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J D Scott

Publications and source records attributed to J D Scott.

At least 55 records · Page 3Linked to original sources
PubMed2000

A mammalian PAR-3-PAR-6 complex implicated in Cdc42/Rac1 and aPKC signalling and cell polarity.

Cellular asymmetry is critical for the development of multicellular organisms. Here we show that homologues of proteins necessary for asymmetric cell division in Caenorhabditis elegans associate with each other in mammalian cells and tissues. mPAR-3 and mPAR-6 exhibit similar expression patterns and subcellular distributions in the CNS and associate through their PDZ (PSD-95/Dlg/ZO-1) domains. mPAR-6 binds to Cdc42/Rac1 GTPases, and mPAR-3 and mPAR-6 bind independently to atypical protein kinase C (aPKC) isoforms. In vitro, mPAR-3 acts as a substrate and an inhibitor of aPKC. We conclude that mPAR-3 and mPAR-6 have a scaffolding function, coordinating the activities of several signalling proteins that are implicated in mammalian cell polarity.

Amino Acid Motifs↗
PubMed2000

External fixation as a bridge to intramedullary nailing for patients with multiple injuries and with femur fractures: damage control orthopedics.

BACKGROUND: The advantages of early fracture fixation in patients with multiple injuries have been challenged recently, particularly in patients with head injury. External fixation (EF) has been used to stabilize pelvic fractures after multiple injury. It potentially offers similar benefits to intramedullary nail (IMN) in long-bone fractures and may obviate some of the risks. We report on the use of EF as a temporary fracture fixation in a group of patients with multiple injuries and with femoral shaft fractures. METHODS: Retrospective review of charts and registry data of patients admitted to our Level 1 trauma center July of 1995 to June of 1998. Forty-three patients initially treated with EF of the femur were compared to 284 patients treated with primary IMN of the femur. RESULTS: Patients treated with EF had more severe injuries with significantly higher Injury Severity Scores (26.8 vs. 16.8) and required significantly more fluid (11.9 vs. 6.2 liters) and blood (1.5 vs. 1.0 liters) in the initial 24 hours. Glasgow Coma Scale score was lower (p < 0.01) in those treated with EF (11 vs. 14.2). Twelve patients (28%) had head injuries severe enough to require intracranial pressure monitoring. All 12 required therapy for intracranial pressure control with mannitol (100%), barbiturates (75%), and/or hyperventilation (75%). Most patients had more than one contraindication to IMN, including head injury in 46% of cases, hemodynamic instability in 65%, thoracoabdominal injuries in 51%, and/or other serious injuries in 46%, most often multiple orthopedic injuries. Median operating room time for EF was 35 minutes with estimated blood loss of 90 mL. IMN was performed in 35 of 43 patients at a mean of 4.8 days after EF. Median operating room time for IMN was 135 minutes with an estimated blood loss of 400 mL. One patient died before IMN. One other patient with a mangled extremity was treated with amputation after EF. There was one complication of EF, i.e., bleeding around a pin site, which was self-limited. Four patients in the EF group died, three from head injuries and one from acute organ failure. No death was secondary to the fracture treatment selected. One patient who had EF followed by IMN had bone infection and another had acute hardware failure. CONCLUSION: EF is a viable alternative to attain temporary rigid stabilization in patients with multiple injuries. It is rapid, causes negligible blood loss, and can be followed by IMN when the patient is stabilized. There were minimal orthopedic complications.

Adolescent↗
PubMed2000

COL2A1 exon 2 mutations: relevance to the Stickler and Wagner syndromes.

