Adrenergic stimulation of adipocytes yields a cAMP signal that activates protein kinase A (PKA). PKA phosphorylates perilipin, a protein localized on the surface of lipid droplets that serves as a gatekeeper to regulate access of lipases converting stored triglycerides to free fatty acids and glycerol in a phosphorylation-dependent manner. Here, we report a new function for optic atrophy 1 (OPA1), a protein known to regulate mitochondrial dynamics, as a dual-specificity A-kinase anchoring protein associated with lipid droplets. By a variety of protein interaction assays, immunoprecipitation and immunolocalization experiments, we show that OPA1 organizes a supramolecular complex containing both PKA and perilipin. Furthermore, by a combination of siRNA-mediated knockdown, reconstitution experiments using full-length OPA1 with or without the ability to bind PKA or truncated OPA1 fused to a lipid droplet targeting domain and cellular delivery of PKA anchoring disruptor peptides, we demonstrate that OPA1 targeting of PKA to lipid droplets is necessary for hormonal control of perilipin phosphorylation and lipolysis.
cAMP inhibits Src-family kinase signalling by PKA (protein kinase A)-mediated phosphorylation and activation of Csk (C-terminal Src kinase). The PKA type I-Csk pathway is assembled and localized in membrane microdomains (lipid rafts) and regulates immune responses activated through the TCR (T-cell receptor). PKA type I is targeted to the TCR-CD3 complex during T-cell activation via an AKAP (A-kinase-anchoring protein) that serves as a scaffold for the cAMP-PKA/Csk pathway in lipid rafts of the plasma membrane during T-cell activation. Displacement of PKA by anchoring disruption peptides prevents cAMP/PKA type I-mediated inhibition of T-cell activation. These findings provide functional evidence that PKA type I regulation of T-cell responses is dependent on AKAP anchoring. Furthermore, we show that upon TCR/CD28 co-ligation, beta-arrestin in complex with PDE4 (phosphodiesterase 4) is recruited to lipid rafts. The CD28-mediated recruitment of PDE4 to lipid rafts potentiates T-cell immune responses and counteracts the local, TCR-induced production of cAMP that produces negative feedback in the absence of a co-receptor stimulus. The specific recruitment of PDE4 thus serves to abrogate the negative feedback by cAMP which is elicited in the absence of a co-receptor stimulus.
The second messenger cAMP is frequently utilized in mammalian cells to regulate a variety of physiological processes. Cyclic AMP is generated at the plasma membrane in response to the occupancy of G-protein-coupled receptors. This ultimately leads to the stimulation of adenylyl cyclases, the enzymes that produce cAMP. The newly synthesized cAMP diffuses into the cell where it is available to activate a variety of effector proteins. These include protein kinase A (PKA) 4 (reviewed in Ref. 1), cAMP-regulated ion channels (2), and Epac guanine nucleotide exchange factors (3). Activation of the PKA holoenzyme occurs upon binding of cAMP to the regulatory (R) subunits. This promotes dissociation of the active catalytic (C) subunits from the tetrameric complex and results in the phosphorylation of substrates in the vicinity of the active kinase (4, 5). PKA holoenzymes are classified as either type I or type II on the basis of their R subunit composition (RI or RII) (6). Four genes encode R subunits (RI␣, RI, RII␣, and RII). These proteins have distinct physical properties and affinities for cAMP (1). Because PKA is a broad specificity serine/threonine protein kinase that regulates a wide range of cellular processes, additional mechanisms have evolved to influence the selectivity of PKA action (7). Specificity in PKA action is maintained in part by interaction with protein kinase A anchoring proteins (AKAPs). This family of structurally diverse but functionally related scaffolding proteins targets PKA and other signaling proteins toward distinct substrates. These protein-protein targeting interactions contribute to spatial and temporal regulation of second messenger signaling events (reviewed in Refs. 7,8).The AKAP family now includes more than 50 members when including splice variants (7,8). Although most of the AKAPs were initially identified on the basis of their ability to bind PKA type II inside cells, it is now recognized that several of these anchoring proteins such as D-AKAP1, D-AKAP2, AKAP220, Ezrin, Merlin, and PAP7 have a dual specificity as they also bind PKA type I (9 -14). Other AKAPs are reported to selectively bind RI such as AKAP CE , myosin, and ␣4 integrins (15-17). However, only two of these dual specificity proteins, the mitochondrial protein PAP7 and Ezrin (12,18), have been shown to preferentially interact with PKA type I in situ (15)(16)(17).Conventional AKAPs contain a conserved amphipathic helix of 14 -18 residues that forms the PKA-anchoring domain (19 -21). This region inserts into a hydrophobic groove formed by the R dimer (22, 23). The RII subunits dimerize at the N terminus in an antiparallel fashion forming an X-type four-helix bundle that is necessary for AKAP binding. RI contains a structur-
