Biomedical subjects
I D Fraser
Publications and source records attributed to I D Fraser.
Regulation of membrane targeting of the G protein-coupled receptor kinase 2 by protein kinase A and its anchoring protein AKAP79.
The beta2 adrenergic receptor (beta2AR) undergoes desensitization by a process involving its phosphorylation by both protein kinase A (PKA) and G protein-coupled receptor kinases (GRKs). The protein kinase A-anchoring protein AKAP79 influences beta2AR phosphorylation by complexing PKA with the receptor at the membrane. Here we show that AKAP79 also regulates the ability of GRK2 to phosphorylate agonist-occupied receptors. In human embryonic kidney 293 cells, overexpression of AKAP79 enhances agonist-induced phosphorylation of both the beta2AR and a mutant of the receptor that cannot be phosphorylated by PKA (beta2AR/PKA-). Mutants of AKAP79 that do not bind PKA or target to the beta2AR markedly inhibit phosphorylation of beta2AR/PKA-. We show that PKA directly phosphorylates GRK2 on serine 685. This modification increases Gbetagamma subunit binding to GRK2 and thus enhances the ability of the kinase to translocate to the membrane and phosphorylate the receptor. Abrogation of the phosphorylation of serine 685 on GRK2 by mutagenesis (S685A) or by expression of a dominant negative AKAP79 mutant reduces GRK2-mediated translocation to beta2AR and phosphorylation of agonist-occupied beta2AR, thus reducing subsequent receptor internalization. Agonist-stimulated PKA-mediated phosphorylation of GRK2 may represent a mechanism for enhancing receptor phosphorylation and desensitization.
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.
Coordination of cAMP signaling events through PKA anchoring.
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Circumcision using bipolar diathermy scissors: a simple, safe and acceptable new technique.
BACKGROUND: Circumcision may be performed by a variety of techniques. Postoperative haemorrhage is a recognised complication of the procedure. METHOD: A method of circumcision using bipolar diathermy scissors is described. Foreskin vessels are coagulated as the foreskin is cut away. RESULTS: Thirty patients underwent this procedure without complication. CONCLUSION: Circumcision using bipolar scissors can be a safe and simple operation.
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.
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.
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.
Modulation of ion channels: a "current" view of AKAPs.
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Osteomyelitis of the temporomandibular joint in patients with malignant otitis externa.
Malignant (invasive) otitis externa is an infection involving the external ear canal, often in elderly diabetic patients, which carries a high morbidity and mortality. It may involve widespread areas of soft tissue around the skull base, and in more advanced cases, may give rise to osteomyelitis and cranial neuropathy. We describe two patients who were treated for malignant otitis externa complicated by destructive osteomyelitis of the temporomandibular joint (TMJ). For both patients, diagnosis was made using magnetic resonance imaging (MRI), and repeat scans were employed during follow-up. Improved scan appearances mirrored improvements in clinical condition in both cases.
Characterization of the functional properties of smooth muscle caldesmon domain 4a: evidence for an independent inhibitory actin-tropomyosin binding domain.
Recent analysis has shown the presence of three sequences in the C-terminal 170 amino acids of human caldesmon (domain 4) which are involved in actin binding and tropomyosin-dependent inhibition of actomyosin ATPase. Two are in domain 4b (amino acids 715-793) and one is in domain 4a (amino acids 636-714). In the present work we have compared recombinant peptides containing either domain 4a or domain 4b to address the question as to whether domain 4a alone has any inhibitory activity. We have produced three new recombinant fragments containing domain 4a: H10 [622-708], H12 [506-708] and H13 [622-726] and we have characterized their functional properties. All three fragments bound to actin and tropomyosin. Caldesmon, but not domain 4b, was able to displace the fragments H10, H12 and H13 from actin. Thus the isolated caldesmon domain 4a peptides bind to the same region on actin as in the whole molecule while domains 4a and 4b occupy different sites on the actin molecule. Unlike domain 4b, none of the domain 4a fragments inhibited the actomyosin ATPase in the absence of tropomyosin. However both domain 4a and 4b fragments displayed an inhibitory activity in the presence of tropomyosin. H13 and H12 were more potent inhibitors than H10. Ca2+-calmodulin bound to H13 and reversed the inhibitory activity of this fragment but did not bind to H10 and H12. We conclude that domain 4a can act as an independent inhibitory actin-tropomyosin binding domain, but its properties are very different from the extreme C-terminal domain 4b.
A novel lipid-anchored A-kinase Anchoring Protein facilitates cAMP-responsive membrane events.
