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B E Kemp

Publications and source records attributed to B E Kemp.

At least 55 records · Page 3Linked to original sources
PubMed1998

Components of a calmodulin-dependent protein kinase cascade. Molecular cloning, functional characterization and cellular localization of Ca2+/calmodulin-dependent protein kinase kinase beta.

Ca2+/calmodulin-dependent protein kinases I and IV (CaMKI and CaMKIV, respectively) require phosphorylation on an equivalent single Thr in the activation loop of subdomain VIII for maximal activity. Two distinct CaMKI/IV kinases, CaMKKalpha and CaMKKbeta, were purified from rat brain and partially sequenced (Edelman, A. M., Mitchelhill, K., Selbert, M. A., Anderson, K. A., Hook, S. S., Stapleton, D., Goldstein, E. G., Means, A. R., and Kemp, B. E. (1996) J. Biol. Chem. 271, 10806-10810). We report here the cloning and sequencing of cDNAs for human and rat CaMKKbeta, tissue and regional brain localization of CaMKKbeta protein, and mRNA and functional characterization of recombinant CaMKKbeta in vitro and in Jurkat T cells. The sequences of human and rat CaMKKbeta demonstrate 65% identity and 80% similarity with CaMKKalpha and 30-40% identity with CaMKI and CaMKIV themselves. CaMKKbeta is broadly distributed among rat tissues with highest levels in CaMKIV-expressing tissues such as brain, thymus, spleen, and testis. In brain, CaMKKbeta tracks more closely with CaMKIV than does CaMKKalpha. Bacterially expressed CaMKKbeta undergoes intramolecular autophosphorylation, is regulated by Ca2+/CaM, and phosphorylates CaMKI and CaMKIV on Thr177 and Thr200, respectively. CaMKKbeta activates both CaMKI and CaMKIV when coexpressed in Jurkat T cells as judged by phosphorylated cAMP response element-binding protein-dependent reporter gene expression. CaMKKbeta activity is enhanced by elevation of intracellular Ca2+, although substantial activity is observed at the resting Ca2+ concentration. The strict Ca2+ requirement of CaMKIV-dependent phosphorylation of cAMP response element-binding protein, is therefore controlled at the level of CaMKIV rather than CaMKK.

Amino Acid Sequence↗
PubMed1998

Crystallization of a trimeric human T cell leukemia virus type 1 gp21 ectodomain fragment as a chimera with maltose-binding protein.

We present a novel protein crystallization strategy, applied to the crystallization of human T cell leukemia virus type 1 (HTLV-1) transmembrane protein gp21 lacking the fusion peptide and the transmembrane domain, as a chimera with the Escherichia coli maltose binding protein (MBP). Crystals could not be obtained with a MBP/gp21 fusion protein in which fusion partners were separated by a flexible linker, but were obtained after connecting the MBP C-terminal alpha-helix to the predicted N-terminal alpha-helical sequence of gp21 via three alanine residues. The gp21 sequences conferred a trimeric structure to the soluble fusion proteins as assessed by sedimentation equilibrium and X-ray diffraction, consistent with the trimeric structures of other retroviral transmembrane proteins. The envelope protein precursor, gp62, is likewise trimeric when expressed in mammalian cells. Our results suggest that MBP may have a general application for the crystallization of proteins containing N-terminal alpha-helical sequences.

ATP-Binding Cassette Transporters↗
PubMed1998

Mutation-directed chemical cross-linking of human immunodeficiency virus type 1 gp41 oligomers.