The optical biosensor technique, based on the surface plasmon resonance (SPR) phenomenon, was used for real-time measurements of the slow conformational transition (isomerization) which occurs in human phenylalanine hydroxylase (hPAH) on the binding/dissociation of L-phenylalanine (L-Phe). The binding to immobilized tetrameric wt-hPAH resulted in a time-dependent increase in the refractive index (up to approx. 3 min at 25 degrees C) with an end point of approx. 75 RU (resonance units)/(pmol subunit/mm(2)). By contrast, the contribution of binding the substrate (165 Da) to its catalytic core enzyme [DeltaN(1-102)/DeltaC(428-452)-hPAH] was only approx. 2 RU/(pmol subunit/mm(2)). The binding isotherm for tetrameric and dimeric wt-hPAH revealed a [S](0.5)-value of 98+/-7 microM (h =1.0) and 158+/-11 microM, respectively, i.e. for the tetramer it is slightly lower than the value (145+/-5 microM) obtained for the co-operative binding (h =1.6+/-0.4) of L-Phe as measured by the change in intrinsic tryptophan fluorescence. The responses obtained by SPR and intrinsic tryptophan fluorescence are both considered to be related to the slow reversible conformational transition which occurs in the enzyme upon L-Phe binding, i.e. by the transition from a low-activity state ('T-state') to a relaxed high-activity state ('R-state') characteristic of this hysteretic enzyme, however, the two methods reflect different elements of the transition. Studies on the N- and C-terminal truncated forms revealed that the N-terminal regulatory domain (residues 1-117) plus catalytic domain (residues 118-411) were required for the full signal amplitude of the SPR response. Both the on- and off-rates for the conformational transition were biphasic, which is interpreted in terms of a difference in the energy barrier and the rate by which the two domains (catalytic and regulatory) undergo a conformational change. The substrate analogue 3-(2-thienyl)-L-alanine revealed an SPR response comparable with that of L-Phe on binding to wild-type hPAH.
The catalytic activity of phenylalanine hydroxylase (PAH, phenylalanine 4-monooxygenase EC 1.14.16.1) is regulated by three main mechanisms, i.e. substrate (L-phenylalanine, L-Phe) activation, pterin cofactor inhibition and phosphorylation of a single serine (Ser16) residue. To address the molecular basis for the inhibition by the natural cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin, its effects on the recombinant tetrameric human enzyme (wt-hPAH) was studied using three different conformational probes, i.e. the limited proteolysis by trypsin, the reversible global conformational transition (hysteresis) triggered by L-Phe binding, as measured in real time by surface plasmon resonance analysis, and the rate of phosphorylation of Ser16 by cAMPdependent protein kinase. Comparison of the inhibitory properties of the natural cofactor with the available threedimensional crystal structure information on the ligand-free, the binary and the ternary complexes, have provided important clues concerning the molecular mechanism for the negative modulatory effects. In the binary complex, the binding of the cofactor at the active site results in the formation of stabilizing hydrogen bonds between the dihydroxypropyl side-chain and the carbonyl oxygen of Ser23 in the autoregulatory sequence. L-Phe binding triggers local as well as global conformational changes of the protomer resulting in a displacement of the cofactor bound at the active site by 2.6 Å (mean distance) in the direction of the iron and Glu286 which causes a loss of the stabilizing hydrogen bonds present in the binary complex and thereby a complete reversal of the pterin cofactor as a negative effector. The negative modulatory properties of the inhibitor dopamine, bound by bidentate coordination to the active site iron, is explained by a similar molecular mechanism including its reversal by substrate binding. Although the pterin cofactor and the substrate bind at distinctly different sites, the local conformational changes imposed by their binding at the active site have a mutual effect on their respective binding affinities.
Subcellular localization of PKA (cAMP-dependent protein kinase or protein kinase A) is determined by protein-protein interactions between its R (regulatory) subunits and AKAPs (A-kinase-anchoring proteins). In the present paper, we report the development of the Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen) as a means to characterize AKAP-based peptide competitors of PKA anchoring. In this assay, the prototypic anchoring disruptor Ht31 efficiently competed in RIIalpha isoform binding with RII-specific and dual-specificity AKAPs (IC50 values of 1.4+/-0.2 nM and 6+/-1 nM respectively). In contrast, RIalpha isoform binding to a dual-specific AKAP was less efficiently competed (IC50 of 156+/-10 nM). Characterization of two RI-selective anchoring disruptors, RIAD (RI-anchoring disruptor) and PV-38 revealed that RIAD (IC50 of 13+/-1 nM) was 20-fold more potent than PV-38 (IC50 of 304+/-17 nM) and did not compete in the RIIalpha-AKAP interaction. We also observed that the kinetics of RII displacement from pre-formed PKA-AKAP complexes and competition of RII-AKAP complex formation by Ht31 differed by an order of magnitude when the component parts were mixed in vitro. No such difference in potency was seen for RIalpha-AKAP complexes. Thus the AlphaScreen assay may prove to be a valuable tool for detailed characterization of a variety of PKA-AKAP complexes.
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