Compartmentalization of protein kinases with substrates is a mechanism that may promote specificity of intracellular phosphorylation events. We have cloned a low-molecular weight A-kinase Anchoring Protein, called AKAP18, which targets the cAMP-dependent protein kinase (PKA) to the plasma membrane, and permits functional coupling to the L-type calcium channel. Membrane anchoring is mediated by the first 10 amino acids of AKAP18, and involves residues Gly1, Cys4 and Cys5 which are lipid-modified through myristoylation and dual palmitoylation, respectively. Transient transfection of AKAP18 into HEK-293 cells expressing the cardiac L-type Ca2+ channel promoted a 34 9% increase in cAMP-responsive Ca2+ currents. In contrast, a targeting-deficient mutant of AKAP18 had no effect on Ca2+ currents in response to the application of a cAMP analog. Further studies demonstrate that AKAP18 facilitates GLP-1-mediated insulin secretion in a pancreatic beta cell line (RINm5F), suggesting that membrane anchoring of the kinase participates in physiologically relevant cAMP-responsive events that may involve ion channel activation.
Structure-activity studies of the regulatory interaction of the 10 kilodalton C-terminal fragment of caldesmon with actin and the effect of mutation of caldesmon residues 691-696.
We have used isotope-edited nuclear magnetic resonance spectroscopy, binding studies, and ATPase activity assays to investigate the interaction with F-actin of the 10 kDa C-terminal 658C fragment of chicken gizzard caldesmon and two site-directed mutants of this fragment. Simultaneous dual-sited contacts with F-actin are observed for the segments of the 658C sequence flanking tryptophan residues 692 and 722. Competition experiments showed that both 658C contacts with actin are displaced by substoichiometric concentrations of the short inhibitory region of troponin-I indicative of different binding sites on actin for these regions of troponin-I and caldesmon. Substitution of caldesmon serine-702 by aspartic acid within the spacer region linking the two actin contacts of 658C led to weaker binding but with retention of equivalent affinity for each interaction site. Differential binding affinity of the two sites was achieved by replacement of the sequence Glu691-Trp-Leu-Thr-Lys-Thr696 by Pro-Gly-His-Tyr-Asn-Asn. Consistent with these data, the concentration of this Cg1 mutant required to achieve 50% inhibition of actin-tropomyosin-activated myosin ATPase was 4-fold greater than found for the 658C fragment. Although calmodulin binding to Cg1 was observed, calmodulin proved ineffective in relieving the inhibition induced by the binding of this mutant to actin. These results are discussed in light of the actin contacts which are involved in the inhibitory activity possessed by different regions of the C-terminus of caldesmon.
Troponin I and troponin T interact with troponin C to produce different Ca2+-dependent effects on actin-tropomyosin filament motility.
We have developed an in vitro motility assay to make a detailed quantitative analysis of Ca2+ control of skeletal-muscle troponin-tropomyosin control of actin-filament movement over immobilized myosin. Ca2+ regulates both filament velocity and the fraction of filaments that are motile. We have demonstrated that the two effects are due to separate interactions of troponin C with troponin I and troponin T. When 64nM of the complex actin-tropomyosin-troponin I-troponin C was added at pCa 5, more than 80% of filaments were moving and their velocity did not change. At pCa 9, more than 20% of the filaments were moving. When 20nM of the complex actin-tropomyosin-troponin T+troponin I+troponin C was added at pCa 5, filament motility remained high, whereas velocity increased. The 30% increase in velocity observed when troponin T was present was also observed when heavy meromyosin fragment 1 labelled with N-ethylmaleimide (NEM S-1) was added after actin-tropomyosin filaments. The NEM S-1 effect was not additive with the troponin T-dependent velocity increase. The pattern of motile behaviour is characteristic of myosin on silicone-treated glass and different from the behaviour on nitrocellulose-coated glass.
Mapping of contact sites in the caldesmon-calmodulin complex.
The interaction of intact calmodulin and its four tryptic peptides with deletion mutants of caldesmon was analysed by native gel electrophoresis, fluorescence spectroscopy and zero-length cross-linking. Deletion mutants H2 (containing calmodulin-binding sites A and B) and H9 (containing sites B and B') interacted with intact calmodulin to form complexes whose stoichiometries varied from 2:1 to 1:1. The N-terminal peptides of calmodulin (TR1C, residues 1-77, and TR2E, residues 1-90) bound H2 with higher affinity than H9. At the same time H2 was less effective than H9 in binding to the C-terminal peptides of calmodulin TR2C (residues 78-148) and TR3E (residues 107-148). The N-terminal peptides of calmodulin (TR1C and TR2E) could be cross-linked to intact caldesmon and its deletion mutants H2 and H9. The similarity in the primary structures of sites A and B' of caldesmon and our measurements of the affinities of H2 and H9 to calmodulin and its peptides strongly indicate an orientation of the protein complex where sites A and B' interact with the N-terminal domain of calmodulin, whereas site B interacts with the C-terminal domain of calmodulin. The spatial organization of contact sites in the caldesmon-calmodulin complex agrees with the earlier proposed two-dimensional model of interaction of the two proteins [Huber, El-Mezgueldi, Grabarek, Slatter, Levine and Marston (1996) Biochem. J. 316, 413-420].
The inhibitory complex of smooth muscle caldesmon with actin and tropomyosin involves three interacting segments of the C-terminal domain 4.
