Aggregation of scaffolding protein DISC1 dysregulates phosphodiesterase 4 in Huntington's disease.

Huntington's disease (HD) is a polyglutamine (polyQ) disease caused by aberrant expansion of the polyQ tract in Huntingtin (HTT). While motor impairment mediated by polyQ-expanded HTT has been intensively studied, molecular mechanisms for nonmotor symptoms in HD, such as psychiatric manifestations, remain elusive. Here we have demonstrated that HTT forms a ternary protein complex with the scaffolding protein DISC1 and cAMP-degrading phosphodiesterase 4 (PDE4) to regulate PDE4 activity.

Mitotic activation of the DISC1-inducible cyclic AMP phosphodiesterase-4D9 (PDE4D9), through multi-site phosphorylation, influences cell cycle progression.

In Rat-1 cells, the dramatic decrease in the levels of both intracellular cyclic 3'5' adenosine monophosphate (cyclic AMP; cAMP) and in the activity of cAMP-activated protein kinase A (PKA) observed in mitosis was paralleled by a profound increase in cAMP hydrolyzing phosphodiesterase-4 (PDE4) activity. The decrease in PKA activity, which occurs during mitosis, was attributable to PDE4 activation as the PDE4 selective inhibitor, rolipram, but not the phosphodiesterase-3 (PDE3) inhibitor, cilostamide, specifically ablated this cell cycle-dependent effect. PDE4 inhibition caused Rat-1 cells to move from S phase into G2/M more rapidly, to transit through G2/M more quickly and to remain in G1 for a longer period.

Elucidation of a structural basis for the inhibitor-driven, p62 (SQSTM1)-dependent intracellular redistribution of cAMP phosphodiesterase-4A4 (PDE4A4).

A survey of PDE4 inhibitors reveals that some compounds trigger intracellular aggregation of PDE4A4 into accretion foci through association with the ubiquitin-binding scaffold protein p62 (SQSTM1). We show that this effect is driven by inhibitor occupancy of the catalytic pocket and stabilization of a "capped state" in which a sequence within the enzyme's upstream conserved region 2 (UCR2) module folds across the catalytic pocket. Only certain inhibitors cause PDE4A4 foci formation, and the structural features responsible for driving the process are defined.

Cyclic AMP controls mTOR through regulation of the dynamic interaction between Rheb and phosphodiesterase 4D.

The mammalian target of rapamycin complex 1 (mTORC1) is a molecular hub that regulates protein synthesis in response to a number of extracellular stimuli. Cyclic AMP (cAMP) is considered to be an important second messenger that controls mTOR; however, the signaling components of this pathway have not yet been elucidated. Here, we identify cAMP phosphodiesterase 4D (PDE4D) as a binding partner of Rheb that acts as a cAMP-specific negative regulator of mTORC1.

Cyclic AMP phosphodiesterase 4D (PDE4D) Tethers EPAC1 in a vascular endothelial cadherin (VE-Cad)-based signaling complex and controls cAMP-mediated vascular permeability.

Vascular endothelial cell (VEC) permeability is largely dependent on the integrity of vascular endothelial cadherin (VE-cadherin or VE-Cad)-based intercellular adhesions. Activators of protein kinase A (PKA) or of exchange protein activated by cAMP (EPAC) reduce VEC permeability largely by stabilizing VE-Cad-based intercellular adhesions. Currently, little is known concerning the nature and composition of the signaling complexes that allow PKA or EPAC to regulate VE-Cad-based structures and through these actions control permeability.

Evolutionarily conserved role of calcineurin in phosphodegron-dependent degradation of phosphodiesterase 4D.

Calcineurin is a widely expressed and highly conserved Ser/Thr phosphatase. Calcineurin is inhibited by the immunosuppressant drug cyclosporine A (CsA) or tacrolimus (FK506). The critical role of CsA/FK506 as an immunosuppressant following transplantation surgery provides a strong incentive to understand the phosphatase calcineurin.

MEK1 binds directly to betaarrestin1, influencing both its phosphorylation by ERK and the timing of its isoprenaline-stimulated internalization.

betaArrestin is a multifunctional signal scaffold protein. Using SPOT immobilized peptide arrays, coupled with scanning alanine substitution and mutagenesis, we show that the MAPK kinase, MEK1, interacts directly with betaarrestin1. Asp(26) and Asp(29) in the N-terminal domain of betaarrestin1 are critical for its binding to MEK1, whereas Arg(47) and Arg(49) in the N-terminal domain of MEK1 are critical for its binding to betaarrestin1.

