Search PubMed⌕ Search

Biomedical subjects

J D Scott

Publications and source records attributed to J D Scott.

At least 91 records · Page 5Linked to original sources
PubMed1997

Regulation of the AKAP79-protein kinase C interaction by Ca2+/Calmodulin.

The A kinase-anchoring protein AKAP79 coordinates the location of the cAMP-dependent protein kinase (protein kinase A), calcineurin, and protein kinase C (PKC) at the postsynaptic densities in neurons. Individual enzymes in the AKAP79 signaling complex are regulated by distinct second messenger signals; however, both PKC and calcineurin are inhibited when associated with the anchoring protein, suggesting that additional regulatory signals must be required to release active enzyme. This report focuses on the regulation of AKAP79-PKC interaction by calmodulin. AKAP79 binds calmodulin with high affinity (KD of 28 +/- 4 nM (n = 3)) in a Ca2+-dependent manner. Immunofluorescence staining shows that both proteins exhibit overlapping staining patterns in cultured hippocampal neurons. Calmodulin reversed the inhibition of PKCbetaII by the AKAP79(31-52) peptide and reduced inhibition by the full-length AKAP79 protein. The effect of calmodulin on inhibition of a constitutively active PKC fragment by the AKAP79(31-52) peptide was shown to be partially dependent on Ca2+. Ca2+/calmodulin reduced PKC coimmunoprecipitated with AKAP79 and resulted in a 2.6 +/- 0.5-fold (n = 6) increase in PKC activity in a preparation of postsynaptic densities. Collectively, these findings suggest that Ca2+/calmodulin competes with PKC for binding to AKAP79, releasing the inhibited kinase from its association with the anchoring protein.

A Kinase Anchor Proteins↗
PubMed1997

The physician's role in minimizing prehospital delay in patients at high risk for acute myocardial infarction: recommendations from the National Heart Attack Alert Program. Working Group on Educational Strategies To Prevent Prehospital Delay in Patients at High Risk for Acute Myocardial Infarction.

Physicians and other health care professionals play an important role in reducing the delay to treatment in patients who have an evolving acute myocardial infarction. A multidisciplinary working group has been convened by the National Heart Attack Alert Program (which is coordinated by the National Heart, Lung, and Blood Institute of the National Institutes of Health) to address this concern. The working group's recommendations target specific groups of patients: those who are known to have coronary heart disease, atherosclerotic disease of the aorta or peripheral arteries, or cerebrovascular disease. The risk for acute myocardial infarction or death in such patients is five to seven times greater than that in the general population. The working group recommends that these high-risk patients be clearly informed about symptoms that they might have during a coronary occlusion, steps that they should take, the importance of contacting emergency medical services, the need to report to an appropriate facility quickly, treatment options that are available if they present early, and rewards of early treatment in terms of improved quality of life. These instructions should be reviewed frequently and reinforced with appropriate written material, and patients should be encouraged to have a plan and to rehearse it periodically. Because of the important role of the bystander in increasing or decreasing delay to treatment, family members and significant others should be included in all instruction. Finally, physicians' offices and clinics should devise systems to quickly assess patients who telephone or present with symptoms of a possible acute myocardial infarction.

Algorithms↗
PubMed1997

Gravin, an autoantigen recognized by serum from myasthenia gravis patients, is a kinase scaffold protein.

BACKGROUND: Subcellular targeting of protein kinases and phosphatases provides a mechanism for co-localizing these enzymes with their preferred substrates. A recently identified mammalian scaffold protein, AKAP79, controls the location of two broad-specificity kinases and a phosphatase. RESULTS: We have identified and characterized another mammalian scaffold protein which coordinates the location of protein kinase A and protein kinase C. We isolated a cDNA encoding a 250 kDa A-skinase anchoring protein (AKAP) called gravin, which was originally identified as a cytoplasmic antigen recognized by myasthenia gravis sera. Sequence homology to proteins that are known to bind protein kinase C suggests that gravin also binds this kinase. Studies of binding in vitro show that residues 1526-1780 of gravin bind the regulatory subunit (RII) of protein kinase A with high affinity, and residues 265-556 bind protein kinase C. Gravin expression in human erythroleukemia cells can be induced with phorbol ester, resulting in the detection of a 250 kDa RII- and PKC-binding protein. Immunolocalization experiments show that gravin is concentrated at the cell periphery and is enriched in filopodia. Gravin staining is coincident with an AKAP detected by an in situ RII-overlay assay, and a PKA-gravin complex can be isolated from human erythroleukemia cells. CONCLUSIONS: We present biochemical evidence that gravin forms part of a signaling scaffold, and propose that protein kinases A and C may participate in the coordination of signal transduction events in the filopodia of human erythroleukemia cells.