The human immunodeficiency virus type 1 transmembrane protein gp41 oligomer anchors the attachment protein, gp120, to the viral envelope and mediates viral envelope-cell membrane fusion following gp120-CD4 receptor-chemokine coreceptor binding. We have used mutation-directed chemical cross-linking with bis(sulfosuccinimidyl)suberate (BS3) to investigate the architecture of the gp41 oligomer. Treatment of gp41 with BS3 generates a ladder of four bands on sodium dodecyl sulfate-polyacrylamide gels, corresponding to monomers, dimers, trimers, and tetramers. By systematically replacing gp41 lysines with arginine and determining the mutant gp41 cross-linking pattern, we observed that gp41 N termini are cross-linked. Lysine 678, which is close to the transmembrane sequence, was readily cross-linked to Lys-678 on other monomers within the oligomeric structure. This arrangement appears to be facilitated by the close packing of membrane-anchoring sequences, since the efficiency of assembly of heterooligomers between wild-type and mutant Env proteins is improved more than twofold if the mutant contains the membrane-anchoring sequence. We also detected close contacts between Lys-596 and Lys-612 in the disulfide-bonded loop/glycan cluster of one monomer and lysines in the N-terminal amphipathic alpha-helical oligomerization domain (Lys-569 and Lys-583) and C-terminal alpha-helical sequence (Lys-650 and Lys-660) of adjacent monomers. Precursor-processing efficiency, gp120-gp41 association, soluble recombinant CD4-induced shedding of gp120 from cell surface gp41, and acquisition of gp41 ectodomain conformational antibody epitopes were unaffected by the substitutions. However, the syncytium-forming function was most dependent on the conserved Lys-569 in the N-terminal alpha-helix. These results indicate that gp160-derived gp41 expressed in mammalian cells is a tetramer and provide information about the juxtaposition of gp41 structural elements within the oligomer.

Binding Sites↗
PubMed1997

Evidence that a low-molecular-mass GTP-binding protein is required for store-activated Ca2+ inflow in hepatocytes.

The roles of a monomeric GTP-binding regulatory protein in the activation of store-activated plasma membrane Ca2+ channels and in the release of Ca2+ from the smooth endoplasmic reticulum (SER) in rat liver parenchymal cells were investigated with the use of freshly isolated rat hepatocytes and rat liver microsomes. A low concentration (approx. 130 microM intracellular) of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) activated Ca2+ inflow in intact hepatocytes in the absence of an agonist, whereas a high concentration (approx. 530 microM intracellular) of GTP-S- or guanosine 5'-[betagamma-imido]triphosphate (p[NH]ppG) inhibited the Ca2+ inflow induced by inhibitors of the activity of the endoplasmic-reticulum Ca2+-ATPase (SERCA) and by vasopressin. GTP (530 microM) prevented the inhibition of Ca2+ inflow by GTP-S- and p[NH]ppG. Brefeldin A and the peptide human Arf-1-(2-17), which inhibit many functions of ADP ribosylation factor (Arf) proteins, inhibited the Ca2+ inflow induced by SERCA inhibitors and vasopressin, and altered the profile of Ca2+ release from the SER. These effects were observed at concentrations of Brefeldin A and Arf-1-(2-17) comparable with those that inhibit the functions of Arf proteins in other systems. Succinylated Arf-1-(2-17) had a negligible effect on Ca2+ inflow. GTP[S] and Arf-1-(2-17) completely inhibited the synergistic action of GTP and Ins(1,4,5)P3 in releasing 45Ca2+ from rat liver microsomes loaded with 45Ca2+. AlF4(-) (under conditions expected to activate trimeric G-proteins) and succinylated Arf-1-(2-17) had no effect on GTP/Ins(1,4,5))3-induced 45Ca2+ release, and a mastoparan analogue caused partial inhibition. Arf-1-(2-17) did not inhibit 45Ca2+ release induced by either thapsigargin or ionomycin. It is concluded that a low-molecular-mass G-protein, most probably a member of the Arf protein family, is required for store-activated Ca2+ inflow in rat hepatocytes. The idea that the role of this G-protein is to maintain a region of the SER in the correct intracellular location is discussed briefly.

ADP-Ribosylation Factors↗
PubMed1997

Solution structure of parathyroid hormone related protein (residues 1-34) containing an Ala substituted for an Ile in position 15 (PTHrP[Ala15]-(1-34)).

