Zeta Inhibitory Peptide Disrupts Electrostatic Interactions That Maintain Atypical Protein Kinase C in Its Active Conformation on the Scaffold p62.

Atypical protein kinase C (aPKC) enzymes signal on protein scaffolds, yet how they are maintained in an active conformation on scaffolds is unclear. A myristoylated peptide based on the autoinhibitory pseudosubstrate fragment of the atypical PKCζ, zeta inhibitory peptide (ZIP), has been extensively used to inhibit aPKC activity; however, we have previously shown that ZIP does not inhibit the catalytic activity of aPKC isozymes in cells (Wu-Zhang, A. X., Schramm, C. L., Nabavi, S., Malinow, R., and Newton, A. C. (2012) J. Biol. Chem. 287, 12879-12885). Here we sought to identify a bona fide target of ZIP and, in so doing, unveiled a novel mechanism by which aPKCs are maintained in an active conformation on a protein scaffold. Specifically, we used protein-protein interaction network analysis, structural modeling, and protein-protein docking to predict that ZIP binds an acidic surface on the Phox and Bem1 (PB1) domain of p62, an interaction validated by peptide array analysis. Using a genetically encoded reporter for PKC activity fused to the p62 scaffold, we show that ZIP inhibits the activity of wild-type aPKC, but not a construct lacking the pseudosubstrate. These data support a model in which the pseudosubstrate of aPKCs is tethered to the acidic surface on p62, locking aPKC in an open, signaling-competent conformation. ZIP competes for binding to the acidic surface, resulting in displacement of the pseudosubstrate of aPKC and re-engagement in the substrate-binding cavity. This study not only identifies a cellular target for ZIP, but also unveils a novel mechanism by which scaffolded aPKC is maintained in an active conformation.

cAMP-specific phosphodiesterases 8A and 8B, essential regulators of Leydig cell steroidogenesis.

Phosphodiesterase (PDE) 8A and PDE8B are high-affinity, cAMP-specific phosphodiesterases that are highly expressed in Leydig cells. PDE8A is largely associated with mitochondria, whereas PDE8B is broadly distributed in the cytosol. We used a new, PDE8-selective inhibitor, PF-04957325, and genetically ablated PDE8A(-/-), PDE8B(-/-) and PDE8A(-/-)/B(-/-) mice to determine roles for these PDEs in the regulation of testosterone production. PF-04957325 treatment of WT Leydig cells or MA10 cells increased steroid production but had no effect in PDE8A (-/-)/B(-/-) double-knockout cells, confirming the selectivity of the drug. Moreover, under basal conditions, cotreatment with PF-04957325 plus rolipram, a PDE4-selective inhibitor, synergistically potentiated steroid production. These results suggest that the pool(s) of cAMP regulating androgen production are controlled by PDE8s working in conjunction with PDE4. Likewise, PDE8A (-/-)/B(-/-) cells had higher testosterone production than cells from either PDE8A(-/-) or PDE8B(-/-) mice, suggesting that both PDE8s work in concert to regulate steroid production. We further demonstrate that combined inhibition of PDE8s and PDE4 greatly increased PKA activity including phosphorylation of cholesterol-ester hydrolase (CEH)/hormone-sensitive lipase (HSL). CEH/HSL phosphorylation also was increased in PDE8A(-/-)/B(-/-) cells compared with WT cells. Finally, combined inhibition of PDE8s and PDE4 increased the expression of steroidogenic acute regulatory (StAR) protein. Together these findings suggest that both PDE8A and PDE8B play essential roles to maintain low cAMP levels, thereby suppressing resting steroidogenesis by keeping CEH/HSL inactive and StAR protein expression low. They also suggest that in order for PDE inhibitor therapy to be an effective stimulator of steroidogenesis, both PDE8 isozymes and PDE4 need to be simultaneously targeted.

The high-affinity cAMP-specific phosphodiesterase 8B controls steroidogenesis in the mouse adrenal gland.

The functions of the phosphodiesterase 8B (PDE8) family of phosphodiesterases have been largely unexplored because of the unavailability of selective pharmacological inhibitors. Here, we report a novel function of PDE8B as a major regulator of adrenal steroidogenesis using a genetically ablated PDE8B mouse model as well as cell lines treated with either a new PDE8-selective inhibitor or a short hairpin RNA (shRNA) construct against PDE8B. We demonstrate that PDE8B is highly enriched in mouse adrenal fasciculata cells, and show that PDE8B knockout mice have elevated urinary corticosterone as a result of adrenal hypersensitivity toward adrenocorticotropin. Likewise, ablation of PDE8B mRNA transcripts by an shRNA construct potentiates steroidogenesis in the commonly used Y-1 adrenal cell line. We also observed that the PDE8-selective inhibitor (PF-04957325) potentiates adrenocorticotropin stimulation of steroidogenesis by increasing cAMP-dependent protein kinase activity in both primary isolated adrenocortical cells and Y-1 cells. It is noteworthy that PDE8s have their greatest control under low adrenocorticotropin-stimulated conditions, whereas other higher K(m) PDE(s) modulate steroidogenesis more effectively when cells are fully stimulated. Finally, both genetic ablation of PDE8B and long-term pharmacological inhibition of PDE8s cause increased expression of steroidogenic enzymes. We conclude that PDE8B is a major regulator of one or more pools of cAMP that promote steroidogenesis via both short- and long-term mechanisms. These findings further suggest PDE8B as a potential therapeutic target for the treatment of several different adrenal diseases.

The roles of cyclic nucleotide phosphodiesterases (PDEs) in steroidogenesis.

The second messenger, cAMP, is one of the most important regulatory signals for control of steroidogenesis. This review focuses on current knowledge about regulation of cyclic nucleotides by phosphodiesterases (PDEs) in steroidogenic tissues. The first PDE known to directly regulate steroidogenesis was PDE2, the cGMP-stimulated PDE. PDE2 mediates ANP/cGMP-induced decreases in aldosterone production. Recently, the PDE8 family has been shown to control steroidogenesis in two tissues. Specifically, PDE8A regulates testosterone production by itself and in concert with additional IBMX-sensitive PDEs. PDE8B modulates basal corticosterone synthesis via acute and chronic mechanisms. In addition to cAMP-dependent pathways, cGMP signaling also can promote steroidogenesis, and PDE5 modulates this process. Finally, PDE mutations may lead to several human diseases characterized by abnormal steroid levels.