EPAC and PKA allow cAMP dual control over DNA-PK nuclear translocation.

We identify a compartmentalized signaling system that identifies a functional role for the GTP exchange factor, exchange protein activated by cAMP (EPAC) coupled to Rap2 in the nucleus. In this system, cAMP regulates the nuclear/cytoplasmic trafficking of DNA-dependent protein kinase (DNA-PK), a critical kinase that acts to repair double-stranded breaks (DSBs) in damaged DNA and to phosphorylate the cell survival kinase, PKB/Akt. Intersecting regulatory inputs for cAMP employ EPAC to transduce positive effects, namely the Rap2-dependent nuclear exit and activation of DNA-PK, whereas protein kinase A (PKA) provides the negative input by antagonizing these actions.

Helix-1 of the cAMP-specific phosphodiesterase PDE4A1 regulates its phospholipase-D-dependent redistribution in response to release of Ca2+.

The unique N-terminal regions of PDE4 cAMP-specific phosphodiesterases confer interaction with distinct signalling and scaffolding proteins. The PDE4A1 isoform is unique in being entirely membrane associated. Its N-terminal region is formed from two helices separated by a mobile hinge, where helix-2 contains a TAPAS1 domain that inserts into the lipid bilayer in a Ca2+-triggered fashion.

Phosphodiesterase-4 influences the PKA phosphorylation status and membrane translocation of G-protein receptor kinase 2 (GRK2) in HEK-293beta2 cells and cardiac myocytes.

Membrane-recruitment of GRK2 (G-protein receptor kinase 2) provides a fundamental step in the desensitization process controlling GPCRs (G-protein-coupled receptors), such as the beta2AR (beta2-adrenergic receptor). In the present paper, we show that challenge of HEK-293beta2 [human embryonic kidney cells stably overexpressing the FLAG-tagged beta2AR-GFP (green fluorescent protein)] cells with the beta-adrenoceptor agonist, isoprenaline, causes GRK2 to become phosphorylated by PKA (cAMP-dependent protein kinase). This action is facilitated when cAMP-specific PDE4 (phosphodiesterase-4) activity is selectively inactivated, either chemically with rolipram or by siRNA (small interfering RNA)-mediated knockdown of PDE4B and PDE4D.

DISC1 and PDE4B are interacting genetic factors in schizophrenia that regulate cAMP signaling.

The disrupted in schizophrenia 1 (DISC1) gene is a candidate susceptibility factor for schizophrenia, but its mechanistic role in the disorder is unknown. Here we report that the gene encoding phosphodiesterase 4B (PDE4B) is disrupted by a balanced translocation in a subject diagnosed with schizophrenia and a relative with chronic psychiatric illness. The PDEs inactivate adenosine 3',5'-monophosphate (cAMP), a second messenger implicated in learning, memory, and mood.

In resting COS1 cells a dominant negative approach shows that specific, anchored PDE4 cAMP phosphodiesterase isoforms gate the activation, by basal cyclic AMP production, of AKAP-tethered protein kinase A type II located in the centrosomal region.

We employ a novel, dominant negative approach to identify a key role for certain tethered cyclic AMP specific phosphodiesterase-4 (PDE4) isoforms in regulating cyclic AMP dependent protein kinase A (PKA) sub-populations in resting COS1 cells. A fraction of PKA is clearly active in resting COS1 cells and this activity increases when cells are treated with the selective PDE4 inhibitor, rolipram. Point mutation of a critical, conserved aspartate residue in the catalytic site of long PDE4A4, PDE4B1, PDE4C2 and PDE4D3 isoforms renders them catalytically inactive.

Identification and characterization of PDE4A11, a novel, widely expressed long isoform encoded by the human PDE4A cAMP phosphodiesterase gene.

PDE4A11 is a novel cAMP-specific phosphodiesterase that is conserved in humans, mouse, rat, pig, and bat. Exon-1(4A11) encodes its unique, 81 amino acid N-terminal region. Reverse-transcriptase polymerase chain reaction performed across the splice junction, plus identification of expressed sequence tags, identifies PDE4A11 as a long isoform possessing UCR1 and UCR2 regulatory domains.

Fluorescence resonance energy transfer-based analysis of cAMP dynamics in live neonatal rat cardiac myocytes reveals distinct functions of compartmentalized phosphodiesterases.