The structure of human parathyroid hormone (PTH) related protein (residues 1-34) containing an Ala substituted for an Ile in position 15 was studied by two-dimensional proton nuclear magnetic resonance spectroscopy. This mutant retains quite high levels of adenylate cyclase activity based on slightly reduced PTH receptor binding capacity. Three segments of helix were revealed extending from His5 to Lys11, Lys13 to Arg19, and from Phe22 to Thr33/Ala34, with a decided kink between the first two helices around Gly12. N- and C-terminal helices were stabilized by charged and hydrophobic side chain interactions between His5 and Glu30, Asp17 and both His9 and His25, and between Leu8 and Ala29, resulting in a globular molecule occupying a single conformation. While the structure of the entire mid-molecule region differed greatly from the structure of the native peptide, the structure of both N- and C-terminal regions remains essentially unaltered. The residues responsible for initiating signal transduction in the mutant are located in the vicinity of the residues responsible for receptor binding. The C-terminal amphipathic helix forming the receptor binding site exhibits reduced binding as a result of the closely applied N-terminal signal transduction-activating region. Although not contributing directly to receptor binding, the N-terminal region can sterically affect hormone binding through modifications to certain N-terminal side chains.

Alanine↗
PubMed1997

Interaction of the recombinant S100A1 protein with twitchin kinase, and comparison with other Ca2+-binding proteins.

The giant myosin-associated twitchin kinase, a member of the Ca2+-regulated protein kinase superfamily, is activated by the EF-hand protein S100A1 in a Ca2+-dependent and Zn2+-enhanced manner. We used recombinant S100A1 to further characterize the interaction between the two proteins. Zn2+ enhanced the binding of Ca2+/S100A1 to twitchin kinase fragments (Kd < 50 nM) in assays using a BIAcore biosensor by reducing the S100A1 off rate. Other Ca2+-binding proteins (S100A6, calmodulin, and the calmodulin-like domain of Ca2+-dependent protein kinase alpha) bound to the kinase but did not activate it. These results indicate that binding of Ca2+-binding proteins alone is insufficient to trigger the intramolecular rearrangement of kinase autoinhibitory contacts required for twitchin kinase activation that is specifically elicited by the S100A1 protein. Kinase fragments that contained only the autoinhibited catalytic sequence or an additional immunoglobulin-like domain had very similar properties, indicating that the tethered immunoglobulin-like domain does not modulate kinase regulation.

Animals↗
PubMed1997

Posttranslational modifications of the 5'-AMP-activated protein kinase beta1 subunit.

The AMP-activated protein kinase (AMPK) consists of catalytic alpha and noncatalytic beta and gamma subunits and is responsible for acting as a metabolic sensor for AMP levels. There are multiple genes for each subunit and the rat liver AMPK alpha1 and alpha2 catalytic subunits are associated with beta1 and gamma1 noncatalytic subunits. We find that the isolated gamma1 subunit is N-terminally acetylated with no other posttranslational modification. The isolated beta1 subunit is N-terminally myristoylated. Transfection of COS cells with AMPK subunit cDNAs containing a nonmyristoylatable beta1 reduces, but does not eliminate, membrane binding of AMPK heterotrimer. The isolated beta1 subunit is partially phosphorylated at three sites, Ser24/25, Ser182, and Ser108. The Ser24/25 and Ser108 sites are substoichiometrically phosphorylated and can be autophosphorylated in vitro. The Ser-Pro site in the sequence LSSS182PPGP is stoichiometrically phosphorylated, and no additional phosphate is incorporated into this site with autophosphorylation. Based on labeling studies in transfected cells, we conclude that alpha1 Thr172 is a major, although not exclusive, site of both basal and stimulated alpha1 phosphorylation by an upstream AMPK kinase.

AMP-Activated Protein Kinases↗
PubMed1997

AMP-activated protein kinase isoenzyme family: subunit structure and chromosomal location.

