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J D Scott

Publications and source records attributed to J D Scott.

At least 37 records · Page 2Linked to original sources
PubMed2001

Multiple interactions within the AKAP220 signaling complex contribute to protein phosphatase 1 regulation.

The phosphorylation status of cellular proteins is controlled by the opposing actions of protein kinases and phosphatases. Compartmentalization of these enzymes is critical for spatial and temporal control of these phosphorylation/dephosphorylation events. We previously reported that a 220-kDa A-kinase anchoring protein (AKAP220) coordinates the location of the cAMP-dependent protein kinase (PKA) and the type 1 protein phosphatase catalytic subunit (PP1c) (Schillace, R. V., and Scott, J. D. (1999) Curr. Biol. 9, 321-324). We now demonstrate that an AKAP220 fragment is a competitive inhibitor of PP1c activity (K(i) = 2.9 +/- 0.7 micrometer). Mapping studies and activity measurements indicate that several protein-protein interactions act synergistically to inhibit PP1. A consensus targeting motif, between residues 1195 and 1198 (Lys-Val-Gln-Phe), binds but does not affect enzyme activity, whereas determinants between residues 1711 and 1901 inhibit the phosphatase. Analysis of truncated PP1c and chimeric PP1/2A catalytic subunits suggests that AKAP220 inhibits the phosphatase in a manner distinct from all known PP1 inhibitors and toxins. Intermolecular interactions within the AKAP220 signaling complex further contribute to PP1 inhibition as addition of the PKA regulatory subunit (RII) enhances phosphatase inhibition. These experiments indicate that regulation of PP1 activity by AKAP220 involves a complex network of intra- and intermolecular interactions.

A Kinase Anchor Proteins↗
PubMed2001

Factors associated with postoperative infection.

OBJECTIVE: We have developed and analyzed a large surgical prophylaxis database and now report the factors significantly associated with early infection, readmission due to infection, and death within 28 days of surgery. This study is intended to be a stepping-stone for further studies using our clinical database. DESIGN AND SETTING: A computerized database of 9,016 surgical patients from a 400-bed community hospital was examined. Multivariate logistic regression and tree-based modeling were used to identify factors associated with the outcomes. Factors considered included surgical procedure, prophylactic antibiotic, age, gender, serum creatinine, and albumin. RESULTS: 12.6% had an early infection, 2.5% were readmitted due to infection, and 2.5% died within 28 days. Most combination prophylactic antibiotics were associated with an increased probability of an early infection. Decreased albumin and increased age were associated with an increased probability of an early infection. Tracheostomy and amputations were associated with an increased probability of an early infection, whereas gallbladder and orthopedic procedures involving the arm were associated with a decreased probability. Factors associated with readmission due to infection included dialysis shunt, vascular repair, and an early infection. Factors associated with increased probability of death within 28 days included age, albumin, serum creatinine, and an early infection. Gallbladder procedures and obstetric-gynecologic procedures were associated with a decreased probability of death within 28 days. DISCUSSION: Older patients and those with a decreased albumin were most likely to have an early infection. To the extent that an early infection was a significant risk factor for readmission due to infection, the impact of age and albumin on the probability of readmission due to infection is demonstrated by their effects on early infections. Interestingly, albumin and age were significantly associated with death within 28 days, in addition to early infection, showing the predictive association between these factors and early death.

Anti-Bacterial Agents↗
PubMed2001

AKAP proteins anchor cAMP-dependent protein kinase to KvLQT1/IsK channel complex.

In cardiac myocytes, the slow component of the delayed rectifier K(+) current (I(Ks)) is regulated by cAMP. Elevated cAMP increases I(Ks) amplitude, slows its deactivation kinetics, and shifts its activation curve. At the molecular level, I(Ks) channels are composed of KvLQT1/IsK complexes. In a variety of mammalian heterologous expression systems maintained at physiological temperature, we explored cAMP regulation of recombinant KvLQT1/IsK complexes. In these systems, KvLQT1/IsK complexes were totally insensitive to cAMP regulation. cAMP regulation was not restored by coexpression with the dominant negative isoform of KvLQT1 or with the cystic fibrosis transmembrane regulator. In contrast, coexpression of the neuronal A kinase anchoring protein (AKAP)79, a fragment of a cardiac AKAP (mAKAP), or cardiac AKAP15/18 restored cAMP regulation of KvLQT1/IsK complexes inasmuch as cAMP stimulation increased the I(Ks) amplitude, increased its deactivation time constant, and negatively shifted its activation curve. However, in cells expressing an AKAP, the effects of cAMP stimulation on the I(Ks) amplitude remained modest compared with those previously reported in cardiac myocytes. The effects of cAMP stimulation were fully prevented by including the Ht31 peptide (a global disruptor of protein kinase A anchoring) in the intracellular medium. We concluded that cAMP regulation of I(Ks) requires protein kinase A anchoring by AKAPs, which therefore participate with the channel protein complex underlying I(Ks).

