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Stephen Shaw

Publications and source records attributed to Stephen Shaw.

At least 19 recordsLinked to original sources
PubMed2006

Comment on "PDK1 nucleates T cell receptor-induced signaling complex for NF-kappaB activation".

We observe that protein kinase C (PKC) is phosphorylated on the activation loop at threonine 538 (Thr-538) before T cell activation. Our results are inconsistent with the conclusions of Lee et al. (Reports, 1 April 2005, p. 114) that the Thr-538 phosphorylation of PKC is regulated by T cell receptor activation. Other mechanisms, such as autophosphorylation of Thr-219, might orchestrate the cellular function of PKC in T cells.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2006

Independence of protein kinase C-delta activity from activation loop phosphorylation: structural basis and altered functions in cells.

Activation loop phosphorylation plays critical regulatory roles for many kinases. Unlike other protein kinase Cs (PKC), PKC-delta does not require phosphorylation of its activation loop (Thr-507) for in vitro activity. We investigated the structural basis for this unusual capacity and its relevance to PKC-delta function in intact cells. Mutational analysis demonstrated that activity without Thr-507 phosphorylation depends on 20 residues N-terminal to the kinase domain and a pair of phenylalanines (Phe-500/Phe-527) unique to PKC-delta in/near the activation loop. Molecular modeling demonstrated that these elements stabilize the activation loop by forming a hydrophobic chain of interactions from the C-lobe to activation loop to N-terminal (helical) extension. In cells PKC-delta mediates both apoptosis and transcription regulation. We found that the T507A mutant of the PKC-delta kinase domain resembled the corresponding wild type in mediating apoptosis in transfected HEK293T cells. But the T507A mutant was completely defective in AP-1 and NF-kappaB reporter assays. A novel assay in which the kinase domain of PKC-delta and its substrate (a fusion protein of PKC substrate peptide with green fluorescent protein) were co-targeted to lipid rafts revealed a major substrate-selective defect of the T507A mutant in phosphorylating the substrate in cells. In vitro analysis showed strong product inhibition on the T507A mutant with particular substrates whose characteristics suggest it contributes to the substrate selective defect of the PKC-delta T507A mutant in cells. Thus, activation loop phosphorylation of PKC-delta may regulate its function in cells in a novel way.

Apoptosis↗
PubMed2006

Development of lithium-induced nephrogenic diabetes insipidus is dissociated from adenylyl cyclase activity.

In antidiuresis, vasopressin (AVP) occupation of V2 receptors in renal collecting ducts activates adenylyl cyclase, resulting in increased intracellular cAMP levels, which activates protein kinase A (PKA). PKA phosphorylates both the cAMP responsive element binding protein, which induces aquaporin-2 (AQP2) transcription, and AQP2, which then is translocated to the apical membrane, allowing urine concentration. Lithium treatment often causes nephrogenic diabetes insipidus (NDI), which coincides with decreased AQP2 expression and which generally is ascribed to reduced adenylyl cyclase activity. However, the underlying mechanism by which lithium causes NDI is poorly understood. This study demonstrated that the mouse cortical collecting duct mpkCCD(c14) cells are a good model; the deamino-8 D-arginine vasopressin (dDAVP)-induced endogenous AQP2 expression and plasma membrane localization was time-dependently reduced by treatment with clinically relevant lithium concentrations. Lithium did not affect AQP2 stability but decreased its mRNA levels. Surprising, the effect of lithium was cAMP independent; it did not alter AVP-stimulated cAMP production or PKA-dependent phosphorylation of AQP2 or cAMP responsive element binding protein. In vivo, kidney tissue of rats with lithium-induced NDI indeed generated less dDAVP-induced cAMP than that of controls, but this could be due to elevated blood AVP levels in rats with lithium-induced NDI. Indeed, Brattleboro rats, which lack endogenous AVP, with clamped blood dDAVP levels, showed no difference in dDAVP-generated cAMP generation between kidneys of rats with lithium-induced NDI and control rats. In conclusion, the first proper cell model to study lithium-induced NDI was developed, and it was demonstrated that the lithium-induced downregulation of AQP2 and development of NDI occur independent of adenylyl cyclase activity in vitro and in vivo.

