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John D Scott

Publications and source records attributed to John D Scott.

63 records · Page 4Linked to original sources
PubMed2002

Rab32 is an A-kinase anchoring protein and participates in mitochondrial dynamics.

A-kinase anchoring proteins (AKAPs) tether the cAMP-dependent protein kinase (PKA) and other signaling enzymes to distinct subcellular organelles. Using the yeast two-hybrid approach, we demonstrate that Rab32, a member of the Ras superfamily of small molecular weight G-proteins, interacts directly with the type II regulatory subunit of PKA. Cellular and biochemical studies confirm that Rab32 functions as an AKAP inside cells. Anchoring determinants for PKA have been mapped to sites within the conserved alpha5 helix that is common to all Rab family members. Subcellular fractionation and immunofluorescent approaches indicate that Rab32 and a proportion of the cellular PKA pool are associated with mitochondria. Transient transfection of a GTP binding-deficient mutant of Rab32 promotes aberrant accumulation of mitochondria at the microtubule organizing center. Further analysis of this mutant indicates that disruption of the microtubule cytoskeleton results in aberrantly elongated mitochondria. This implicates Rab32 as a participant in synchronization of mitochondrial fission. Thus, Rab32 is a dual function protein that participates in both mitochondrial anchoring of PKA and mitochondrial dynamics.

Amino Acid Sequence↗
PubMed2002

Trafficking of L-type calcium channels mediated by the postsynaptic scaffolding protein AKAP79.

Accurate calcium signaling requires spatial and temporal coordination of voltage-gated calcium channels (VGCCs) and a variety of signal transduction proteins. Accordingly, regulation of L-type VGCCs involves the assembly of complexes that include the channel subunits, protein kinase A (PKA), protein kinase A anchoring proteins (AKAPs), and beta2-adrenergic receptors, although the molecular details underlying these interactions remain enigmatic. We show here, by combining extracellular epitope splicing into the channel pore-forming subunit and immunoassays with whole cell and single channel electrophysiological recordings, that AKAP79 directly regulates cell surface expression of L-type calcium channels independently of PKA. This regulation involves a short polyproline sequence contained specifically within the II-III cytoplasmic loop of the channel. Thus we propose a novel mechanism whereby AKAP79 and L-type VGCCs function as components of a biosynthetic mechanism that favors membrane incorporation of organized molecular complexes in a manner that is independent of PKA phosphorylation events.

A Kinase Anchor Proteins↗
PubMed2002

Regulation of GluR1 by the A-kinase anchoring protein 79 (AKAP79) signaling complex shares properties with long-term depression.

Second messengers regulate synaptic plasticity by influencing the balance between kinase and phosphatase activity. One target of this balance is the phosphorylation state of the AMPA receptor glutamate receptor 1 (GluR1) subunit. Hippocampal long-term depression (LTD) is a calcium-dependent downregulation of synaptic AMPA receptor currents associated with dephosphorylation of Ser845, a cAMP-dependent protein kinase (PKA) site on GluR1. Recruitment of kinases and phosphatases to the AMPA receptor might enable modulation of AMPA receptor function. The neuronal A-kinase anchoring protein AKAP79/150 interacts with PKA and the calcium-dependent protein phosphatase PP2B and is linked to the AMPA receptor GluR1 subunit by synapse-associated protein 97 (SAP97), a membrane-associated guanylate kinase family protein. Here we demonstrate that AKAP79 not only promotes basal phosphorylation of Ser845 but also confers a calcium- and PP2B-mediated downregulation to GluR1 receptor currents. This AKAP79-dependent downregulation is contingent on the local presence of PKA, Ser845 of GluR1, and a PDZ (postsynaptic density 95/Discs large/zona occludens 1)-domain interaction between GluR1 and SAP97, all of which support basal phosphorylation of the receptor. These findings suggest that the AKAP79 signaling complex is sufficient to couple intracellular calcium levels to the PKA phosphorylation state of GluR1. Thus, the integration of intracellular signals relevant for LTD may be transduced to GluR1 by the AKAP79 signaling complex.

A Kinase Anchor Proteins↗
PubMed2002

Signaling complexes: junctions on the intracellular information super highway.

