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Biomedical subjects

B E Kemp

Publications and source records attributed to B E Kemp.

At least 19 recordsLinked to original sources
PubMed1992

Regulation of intrasteric inhibition of the multifunctional calcium/calmodulin-dependent protein kinase.

A regulatory region involved in both autoinhibition and calmodulin (CaM) binding has previously been identified in the multifunctional Ca2+/CaM-dependent protein kinase (CaM kinase II). We have tested the role of various segments of the regulatory region in autoinhibition by the analysis of a series of truncation, substitution, and deletion mutants of the CaM kinase II alpha subunit (CaM kinase II alpha). Unexpectedly, the sequence Lys-Lys-Phe-Asn at positions 291-294, adjacent to the CaM binding domain, was found to be sufficient to maintain an inhibited state in a truncated form of the kinase. However, these residues are not essential in the context of the full-length protein, indicating the importance of additional residues from the overlapping CaM binding domain. We propose here a molecular model for CaM kinase II alpha based on the three-dimensional structure of the cAPK-PKI-(5-24) (protein kinase inhibitor fragment) complex. It is predicted from this model that autoinhibition is of the pseudosubstrate variety and that autophosphorylation of Thr-286 could occur by an intersubunit reaction in the holoenzyme complex.

Allosteric Regulation↗
PubMed1992

Structural basis of the intrasteric regulation of myosin light chain kinases.

The smooth muscle myosin light chain kinase (smMLCK) catalytic core was modeled by using the crystallographic coordinates of the cyclic AMP-dependent protein kinase catalytic subunit (cAPK) and a bound pseudosubstrate inhibitor peptide, PKI(5-24). Despite only 30% identity in amino acid sequence, the MLCK sequence can be readily accommodated in this structure. With the exception of the short B-helix, all major elements of secondary structure in the core are very likely conserved. The active site of the modeled MLCK complements the known requirements for peptide substrate recognition. MLCK contains a pseudosubstrate sequence that overlaps the calmodulin binding domain and has been proposed to act as an intrasteric inhibitor and occupy the substrate binding site in the absence of Ca(2+)-calmodulin. The pseudosubstrate sequence can be modeled easily into the entire backbone of PKI(5-24). The results demonstrate that the intrasteric model for regulation of MLCK by intramolecular competitive inhibition is structurally plausible.

Amino Acid Sequence↗
PubMed1992

Insulin activation of acetyl-CoA carboxylase accompanied by inhibition of the 5'-AMP-activated protein kinase.

The activity of acetyl-CoA carboxylase (ACC), a rate-limiting enzyme of fatty acid biosynthesis and malonyl-CoA production, can be regulated by several mechanisms, including multisite covalent phosphorylation, both in vitro and in intact cells. Evidence has been presented by others to indicate that a 5'-AMP-activated protein kinase (AMPK) is likely the major regulatory kinase active on ACC. While insulin is known to activate ACC in several cell types, accompanied by changes in ACC phosphorylation, the mechanism underlying this activation has been obscure. In the present study, we have examined, in Fao hepatoma cells, the effects of insulin on ACC and AMPK activity, the latter measured with a synthetic peptide corresponding to one of the phosphorylation sites on ACC for AMPK. Our results show that insulin leads to inhibition of kinase activity prior to the onset of ACC activation; the peak of maximal kinase inhibition (approximately 35% at 10 min) is seen to precede the onset of ACC activation (20 min). The inhibition of kinase activity due to insulin is observed both in the absence and presence of varying stimulating concentrations of added 5'-AMP. Both kinase inhibition and ACC activation display similar insulin sensitivity (A50 0.3 nM). Preservation of this insulin-induced kinase inhibition requires the presence of protein phosphatase inhibitors in the cell lysis buffer, suggesting that AMPK itself might be regulated by insulin-stimulated changes in kinase phosphorylation. Taken together, these data are consistent with the hypothesis that the 5'-AMP-activated protein kinase is a regulated component of the insulin signal transduction pathway and may be the major target for insulin regulation of ACC.