Adenylyl Cyclases↗
PubMed2006

An open study to assess the efficacy clinically and immunologically of M. vaccae vaccine in patients with atopic dermatitis.

OBJECTIVE: To assess clinical and immunological changes in a group of atopic dermatitis patients treated with Mycobacterium vaccae (M. vaccae). METHODS: In an open, randomized, placebo-controlled, clinical observer-blinded trial, 20 patients with moderately severe to severe atopic dermatitis were treated with two subcutaneous M. vaccae injections 3 months apart. Clinical scoring and total serum IgE estimation were performed at the start, during and 3 months after the end of the study. RESULTS: Modest clinical improvement was seen in the patients after M. vaccae immunotherapy. There was no significant change in patients' total serum IgE levels. CONCLUSIONS: This study has shown limited improvement clinically in a group of patients with atopic dermatitis treated with two M. vaccae injections. It is suggested that further trials using four to six injections would produce much greater benefit.

Adolescent↗
PubMed2005

A single pair of acidic residues in the kinase major groove mediates strong substrate preference for P-2 or P-5 arginine in the AGC, CAMK, and STE kinase families.

Most basophilic serine/threonine kinases preferentially phosphorylate substrates with Arg at P-3 but vary greatly in additional strong preference for Arg at P-2 or P-5. The structural basis for P-2 or P-5 preference is known for two AGC kinases (family of protein kinases A, G, and C) in which it is mediated by a single pair of acidic residues (PEN+1 and YEM+1). We sought a general understanding of P-2 and P-5 Arg preference. The strength of Arg preference at each position was assessed in 15 kinases using a new degenerate peptide library approach. Strong P-2 or P-5 Arg preference occurred not only in AGC kinases (7 of 8 studied) but also in calmodulin-dependent protein kinase (CAMK, 1 of 3) and Ste20 (STE) kinases (2 of 4). Analysis of sequence conservation demonstrated almost perfect correlation between (a) strong P-2 or P-5 Arg preference and (b) acidic residues at both PEN+1 and YEM+1. Mutation of two kinases (PKC-theta and p21-activated kinase 1 (PAK1)) confirmed critical roles of both PEN+1 and YEM+1 residues in determining strong R-2 Arg preference. PAK kinases were unique in having exceptionally strong Arg preference at P-2 but lacking strong Arg preference at P-3. Preference for Arg at P-2 was so critical to PAK recognition that PAK1 activity was virtually eliminated by mutating the PEN+1 or YEM+1 residues. The fact that this specific pair of acidic residues has been repeatedly and exclusively used by evolution for conferring strong Arg preference at two different substrate positions in three different kinase families implies it is uniquely well suited to mediate sufficiently good substrate binding without unduly restricting product release.

Amino Acid Sequence↗
PubMed2005

Exceptional disfavor for proline at the P + 1 position among AGC and CAMK kinases establishes reciprocal specificity between them and the proline-directed kinases.

To precisely regulate critical signaling pathways, two kinases that phosphorylate distinct sites on the same protein substrate must have mutually exclusive specificity. Evolution could assure this by designing families of kinase such as basophilic kinases and proline-directed kinase with distinct peptide specificity; their reciprocal peptide specificity would have to be very complete, since recruitment of substrate allows phosphorylation of even rather poor phosphorylation sites in a protein. Here we report a powerful evolutionary strategy that assures distinct substrates for basophilic kinases (PKA, PKG and PKC (AGC) and calmodulin-dependent protein kinase (CAMK)) and proline-directed kinase, namely by the presence or absence of proline at the P + 1 position in substrates. Analysis of degenerate and non-degenerate peptides by in vitro kinase assays reveals that proline at the P + 1 position in substrates functions as a "veto" residue in substrate recognition by AGC and CAMK kinases. Furthermore, analysis of reported substrates of two typical basophilic kinases, protein kinase C and protein kinase A, shows the lowest occurrence of proline at the P + 1 position. Analysis of crystal structures and sequence conservation provides a molecular basis for this disfavor and illustrate its generality.