AIMS: To compare the clinical and molecular genetic features of two phenotypically distinct subgroups of families with type 1 Stickler syndrome. BACKGROUND: Stickler syndrome (hereditary arthro-ophthalmopathy, McKusick Nos 108300 and 184840) is a dominantly inherited disorder of collagen connective tissue, resulting in an abnormal vitreous, myopia, and a variable degree of orofacial abnormality, deafness, and arthropathy. Stickler syndrome is the commonest inherited cause of rhegmatogenous retinal detachment in childhood with a risk of giant retinal tear (GRT) which is commonly bilateral and a frequent cause of blindness. METHOD: Pedigrees were identified from the vitreoretinal service database and subclassified according to vitreoretinal phenotype. Ophthalmic, skeletal, auditory, and orofacial features were assessed. Linkage analysis was carried out with markers for the candidate genes COL2A1, COL11A1, and COL11A2. The COL2A1 gene was amplified as five overlapping PCR products. Direct sequencing of individual exons identified mutations. RESULTS: Eight families exhibiting the type 1 vitreous phenotype were studied. Seven were consistent for linkage to COL2A1, with lod scores ranging from 2.1 to 0.3. In most instances linkage to COL11A1 and COL11A2 could be excluded. One family was analysed without prior linkage analysis. Three of the families exhibited a predominantly ocular phenotype with minimal or absent systemic involvement and were found to have mutations in exon 2 of COL2A1. Five other pedigrees with an identical ocular phenotype plus orofacial, auditory, and articular involvement had mutations in others regions of the COL2A1 gene. None of the pedigrees exhibited the characteristic lenticular, retinal pigment epithelial, or choroidal changes seen in Wagner syndrome. CONCLUSIONS: These data confirm that type 1 Stickler syndrome is caused by mutations in the gene encoding type II collagen (COL2A1). In addition, data are submitted showing that mutations involving exon 2 of COL2A1 are characterised by a predominantly ocular variant of this disorder, consistent with the major form of type II procollagen in non-ocular tissues having exon 2 spliced out. Such patients are all at high risk of retinal detachment. This has important implications for counselling patients with regard to the development of systemic complications. It also emphasises the importance and reliability of the ophthalmic examination in the differential diagnosis of this predominantly ocular form of Stickler syndrome from Wagner's vitreoretinopathy.

Collagen↗
PubMed1999

Alternative splicing regulates the subcellular localization of A-kinase anchoring protein 18 isoforms.

The cAMP-dependent protein kinase (PKA) is localized to specific subcellular compartments by association with A-kinase anchoring proteins (AKAPs). AKAPs are a family of functionally related proteins that bind the regulatory (R) subunit of PKA with high affinity and target the kinase to specific subcellular organelles. Recently, AKAP18, a low molecular weight plasma membrane AKAP that facilitates PKA-mediated phosphorylation of the L-type Ca(2+) channel, was cloned. We now report the cloning of two additional isoforms of AKAP18, which we have designated AKAP18beta and AKAP18gamma, that arise from alternative mRNA splicing. The AKAP18 isoforms share a common R subunit binding site, but have distinct targeting domains. The original AKAP18 (renamed AKAP18alpha) and AKAP18beta target the plasma membrane when expressed in HEK-293 cells, while AKAP18gamma is cytosolic. When expressed in epithelial cells, AKAP18alpha is targeted to lateral membranes, whereas AKAP18beta is accumulated at the apical membrane. A 23-amino acid insert, following the plasma membrane targeting domain, facilitates the association of AKAP18beta with the apical membrane. The data suggest that AKAP18 isoforms are differentially targeted to modulate distinct intracellular signaling events. Furthermore, the data suggest that plasma membrane AKAPs may be targeted to subdomains of the cell surface, adding additional specificity in intracellular signaling.

A Kinase Anchor Proteins↗
PubMed1999

Identification of cAMP-dependent protein kinase holoenzymes in preantral- and preovulatory-follicle-enriched ovaries, and their association with A-kinase-anchoring proteins.

