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B E Kemp

Publications and source records attributed to B E Kemp.

At least 19 recordsLinked to original sources
PubMed1996

Regulation of 5'-AMP-activated protein kinase activity by the noncatalytic beta and gamma subunits.

The mammalian 5'-AMP-activated protein kinase is a heterotrimer consisting of an alpha catalytic subunit and beta and gamma noncatalytic subunits, each of which is represented in a larger isoprotein family, related to the SNF1 kinase and its interacting proteins in yeast. In this study, we have used mammalian cell transfection to compare the activities of the two alpha subunit isoforms, alpha-1 and alpha-2, and to study the influence of the noncatalytic subunits on enzyme subunit association and activity. Expression of epitope-tagged protein subunits in COS7 cells indicates detectable but low level kinase activity for each of the two catalytic alpha subunits. Co-expression of alpha subunits with the beta or gamma subunits modestly increases kinase activity accompanied by the formation of alpha/beta or alpha/gamma heterodimers. Co-expression of all three subunits, however, is accompanied by a 50-110-fold increase in kinase activity with the formation of a heterotrimeric complex. In addition to binding of each noncatalytic subunit to the alpha subunit, the beta and gamma subunits bind to each other, likely resulting in a more stable heterotrimeric complex. The increase in kinase activity associated with expression of this heterotrimer is due both to an increase in enzyme-specific activity (units/enzyme mass) and to an apparent enhanced alpha subunit expression. Co-expression of a catalytically defective alpha subunit or the beta/gamma-binding COOH-terminal domain of the alpha subunit results in reduced heterotrimeric kinase activity. The synergistic positive regulatory roles for both the noncatalytic beta and gamma subunits of 5'-AMP-activated protein kinase contrasts with the Snf1p kinase, where only heterodimers of Snf1p and Snf4p seem to be required for maximum kinase activity.

AMP-Activated Protein Kinases↗
PubMed1996

Pseudosubstrate flexibility in chicken smooth muscle myosin light chain kinase.

Chicken smooth muscle myosin light chain kinase pseudosubstrate is contained within the C-terminal region of the sequence MLCK(774-807)amide. It forms a helical conformation which interacts with the connecting peptide contained within the N-terminal portion of the sequence. These regions are linked by a well-defined turn around Ser-787 which supports a model of pseudosubstrate regulation involving the hinging of the pseudosubstrate or autoregulatory recognition sequence away from the active site of the catalytic core following the binding of calmodulin to the kinase.

Amino Acid Sequence↗
PubMed1996

Multiple Ca(2+)-calmodulin-dependent protein kinase kinases from rat brain. Purification, regulation by Ca(2+)-calmodulin, and partial amino acid sequence.

We have purified to near homogeneity from rat brain two Ca(2+)-calmodulin-dependent protein kinase I (CaM kinase I) activating kinases, termed here CaM kinase I kinase-alpha and CaM kinase I kinase-beta (CaMKIK alpha and CaMKIK beta, respectively). Both CaMKIK alpha and CaMKIK beta are also capable of activating CaM kinase IV. Activation of CaM kinase I and CaM kinase IV occurs via phosphorylation of an equivalent Thr residue within the "activation loop" region of both kinases, Thr-177 and Thr-196, respectively. The activities of CaMKIK alpha and CaMKIK beta are themselves strongly stimulated by the presence of Ca(2+)-CaM, and both appear to be capable of Ca(2+)-CaM-dependent autophosphorylation. Automated microsequence analysis of the purified enzymes established that CaMKIK alpha and -beta are the products of distinct genes. In addition to rat, homologous nucleic acids corresponding to these CaM kinase kinases are present in humans and the nematode, Caenorhabditis elegans. CaMKIK alpha and CaMKIK beta are thus representatives of a family of enzymes, which may function as key intermediaries in Ca(2+)-CaM-driven signal transduction cascades in a wide variety of eukaryotic organisms.

Amino Acid Sequence↗
PubMed1996

Ca2+/S100 regulation of giant protein kinases.

Protein phosphorylation by protein kinases plays a central regulatory role in cellular processes and these kinases are themselves tightly regulated. One common mechanism of regulation involves Ca2+-binding proteins (CaBP) such as calmodulin (CaM). Here we report a Ca2+-effector mechanism for protein kinase activation by demonstrating the specific and >1,000-fold activation of the myosin-associated giant protein kinase twitchin by Ca2+/S100A1(2). S100A1(2) is a member of a large CaBP family that is implicated in various cellular processes, including cell growth, differentiation and motility, but whose molecular actions are largely unknown. The S100A1(2)-binding site is a part of the autoregulatory sequence positioned in the active site that is responsible for intrasteric autoinhibition of twitchin kinase; the mechanism of autoinhibition based on the crystal structures of two twitchin kinase fragments is described elsewhere. Ca2+/S100 represents a likely physiological activator for the entire family of giant protein kinases involved in muscle contractions and cytoskeletal structure.