An enigmatic yet fundamental principle of signal transduction is that parallel signaling pathways assembled from a common repertoire of enzymes are able to propagate diverse physiological responses. A key feature of such a mechanism is that separate signaling pathways are organized into localized transduction units, each tailored to respond optimally to a particular signal. Protein-protein interactions maintained by anchoring, adapter and scaffolding proteins provide the molecular glue that holds these signal transduction units together. A major objective of the signaling community is to ascertain how signals flow through compartmentalized transduction units that contain transmembrane receptors, protein kinases, phosphatases and their substrates.

Animals↗
PubMed2002

The WRP component of the WAVE-1 complex attenuates Rac-mediated signalling.

WAVE-1, which is also known as Scar, is a scaffolding protein that directs actin reorganization by relaying signals from the GTPase Rac to the Arp2/3 complex. Although the molecular details of WAVE activation by Rac have been described, the mechanisms by which these signals are terminated remain unknown. Here we have used tandem mass spectrometry to identify previously unknown components of the WAVE signalling network including WRP, a Rac-selective GTPase-activating protein. WRP binds directly to WAVE-1 through its Src homology domain 3 and specifically inhibits Rac function in vivo. Thus, we propose that WRP is a binding partner of WAVE-1 that functions as a signal termination factor for Rac.

Amino Acid Sequence↗
PubMed2002

Electrostatic properties of the structure of the docking and dimerization domain of protein kinase A IIalpha.

The structure of the N-terminal docking and dimerization domain of the type IIalpha regulatory subunit (RIIalpha D/D) of protein kinase A (PKA) forms a noncovalent stand-alone X-type four-helix bundle structural motif, consisting of two helix-loop-helix monomers. RIIalpha D/D possesses a strong hydrophobic core and two distinct, exposed faces. A hydrophobic face with a groove is the site of protein-protein interactions necessary for subcellular localization. A highly charged face, opposite to the former, may be involved in regulation of protein-protein interactions as a result of changes in phosphorylation state of the regulatory subunit. Although recent studies have addressed the hydrophobic character of packing of RIIalpha D/D and revealed the function of the hydrophobic face as the binding site to A-kinase anchoring proteins (AKAPs), little attention has been paid to the charges involved in structure and function. To examine the electrostatic character of the structure of RIIalpha D/D we have predicted mean apparent pKa values, based on Poisson-Boltzmann electrostatic calculations, using an ensemble of calculated dimer structures. We propose that the helix promoting sequence Glu34-X-X-X-Arg38 stabilizes the second helix of each monomer, through the formation of a (i, i +4) side chain salt bridge. We show that a weak inter-helical hydrogen bond between Tyr35-Glu19 of each monomer contributes to tertiary packing and may be responsible for discriminating from alternative quaternary packing of the two monomers. We also show that an inter-monomer hydrogen bond between Asp30-Arg40 contributes to quaternary packing. We propose that the charged face comprising of Asp27-Asp30-Glu34-Arg38-Arg40-Glu41-Arg43-Arg44 may be necessary to provide flexibility or stability in the region between the C-terminus and the interdomain/autoinhibitory sequence of RIIalpha, depending on the activation state of PKA. We also discuss the structural requirements necessary for the formation of a stacked (rather than intertwined) dimer, which has consequences for the orientation of the functionally important and distinct faces.

Binding Sites↗
PubMed2002

AKAP mediated signal transduction.

Compartmentalization of cyclic AMP-dependent protein kinase (PKA) is achieved through association with A-kinase anchoring proteins (AKAPs). AKAPs are a group of structurally diverse proteins with the common function of binding to the regulatory subunit of PKA and confining the holoenzyme to discrete locations within the cell. This mode of regulation ensures that PKA is exposed to isolated cAMP gradients, which allows for efficient catalytic activation and accurate substrate selection. Several AKAPs coordinate multiple members of signaling cascades, effectively assembling upstream activators and downstream effectors within the same macromolecular complex. Consequently, AKAPs may serve as points of integration for numerous signaling pathways. This review details the most recent advances in our understanding of the various biological functions dependent upon AKAP-anchored signaling complexes.

A Kinase Anchor Proteins↗
PubMed2002

Intracellular targeting of protein kinases and phosphatases.

Compartmentalization of kinases and phosphatases is a key determinant in the specificity of second messenger-mediated signaling events. Localization of the cAMP-dependent protein kinase (PKA) and other signaling enzymes is mediated by interaction with A-kinase anchoring proteins (AKAPs). This study focused on recent advances that further our understanding of AKAPs, with particular emphasis on the bidirectional regulation of signaling events by AKAP signaling complexes and their contribution to the control of actin reorganization events.

Actins↗