Adenylate control in cAMP signaling: implications for adaptation in signalosomes.

In cAMP-Protein Kinase A (PKA) signaling, A-kinase anchoring protein scaffolds assemble PKA in close proximity to phosphodiesterases (PDE), kinase-substrates to form signaling islands or 'signalosomes'. In its basal state, inactive PKA holoenzyme (R2:C2) is activated by binding of cAMP to regulatory (R)-subunits leading to dissociation of active catalytic (C)-subunits. PDEs hydrolyze cAMP-bound to the R-subunits to generate 5'-AMP for termination and resetting the cAMP signaling. Mechanistic basis for cAMP signaling has been derived primarily by focusing on the proteins in isolation. Here, we set out to simulate cAMP signaling activation-termination cycles in a signalosome-like environment with PDEs and PKA subunits in close proximity to each other. Using a combination of fluorescence polarization and amide hydrogen exchange mass spectrometry with regulatory (RIα), C-subunit (Cα) and PDE8 catalytic domain, we have tracked movement of cAMP through activation-termination cycles. cAMP signaling operates as a continuum of four phases: (1) Activation and dissociation of PKA into R- and C-subunits by cAMP and facilitated by substrate (2) PDE recruitment to R-subunits (3) Hydrolysis of cAMP to 5'-AMP (4) Reassociation of C-subunit to 5'-AMP-bound-RIα in the presence of excess ATP to reset cAMP signaling to form the inactive PKA holoenzyme. Our results demonstrate that 5'-AMP is not merely a passive hydrolysis end-product of PDE action. A 'ligand-free' state R subunit does not exist in signalosomes as previously assumed. Instead the R-subunit toggles between cAMP- or 5'-AMP bound forms. This highlights, for the first time, the importance of 5'-AMP in promoting adaptation and uncovers adenylate control in cAMP signaling.

Synergistic Allostery in Multiligand-Protein Interactions.

Amide hydrogen-deuterium exchange mass spectrometry is powerful for describing combinatorial coupling effects of a cooperative ligand pair binding at noncontiguous sites: adenosine at the ATP-pocket and a docking peptide (PIFtide) at the PIF-pocket, on a model protein kinase PDK1. Binding of two ligands to PDK1 reveal multiple hotspots of synergistic allostery with cumulative effects greater than the sum of individual effects mediated by each ligand. We quantified this synergism and ranked these hotspots using a difference in deuteration-based approach, which showed that the strongest synergistic effects were observed at three of the critical catalytic loci of kinases: the αB-αC helices, and HRD-motif loop, and DFG-motif. Additionally, we observed weaker synergistic effects at a distal GHI-subdomain locus. Synergistic changes in deuterium exchange observed at a distal site but not at the intermediate sites of the large lobe of the kinase reveals allosteric propagation in proteins to operate through two modes. Direct electrostatic interactions between polar and charged amino acids that mediate targeted relay of allosteric signals, and diffused relay of allosteric signals through soft matter-like hydrophobic core amino acids. Furthermore, we provide evidence that the conserved ß-3 strand lysine of protein kinases (Lys111 of PDK1) functions as an integrator node to coordinate allosteric coupling of the two ligand-binding sites. It maintains indirect interactions with the ATP-pocket and mediates a critical salt bridge with a glutamate (Glu130) of αC helix, which is conserved across all kinases. In summary, allosteric propagation in cooperative, dual-liganded enzyme targets is bidirectional and synergistic and offers a strategy for combinatorial drug development.

Impaired cAMP processivity by phosphodiesterase-protein kinase A complexes in acrodysostosis.