Biotinylation↗
PubMed2005

Protein kinase specificity. A strategic collaboration between kinase peptide specificity and substrate recruitment.

Specificity of phosphorylation by protein kinases is essential to the integrity of biological signal transduction. Specificity is determined by two critical elements: (1) peptide specificity of the kinase, i. e., preferential phosphorylation of S/T/Y residues surrounded by particular patterns of amino acids; and (2) recruitment, i. e., increasing the frequency of encounter between kinase and substrate. Historically, the importance of peptide specificity was studied first, but it has been somewhat overshadowed by emerging emphasis on the importance of recruitment. Recent studies confirm and extend understanding of the relative contribution of these two elements. Peptide specificity always constrains the range of sites that can be phosphorylated by a kinase. Only when recruitment is very strong, as in the case with autophosphorylation, can markedly suboptimal substrates be phosphorylated.

Amino Acid Sequence↗
PubMed2005

Conduit for privileged communications in the lymph node.

A study by Sixt et al. in this issue of Immunity identifies conduit-associated dendritic cells whose privileged access to antigen arriving by the conduit enables uptake and processing of antigen within 90 min of antigen inoculation, long before the arrival of dendritic cells from skin.

Animals↗
PubMed2005

Selective inhibition by rottlerin of macropinocytosis in monocyte-derived dendritic cells.

We present here the analysis of fluid-phase endocytosis (FPE) in human blood monocytes and monocyte-derived dendritic cells (MDDC) facilitated by our serendipitous identification of rottlerin as an efficient inhibitor of dendritic cell FPE (IC(50) of 0.4 microM). Rottlerin was found to be an excellent tool for FPE analysis: rapid-acting, irreversible and selective for FPE (as opposed to receptor-mediated endocytosis) at concentrations of 3 microM and below. The inhibitory effect was not due to toxicity or visible change in membrane ruffles, but affects on cytoskeletal reorganization were evident in MDDC treated with relevant rottlerin concentrations during adhesion. A marked increase in FPE was observed in 1 hr interleukin (IL)-4 and granulocyte macrophage-colony stimulating factor (GM-CSF)-stimulated monocytes. Moreover, rottlerin inhibited the augmented FPE of 1-day cytokine treated monocytes and their augmented ability to induce T cell proliferative responses to tetanus toxoid. We conclude that rottlerin is a useful tool for investigating FPE and its functional importance.

Acetophenones↗
PubMed2005

Junctional disc herniation syndrome in post spinal fusion treated with endoscopic spine surgery.

Fusions of the cervical and lumbar spine are often followed within months or several years by protrusion of discs at the adjacent level or levels. Biomechanical alterations and mobility lost at the fused levels are thought to be transferring the stress to the adjacent segments or discs, which results in accelerated degeneration of the discs and causes disc protrusion. This post-spinal fusion "junctional disc herniation syndrome" (JDHS), or the post-spinal fusion "adjacent segment disease (ASD)" can occur from 15% to 52% of post-spinal fusion, in both superior and/or inferior adjacent levels. The ways in which endoscopic minimally invasive spinal discectomy procedure can be used to treat this JDHS and preserve spinal segmental motion are discussed herein. Also, case illustrations are presented.

Adult↗
PubMed2004

N-ethylmaleimide causes aquaporin-2 trafficking in the renal inner medullary collecting duct by direct activation of protein kinase A.

The antidiuretic hormone arginine vasopressin increases the osmotic water permeability of the renal collecting ducts by inducing the shuttling of aquaporin-2 (AQP2) water channels from intracellular vesicles to the apical plasma membrane of the principal cells. This process has been demonstrated to be dependent on the cytoskeleton and protein kinase A (PKA). Previous studies in the toad urinary bladder, a functional homologue of the renal collecting duct, have demonstrated that the sulfhydryl reagent N-ethylmaleimide (NEM) is also able to activate the vasopressin-sensitive water permeability pathway in this tissue. The aim of the present study was to investigate the effects of NEM on AQP2 trafficking in a mammalian system. We show that NEM causes translocation of AQP2 from the cytosol to the plasma membrane in rat inner medullary collecting ducts; like the response to arginine vasopressin, this action was also dependent on an intact cytoskeleton and PKA. This effect is not mediated by cAMP but results from direct activation of PKA by NEM.