3T3 Cells↗
PubMed2001

AQP2 is a substrate for endogenous PP2B activity within an inner medullary AKAP-signaling complex.

We have demonstrated that inner medullary collecting duct (IMCD) heavy endosomes purified from rat kidney IMCD contain the type II protein kinase A (PKA) regulatory subunit (RII), protein phosphatase (PP)2B, PKCzeta, and an RII-binding protein (relative molecular mass ~90 kDa) representing a putative A kinase anchoring protein (AKAP). Affinity chromatography of detergent-solubilized endosomes on cAMP-agarose permits recovery of a protein complex consisting of the 90-kDa AKAP, RII, PP2B, and PKCzeta. With the use of small-particle flow cytometry, RII and PKCzeta were localized to an identical population of endosomes, suggesting that these proteins are components of an endosomal multiprotein complex. (32)P-labeled aquaporin-2 (AQP2) present in these PKA-phosphorylated endosomes was dephosphorylated in vitro by either addition of exogenous PP2B or by an endogenous endosomal phosphatase that was inhibited by the PP2B inhibitors EDTA and the cyclophilin-cyclosporin A complex. We conclude that IMCD heavy endosomes possess an AKAP multiprotein-signaling complex similar to that described previously in hippocampal neurons. This signaling complex potentially mediates the phosphorylation of AQP2 to regulate its trafficking into the IMCD apical membrane. In addition, the PP2B component of the AKAP-signaling complex could also dephosphorylate AQP2 in vivo.

Animals↗
PubMed2001

Targeted protein kinase A and PP-2B regulate insulin secretion through reversible phosphorylation.

Protein kinases and phosphatases play key roles in integrating signals from various insulin secretagogues. In this study, we show that the activities of the cAMP-dependent protein kinase (PKA) and the calcium/calmodulin-dependent phosphatase, PP-2B are coordinated resulting in the regulation of insulin secretion. Transient inhibition of PP-2B, using the immunosuppressant FK506, increased forskolin stimulated insulin secretion by 2.5-fold +/- 0.3 (n = 6) in rat islets and RINm5F cells. Surprisingly, forskolin treatment resulted in the dephosphorylation of the vesicle-associated protein synapsin 1 and increased PP-2B activity by 2.98 +/- 0.97-fold (n = 4). One potential explanation for the observed coordination of PKA and PP-2B activity is their colocalization through a mutual anchoring protein, AKAP79/150. Accordingly, RINm5F cells expressing AKAP79 exhibited decreased insulin secretion, reduced PP-2B activity and were insensitive to FK506. This suggests that AKAP targeting of PKA and PP-2B maintains a signal transduction complex that may regulate reversible phosphorylation events involved in insulin secretion.

Animals↗
PubMed2001

AKAP signaling complexes at the cytoskeleton.

Targeting of protein kinases and phosphatases to the cytoskeleton enhances the regulation of signal transduction events. The assembly of cytoskeletal signaling complexes facilitates the relay of messages from membrane receptors to specific sites on the actin cytoskeleton. These signals influence fundamental cell properties, such as shape, movement and division. Targeting of the cAMP-dependent kinase (PKA) to the cytoskeleton is achieved through interaction with A-kinase anchoring proteins (AKAPs). AKAPs maintain multivalent signaling complexes by binding additional enzymes, including kinases and phosphatases.

A Kinase Anchor Proteins↗
PubMed2001

Birds disperse ixodid (Acari: Ixodidae) and Borrelia burgdorferi-infected ticks in Canada.