AMP-Activated Protein Kinases↗
PubMed1992

CD8+ T-cells from HIV-infected patients can either augment or abrogate HIV-specific lymphoproliferation.

Despite the presence of activated CD8+ T-cells that have been identified in infected individuals, these cells do not overcome natural HIV infection. To understand this better, we analyzed the CD8+ cell-dependent HIV-specific lymphoproliferation that occurs after HIV infection. Our study group of 36 individuals included 11 asymptomatic and 16 symptomatic patients (12 ARC and 4 AIDS), as well as HIV-seronegative controls. After CD8+ cell depletion of PBMC cultures, the remaining cells were tested for proliferation during culture with a well-defined and immunodominant gp41-derived HIV analog, gp41(8). After CD8+ cell depletion, three functional outcomes, which differed in accordance with the disease status of the individual, were consistently recorded, namely (i) an "abrogation effect," (ii) an "augmentation effect," or (iii) "no effect." First, removal of CD8+ cells from PBMC cultures abrogated gp41(8)-specific lymphoproliferation in gp41(8)-specific responders. Paradoxically, in other patients, including 5 symptomatics, the same inhibition of CD8+ cell function caused significant augmentation of gp41(8)-specific lymphoproliferation. These results suggest that the subpopulations of CD8+ T-cells that predominate at different stages of HIV-induced disease have different functional properties, including the ability to modulate HIV-specific cell-mediated immunity.

CD8 Antigens↗
PubMed1992

Antibody epitopes sensitive to the state of human immunodeficiency virus type 1 gp41 oligomerization map to a putative alpha-helical region.

Two antibodies, affinity-purified from human immunodeficiency virus-positive human plasma with synthetic peptides in the region gp41(566-596), were found to recognize oligomeric gp41 more strongly than the monomeric form in an immunoblot assay. In contrast, a murine anti-gp160 monoclonal antibody, which maps within this sequence to gp41(581-596), recognized only monomeric gp41 after disruption of the oligomer with sodium dodecyl sulfate. This monoclonal anti-gp160 antibody did not recognize chemically crosslinked oligomeric gp41 that had been treated with similar conditions used to disrupt the gp41 oligomer. These results indicate that this epitope is inaccessible to binding by this antibody when gp41 is oligomeric. Cyanogen bromide cleavage of gp41 resulted in a 17-kD fragment Thr-541-Met-631. A significant proportion of this fragment was oligomeric when derived from chemically crosslinked gp41. The region Ala-566-Gln-596, within the cyanogen bromide fragment, contains the oligomerization-sensitive epitopes as well as two lysine residues available for crosslinkage. This region is relatively conserved and has the propensity to form an amphipathic alpha-helix.

Amino Acid Sequence↗
PubMed1992

Heterogeneous effects of exogenous IL-2 on HIV-specific cell-mediated immunity (CMI).

A characteristic feature associated with HIV-1 infection of the human host is a chronic decline in circulating CD4+ T helper/inducer cell numbers. Impaired cell-mediated immune functions usually occur in parallel with the decline in CD4+ T cells. Activated CD4+ T helper cells are a major source of endogenous IL-2 which is required for the immunoregulation of both antigen-specific B cells and CD8+ T cells. HIV-specific T cell proliferative responses are said to be weak and inconsistent, even during the asymptomatic phase of disease. We thus wished to determine how exogenous IL-2 affected HIV-specific T cell proliferation at different stages of the disease. Our cohort of 81 included both asymptomatic and symptomatic HIV-infected patients as well as uninfected normal donors. Proliferative responses of peripheral blood mononuclear cells (PBMC) that were elicited during culture with an immunodominant gp41-derived synthetic peptide, gp41[8], and which were known to be CD8+ cell-associated in asymptomatics only, were used to analyse the effects of exogenous IL-2. IL-2 had three main effects on HIV-specific proliferation, namely (i) an additive effect, (ii) a synergistic effect, and (iii) an induced effect. More specifically, low dose exogenous IL-2 frequently augmented lymphoproliferation in both asymptomatic and symptomatic gp41[8] responders. In most symptomatics, however, who were predominantly gp41[8] non-responders, exogenous IL-2 induced lymphoproliferation. Flow cytometric analyses using dual immunofluorescence were used to analyse the T cell subset distribution of proliferating PBMC cultures. During culture with gp41[8], both CD4+ and CD8+ T cell numbers increased. However, after the addition of exogenous IL-2 to gp41[8]-containing cultures, CD8+ cell-associated lymphoproliferative responses were preferentially augmented. These results suggest that in symptomatics there is an inadequate supply of endogenous IL-2 to help maintain the strong and effective CD8+ cell-associated anti-viral immunity, and an exogenous supply of IL-2 may be required.