Cardiac myocytes have provided a key paradigm for the concept of the compartmentalized cAMP generation sensed by AKAP-anchored PKA. Phosphodiesterases (PDEs) provide the sole route for degrading cAMP in cells and are thus poised to regulate intracellular cAMP gradients. PDE3 and PDE4 represent the major cAMP degrading activities in rat ventriculocytes.

Expression, intracellular distribution and basis for lack of catalytic activity of the PDE4A7 isoform encoded by the human PDE4A cAMP-specific phosphodiesterase gene.

PDE4A7 is an isoform encoded by the human PDE4A cAMP-specific phosphodiesterase gene that fails to hydrolyse cAMP and whose transcripts are widely expressed. Removal of either the N- or C-terminal unique portions of PDE4A7 did not reconstitute catalytic activity, showing that they did not exert a chronic inhibitory effect. A chimera (Hyb2), formed by swapping the unique N-terminal portion of PDE4A7 with that of the active PDE4A4C form, was not catalytically active.

The unique amino-terminal region of the PDE4D5 cAMP phosphodiesterase isoform confers preferential interaction with beta-arrestins.

Isoproterenol challenge of Hek-B2 cells causes a transient recruitment of the endogenous PDE4D isoforms found in these cells, namely PDE4D3 and PDE4D5, to the membrane fraction. PDE4D5 provides around 80% of the total PDE4D protein so recruited, although it only comprises about 40% of the total PDE4D protein in Hek-B2 cells. PDE4D5 provides about 80% of the total PDE4D protein found associated with beta-arrestins immunopurified from Hek-B2, COS1, and A549 cells as well as cardiac myocytes, whereas its overall level in these cells is between 15 and 50% of the total PDE4D protein.

Occupancy of the catalytic site of the PDE4A4 cyclic AMP phosphodiesterase by rolipram triggers the dynamic redistribution of this specific isoform in living cells through a cyclic AMP independent process.

In cells transfected to express wild-type PDE4A4 cAMP phosphodiesterase (PDE), the PDE4 selective inhibitor rolipram caused PDE4A4 to relocalise so as to form accretion foci. This process was followed in detail in living cells using a PDE4A4 chimera formed with Green Fluorescent Protein (GFP). The same pattern of behaviour was also seen in chimeras of PDE4A4 formed with various proteins and peptides, including LimK, RhoC, FRB and the V5-6His tag.

Attenuation of the activity of the cAMP-specific phosphodiesterase PDE4A5 by interaction with the immunophilin XAP2.

The cyclic AMP-specific phosphodiesterase (PDE4) isoform PDE4A5 interacted with the immunophilin XAP2 in a yeast two-hybrid assay. The interaction was confirmed in biochemical pull-down analyses. The interaction was specific, in that PDE4A5 did not interact with the closely related immunophilins AIPL1, FKBP51, or FKBP52.

TAPAS-1, a novel microdomain within the unique N-terminal region of the PDE4A1 cAMP-specific phosphodiesterase that allows rapid, Ca2+-triggered membrane association with selectivity for interaction with phosphatidic acid.

Here we identify an 11-residue helical module in the unique N-terminal region of the cyclic AMP-specific phosphodiesterase PDE4A1 that determines association with phospholipid bilayers and shows a profound selectivity for interaction with phosphatidic acid (PA). This module contains a core bilayer insertion unit that is formed by two tryptophan residues, Trp(19) and Trp(20), whose orientation is optimized for bilayer insertion by the Leu(16):Val(17) pairing. Ca(2+), at submicromolar levels, interacts with Asp(21) in this module and serves to gate bilayer insertion, which is completed within 10 ms.

In addition to the SH3 binding region, multiple regions within the N-terminal noncatalytic portion of the cAMP-specific phosphodiesterase, PDE4A5, contribute to its intracellular targeting.

The long cyclic AMP (cAMP)-specific phosphodiesterase isoform, PDE4A5 (PDE4A subfamily isoform variant 5), when transiently expressed in COS-7 cells, was shown in subcellular fractionation studies to be associated with both membrane and cytosol fractions, with immunofluorescence analyses identifying PDE4A5 as associated both with ruffles at the cell margin and also at a distinct perinuclear localisation. Deletion of the first nine amino acids of PDE4A5 (1) ablated its ability to interact with the SH3 domain of the tyrosyl kinase, LYN; (2) reduced, but did not ablate, membrane association; and (3) disrupted the focus of PDE4A5 localisation within ruffles at the cell margin. This deleted region contained a Class I SH3 binding motif of similar sequence to those identified by screening a phage display library with the LYN-SH3 domain.