A total of 152 ixodid ticks (Acari: Ixodidae) consisting of nine species was collected from 82 passerine birds (33 species) in 14 locations in Canada from 1996 to 2000. The Lyme disease spirochete Borrelia burgdorferi Johnson, Schmidt, Hyde, Steigerwaldt & Brenner was cultured from the nymph of a blacklegged tick, Ixodes scapularis Say, that had been removed from a common yellowthroat, Geothlypis trichas L., from Bon Portage Island, Nova Scotia. As a result of bird movement, a nymphal I. scapularis removed from a Swainson's thrush, Catharus ustulatus incanus (Godfrey), at Slave Lake, Alberta, during spring migration becomes the new, most western and northern record of this tick species in Canada. Amblyomma longirostre Koch, Amblyomma sabanerae Stoll, and Ixodes baergi Cooley & Kohls are reported for the first time in Canada. Similarly, Amblyomma americanum L., Arnblyomma maculatum Koch, and ixodes muris Bishopp & Smith are reported for the first time on birds in Canada. After removal of an I. muris gravid female from a song sparrow, Melospiza melodia Wilson, at St. Andrews, New Brunswick, eggs were laid, which developed into larvae, and this new tick-host record demonstrates that birds have the potential to start a new tick population. We conclude that passerine birds disperse several species of ixodid ticks in Canada, and during spring migration translocate ticks from the United States, and Central and South America, some of which are infected with B. burgdorferi.

Animals↗
PubMed2000

Variation in the vitreous phenotype of Stickler syndrome can be caused by different amino acid substitutions in the X position of the type II collagen Gly-X-Y triple helix.

Stickler syndrome is a dominantly inherited disorder characterized by arthropathy, midline clefting, hearing loss, midfacial hypoplasia, myopia, and retinal detachment. These features are highly variable both between and within families. Mutations causing the disorder have been found in the COL2A1 and COL11A1 genes. Premature termination codons in COL2A1 that result in haploinsufficiency of type II collagen are a common finding. These produce a characteristic congenital "membranous" anomaly of the vitreous of all affected individuals. Experience has shown that vitreous slit-lamp biomicroscopy can distinguish between patients with COL2A1 mutations and those with dominant negative mutations in COL11A1, who produce a different "beaded" vitreous phenotype. Here we characterize novel dominant negative mutations in COL2A1 that result in Stickler syndrome. Both alter amino acids in the X position of the Gly-X-Y triple-helical region. A recurrent R365C mutation occurred in two unrelated sporadic cases and resulted in the membranous vitreous anomaly associated with haploinsufficiency. In a large family with linkage to COL2A1, with a LOD score of 2.8, a unique L467F mutation produced a novel "afibrillar" vitreous gel devoid of all normal lamella structure. These data extend the mutation spectrum of the COL2A1 gene and help explain the basis for the different vitreous phenotypes seen in Stickler syndrome.

Abnormalities, Multiple↗
PubMed2000

Scar/WAVE-1, a Wiskott-Aldrich syndrome protein, assembles an actin-associated multi-kinase scaffold.

WAVE proteins are members of the Wiskott-Aldrich syndrome protein (WASP) family of scaffolding proteins that coordinate actin reorganization by coupling Rho-related small molecular weight GTPases to the mobilization of the Arp2/3 complex. We identified WAVE-1 in a screen for rat brain A kinase-anchoring proteins (AKAPs), which bind to the SH3 domain of the Abelson tyrosine kinase (Abl). Recombinant WAVE-1 interacts with cAMP-dependent protein kinase (PKA) and Abl kinases when expressed in HEK-293 cells, and both enzymes co-purify with endogenous WAVE from brain extracts. Mapping studies have defined binding sites for each kinase. Competition experiments suggest that the PKA-WAVE-1 interaction may be regulated by actin as the kinase binds to a site overlapping a verprolin homology region, which has been shown to interact with actin. Immunocytochemical analyses in Swiss 3T3 fibroblasts suggest that the WAVE-1 kinase scaffold is assembled dynamically as WAVE, PKA and Abl translocate to sites of actin reorganization in response to platelet-derived growth factor treatment. Thus, we propose a previously unrecognized function for WAVE-1 as an actin-associated scaffolding protein that recruits PKA and Abl.

3T3 Cells↗
PubMed2000

AKAP79 and the evolution of the AKAP model.

A molecular explanation for the specificity of the cAMP-dependent protein kinase (PKA) can be provided by its compartmentalization through association with A-kinase-anchoring proteins (AKAPs). Structural and functional studies have led to the development of an anchoring model proposing that AKAPs contain a common PKA binding domain and a unique subcellular targeting domain. The discovery that AKAPs can bind other signaling enzymes led to the addition of a third property, that of scaffolding molecule. Recent research has now expanded the role of AKAPs to members of multiunit complexes containing both upstream activators and downstream targets.

A Kinase Anchor Proteins↗
PubMed2000

Assembly of an A kinase-anchoring protein-beta(2)-adrenergic receptor complex facilitates receptor phosphorylation and signaling.