The AMP-activated protein kinase (AMPK) consists of catalytic alpha and non-catalytic, beta and gamma (38 kDa) subunits and is responsible for acting as a metabolic sensor for AMP levels. There are multiple genes for each subunit and we find that rat liver AMPK-alpha2 isoform catalytic subunit is associated with beta1 and gamma1 and not with beta2 or gamma2 subunit isoforms. The beta1 and gamma1 isoforms are also subunits of the alpha1 isoform. The sequence of cloned human AMPK-beta1 is 95% identical in amino acid sequence with rat beta1. Human chromosomal localizations were determined for AMPK-alpha1 (5p11-p14), AMPK-beta1 (12q24.1-24.3) and AMPK-gamma1 (12q12-q14), respectively.

AMP-Activated Protein Kinases↗
PubMed1997

Contraction-induced changes in acetyl-CoA carboxylase and 5'-AMP-activated kinase in skeletal muscle.

The concentration of malonyl-CoA, a negative regulator of fatty acid oxidation, diminishes acutely in contracting skeletal muscle. To determine how this occurs, the activity and properties of acetyl-CoA carboxylase beta (ACC-beta), the skeletal muscle isozyme that catalyzes malonyl-CoA formation, were examined in rat gastrocnemius-soleus muscles at rest and during contractions induced by electrical stimulation of the sciatic nerve. To avoid the problem of contamination of the muscle extract by mitochondrial carboxylases, an assay was developed in which ACC-beta was first purified by immunoprecipitation with a monoclonal antibody. ACC-beta was quantitatively recovered in the immunopellet and exhibited a high sensitivity to citrate (12-fold activation) and a Km for acetyl-CoA (120 microM) similar to that reported for ACC-beta purified by other means. After 5 min of contraction, ACC-beta activity was decreased by 90% despite an apparent increase in the cytosolic concentration of citrate, a positive regulator of ACC. SDS-polyacrylamide gel electrophoresis of both homogenates and immunopellets from these muscles showed a decrease in the electrophoretic mobility of ACC, suggesting that phosphorylation could account for the decrease in ACC activity. In keeping with this notion, citrate activation of ACC purified from contracting muscle was markedly depressed. In addition, homogenization of the muscles in a buffer free of phosphatase inhibitors and containing the phosphatase activators glutamate and MgCl2 or treatment of immunoprecipitated ACC-beta with purified protein phosphatase 2A abolished the decreases in both ACC-beta activity and electrophoretic mobility caused by contraction. The rapid decrease in ACC-beta activity after the onset of contractions (50% by 20 s) and its slow restoration to initial values during recovery (60-90 min) were paralleled temporally by reciprocal changes in the activity of the alpha2 but not the alpha1 isoform of 5'-AMP-activated protein kinase (AMPK). In conclusion, the results suggest that the decrease in ACC activity during muscle contraction is caused by an increase in its phosphorylation, most probably due, at least in part, to activation of the alpha2 isoform of AMPK. They also suggest a dual mechanism for ACC regulation in muscle in which inhibition by phosphorylation takes precedence over activation by citrate. These alterations in ACC and AMPK activity, by diminishing the concentration of malonyl-CoA, could be responsible for the increase in fatty acid oxidation observed in skeletal muscle during exercise.

AMP-Activated Protein Kinases↗
PubMed1997

The myosin-I-binding protein Acan125 binds the SH3 domain and belongs to the superfamily of leucine-rich repeat proteins.

