A −/− ) mice. Similarly, PDE8 deletion in Drosophila melanogaster reduced basal ERK activation and sensitized flies to stress-induced death. We propose that PDE8A is a physiological regulator of Raf-1 signaling in some cells.

Cell signalling mechanisms for insect stress tolerance

“…Critically, this biochemical interaction was shown to be relevant to oxidative stress tolerance in the organism, especially given the putative localisation of PDE8 in mitochondria as well as the role of the tubules in stress resistance. PDE8 deletion mutants are significantly more susceptible to oxidative stress imposed by either H 2 O 2 or paraquat feeding, compared with parental controls (Brown et al, 2013). This work demonstrates for the first time in insects that cAMP and ERK signalling mediates oxidative stress tolerance.…”

“…The PDE8A isoform is localised to mitochondria (Tsai and Beavo, 2011), and in D. melanogaster, DmPDE8 is the orthologue of mammalian PDE8A (Davies and Day, 2007). In a recent study, PDE8A was shown to have a novel binding partner, Raf-1, which allows modulation of downstream extracellular signal-regulated kinase (ERK) signalling via phosphorylation of ERK by Raf-1 kinase (Brown et al, 2013). DmPDE8 is abundantly expressed in epithelia, including the tubules, and PDE8 deletion mutants show reduced phospho-ERK signals.…”

“…Populations of mitochondria (M; in grey) are illustrated in the vicinity of the basolateral and apical membrane . Cell signalling components that have been experimentally determined to act in the principal cells for organismal stress tolerance are indicated by light blue shading as follows: oxidative stress (Brown et al, 2013;Terhzaz et al, 2010a;Terhzaz et al, 2010b), immune stress McGettigan et al, 2005), salt stress and desiccation stress Although the only route for degradation of cyclic nucleotides is via the PDEs (Bender and Beavo, 2006), extrusion of cyclic nucleotides in some cell types also contributes to reduction of cyclic nucleotide levels. In tubule principal cells, DmPDE5/6 is expressed at the apical membrane, and controls cGMP efflux into the lumen (Day et al, 2006), suggesting that control of PDE levels in tubule principal cells is not only due to breakdown by PDEs.…”

PDE8A runs interference to limit PKA inhibition of Raf-1

“…Of import, this system was shown to be operational and to control basal Raf-1-mediated phosphorylation of ERK when tested in wild-type and PDE8-null Drosophila, as well as in Leydig cells isolated from wild-type and PDE8-null mice. Although previous studies have shown that PDE3-or PDE4-family enzymes can be counted on to control localized cAMPdependent events in cells (8-12), Brown et al's study (3) shows that local control of Raf-1 activity can be added to the list of important signaling events that are regulated in cells by the hyperlocalized hydrolysis of cAMP by compartmented PDEs.…”

“…Using several complementary approaches, including peptide array-based binding studies, Brown et al (3) demonstrate that Raf-1 and PDE8A interact directly without the need of accessory proteins or lipids, and identify a peptide motif in PDE8A (R454 RLSGNEY461) critical for this interaction. Interestingly, although the affinity of many protein-protein interactions tends to be weak, in this instance PDE8A/Raf-1 binding was very tight (K d < 61 pM), and was not affected by changes in cellular cAMP concentrations or the presence in cells of PDE8 cAMP Fig.…”