Flow Cytometry↗
PubMed1992

Definition of an immunodominant T cell epitope contained in the envelope gp41 sequence of HIV-1.

The majority of the immunodominant amino acid sequences of HIV-1 that have been characterized to date are coded for by hypervariable gene sequences. These variable sequences are however interspersed with sequences that are highly conserved between HIV strains. Immunogenic viral products with amino acid sequences that vary minimally between strains, and that consistently elicit both humoral and cellular immune responses, may be ideal for inclusion in a subunit vaccine. We studied HIV-seronegative and HIV-infected persons, classified as asymptomatic (AS), ARC or AIDS. Initially, we assessed the cellular immune status of each subject from results of T cell phenotype analyses, assays for serum levels of surrogate markers of disease progression, and responses to mitogens and recall antigen. In addition, we tested whether three short synthetic peptides derived from the conserved sequences of the envelope gp120 (aa 262-284) and gp41 (aa 579-601), and core p17 (aa 106-125) regions of the HTLV-IIIB isolate, could elicit B cell as well as T cell responses in HIV-infected subjects. Only the gp41-derived sequence was immunogenic at both B and T cell levels. To further characterize the gp41 epitope, we used a series of overlapping synthetic peptides derived from a conserved region of the envelope gp41 (aa 572-613). We thus identified an immunodominant 12-mer peptide sequence, gp41(8)(aa 593-604), which consistently elicited both T cell blastogenic and B cell (antibody) responses in AS HIV-seropositive individuals but not in ARC and AIDS patients. Linear regression analysis showed that in AS persons there was a strong positive correlation (P less than 0.0005) between the absolute CD8+ T cell numbers and the magnitude of blastogenic responses to the gp41(8)(aa 593-604). Furthermore, those AS subjects with T cells that proliferated in response to this gp41 analogue also had significantly greater serum levels of antibody to the same short peptide sequence than symptomatic ARC and AIDS patients. These results suggest that cellular responses to the immunodominant and highly conserved envelope sequences of HIV-1, associated with increased CD8+ T cells, may be important in the pathogenesis of HIV disease.

Amino Acid Sequence↗
PubMed1992

ADR1c mutations enhance the ability of ADR1 to activate transcription by a mechanism that is independent of effects on cyclic AMP-dependent protein kinase phosphorylation of Ser-230.

Four ADR1c mutations that occur close to Ser-230 of the Saccharomyces cerevisiae transcriptional activator ADR1 and which greatly enhance the ability of ADR1 to activate ADH2 expression under glucose-repressed conditions have been shown to reduce or eliminate cyclic AMP-dependent protein kinase (cAPK) phosphorylation of Ser-230 in vitro. In addition, unregulated cAPK expression in vivo blocks ADH2 depression in an ADR1-dependent fashion in which ADR1c mutations display decreased sensitivity to unregulated cAPK activity. Taken together, these data have suggested that ADR1c mutations enhance ADR1 activity by blocking cAPK phosphorylation and inactivation of Ser-230. We have isolated and characterized an additional 17 ADR1c mutations, defining 10 different amino acid changes, that were located in the region defined by amino acids 227 through 239 of ADR1. Three observations, however, indicate that the ADR1c phenotype is not simply equivalent to a lack of cAPK phosphorylation. First, only some of these newly isolated ADR1c mutations affected the ability of yeast cAPK to phosphorylate corresponding synthetic peptides modeled on the 222 to 234 region of ADR1 in vitro. Second, we observed that strains lacking cAPK activity did not display enhanced ADH2 expression under glucose growth conditions. Third, when Ser-230 was mutated to a nonphosphorylatable residue, lack of cAPK activity led to a substantial increase in ADH2 expression under glucose-repressed conditions. Thus, while cAPK controls ADH2 expression and ADR1 is required for this control, cAPK acts by a mechanism that is independent of effects on ADR1 Ser-230. It was also observed that deletion of the ADR1c region resulted in an ADR1c phenotype. The ADR1c region is, therefore, involved in maintaining ADR1 in an inactive form. ADR1c mutations may block the binding of a repressor to ADR1 or alter the structure of ADR1 so that transcriptional activation regions become unmasked.