Undifferentiated cells from preantral (PA) follicles respond to high levels of cAMP in a different manner than do differentiated cells from preovulatory (PO) follicles. We hypothesized that this differential response of PA and PO cells to cAMP could be due, in part, to either a difference in the profile of isoforms that comprise the cAMP-dependent protein kinase (PKA) holoenzymes and/or a difference in the interaction of PKA with A-kinase-anchoring proteins (AKAPs). To test these hypotheses, PKA activity, PKA holoenzymes, PKA subunits and AKAPs from PA and PO ovaries were compared. Soluble PKA holoenzymes and regulatory (R) subunits were separated by DEAE-cellulose chromatography and sucrose-density-gradient centrifugation. PKA R subunits were distinguished by photoaffinity labelling, autophosphorylation, size, isoelectric point and immunoreactivity. AKAPs were identified by RII subunit overlay assays and immunoreactivity. The results showed that extracts from PA and PO ovaries exhibited equivalent PKA holoenzyme profiles and activities, characterized by low levels of PKA type I (PKAI) holoenzyme and two distinct PKAII holoenzyme peaks, one containing only RIIbeta subunits (PKAIIbeta) and one containing both PKAIIbeta and PKAIIalpha holoenzymes. Both PA and PO ovarian extracts also contained PKA catalytic (C)-subunit-free RIalpha, while only PO ovaries exhibited C-subunit-free RIIbeta. Consistent with the elevated levels of C-subunit-free RIIbeta in PO cells, PKA activation in PO cells required higher concentrations of forskolin than that in PA cells. While extracts of PA and PO ovaries exhibited a number of similar AKAPs, including four prominent ones reactive with anti-AKAP-KL antisera (where AKAP-KL is an AKAP especially abundant in kidney and liver), cAMP-agarose affinity chromatography revealed two major differences in AKAP binding to purified R subunits. PO ovaries contained increased levels of AKAP80 (AKAP of 80 kDa) bound selectively to R subunits in DEAE-cellulose peak 2 (comprising PKAIIbeta and RIalpha), but not to R subunits in DEAE-cellulose peak 3 (comprising PKAIIalpha, PKAIIbeta and RIIbeta). PO ovaries also showed increased binding of R subunits to AKAPs reactive with anti-AKAP-KL antisera at 210, 175, 150 and 115 kDa. Thus in PO ovaries, unlike in PA ovaries, the majority of AKAPs are bound to R subunits. These results suggest that altered PKA-AKAP interactions may contribute to the distinct responses of PA and PO follicles to high levels of cAMP, and that higher cAMP levels are required to activate PKA in PO ovaries.

Animals↗
PubMed1999

Mechanism of A-kinase-anchoring protein 79 (AKAP79) and protein kinase C interaction.

The A-kinase-anchoring protein AKAP79 co-ordinates the location of cAMP-dependent protein kinase, phosphatase 2B (PP2B/calcineurin) and protein kinase C (PKC) at postsynaptic sites in neurons. In this report we focus on the mechanism of interaction between AKAP79 and PKC. We show that neither lipid activators nor kinase activation are required for association with AKAP79. The anchoring protein binds and inhibits the conserved catalytic core of PKCbetaII. AKAP79 also associates with conventional, novel and atypical isoforms of PKC in vitro and in vivo, and immunofluorescence staining of rat hippocampal neurons demonstrates that the murine anchoring-protein homologue AKAP150 is co-distributed with PKCalpha/beta, PKCepsilon or PKCiota. Binding of the AKAP79(31-52) peptide, which inhibits kinase activity, exposes the pseudosubstrate domain of PKCbetaII, allowing endoproteinase Arg-C proteolysis in the absence of kinase activators. Reciprocal experiments have identified two arginine residues at positions 39 and 40 that are essential for AKAP79(31-52) peptide inhibition of PKCbetaII. Likewise, the same mutations in the full-length anchoring protein reduced inhibition of PKCbetaII. Thus AKAP79 associates with multiple PKC isoforms through a mechanism involving protein-protein interactions at the catalytic core where binding of the anchoring protein inhibits kinase activity through displacement of the pseudosubstrate.

A Kinase Anchor Proteins↗
PubMed1999

Regulation of NMDA receptors by an associated phosphatase-kinase signaling complex.