A Kinase Anchor Proteins↗
PubMed1997

Integrated RF coil with stabilization for fMRI human cortex.

Functional brain imaging of the human cortex is limited by poor contrast to noise ratio (CNR) and image degradation due to subject motion during the acquisition period. The work described here combines the use of closely coupled phased array receiver coils with a stabilization system to address these needs. Several phased array designs are evaluated and compared with the conventional "birdcage" design. Coil performance is reported in terms of relative SNR and fMRI results. Relative improvements of up to 360% are obtained for the occipital region and 180% in the temporal region. More modest gains of 10-30% were obtained for a "dome"-shaped birdcage volume coil covering the entire cortex.

Cerebral Cortex↗
PubMed1997

cAMP-dependent regulation of cardiac L-type Ca2+ channels requires membrane targeting of PKA and phosphorylation of channel subunits.

The cardiac L-type Ca2+ channel is a textbook example of an ion channel regulated by protein phosphorylation; however, the molecular events that underlie its regulation remain unknown. Here, we report that in transiently transfected HEK293 cells expressing L-type channels, elevations in cAMP resulted in phosphorylation of the alpha1C and beta2a channel subunits and increases in channel activity. Channel phosphorylation and regulation were facilitated by submembrane targeting of protein kinase A (PKA), through association with an A-kinase anchoring protein called AKAP79. In transfected cells expressing a mutant AKAP79 that is unable to bind PKA, phosphorylation of the alpha1C subunit and regulation of channel activity were not observed. Furthermore, we have demonstrated that the association of an AKAP with PKA was required for beta-adrenergic receptor-mediated regulation of L-type channels in native cardiac myocytes, illustrating that the events observed in the heterologous expression system reflect those occurring in the native system. Mutation of Ser1928 to alanine in the C-terminus of the alpha1C subunit resulted in a complete loss of cAMP-mediated phosphorylation and a loss of channel regulation. Thus, the PKA-mediated regulation of L-type Ca2+ channels is critically dependent on a functional AKAP and phosphorylation of the alpha1C subunit at Ser1928.

Animals↗
PubMed1997

Techniques for determining electron losses at a 1.5 GeV synchrotron light source.

A computer code was developed to simulate the bremsstrahlung dose rate distribution patterns produced by the CAMD electron storage ring. These bremsstralung dose rate distributions were measured along the interior surface of the shield walls (short walls perpendicular to the bending magnets) with TLD chips. Electron losses for each specific magnet was determined by running the computer code and varying the electron loss parameters to fit the measured dose rate distributions. It was determined that (1) bending magnet No. 1 (closest to the injection septum) loses 2.7 x 1010 electrons per minute during electron injection. The loss rate for each of the subsequent seven bending magnets during injection varies from about 25% to 33% of this number; (2) the magnet immediately subsequent to the bending magnet loses more electrons than the magnet immediately prior to the bending magnet does; and (3) the computer code may be used to predict potential problems such as misalignment.

Computer Simulation↗
PubMed1997

Dissection of protein kinase and phosphatase targeting interactions.

Protein phosphorylation is a primary means of mediating signal transduction events that control cellular processes. Accordingly, the activities of protein kinases and phosphatases are highly regulated. One level of regulation is that the subcellular distribution of several kinases and phosphatases is restricted by association with targeting proteins or subunits. This mechanism promotes rapid and preferential modulation of specific targets within a defined microenvironment in response to diffusible second messengers. The type II cAMP-dependent protein kinase (PKA) is targeted by association of its regulatory subunit (RII) with A-kinase anchoring proteins (AKAPs). To date, 36 unique AKAPs have been identified. Each of these proteins contains a conserved amphipathic helix responsible for AKAP association with cellular structures. Disruption of PKA/AKAP interaction with peptides patterned after the amphipathic helix region blocks certain cAMP responses, including the modulation of glutamate receptor ion-channel activity in neurons and transcription of cAMP-responsive genes. Yeast two-hybrid screening methods have identified neuronal specific AKAP79-binding proteins including the beta isoform of the phosphatase 2B, calcineurin. Biochemical and immunological studies have confirmed the two-hybrid results and identified additional members of this multienzyme signaling complex, including certain protein kinase C isoforms. These findings are consistent with colocalization of CaN, PKC, and type II PKA by AKAP79 and suggest a novel model for reversible phosphorylation in which the opposing kinase and phosphatase actions are colocalized in a signal transduction complex by association with a common anchor protein.