Animals↗
PubMed2004

Rac1 mediates collapse of microvilli on chemokine-activated T lymphocytes.

Lymphocytes circulate in the blood and upon chemokine activation rapidly bind, where needed, to microvasculature to mediate immune surveillance. Resorption of microvilli is an early morphological alteration induced by chemokines that facilitates lymphocyte emigration. However, the antecedent molecular mechanisms remain largely undefined. We demonstrate that Rac1 plays a fundamental role in chemokine-induced microvillar breakdown in human T lymphocytes. The supporting evidence includes: first, chemokine induces Rac1 activation within 5 s via a signaling pathway that involves Galphai. Second, constitutively active Rac1 mediates microvilli disintegration. Third, blocking Rac1 function by cell permeant C-terminal "Trojan" peptides corresponding to Rac1 (but not Rac2, Rho, or Cdc42) blocks microvillar loss induced by the chemokine stromal cell-derived factor 1alpha (SDF-1alpha). Furthermore, we demonstrate that the molecular mechanism of Rac1 action involves dephosphorylation-induced inactivation of the ezrin/radixin/moesin (ERM) family of actin regulators; such inactivation is known to detach the membrane from the underlying actin cytoskeleton, thereby facilitating disassembly of actin-based peripheral processes. Specifically, ERM dephosphorylation is induced by constitutively active Rac1 and stromal cell-derived factor 1alpha-induced ERM dephosphorylation is blocked by either the dominant negative Rac1 construct or by Rac1 C-terminal peptides. Importantly, the basic residues at the C terminus of Rac1 are critical to Rac1's participation in ERM dephosphorylation and in microvillar retraction. Together, these data elucidate new roles for Rac1 in early signal transduction and cytoskeletal rearrangement of T lymphocytes responding to chemokine.

Actins↗
PubMed2004

A novel mutation in IFN-gamma receptor 2 with dominant negative activity: biological consequences of homozygous and heterozygous states.

We identified two siblings homozygous for a single base pair deletion in the IFN-gammaR2 transmembrane domain (791delG) who presented with multifocal Mycobacterium abscessus osteomyelitis (patient 1) and disseminated CMV and Mycobacterium avium complex infection (patient 2), respectively. Although the patients showed no IFN-gammaR activity, their healthy heterozygous parents showed only partial IFN-gammaR activity. An HLA-identical bone marrow transplant from the mother led patient 1 to complete hemopoietic reconstitution, but only partial IFN-gammaR function. We cloned and expressed fluorescent fusion proteins of the wild-type IFN-gammaR2, an IFN-gammaR2 mutant previously described to produce a complete autosomal recessive deficiency (278del2), and of 791delG to determine whether the intermediate phenotype in the 791delG heterozygous state was caused by haploinsufficiency or a dominant negative effect. When cotransfected together with the wild-type vector into IFN-gammaR2-deficient fibroblasts, the fusion protein with 791delG inhibited IFN-gammaR function by 48.7 +/- 5%, whereas fusion proteins with 278del2 had no inhibitory effect. Confocal microscopy of 791delG fusion proteins showed aberrant diffuse intracellular accumulation without plasma membrane localization. The fusion protein created by 791delG did not complete Golgi processing, and was neither expressed on the plasma membrane, nor shed extracellularly. The mutant construct 791delG exerts dominant negative effects on IFN-gamma signaling without cell surface display, suggesting that it is acting on pathways other than those involved in cell surface recognition of ligand.

Cell Line↗
PubMed2004

Kinase peptide specificity: improved determination and relevance to protein phosphorylation.