The SH3 domains of src and other nonreceptor tyrosine kinases have been shown to associate with the motif PXXP, where P and X stand for proline and an unspecified amino acid, but a motif that binds to the SH3 domain of myosin has thus far not been characterized. We previously showed that the SH3 domain of Acanthamoeba myosin-IC interacts with the protein Acan125. We now report that the Acan125 protein sequence contains two tandem consensus PXXP motifs near the C terminus. To test for binding, we expressed a polypeptide, AD3p, which includes 344 residues of native C-terminal sequence and a mutant polypeptide, AD3delta977-994p, which lacks the sequence RPKPVPPPRGAKPAPPPR containing both PXXP motifs. The SH3 domain of Acanthamoeba myosin-IC bound AD3p and not AD3delta977-994p, showing that the PXXP motifs are required for SH3 binding. The sequence of Acan125 is related overall to a protein of unknown function coded by Caenorhabditis elegans gene K07G5.1. The K07G5.1 gene product contains a proline-rich segment similar to the SH3 binding motif found in Acan125. The aligned sequences show considerable conservation of leucines and other hydrophobic residues, including the spacing of these residues, which matches a motif for leucine-rich repeats (LRRs). LRR domains have been demonstrated to be sites for ligand binding. Having an LRR domain and an SH3-binding domain, Acan125 and the C. elegans homologue define a novel family of bifunctional binding proteins.

Acanthamoeba↗
PubMed1997

Regulation and crystallization of phosphorylated and dephosphorylated forms of truncated dimeric phenylalanine hydroxylase.

Phenylalanine hydroxylase is regulated in a complex manner, including activation by phosphorylation. It is normally found as an equilibrium of dimeric and tetrameric species, with the tetramer thought to be the active form. We converted the protein to the dimeric form by deleting the C-terminal 24 residues and show that the truncated protein remains active and regulated by phosphorylation. This indicates that changes in the tetrameric quaternary structure of phenylalanine hydroxylase are not required for enzyme activation. Truncation also facilitates crystallization of both phosphorylated and dephosphorylated forms of the enzyme.

Animals↗
PubMed1997

Human immunodeficiency virus type 1 envelope glycoprotein oligomerization requires the gp41 amphipathic alpha-helical/leucine zipper-like sequence.

Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) oligomerization was investigated by coexpressing wild-type and truncated envelope glycoproteins to determine the minimum sequence required for mutant-wild-type hetero-oligomerization. The gp41 putative amphipathic alpha-helix, Leu-550 to Leu-582, was essential for hetero-oligomer formation. Alanine substitution of 9 of the 10 residues composing the gp41 amphipathic alpha-helix 4-3 hydrophobic repeat sequence was required to inhibit mutant-wild-type hetero-oligomerization and to render the envelope glycoprotein precursor, gp160, monomeric. This indicates that multiple hydrophobic contacts contribute to the stable envelope glycoprotein oligomeric structure. Single alanine substitutions within the hydrophobic repeat sequence did not affect gp160 oligomeric structure but abolished syncytium-forming function. Some mutations also diminished gp160 processing efficiency and the association between gp120 and gp41 in a position-dependent manner. These results indicate that the gp41 amphipathic alpha-helix 4-3 hydrophobic repeat sequence plays a central role in HIV-1 envelope glycoprotein oligomerization and fusion function.

Alanine↗
PubMed1997

Human immunodeficiency virus type 1 and 2 envelope glycoproteins oligomerize through conserved sequences.

Hetero-oligomerization between human immunodeficiency virus type 2 (HIV-2) envelope glycoprotein (Env) truncation mutants and epitope-tagged gp160 is dependent on the presence of gp41 transmembrane protein (TM) amino acids 552 to 589, a putative amphipathic alpha-helical sequence. HIV-2 Env truncation mutants containing this sequence were also able to form cross-type hetero-oligomers with HIV-1 Env. HIV-2/HIV-1 hetero-oligomerization was, however, more sensitive to disruption by mutagenesis or increased temperature. The conservation of the Env oligomerization function of the HIV-1 and HIV-2 alpha-helical sequences suggests that retroviral TM alpha-helical motifs may have a universal role in oligomerization.

Amino Acid Sequence↗
PubMed1996

Giant protein kinases: domain interactions and structural basis of autoregulation.