Acrodysostosis represents a group of rare genetic disorders characterized by defective skeletal development and is often accompanied by intellectual disabilities. Mutations in the 3'5'cyclic AMP (cAMP)-dependent protein kinase (PKA) type I regulatory subunit isoform α (RIα) and phosphodiesterase (PDE) PDE4D have both been implicated in impaired PKA regulation in acrodysostosis. How mutations on PDEs and RIα interfere with the regulation of cAMP-PKA signaling is not understood. cAMP-PKA signaling can be described in two phases. In the activation phase, cAMP binding to RIα dissociates the free C-subunit (Catalytic subunit). PDEs hydrolyze cAMP bound to RIα, priming the cAMP-free RIα for reassociation with the C-subunit, thereby completing one PKA activation cycle. Signal termination is thus critical for resetting PKA to its basal state and promoting adaptation to hormonal hyperstimulation. This proceeds through formation of a transient signal termination RIα: PDE complex that facilitates cAMP channeling from the cAMP-binding domain of RIα to the catalytic site of PDE. Signal termination of cAMP-PKA proceeds in three steps: Step 1) Channeling: translocation of cAMP from the CNB of RIα to the PDE catalytic site for hydrolysis. Step 2) Processivity: binding of free cAMP from the cytosol at both CNBs of RIα. Step 3) Product (5'AMP) release from the PDE hydrolysis site through competitive displacement by a new molecule of cAMP that triggers subsequent activation cycles of PKA. We have identified the molecular basis for two acrodysostosis mutants, PDE (PDE8 T690P) and RIα (T207A), that both allosterically impair cAMP-PKA signal termination. A combination of amide hydrogen/deuterium exchange mass spectrometry (HDXMS) and fluorescence polarization (FP) reveals that PDE8 T690P and RIα T207A both blocked processive hydrolysis of cAMP by interfering with competitive displacement of product 5'AMP release from the nucleotide channel at the end of each round of cAMP hydrolysis. While T690P blocked product 5'AMP release from the PDE, T207A greatly slowed the release of the substrate from RIα. These results highlight the role of processivity in cAMP hydrolysis by RIα: PDE termination complexes for adaptation to cAMP from GPCR hyperstimulation. Impairment of the signal termination process provides an alternate molecular basis for acrodysostosis.

Modulation of the substrate specificity of the kinase PDK1 by distinct conformations of the full-length protein.

The activation of at least 23 different mammalian kinases requires the phosphorylation of their hydrophobic motifs by the kinase PDK1. A linker connects the phosphoinositide-binding PH domain to the catalytic domain, which contains a docking site for substrates called the PIF pocket. Here, we used a chemical biology approach to show that PDK1 existed in equilibrium between at least three distinct conformations with differing substrate specificities. The inositol polyphosphate derivative HYG8 bound to the PH domain and disrupted PDK1 dimerization by stabilizing a monomeric conformation in which the PH domain associated with the catalytic domain and the PIF pocket was accessible. In the absence of lipids, HYG8 potently inhibited the phosphorylation of Akt (also termed PKB) but did not affect the intrinsic activity of PDK1 or the phosphorylation of SGK, which requires docking to the PIF pocket. In contrast, the small-molecule valsartan bound to the PIF pocket and stabilized a second distinct monomeric conformation. Our study reveals dynamic conformations of full-length PDK1 in which the location of the linker and the PH domain relative to the catalytic domain determines the selective phosphorylation of PDK1 substrates. The study further suggests new approaches for the design of drugs to selectively modulate signaling downstream of PDK1.

Proapoptotic Cyclic Peptide BC71 Targets Cell-Surface GRP78 and Functions as an Anticancer Therapeutic in Mice.

Glucose regulated protein 78 kDa (GRP78) is a recently emerged target for cancer therapy and a biomarker for cancer prognosis. Overexpression of GRP78 is observed in many types of cancers, with the cell-surface GRP78 being preferentially present in cancer cells and cancer blood vessel endothelial cells. Isthmin (ISM) is a secreted high-affinity proapoptotic protein ligand of cell-surface GRP78 that suppresses angiogenesis and tumor growth in mice. The C-terminal AMOP (adhesion-associated domain in MUC4 and other proteins) domain of ISM is critical in mediating its interaction with human umbilical vein endothelial cells (HUVECs). In this work, we report novel cyclic peptides harboring the RKD motif in the ISM AMOP domain that function as proapoptotic ligands of cell-surface GRP78. The most potent peptide, BC71, binds to GRP78 and converge to tumor in mice. Intravenous administration of BC71 suppressed xenograft tumor growth in mice as a single agent, with significant reduction in tumor angiogenesis and upsurge in apoptosis. Fluorescent-labeled BC71 accumulates in tumor in mice by targeting cell-surface GRP78. We show that BC71 triggers apoptosis via cell-surface GRP78 and activates caspase-8 and p53 signaling pathways in HUVECs. Using amide hydrogen-deuterium exchange mass spectrometry (HDXMS), we identified that BC71 preferentially binds to ATP-bound GRP78 via amino acid residues 244-257 of GRP78. Hence, BC71 serves as a valuable prototype for further development of peptidomimetic anticancer drugs targeting cell-surface GRP78 as well as PET imaging agents for cancer prognosis.