Amino Acid Sequence↗
PubMed1992

Intrasteric regulation of myosin light chain kinase: the pseudosubstrate prototope binds to the active site.

We previously proposed a molecular mechanism for the activation of smooth muscle myosin light chain kinase (smMLCK) by calmodulin (CaM). According to this model, smMLCK is autoinhibited in the absence of Ca2+/CaM due to the interaction of a pseudosubstrate prototope, contained within the CaM binding/regulatory region, with the active site of the enzyme. Binding of Ca2+/CaM releases the autoinhibition and allows access of the protein substrate to the active site of the enzyme, resulting in phosphorylation of the myosin light chains. We now provide direct experimental evidence that the pseudosubstrate prototope can associate with the active site. We constructed a smMLCK mutant in which the five-amino acid phosphorylation site of the myosin light chain substrate was inserted into the pseudosubstrate sequence of the CaM binding domain without disrupting the ability of the enzyme to bind Ca2+/CaM. We demonstrate that this mutant undergoes intramolecular autophosphorylation at the appropriate inserted serine residue in the absence of CaM and that this autophosphorylation activates the enzyme. Binding of Ca2+/CaM to the mutant enzyme stimulated myosin light chain substrate phosphorylation but strongly inhibited autophosphorylation, presumably by removing the pseudosubstrate from the active site. These results confirm that the pseudosubstrate sequence has access to the catalytic site and that the activation of the enzyme is accompanied by its removal from this position due to Ca2+/CaM binding as predicted by the model.

Amino Acid Sequence↗
PubMed1991

Substrate specificities for yeast and mammalian cAMP-dependent protein kinases are similar but not identical.

The substrate specificity of the cAMP-dependent protein kinase (cAPK) from Saccharomyces cerevisiae has been investigated using synthetic peptides corresponding to the local phosphorylation site sequence around Ser-230 in the yeast transcriptional activator ADR1. ADR1 is required for the expression of the glucose-repressible alcohol dehydrogenase. Yeast cAPK (encoded by the TPK1 gene) phosphorylated Ser-230 in the synthetic peptide ADR1-217-234, VRKRYLKKLTRRASFSAQ-NH2, with a Km of 5.3 microM compared with 46 microM for LRRASLG (Kemptide). Porcine heart cAPK phosphorylated the ADR1 peptide and Kemptide with the considerable lower Km values of 0.23 and 1.6 microM, respectively. These results indicate that the ADR1 peptide is an excellent substrate for cAPK. Both the yeast and mammalian protein kinases qualitatively shared a number of substrate specificity determinants in common involving residues on the proximal NH2-terminal side and up to the +4 position of the COOH-terminal side of the phosphoacceptor. The mammalian enzyme, however, had a much higher affinity for its substrates than did the yeast enzyme. In addition, the yeast and mammalian enzymes displayed several quantitative differences in their preferences for particular peptide substrates. In particular, the mammalian enzyme strongly preferred substrates with NH2-terminal extensions beyond the -4 position relative to the phosphoacceptor. These results suggest that all eukaryotic cAPKs recognize similar but not identical substrate specificity determinants. They also suggest that the different affinities for substrates that inhere to the individual enzymes could influence their physiological roles.

Animals↗
PubMed1991

Proteolytic cleavage sites in smooth muscle myosin-light-chain kinase and their relation to structural and regulatory domains.