Specificity of phosphorylation is critical to signal transduction. Recent emphasis on colocalization of substrate and kinase has eclipsed emphasis on peptide specificity, i.e., kinase preference for particular amino acids surrounding the phosphorylation site. We describe an approach to determining peptide specificity by using positional scanning of biotinylated oriented peptide libraries and insights emerging from those determinations. We accurately determine preference (or disfavor) for residues at a given substrate position (such as P+2) by comparison of in vitro phosphorylation of peptide libraries differing by a single residue at that position. By analysis of all positions near the phosphorylation site, position-specific scoring matrices are generated and used both to understand the basis of specificity and to predict phosphorylation. PKC-delta and -zeta predictions have been validated rigorously by comparisons with measured phosphorylation. The results demonstrate specificity and sensitivity (80-90%) much better than the previous predictive method. These predictions can be accessed at http://mpr.nci.nih.gov. The accuracy of the specificity determination allows identification of an important difference in peptide specificity between these closely related kinases; Ile/Leu at the P-1 position is disfavored by PKC-zeta but not PKC-delta. Our findings and visual representation of peptide specificity highlight the importance of disfavored residues. Finally, analysis of 124 experimentally determined PKC sites from the literature demonstrates a very strong role of peptide specificity in many of those sites. Thus, position-specific scoring matrices generated by this method provide a foundation for quantitative analyses of kinase specificity and improved predictions of previously determined physiologically relevant phosphorylation sites.

Cyclic AMP-Dependent Protein Kinases↗
PubMed2003

Chemokine stimulation of human peripheral blood T lymphocytes induces rapid dephosphorylation of ERM proteins, which facilitates loss of microvilli and polarization.

Lymphocyte microvilli mediate initial rolling-adhesion along endothelium but are lost during transmigration from circulation to tissue. However, the mechanism for resorption of lymphocyte microvilli remains unexplored. We show that chemokine stimulation of human peripheral blood T (PBT) cells is sufficient to induce rapid resorption of microvilli. Microvilli in other cells are regulated by ezrin/radixin/moesin (ERM) proteins, which link the plasma membrane to the cortical F-actin cytoskeleton; maintenance of these linkages requires ERM activation, reflected by phosphorylation at a specific carboxy-terminal threonine residue. Carboxyphosphorylated-ERM (cpERM) proteins in resting PBT cells show a punctate peripheral distribution consistent with localization to microvilli. cpERM dephosphorylation begins within seconds of stimulation by chemokines (stromal derived factor 1 alpha [SDF-1 alpha] or secondary lymphoid tissue cytokine), and ERM proteins lose their punctate distribution with kinetics paralleling the loss of microvilli. The cpERM proteins are preferentially associated with the cytoskeleton at rest and this association is lost with chemokine-induced dephosphorylation. Transfection studies show that a dominant-negative ERM construct destroys microvilli, whereas a construct mimicking cpERM facilitates formation of microvilli, retards chemokine-induced loss of microvilli, and markedly impairs chemokine-induced polarization. Thus, chemokine induces rapid dephosphorylation and inactivation of cpERM, which may in turn facilitate 2 aspects of cytoskeletal reorganization involved in lymphocyte recruitment: loss of microvilli and polarization.

Blood Proteins↗
PubMed2003

Dispositional pessimism predicts delayed return to normal activities after inguinal hernia operation.

BACKGROUND: The traditional outcome measure to assess effectiveness of inguinal hernia operation was recurrence. Open mesh repair has reduced recurrence rates and attention is now turning to outcomes other than recurrence. The factors influencing the large variation in reported times of resumption of normal activities after inguinal hernia repair are many and diverse. The human factors influencing resumption of normal activity are rarely reported. METHODS: We undertook a prospective study of 206 patients undergoing primary inguinal hernia repair in an ambulatory setting in a public hospital to ascertain whether dispositional outlook on life affected resumption of normal activity after hernia repair. Outlook on life was assessed using the life orientation test. An independent observer assessed the patients preoperatively and determined timing of resumption of normal activities. RESULTS: Regression analysis showed a highly significant relationship between delayed return to normal activity and dispositional pessimism (P =.0004). DISCUSSION: Dispositional pessimism correlates strongly with delayed return to normal activities. Further studies of this kind will help to elucidate the human factors that affect recovery after operation. Studies which use return to normal activities as a measure of the outcome of a surgical technique should include an assessment of the patient's preoperative outlook on life. Surgeons should consider the personality of the patient presenting for groin hernia repair and may wish to tailor their counseling accordingly.

Activities of Daily Living↗
PubMed2002

CD antigens 2002.

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Antibodies, Monoclonal↗