The myosin-associated giant protein kinases twitchin and titin are composed predominantly of fibronectin- and immunoglobulin-like modules. We report the crystal structures of two autoinhibited twitchin kinase fragments, one from Aplysia and a larger fragment from Caenorhabditis elegans containing an additional C-terminal immunoglobulin-like domain. The structure of the longer fragment shows that the immunoglobulin domain contacts the protein kinase domain on the opposite side from the catalytic cleft, laterally exposing potential myosin binding residues. Together, the structures reveal the cooperative interactions between the autoregulatory region and the residues from the catalytic domain involved in protein substrate binding, ATP binding, catalysis and the activation loop, and explain the differences between the observed autoinhibitory mechanism and the one found in the structure of calmodulin-dependent kinase I.

Amino Acid Sequence↗
PubMed1996

Substrate specificity and inhibitor sensitivity of Ca2+/S100-dependent twitchin kinases.

Myosin-associated giant protein kinases of the titin/witchin-like superfamily have previously been implicated in the regulation of muscle function, based on genetic and physiological studies. We find that recombinant constitutively active Caenorhabditis elegans and Aplysia twitchin kinase fragments differ in their catalytic activities and peptide-substrate specificities, as well as in their sensitivities to the naphthalene sulfonamide inhibitors 1-(5-chloronaphthalenesulfonyl)-1H-hexahydro-1,4-diazepine (ML-7) and 1-(5-iodonaphthalenesulfonyl)-1H-hexahydro-1,4-diazepine (ML-9). The constitutively active Aplysia twitchin kinase fragment has a remarkably high activity (Vmax > 100 mumol.min-1.mg-1) towards some substrate peptides. The autoinhibited forms of these twitchin kinases can be activated in a Ca(2+)-dependent manner by the dimeric form of the S100A1 protein (S100A1(2)). The twitchin kinase S100A1(2)-binding site can also bind Ca2+/calmodulin but neither kinase is activated by calmodulin. The data provide a functional basis for the ongoing crystallographic study of twitchin kinase fragments.

Amino Acid Sequence↗
PubMed1996

Isoform-specific purification and substrate specificity of the 5'-AMP-activated protein kinase.

The 5'-AMP-activated protein kinase (AMPK) mediates several cellular responses to metabolic stress. Rat liver contains at least two isoforms of this enzyme, either alpha1 or alpha2 catalytic subunits together with beta and gamma noncatalytic subunits in a trimeric complex. The alpha1 isoform is purified using a peptide substrate affinity chromatography column with ADR1 (222-234)P229 (LKKLTRRPSFSAQ), corresponding to the cAMP-dependent protein kinase phosphorylation site in the yeast transcriptional activator of the ADH2 gene, ADR1. This peptide is phosphorylated at Ser230 by AMPK alpha1 with a Km of 3.8 microM and a Vmax of 4.8 micromol/min/mg compared to the commonly used rat acetyl-CoA carboxylase (73-87)A77R86-87 peptide substrate, HMRSAMSGLHLVKRR, with a Km of 33.3 microM and a Vmax of 8.1 micromol/min/mg. Thus, the AMPK exhibits some overlapping specificity with the cAMP-dependent protein kinase. The rat liver AMPK alpha1 isoform has a Kcat approximately 250-fold higher than the AMPK alpha2 isoform isolated from rat liver. The AMPK alpha1 isoform readily phosphorylates peptides corresponding to the reported AMPK phosphorylation sites in rat, chicken, and yeast acetyl-CoA carboxylase and rat hydroxymethylglutaryl-CoA reductase but not phosphorylase kinase. Based on previous peptide substrate specificity studies (Dale, S., Wilson, W. A., Edelman, A. M., and Hardie, G. (1995) FEBS Lett. 361, 191-195) using partially purified enzyme and variants of the peptide AMARAASAAALARRR, it was proposed that the AMPK preferred the phosphorylation site motif Phi(X, beta)XXS/TXXXPhi (Phi, hydrophobic; beta, basic). In good AMPK alpha1 peptide substrates, a hydrophobic residue at the P-5 position is conserved but not at the P+4 position. Oxidation of the Met residues in the rat acetyl-CoA carboxylase (73-87)A77R86-87 peptide increased the Km 6-fold and reduced the Vmax to 4% of the reduced peptide.

AMP-Activated Protein Kinases↗