Amino Acid Sequence↗
PubMed1996

Non-catalytic beta- and gamma-subunit isoforms of the 5'-AMP-activated protein kinase.

The mammalian 5'-AMP-activated protein kinase (AMPK) is a heterotrimeric protein consisting of alpha-, beta-, and gamma-subunits. The alpha-subunit is the catalytic subunit and is related to the yeast Snf1p kinase. In this study, we report the cloning of full-length cDNAs for the non-catalytic beta- and gamma-subunits. The rat liver AMPK beta-subunit clone predicts a protein of 30,464 Da, which is related to the Sip1p, Sip2p, and Gal83p subfamily of yeast proteins that interact with Snf1p and are involved in glucose regulation of gene expression. The AMPK beta-subunit, when expressed in bacteria and in mammalian cells, migrates anomalously on SDS gels at an apparent molecular mass of 40 kDa. Rat and human liver AMPK gamma-subunit clones predict a protein of 37,577 Da (AMPK-gamma1), which is related to the yeast Snf4p protein that copurifies with Snf1p and to a larger family of other human AMPK gamma-isoforms. The mRNAs for both AMPK- beta and AMPK-gamma1 are widely expressed in rat tissues, consistent with a broad role for AMPK in cellular regulation. These data reveal a mammalian multisubunit protein kinase strikingly similar to the multisubunit glucose-sensing Snf1 kinase complex. The identification of isoform families for the AMPK subunits indicates the potential diversity of the roles of this highly conserved signaling system in nutrient regulation and utilization in mammalian cells.

AMP-Activated Protein Kinases↗
PubMed1996

Evidence that the PTH receptor binding site on PTHrP(1-34) can hinge at Arg19/Arg20.

The structure of the biologically-active mutant of human parathyroid hormone related protein (residues 1-34) containing an Ala substituted for a His in position 9 reveals two segments of helix extending from Glu4 to Lys13 and from Phe22 to Ala34, with a reverse turn from Gln16 to Arg19. The C-terminal region contains the bulk of the PTH receptor binding site in an amphipathic helix and is capable of hinging at Arg19/Arg20. The region of the molecule containing full antagonist properties is thus confined to the C-terminal helix.

Algorithms↗
PubMed1996

Protein kinase CK2: biphasic kinetics with peptide substrates.

The regulatory beta-subunit of the protein kinase CK2 modulates the salt optimum for alpha-subunit activity. In the presence of salt the beta-subunit is stimulatory while in the absence of salt it is inhibitory. In the presence of 150 mM NaCl CK2 has linear kinetics (Lineweaver-Burk) for the synthetic substrate RRRDDDSDDD with an apparent Km of 60 microM. In contrast, CK2 displayed biphasic kinetics for the peptide substrate when assayed in the absence of added NaCl. Biphasic kinetics were also obtained for other peptides but not for calsequestrin or casein. Recombinant alpha-subunit had strictly linear kinetics in the absence of added NaCl with an apparent Km of 104 microM. Preincubation of CK2 with ATP/Mg2+ or GTP/Mg2+, but not adenosine/Mg2+ or Mg2+ alone, resulted in kinetics that were near linear. This change in kinetics was dependent on enzyme conditions of low salt CK2 displays biphasic kinetics for peptide substrates, the biphasic kinetics require the presence of the beta-subunit, and ATP/Mg2+ binding reverses the effect.

Adenosine Triphosphate↗
PubMed1996

Mammalian AMP-activated protein kinase subfamily.

The mammalian 5'-AMP-activated protein kinase (AMPK) is related to a growing family of protein kinases in yeast and plants that are regulated by nutritional stress. We find the most prominent expressed form of the hepatic AMPK catalytic subunit (alpha 1) is distinct from the previously cloned kinase subunit (alpha 2). The alpha 1 (548 residues) and alpha 2 (552 residues) isoforms have 90% amino acid sequence identity within the catalytic core but only 61% identity elsewhere. The tissue distribution of the AMPK activity most closely parallels the low abundance 6-kilobase alpha 1 mRNA distribution and the alpha 1 immunoreactivity rather than alpha 2, with substantial amounts in kidney, liver, lung, heart, and brain. Both alpha 1 and alpha 2 isoforms are stimulated by AMP and contain noncatalytic beta and gamma subunits. The liver alpha 1 isoform accounts for approximately 94% of the enzyme activity measured using the SAMS peptide substrate. The tissue distribution of the alpha 2 immunoreactivity parallels the alpha 2 8.5-kilobase mRNA and is most prominent in skeletal muscle, heart, and liver. Isoforms of the beta and gamma subunits present in the human genome sequence reveal that the AMPK consists of a family of isoenzymes.