Regulation of N-methyl-D-aspartate (NMDA) receptor activity by kinases and phosphatases contributes to the modulation of synaptic transmission. Targeting of these enzymes near the substrate is proposed to enhance phosphorylation-dependent modulation. Yotiao, an NMDA receptor-associated protein, bound the type I protein phosphatase (PP1) and the adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (PKA) holoenzyme. Anchored PP1 was active, limiting channel activity, whereas PKA activation overcame constitutive PP1 activity and conferred rapid enhancement of NMDA receptor currents. Hence, yotiao is a scaffold protein that physically attaches PP1 and PKA to NMDA receptors to regulate channel activity.

A Kinase Anchor Proteins↗
PubMed1999

Association of the type 1 protein phosphatase PP1 with the A-kinase anchoring protein AKAP220.

The cyclic AMP (cAMP)-dependent protein kinase (PKA) and the type 1 protein phosphatase (PP1) are broad-specificity signaling enzymes with opposing actions that catalyze changes in the phosphorylation state of cellular proteins. Subcellular targeting to the vicinity of preferred substrates is a means of restricting the specificity of each enzyme [1] [2]. Compartmentalization of the PKA holoenzyme is mediated through association of the regulatory subunits with A-kinase anchoring proteins (AKAPs), whereas a diverse family of phosphatase-targeting subunits directs the location of the PP1 catalytic subunit (PP1c) [3] [4]. Here, we demonstrate that the PKA-anchoring protein, AKAP220, binds PP1c with a dissociation constant (KD) of 12.1 +/- 4 nM in vitro. Immunoprecipitation of PP1 from cell extracts resulted in a 10.4 +/- 3.8-fold enrichment of PKA activity. AKAP220 co-purified with PP1c by affinity chromatography on microcystin sepharos Immunocytochemical analysis demonstrated that the kinase, the phosphatase and the anchoring protein had distinct but overlapping staining patterns in rat hippocampal neurons. Collectively, these results provide the first evidence that AKAP220 is a multivalent anchoring protein that maintains a signaling scaffold of PP1 and the PKA holoenzyme.

A Kinase Anchor Proteins↗
PubMed1999

Carboxyl-terminal phosphorylation regulates the function and subcellular localization of protein kinase C betaII.

Protein kinase C is processed by three phosphorylation events before it is competent to respond to second messengers. Specifically, the enzyme is first phosphorylated at the activation loop by another kinase, followed by two ordered autophosphorylations at the carboxyl terminus (Keranen, L. M., Dutil, E. M., and Newton, A. C. (1995) Curr. Biol. 5, 1394-1403). This study examines the role of negative charge at the first conserved carboxyl-terminal phosphorylation position, Thr-641, in regulating the function and subcellular localization of protein kinase C betaII. Mutation of this residue to Ala results in compensating phosphorylations at adjacent sites, so that a triple Ala mutant was required to address the function of phosphate at Thr-641. Biochemical and immunolocalization analyses of phosphorylation site mutants reveal that negative charge at this position is required for the following: 1) to process catalytically competent protein kinase C; 2) to allow autophosphorylation of Ser-660; 3) for cytosolic localization of protein kinase C; and 4) to permit phorbol ester-dependent membrane translocation. Thus, phosphorylation of Thr-641 in protein kinase C betaII is essential for both the catalytic function and correct subcellular localization of protein kinase C. The conservation of this residue in every protein kinase C isozyme, as well as other members of the kinase superfamily such as protein kinase A, suggests that carboxyl-terminal phosphorylation serves as a key molecular switch for defining kinase function.

Amino Acid Sequence↗
PubMed1999

Dynamic complexes of beta2-adrenergic receptors with protein kinases and phosphatases and the role of gravin.