Proteolysis of the smooth muscle myosin-light-chain kinase with either thermolysin or endoproteinase Lys-C cleaves the enzyme towards the amino-terminus between the first and second unc domains, unc-II-1 and unc-II-2, and in the calmodulin-binding domain. The thermolytic fragment extends 532 residues from Ser275 to Ala806 and is resistant to further digestion. It is catalytically inactive and does not bind calmodulin. Further proteolysis of the thermolytic fragment with trypsin generates a constitutively active fragment. Digestion with endoproteinase Lys-C initially results in an inactive fragment of 516 residues, Ala287 to Lys802. Further digestion with Lys-C endoproteinase results in a constitutively active 474-residue fragment with the same amino-terminus, but a carboxyl-terminus at Lys760, near Arg762, the last conserved residue of protein kinase catalytic domains. There is no cleavage in the acidic-residue-rich connecting peptide between the amino-terminus of the catalytic domain and the unc-I domain, nor within the unc-II or unc-I domains or between the adjacent unc-II-2 and unc-I domains. The pattern of cleavages by these proteases reflects well the predicted domain structure of the myosin-light-chain kinase and further delineates the regulatory pseudosubstrate region. A synthetic peptide corresponding to the pseudosubstrate sequence, MLCK(787-807) was a more potent inhibitor by three orders of magnitude than the overlapping peptide MLCK(777-793) proposed by Ikebe et al. (1989) [Ikebe, M., Maruta, S. & Reardon, S. (1989) J. Biol. Chem. 264, 6967-6971] to be important in autoregulation of the myosin-light-chain kinase.

Amino Acid Sequence↗
PubMed1991

Intrasteric regulation of protein kinases and phosphatases.

Protein kinases and protein phosphatases are the pre-eminent regulators of cellular processes. Many of these enzymes are present in latent forms that are activated by various modulators. The inhibited form is maintained by autoinhibitory domains either within these proteins or in some instances by separate inhibitory subunits. A number of these autoinhibitory structures have been identified because of structural similarity to their enzyme's substrate. These findings indicate that the enzyme's active site may recognize either substrates or pseudosubstrate autoinhibitory structures that turn them off. Because this form of regulation is directed at the active site it is termed intrasteric control.

Amino Acid Sequence↗
PubMed1991

Localization of cofactor binding sites with monoclonal anti-idiotype antibodies: phenylalanine hydroxylase.

A monoclonal anti-idiotype antibody, NS7, previously shown to mimic the binding of the pterin cofactor of phenylalanine hydroxylase (phenylalanine 4-monooxygenase, EC 1.14.16.1) has been used to localize the cofactor binding site within the phenylalanine hydroxylase catalytic domain to a 27-amino-acid sequence that is highly conserved among the three aromatic amino acid hydroxylases. The binding of NS7 to a synthetic peptide corresponding to the phenylalanine hydroxylase sequence from residue 263 to residue 289 was blocked by the competitive inhibitor of phenylalanine hydroxylase enzyme activity, 7,8-dihydro-6,7-dimethylpterin. In addition this peptide competed with native phenylalanine hydroxylase for binding to 6,7-dimethyl-5,6,7,8-tetrahydropterin conjugated to a polyglutamate carrier. Application of this simple and direct approach to other enzymes is likely to greatly facilitate the identification of ligand binding sites on enzymes, which will significantly contribute to the understanding of enzyme structure-function relationships.

Amino Acid Sequence↗
PubMed1991

Rapid whole blood assay for HIV-1 seropositivity using an Fab-peptide conjugate.

A rapid whole blood test has been developed for circulating antibodies to human immunodeficiency virus type 1 (HIV-1), based on agglutination of autologous red blood cells. Evaluation of the test revealed that 100% of seropositive HIV-1 patients (both asymptomatic and AIDS cases) were detected (n = 94) with a specificity of 99.5% in healthy blood donors (n = 596). The assay uses an Fab fragment of a monoclonal antibody specifically directed against glycophorin (a transmembrane glycoprotein present on the surface of human red blood cells). This anti-red blood cell Fab is conjugated via the inter-heavy chain cysteines to a synthetic peptide corresponding to the immunodominant epitope of the HIV-1 viral coat protein gp41 (579-613). Addition of this reagent to 10 microliters of whole blood results in the Fab-peptide conjugate coating the red blood cells with peptide. In the presence of circulating antibodies to the HIV-1 peptide, red cell agglutination occurs within 2 min. The sensitivity and specificity of this reagent indicate that it is appropriate for use as a rapid diagnostic test for HIV-1 seropositivity.