Amino Acid Sequence↗
PubMed1996

Structure of the pseudosubstrate recognition site of chicken smooth muscle myosin light chain kinase.

The structure of the chicken smooth muscle myosin light chain kinase pseudosubstrate sequence MLCK(774-807)amide was studied using two-dimensional proton NMR spectroscopy. Resonance assignments were made with the aid of totally correlated and nuclear Overhauser effect spectroscopy. A distance geometry algorithm was used to process the body of NMR distance and angle data and the resulting family of structures was further refined using dynamic simulated annealing. The major structural features determined include two helical segments extending from Asp-777 to Lys-785 and from Arg-790/Met-791 to Trp-800 connected by a turn region from Leu-786 to Asp-789 enabling the helices to interact in solution. The C-terminal helix incorporates the bulk of the pseudosubstrate recognition site which is partially overlapped by the calmodulin binding site while the N-terminal helix forms the bulk of the connecting peptide. The demonstrated turn between the helices may assist in enabling the autoregulatory or pseudosubstrate recognition sequence to be rotated out of the active site of the catalytic core following calmodulin binding.

Amino Acid Sequence↗
PubMed1995

Evidence that the pertussis toxin-sensitive trimeric GTP-binding protein Gi2 is required for agonist- and store-activated Ca2+ inflow in hepatocytes.

The role of a trimeric GTP-binding protein (G-protein) in the mechanism of vasopressin-dependent Ca2+ inflow in hepatocytes was investigated using both antibodies against the carboxyl termini of trimeric G-protein alpha subunits, and carboxyl-terminal alpha-subunit synthetic peptides. An anti-Gi1-2 alpha antibody and a Gi2 alpha peptide (Gi2 alpha) Ile345-Phe355), but not a Gi3 alpha peptide (Gi3 alpha Ile344-Phe354), inhibited vasopressin- and thapsigargin-stimulated Ca2+ inflow, had no effect on vasopressin-stimulated release of Ca2+ from intracellular stores, and caused partial inhibition of thapsigargin-stimulated release of Ca2+. An anti-Gq alpha antibody also inhibited vasopressin-stimulated Ca2+ inflow and partially inhibited vasopressin-induced release of Ca2+ from intracellular stores. Immunofluorescence measurements showed that Gi2 alpha is distributed throughout much of the interior of the hepatocyte as well as at the periphery of the cell. By contrast, Gq/11 alpha was found principally at the cell periphery. It is concluded that the trimeric G-protein, Gi2, is required for store-activated Ca2+ inflow in hepatocytes and acts between the release of Ca2+ from the endoplasmic reticulum (presumably adjacent to the plasma membrane) and the receptor-activated Ca2+ channel protein(s) in the plasma membrane.

Amino Acid Sequence↗
PubMed1995

Insert regions in domain X of the casein kinase II catalytic subunit.

Casein kinase II, cyclin-dependent kinases, and glycogen synthase kinase-3 are members of the protein kinase subfamily with a prominent insert in domain X of their catalytic subunit sequence. The function of the insert sequence in casein kinase II was investigated utilising synthetic peptides corresponding to the insert, cross-linking experiments, and the generation of casein kinase II insert region mutants. The mutation of basic residues (R276-->A, R278-->A, R281-->A, K277-->A) within the major insert sequence (PRFHDILQRHSRKRWERFVHSDNQHL, positions 265-290) did not affect alpha/beta subunit association, enzyme tetramerisation, thermal stability, and peptide (RRRDDDSDDD-NH2) phosphorylation. Similarly, replacement of residues 276-290 within the major insert with the corresponding residues from the cell-cycle kinase cyclin-dependent kinase 2 (CDK2) (FPKWKPGSLASHVKN) had no significant effect. The mutation of charged residues (H232-->A, H234-->A, D235-->A) within a nearby minor insert sequence (HGHDNY, positions 232-237), or replacement of residues 234-237 with the corresponding residues from CDK2 (DSEI) also did not affect alpha/beta subunit association and tetramerisation, but reduced enzyme thermal stability to more closely resemble the stability of the isolated alpha-subunit. In addition, mutations within the minor insert caused approximately a threefold increase in the apparent Km for peptide substrate. The results indicate that the major and minor inserts are not essential for alpha/beta subunit association, but the minor insert region influences substrate binding and thermal stability.