Signals mediated by G-protein-linked receptors display agonist-induced attenuation and recovery involving both protein kinases and phosphatases. The role of protein kinases and phosphatases in agonist-induced attenuation and recovery of beta-adrenergic receptors was explored by two complementary approaches, antisense RNA suppression and co-immunoprecipitation of target elements. Protein phosphatases 2A and 2B are associated with the unstimulated receptor, the latter displaying a transient decrease followed by a 2-fold increase in the levels of association at 30 min following challenge with agonist. Protein kinase A displays a robust, agonist-induced association with beta-adrenergic receptors over the same period. Suppression of phosphatases 2A and 2B with antisense RNA or inhibition of their activity with calyculin A and FK506, respectively, blocks resensitization following agonist removal. Recycling of receptors to the plasma membrane following agonist-promoted sequestration is severely impaired by loss of either phosphatase 2B or protein kinase C. In addition, loss of protein kinase C diminishes association of phosphatase 2B with beta-adrenergic receptors. Overlay assays performed with the RII subunit of protein kinase A and co-immunoprecipitations reveal proteins of the A kinase-anchoring proteins (AKAP) family, including AKAP250 also known as gravin, associated with the beta-adrenergic receptor. Suppression of gravin expression disrupts recovery from agonist-induced desensitization, confirming the role of gravin in organization of G-protein-linked signaling complexes. The Ht31 peptide, which blocks AKAP protein-protein interactions, blocks association of beta-adrenergic receptors with protein kinase A. These data are the first to reveal dynamic complexes of beta-adrenergic receptors with protein kinases and phosphatases acting via an anchoring protein, gravin.

A Kinase Anchor Proteins↗
PubMed1999

AKAPs: from structure to function.

Compartmentalization of signalling molecules through association with anchoring proteins ensures specificity in signal transduction by placing enzymes close to their appropriate effectors and substrates. For example, 'A-kinase anchoring proteins' (AKAPs) bind to the regulatory subunit of cAMP-dependent protein kinase (PKA) to direct the kinase to discrete intracellular locations. Recently, functional studies aimed at disrupting AKAP-PKA complexes have demonstrated a role for anchored PKA in various cellular processes, including gene transcription, hormone-mediated insulin secretion and ion-channel modulation. By binding to additional signalling molecules, AKAPs might function to coordinate multiple components of signal-transduction pathways.

Carrier Proteins↗
PubMed1999

Phosphorylation and inactivation of BAD by mitochondria-anchored protein kinase A.

Signaling pathways between cell surface receptors and the BCL-2 family of proteins regulate cell death. Survival factors induce the phosphorylation and inactivation of BAD, a proapoptotic member. Purification of BAD kinase(s) identified membrane-based cAMP-dependent protein kinase (PKA) as a BAD Ser-112 (S112) site-specific kinase. PKA-specific inhibitors blocked the IL-3-induced phosphorylation on S112 of endogenous BAD as well as mitochondria-based BAD S112 kinase activity. A blocking peptide that disrupts type II PKA holoenzyme association with A-kinase-anchoring proteins (AKAPs) also inhibited BAD phosphorylation and eliminated the BAD S112 kinase activity at mitochondria. Thus, the anchoring of PKA to mitochondria represents a focused subcellular kinase/substrate interaction that inactivates BAD at its target organelle in response to a survival factor.

Animals↗
PubMed1999

The molecular basis for protein kinase A anchoring revealed by solution NMR.

Compartmentalization of signal transduction enzymes into signaling complexes is an important mechanism to ensure the specificity of intracellular events. Formation of these complexes is mediated by specialized protein motifs that participate in protein-protein interactions. The adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) is localized through interaction of the regulatory (R) subunit dimer with A-kinase-anchoring proteins (AKAPs). We now report the solution structure of the type II PKA R-subunit fragment RIIalpha(1-44), which encompasses both the AKAP-binding and dimerization interfaces. This structure incorporates an X-type four-helix bundle dimerization motif with an extended hydrophobic face that is necessary for high-affinity AKAP binding. NMR data on the complex between RIIalpha(1-44) and an AKAP fragment reveals extensive contacts between the two proteins. Interestingly, this same dimerization motif is present in other signaling molecules, the S100 family. Therefore, the X-type four-helix bundle may represent a conserved fold for protein-protein interactions in signal transduction.

Amino Acid Sequence↗