Amino Acid Sequence↗
PubMed1997

Anchoring and scaffold proteins for kinases and phosphatases.

Many hormones mediate their intracellular actions by triggering signal transduction pathways that alter the phosphorylation state of key regulatory proteins. Protein phosphorylation is a reversible process involving two classes of signaling enzymes: protein kinases, which catalyze the transfer of phosphate from ATP onto substrate proteins, and phosphoprotein phosphatases, which perform the dephosphorylation step. To insure tight control of hormonally initiated phosphorylation events, the activity of multifunctional kinases and phosphatases is precisely regulated and responds to fluctuations in diffusible second messengers such as Ca2+, phospholipid, and cAMP. Another mechanism that contributes to their regulation is to restrict the location of these enzymes to certain subcellular compartments. Subcellular targeting enhances the selectivity of serine/threonine phosphatases and kinases by favoring their accessibility to certain substrate proteins. Compartmentalization is achieved through a "targeting moiety," which is defined as that part of a phosphatase or kinase that directs the catalytic subunit to a certain subcellular environment. The targeting moiety restricts the location of a phosphatase or kinase through association with a "targeting locus." These are often structural membrane proteins, cytoskeletal components, or cellular organelles. Targeting subunits for the type I phosphatase and protein kinase C have been identified; however, the focus of this chapter centers around a family of anchoring proteins, called AKAPs, that localize the type II cAMP-dependent protein kinase (PKA). Structure-function analysis suggest that each anchoring protein binds to the RII dimer through a conserved amphipathic helix region and is tethered to specific subcellular sites via association of a targeting domain with structural proteins or cellular organelles. Peptides patterned after the amphipathic region have been used to probe the functional significance of PKA anchoring inside cells and have begun to be established by that disruption RII/AKAP interaction in vivo has concomitant effects on certain PKA-mediated phosphorylation events. In addition, multivalent binding proteins such as AKAP79 and AKAP250 have been characterized and appear to serve as platforms for the assembly of kinase/phosphatase signaling complexes. Collectively, these studies suggest that the AKAPs represent a growing family of regulatory proteins that provide a molecular architecture that organizes the intracellular location of a single or multiple multifunctional kinase.

A Kinase Anchor Proteins↗
PubMed1996

Seroreactivity to HPV16 virus-like particles as a marker for cervical cancer risk in high-risk populations.

Sexually transmitted genital human papillomavirus (HPV) infection, most often HPV 16, is considered the major etiologic determinant of cervical cancer. However, some studies have found relatively low prevalences of genital tract HPV DNA in some geographical areas, such as Greenland, that have high rates of cervical cancer. We sought to evaluate HPV 16 infection in high-risk cohorts using a serologic assay that assesses prior exposure as well as current infection and to compare the results with those obtained using a sensitive PCR-based HPV DNA assay. An ELISA based on HPV 16 virus-like particles was used to detect IgG serum antibodies in women attending sexually transmitted disease (STD) clinics in Nuuk, Greenland and Copenhagen, Denmark. Using a preassigned cut-off, 56% of Greenlandic and 41% of Danish women were seropositive (p = 0.02). In Greenlandic women, there was a non-significant increase in seropositivity with age, and odds ratios for seropositivity were similar for women with more than 5 lifetime sex partners. Seropositivity in the Danish women, however, increased linearly with increases in these 2 factors, which are likely correlates of lifetime exposure to genital HPVs. In contrast, any genital HPV DNA (HPV16 specifically) was detected in 24% and 36% of Greenlandic and Danish women, respectively and was most frequently detected in women below 20. The finding that HPV DNA prevalences, unlike seroprevalences, tended to decrease with increased lifetime risk of infection, provides an explanation for the lack of correlation between HPV DNA prevalences and cervical cancer risk in previous studies of high-risk populations.

Adult↗
PubMed1996

Mutational analysis of the A-kinase anchoring protein (AKAP)-binding site on RII. Classification Of side chain determinants for anchoring and isoform selective association with AKAPs.