We have designed a series of recombinant peptides derived from the C-terminus of human caldesmon (amino acids 663-793, domain 4) to determine the structural basis of the multiple-sited caldesmon-actin-tropomyosin interaction. All the recombinant peptides are able to bind to actin and inhibit actin-activated myosin ATPase activity; 1 mol of peptide is bound per actin for >90% inhibition. However, equivalent inhibition of actin-tropomyosin activation of myosin ATPase requires less than one peptide per seven actin to be bound. We have found two sequences, H2 (amino acids 683-767) and H2+12 (amino acids 683-779), from the center of domain 4 which potentiate actin-tropomyosin filament activity; i.e., their effect is opposite to caldesmon. Maximum potentiation correlates with one H2 or H2+12 bound per four actin. This effect is completely dependent upon the presence of tropomyosin on the actin filament. H2 and H2+12 also increase actin-tropomyosin filament velocity in the in vitro motility assay. If the H2 sequence is extended by 20 amino acids at the N-terminal end to the N-terminus of domain 4, the peptide becomes an inhibitor. If H2 is extended by 19 amino acids at its C terminus, it becomes a tropomyosin-dependent inhibitor, and with a further extension of 7 amino acids to reach the C-terminus of human caldesmon (H2+26), inhibition is more potent. We conclude that three regions in domain 4 of caldesmon contribute to tropomyosin-dependent inhibition of actomyosin ATPase: a central segment [747-767 (690-710 in the chicken sequence)], which is essential but not sufficient for tropomyosin-dependent inhibition of actomyosin ATPase; and two actin binding segments N-terminal and C-terminal to this segment, 663-682 (606-625) and 770-793 (713-737). If only the central segment is present (H2, H2+12), the actin-tropomyosin-caldesmon peptide complex is not inhibitory, and its properties resemble actin-tropomyosin-caldesmon-Ca2+ x calmodulin.
A simple method for automatic tracking of actin filaments in the motility assay.
A great deal of quantitative information about the actomyosin interaction can be obtained from the basic Kron and Spudich in vitro motility assay provided that care is taken to obtain consistency between experiments and that the data is examined comprehensively and not selectively. From observations of filament movement under a wide variety of conditions we have formulated the hypothesis that a large number of filaments moving over a short time period is indistinguishable from fewer filaments moving over a longer sequence of frames. This has been used to devise a simple automation of filament detection procedures. A sequence of images is digitized through a frame-grabber. If successive pairs of frames are compared the program will search for and detect the new position of every filament and show its vector on screen. Velocity is calculated and shown as a frequency histogram. The program regularly detects over 100 filaments moving in each pair of frames; usually a sequence of up to 15 pairs of frames are studied yielding 500-1000 vectors in total. The algorithm cannot deal with filaments that meet, cross or divide, however, when filaments are moving less than 2 microns between frames this is only a small proportion of the whole. The program outputs fraction of filaments motile, mean velocity with standard deviation and density of filaments (filaments microns-2). A cumulative frequency histogram gives an immediate visual indication of the performance of the population of filaments. Direct comparisons show that the data produced by automatic tracking is indistinguishable from manual tracking apart from the small apparent velocity of non-mobile filaments. The detection process takes about 5 min and requires little skill or judgement. This can lead to great increases in the rate of data analysis in motility work.
A prospective, randomized study of the use of platelet concentrates irradiated with ultraviolet-B light in patients with hematologic malignancy.
BACKGROUND: Irradiation of platelet concentrates (PCs) with ultraviolet-B (UVB) light inactivates the contaminating white cells and might be an alternative to filtration for the prevention of alloimmunization to HLA antigens and subsequent refractoriness to further platelet transfusions in multiply transfused patients with bone marrow failure. STUDY DESIGN AND METHODS: Patients with hematologic malignancy, mainly acute myeloid leukemia, were prospectively assigned in a random manner to receive either UVB-irradiated or control, nonirradiated PCs. All patients were given red cells that were white cell reduced by filtration. Transfusion efficacy and alloimmunization were assessed by means of corrected count increments, requirement for red cells and PCs, and measurement of lymphocyte-reactive antibodies. RESULTS: UVB-irradiated PCs had a clinical efficacy similar to controls as judged by corrected count increments at 1 to 6 and 12 to 24 hours and by the median requirement for red cell and platelet transfusions. Alloimmunization determined by measurements of lymphocyte-reactive antibodies using both conventional and antiglobulin-augmented lymphocytotoxicity techniques was not abolished in recipients of UVB-irradiated PCs (4/30, 13%) but was less than that in controls (5/20, 25%; p = NS). The mean number of platelet transfusion episodes prior to the occurrence of alloimmunization was greater in the control group (27 vs. 10; p = 0.017). CONCLUSION: In this trial, UVB irradiation did not diminish the clinical efficacy of platelet transfusions. There was a small but nonsignificant reduction alloimmunization, but no difference in refractoriness of the two groups was observed. Larger prospective randomized studies are required to confirm these findings and to compare UVB irradiation with white cell reduction.