Amino Acid Sequence↗
PubMed1995

Catalytic subunits of the porcine and rat 5'-AMP-activated protein kinase are members of the SNF1 protein kinase family.

The 5'-AMP-activated protein kinase (AMPK) regulates the fatty acid and sterol synthesizing pathways via phosphorylation of acetyl-CoA carboxylase and HMG-CoA reductase, respectively. Highly purified kinase from porcine liver contains three apparent subunits of molecular mass 63 kDa, 40 kDa and 38 kDa. Peptide sequencing of the 63 kDa protein (AMPK63cat) revealed that this polypeptide is the catalytic subunit of the kinase. Porcine peptide sequences were used to clone by RT-PCR partial length cDNAs for the catalytic domains of the porcine AMPK63cat, and its rat homolog, which were virtually identical in deduced amino acid sequence. Screening of a rat liver cDNA library with these partial length cDNAs and with degenerate oligonucleotides yielded several unique clones, some of which had a 142 bp deletion in the catalytic domain of the kinase. A consensus full-length sequence with a 1.7 kb open reading frame has been constructed from overlapping library and PCR-derived clones. A large mRNA for rat AMPK63cat (8.5 kb) is expressed in nearly all rat tissues, with highest levels detectable in heart and skeletal muscle. Using PCR, the presence of two mRNA species with or without the 142 bp deletion in the catalytic domain was noted in all rat tissues examined. Comparison of the deduced protein sequence of AMPK63cat reveals highly conserved homologies in both the catalytic and non-catalytic domains to several members of the SNF1 kinase family, including kinases from Arabidopsis, barley, rye, and S. cerevesiae, as well as to other mammalian kinases and to a C. elegans kinase. The high evolutionary conservation of both kinase structure and function (metabolite sensing) coupled with their pattern of tissue/organism expression suggest that the mammalian members of this kinase family likely play wider roles than the regulation of cellular lipid metabolism.

AMP-Activated Protein Kinases↗
PubMed1995

Arg21 is the preferred kexin cleavage site in parathyroid-hormone-related protein.

Parathyroid-hormone-related protein (PTHrP) contains several potential sites for proteolytic processing. Although there is considerable evidence for the existence of cleaved products in vivo, little is known about the post-translational processing of PTHrP. We have used purified kexin (Kex2) protease to identify which cleavage sites in recombinant PTHrP(1-141) might be of physiological significance. Cleavage products were identified by N-terminal sequencing. Kex2 preferentially cleaved PTHrP(1-141) carboxy to the triplet arginine site Arg-Arg-Arg21 with a Km of 3.3 +/- 1.7 microM and a kcat of 6 +/- 1.2 s-1. Substitution of alanine for Arg19 resulted in substantially reduced conversion, while no detectable cleavage occurred when alanine was substituted for either Arg20 or Arg21. In contrast, the degree of Kex2 cleavage at Arg21 in PTHrP(1-34) was lower. No detectable cleavage occurred in an unrelated synthetic peptide containing both double and triple arginine sites. Low levels of cleavage also took place carboxy to Lys-Arg97, Lys-Arg105, Arg-Arg106 and Thr-Arg108. Cleavage carboxy to Lys-Arg105, the best of these minor sites, occurred with a Km of 8.4 +/- 2.7 microM and a kcat of 0.8 +/- 0.2 s-1. These studies indicate that the preferred Kex2 cleavage site in PTHrP(1-141) is carboxy to Arg-Arg-Arg21, which effectively destroys its parathyroid-hormone-like biological activity. Cleavage of this site by Kex2-related mammalian convertases in vivo may be an important mechanism for full elaboration of the non-parathyroid-hormone-like paracrine actions of PTHrP in a tissue-specific manner.

Arginine↗
PubMed1995

Regulation of membrane-associated tyrosine phosphatases in UMR 106.06 osteoblast-like cells.