Phosphorylation of G-protein-coupled receptors by second-messenger-stimulated kinases is central to the process of receptor desensitization [1-3]. Phosphorylation of the beta(2)-adrenergic receptor (beta(2)-AR) by protein kinase A (PKA), in addition to uncoupling adenylate cyclase activation, is obligatory for receptor-mediated activation of mitogen-activated protein kinase (MAP kinase) cascades [4] [5]. Although mechanisms for linking G-protein-coupled receptor kinases to the activated receptor are well established, analogous mechanisms for targeting second messenger kinases to the beta(2)-AR at the plasma membrane have not been elucidated. Here we show that the A-kinase-anchoring protein, AKAP79/150, co-precipitates with the beta(2)-AR in cell and tissue extracts, nucleating a signaling complex that includes PKA, protein kinase C (PKC) and protein phosphatase PP2B. The anchoring protein directly and constitutively interacts with the beta(2)-AR and promotes receptor phosphorylation following agonist stimulation. Functional studies show that PKA anchoring is required to enhance beta(2)-AR phosphorylation and to facilitate downstream activation of the MAP kinase pathway. This defines a role for AKAP79/150 in the recruitment of second-messenger-regulated signaling enzymes to a G-protein-coupled receptor.

A Kinase Anchor Proteins↗
PubMed2000

Pericentrin anchors protein kinase A at the centrosome through a newly identified RII-binding domain.

Centrosomes orchestrate microtubule nucleation and spindle assembly during cell division [1,2] and have long been recognized as major anchoring sites for cAMP-dependent protein kinase (PKA) [3,4]. Subcellular compartmentalization of PKA is achieved through the association of the PKA holoenzyme with A-kinase anchoring proteins (AKAPs) [5,6]. AKAPs have been shown to contain a conserved helical motif, responsible for binding to the type II regulatory subunit (RII) of PKA, and a specific targeting motif unique to each anchoring protein that directs the kinase to specific intracellular locations. Here, we show that pericentrin, an integral component of the pericentriolar matrix of the centrosome that has been shown to regulate centrosome assembly and organization, directly interacts with PKA through a newly identified binding domain. We demonstrate that both RII and the catalytic subunit of PKA coimmunoprecipitate with pericentrin isolated from HEK-293 cell extracts and that PKA catalytic activity is enriched in pericentrin immunoprecipitates. The interaction of pericentrin with RII is mediated through a binding domain of 100 amino acids which does not exhibit the structural characteristics of similar regions on conventional AKAPs. Collectively, these results provide strong evidence that pericentrin is an AKAP in vivo.

Antigens↗
PubMed2000

Targeting of PKA to glutamate receptors through a MAGUK-AKAP complex.

Compartmentalization of glutamate receptors with the signaling enzymes that regulate their activity supports synaptic transmission. Two classes of binding proteins organize these complexes: the MAGUK proteins that cluster glutamate receptors and AKAPs that anchor kinases and phosphatases. In this report, we demonstrate that glutamate receptors and PKA are recruited into a macromolecular signaling complex through direct interaction between the MAGUK proteins, PSD-95 and SAP97, and AKAP79/150. The SH3 and GK regions of the MAGUKs mediate binding to the AKAP. Cell-based studies indicate that phosphorylation of AMPA receptors is enhanced by a SAP97-AKAP79 complex that directs PKA to GluR1 via a PDZ domain interaction. As AMPA receptor phosphorylation is implicated in regulating synaptic plasticity, these data suggest that a MAGUK-AKAP complex may be centrally involved.

Adaptor Proteins, Signal Transducing↗
PubMed2000

A-kinase anchoring proteins: protein kinase A and beyond.

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). In the past year there have been many advances in our understanding of AKAPs, particularly in the field of the functional consequences of PKA anchoring.

A Kinase Anchor Proteins↗
PubMed2000

Synthesis of a netropsin conjugate of a water-soluble epi-quinocarcin analogue: the importance of stereochemistry at nitrogen.

The efficient synthesis of a water-soluble C11a-epi-analogue (6b) of quinocarcin is described. This substance, and a netropsin amide conjugate (8) lack the capacity to inflict oxidative damage on DNA due to the stereoelectronic geometry of their oxazolidine nitrogen atoms. The capacity of these substances to alkylate DNA through the generation of an iminium species has been examined. Both compounds were found to be unreactive as DNA alkylating agents. The results of this study are discussed in the context of previous proposals on the mode of action of this family of antitumor alkaloids.

Alkylating Agents↗