Cilomilast phosphodiesterase(pde) inhibitor Whereas the association of p110γ with PKA is direct, the interaction with PDE3B is mediated by the p84

p110γ. Whereas the association of p110γ with PKA is direct, the interaction with PDE3B is mediated by the p84/87 PI3Kγregulatory subunit. This supports the selective involvement of p84/87, and not of p101, in constraining the assembly of this ternary complex. A broader implication of our results is that multiprotein assemblies involving p84/87/p110γ,PDE3B, and PKA coordinate the Cilomilast phosphodiesterase(pde) inhibitor spatial and temporal modulation of cAMP signaling in the myocardium, acting in a manner similar to other AKAPs such as mAKAP, AKAP350, and gravin. These signaling complexes tether PKA in proximity to PDEs to locally modulate cAMP signaling, thereby Perino et al. Page 6 Mol Cell. Author manuscript; available in PMC 2012 January 24. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript optimizing signal termination.
In respect to what has been shown for other AKAPs, an important finding of the present study is that we provide evidence of the colocalization of PKA and PDE3B in a macromolecular complex. By interacting with PKA and PDE3B, the p84/87/p110γ heterodimer appears involved in a crucial negative feedback controlling the cAMP pathway. In p110γ-deficient animals, BMS-790052 HCV protease inhibitor loss of this feedback leads to cAMP accumulation in resting conditions and to cAMP-mediated cardiac damage under stress. While p110γ appears to behave like an AKAP in that it directly binds the RIIα subunit, its PKA-anchoring site appears to be atypical. Classical AKAPs bind to PKA RIIα through a conserved amphipathic helix , and their association can be disrupted by synthetic peptides designed to reproduce this helical structure.
As expected, the p110γ/PKA RIIα interaction could also be disrupted by AKAP-IS, a consensus RII-anchoring disruptor peptide. However, the p110γ sequence defined by the peptide array is not predicted to form a helical domain, and the interaction with RIIα appears to rely on two positively charged residues. Nonetheless, these findings are in line with the notion that the family of AKAPs, which currently includes 45 genes and their splice variants, exhibits substantial heterogeneity in sequence, yet always featuring the ability to tether PKA at subcellular locations. The PKA associated with p110γ not only influences the catalytic activity of PDE3B, but also modulates the lipid kinase activity of p110γ itself.
Indeed, the proximity of PKA and p110γwithin the same macromolecular complex allows active PKA to phosphorylate both PDE3B and p110γ. The phosphorylation of p110γ by PKA on T1024 results in a negative modulation of p110γ kinase activity. T1024 resides in an α helix situated in close proximity to the ATP-binding pocket, and therefore the functional effects of this phosphorylation on the kinase activity of p110γ may derive from a conformational change disturbing the catalytic pocket. This mechanism is supported by our findings with the phosphomimetic T1024D mutant, which resulted in decreased lipid kinase activity. T1024 of p110γ is highly conserved among species and is not represented in the other class I PI3K isoforms, which are, however, inhibited by their autophosphorylation within the catalytic domain.
Modulation of p110γ by PKA has relevant functional implications in vivo in the myocardium. While the β-AR/cAMP pathway that activates PKA also triggers the PI3K pathway , our results indicate that in physiological conditions, p110γ activity is negligible, owing to its low expression levels and to the inhibitory phosphorylation by PKA. Instead, other G protein-coupled p110 isoforms, such as p110β , appear to be the main PI3K catalytic subunits responsible for the production of PtdIns P3 and the consequent activation of Akt upon β-AR stimulation.