Agglutination Tests↗
PubMed1991

Reversible calcitonin binding to solubilized sheep brain binding sites.

In this study we have solubilized and characterized binding sites for calcitonin (CT) from sheep brainstem. Autoradiography of 125I-labelled salmon CT (125I-sCT) binding to sheep diencephalon revealed a similar pattern of binding to that seen in other species, although the extent of distribution was greater in the sheep. CT binding activity could be extracted from membranes with either CHAPS or digitonin, but not with beta-octyl glucoside, 125I-sCT binding was saturable, with a dissociation constant for CHAPS-solubilized membranes of 2.8 +/- 0.5 nM and a maximum binding site concentration of 6.2 +/- 1.6 pmol/mg of protein. In competition binding studies, various CTs and their analogues demonstrated a similar rank order of potency to that seen in other CT receptor systems, Optimal binding occurred in the pH range 6.5-7.5, and was decreased in the presence of NaCl concentrations greater than 200 mM. In contrast with most other CT receptor binding systems, in which binding is poorly reversible, the binding of 125I-sCT to sheep brain binding sites underwent substantial dissociation upon addition of excess unlabelled sCT, with 40% and 46% dissociation after 2 h at 4 degree C in particulate and solubilized membranes respectively. Photoaffinity labelling of the binding site with the biologically active analogue 125I-[Arg11,18,4-azidobenzoyl-Lys14]sCT and analysis on SDS/PAGE under reducing conditions revealed a specific protein band of Mr approximately solubilized and particulate brain membranes. This is in accordance with the molecular size of CT receptors in other tissues where two species of receptor have been identified. one of Mr approximately 71,000 and another of Mr approximately 88,000. These results demonstrate the presence of high concentrations of CT binding sites in sheep brain which display different kinetic properties to those of CT receptors found in other tissues.

Affinity Labels↗
PubMed1991

Regulation of smooth muscle myosin light chain kinase by calmodulin.

The mutagenesis work described in this paper has been instrumental in furthering our understanding of how CaM binds to and activates MLCK. Figure 2 schematically represents this interaction. The inactive MLCK appears to have a catalytic domain that is repressed by a substrate inhibitory domain that overlaps with the CaM binding domain, a basic amphipathic helix. In the presence of Ca2+, CaM undergoes a conformational change that exposes two hydrophobic pockets, one in each globular lobe, that are important for binding to MLCK. Upon binding CaM, MLCK undergoes a conformational change that derepresses the catalytic site, allows substrate access and light chain phosphorylation. Calmodulin antagonist drugs intercalate within these hydrophobic pockets to interfere with target enzyme binding. The total loss of activity if W800 is altered to A illustrates the importance of these hydrophobic interactions within the enzyme. The basic residues are also important; most of the basic residues in the binding domain of MLCK appear to aid in CaM binding but are not in themselves crucial, this includes the RRK triad. However, a specific electrostatic interaction between R812 of MLCK and CaM is suggested by the complete failure in MLCK activation if this residue is changed to an A. Electrostatic interactions between MLCK and CaM are also indicated by the TaM-BM1 mutant. This mutant can bind to but not activate MLCK. It is hypothesized that TaM-BM1 will bind to the basic amphipathic helix of MLCK but that the alterations in the surface charges (especially E14 and T34) and/or hydrophobicity (S38) prevent the proper conformational change in MLCK necessary for light chain phosphorylation. The resulting MLCK-CaM complex is therefore, inactive but can bind TaM-BM1. The exact interaction of these amino acids in CaM with MLCK will have to await the elucidation of a CaM-MLCK co-crystal.

Amino Acid Sequence↗