Protein tyrosine phosphatases play an important role in cell metabolism. Three distinct protein tyrosine phosphatase activities have been identified in an osteoblast-like cell line, UMR 106.06. These activities comprised two membrane-associated phosphatases and one cytosolic phosphatase of apparent molecular mass > 153 kDa, 80 kDa and 40 kDa respectively, estimated by gel filtration. On the basis of differences in apparent molecular mass, proteolytic-digestion profiles, substrate specificities and responses to a range of extracellular influences and inhibitor molecules, the two membrane-associated tyrosine phosphatases are distinct proteins. Tyrosine phosphatase activity in UMR 106.06 cells was sensitive to cell density. Cells at confluence contained membrane protein tyrosine phosphatase with specific activity 9-fold higher than cells at medium or low cell density. This elevation in membrane tyrosine phosphatase activity was due specifically to an increase in the high-molecular-mass enzyme. This phosphatase was also responsive to extracellular matrix components. This activity was elevated in cells grown on a collagen type-I matrix independently of cell density. Membrane and cytosolic protein tyrosine phosphatases were differentially regulated by a variety of agents including phorbol 12-myristate 13-acetate, parathyroid hormone, epidermal growth factor, okadaic acid and transforming growth factor beta. These observations suggest that regulatory influences control tyrosine phosphorylation in UMR 106.06 cells including cell-cell contact, cell-matrix contact and signal transduction involving tyrosine and serine/threonine phosphorylation events.

Animals↗
PubMed1995

Synthetic peptides representing sequences within gp41 of HIV as immunogens for murine T- and B-cell responses.

Within the gp41 glycoprotein of the human immunodeficiency virus type 1 (HIV-1) there is a relatively conserved region which appears accessible to the immune system during the course of HIV infection and is recognised by antibody from virtually all patients with AIDS. This region has also been shown to function as a target for human T cells. We have examined synthetic peptides spanning this sequence, between residues 572 and 604, with a view to evaluating their potential as immunogens. Peptides 572GIKQLQARILAVERYLKDQQ591 and 579RILAVERYLKDQQLLGGIWGCSGK601 were good immunogens in two different strains of mice while peptide 576LQARILAVERYLKDQQ591 was an inferior immunogen, and peptide 593LGIWGCSGKLIC604 was non-immunogenic unless coupled to a carrier protein. For both antibody and T cell responses it was apparent that sequences that could function as determinants within one peptide could not do so in the context of a different peptide immunogen. It follows that by judicious choice of immunogen sequence it may be possible to direct the immune response towards a desired fine specificity. Unwanted responses by CD4+ T cells isolated from certain peptide-primed animals were also observed. These T cells showed an unusual reactivity in that they were incapable of recognising their determinant AVERYLKDQQ if it was extended at the C-terminal end with the native sequence and as such would not be expected to recognise the native molecule unless processing created the identical C-terminus.

Amino Acid Sequence↗
PubMed1995

Determinants of human immunodeficiency virus type 1 envelope glycoprotein oligomeric structure.

Oligomerization of the human immunodeficiency virus type 1 envelope (env) glycoproteins is mediated by the ectodomain of the transmembrane glycoprotein gp41. We report that deletion of gp41 residues 550 to 561 resulted in gp41 sedimenting as a monomer in sucrose gradients, while the gp160 precursor sedimented as a mixture of monomers and oligomers. Deletion of the nearby residues 571 to 582 did not affect the oligomeric structure of gp41 or gp160, but deletion of both sequences resulted in monomeric gp41 and predominantly monomeric gp160. Deletion of residues 655 to 665, adjacent to the membrane-spanning sequence, partially dissociated the gp41 oligomer while not affecting the gp160 oligomeric structure. In contrast, deletion of residues 510 to 518 from the fusogenic hydrophobic N terminus of gp41 did not affect the env glycoprotein oligomeric structure. Even though the mutant gp160 and gp120 molecules were competent to bind CD4, the mutations impaired fusion function, gp41-gp120 association, and gp160 processing. Furthermore, deletion of residues 550 to 561 or 550 to 561 plus 571 to 582 modified the antigenic properties of the proximal residues 586 to 588 and the distal residues 634 to 664. Our results indicate that residues 550 to 561 are essential for maintaining the gp41 oligomeric structure but that this sequence and additional sequences contribute to the maintenance of gp160 oligomers. Residues 550 to 561 map to the N terminus of a putative amphipathic alpha-helix (residues 550 to 582), whereas residues 571 to 582 map to the C terminus of this sequence.

Amino Acid Sequence↗
PubMed1995

A rapid test for endotoxin in whole blood.

A rapid whole blood agglutination test has been developed for the detection of endotoxin. The test reagent consists of polymyxin B (PmB) conjugated to the Fab fragment of the anti-glycophorin antibody 1C3/86. After addition of reagent to whole blood, red cell agglutination occurs within two minutes in samples from endotoxaemic patients or with the addition of either whole Gram negative bacteria, supernatants from Gram negative bacterial cultures or purified endotoxin. In clinical samples there was a strong correlation (r = 0.83) between the strength of agglutination and the level of endotoxin measured by the Limulus amaebolysate test (LAL). The prospect of a rapid and accurate test for endotoxin may enable better clinical management of Gram negative sepsis.

Adult↗