Compartmentalization of the type II cAMP-dependent protein kinase is conferred by interaction of the regulatory subunit (RII) with A-Kinase Anchoring Proteins (AKAPs). The AKAP-binding site involves amino-terminal residues on each RII protomer and is formed through dimerization. A site-directed mutagenesis strategy was utilized to assess the contribution of individual residues in either RII isoform, RIIalpha or RIIbeta, for interaction with various anchoring proteins. Substitution of long-chain or bulky hydrophobic groups (leucines or phenylalanines) for isoleucines at positions 3 and 5 in RIIalpha decreased AKAP-binding up to 24 +/- 3 (n = 8)-fold, whereas introduction of valines had minimal effects. Replacement with hydrophilic residues (serine or asparigine) at both positions abolished AKAP binding. Mutation of proline 6 in RIIalpha reduced binding for four AKAPs (Ht31, MAP2, AKAP79, and AKAP95) from 2.3 to 20-fold (n = 4) whereas introduction of an additional proline at position 6 in RIIbeta increased or conferred binding toward these anchoring proteins. Therefore, we conclude that beta-branched side chains at positions 3 and 5 are favored determinants for AKAP-binding and prolines at positions 6 and 7 increase or stabilize RIIalpha interaction with selected anchoring proteins.

A Kinase Anchor Proteins↗
PubMed1996

Nuclear localization of type II cAMP-dependent protein kinase during limb cartilage differentiation is associated with a novel developmentally regulated A-kinase anchoring protein.

Differentiation of chicken limb cartilage is accompanied by a rise in intracellular cyclic AMP, an inducer of cartilage-specific gene expression. A basic approximately 35-kDa protein, designated p35, is the major nuclear substrate for cAMP-dependent protein kinase (PKA) during this process. Here we show that whereas both precartilage and cartilage nuclei contain p35, only precartilage nuclei contain PKA. The phosphorylation of p35 in isolated cell fractions was used as an index of changes in the cellular compartmentalization of components of PKA during chondrogenesis. Both the catalytic subunit and type II regulatory subunit (RII) of PKA were present in the precartilage nuclear fraction, but were undetectable or present in only trace amounts in the cartilage nuclear fraction. Furthermore, a novel approximately 150-kDa A-kinase anchoring protein (AKAP), which binds to RII, was detected in the nuclear matrix of precartilage nuclei but, like RII, was virtually absent in the nuclei of fully differentiated cartilage cells. In limb mesenchymal cells undergoing chondrogenesis in culture a corresponding set of changes occurred: cartilage differentiation was accompanied by a marked reduction in the amounts of both nuclear RII and nAKAP150. These observations indicate that type II PKA holoenzyme is imported into the mesenchymal cell nucleus prior to chondrogenesis, an event that appears to depend on the activity of the developmentally regulated nAKAP150.

Affinity Labels↗
PubMed1996

Cloning and characterization of a novel A-kinase anchoring protein. AKAP 220, association with testicular peroxisomes.

Compartmentalization of the type II cyclic AMP-dependent kinase (PKA) is achieved through association of the regulatory subunit (RII) with A-kinase anchoring proteins (AKAPs). Using an interaction cloning strategy with RIIalpha as a probe, we have isolated cDNAs encoding a novel 1129-amino acid protein that contains both a PKA binding region and a peroxisome targeting motif. Northern analysis detected mRNAs of 9.7 and 7.3 kb in several rat tissues with the highest levels present in the brain and testis. Western analysis and RII overlay experiments showed that the protein is approximately 220 kDa and was, therefore, named AKAP 220. Immunoprecipitation of AKAP 220 from rat testis extracts resulted in co-purification of the type II PKA holoenzyme. The specific activity of PKA increased 458-fold from 7.2 pmol/min/mg in the cell lysate to 3.3 nmol/min/mg in the immunoprecipitate. Immunohistochemical analysis of rat testicular TM4 cells showed that AKAP 220 and a proportion of RII were co-localized in microbodies that appear to be a subset of peroxisomes. Collectively, these results suggest that AKAP 220 may play a role in targeting type II PKA for cAMP-responsive peroxisomal events.

A Kinase Anchor Proteins↗
PubMed1996

Coordination of three signaling enzymes by AKAP79, a mammalian scaffold protein.

Multivalent binding proteins, such as the yeast scaffold protein Sterile-5, coordinate the location of kinases by serving as platforms for the assembly of signaling units. Similarly, in mammalian cells the cyclic adenosine 3',5'-monophosphate-dependent protein kinase (PKA) and phosphatase 2B [calcineurin (CaN)] are complexed by an A kinase anchoring protein, AKAP79. Deletion analysis and binding studies demonstrate that a third enzyme, protein kinase C (PKC), binds AKAP79 at a site distinct from those bound by PKA or CaN. The subcellular distributions of PKC and AKAP79 were similar in neurons. Thus, AKAP79 appears to function as a scaffold protein for three multifunctional enzymes.

A Kinase Anchor Proteins↗