Design and use of peptide substrates for protein kinases.
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Amino Acid Sequence↗
Biomedical subjects
B E Kemp
Publications and source records attributed to B E Kemp.
Pseudosubstrate-based peptide inhibitors.
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Amino Acid Sequence↗
N-terminal residues 105-117 of HIV-1 gp120 are not involved in CD4 binding.
Syu et al. recently reported that deletion of residues Ile-108 to Leu-116 from the amino terminus of gp120 abolished CD4 binding. The authors have investigated the role of this region using a monospecific antipeptide antibody. As assessed by a microtiter plate-based radioimmunoassay, the antibody, raised in sheep against a synthetic peptide encompassing this deleted region, does not inhibit the gp120-CD4 association. The reported loss of CD4 binding ability, resulting from the deletion in this region of gp120, is likely to be due to indirect structural changes in gp120 rather than representing an integral part of the CD4 binding domain.
Amino Acid Sequence↗
A carboxyl-terminal peptide from the parathyroid hormone-related protein inhibits bone resorption by osteoclasts.
PTH-related protein (PTHrP) interacts, via its amino-terminal 34 residues, with PTH receptors on osteoblasts to stimulate osteoclastic bone resorption indirectly. We now report that mature hPTHrP-(1-141) (EC50, approximately 10(-11) M) and a carboxyl-terminal fragment, PTHrP-(107-139) (EC50, approximately 10(-15) M), are potent inhibitors of resorption in an isolated rat osteoclast bone resorption assay, whereas hPTHrP-(1-108) and hPTHrP-(1-34) are inactive in this respect. PTHrP-(107-139) also inhibits resorption in a rat long bone organ culture system and reduces osteoclast spreading. PTHrP-(107-139) does not act on osteoclasts via a cAMP signal transduction mechanism, but its effects may be mediated by protein kinase-C. This previously unrecognized action of PTHrP, to inhibit osteoclastic bone resorption directly, indicates that PTHrP may be a precursor of multiple biologically active peptides with differing physiological functions.
Amino Acid Sequence↗
A potent inhibitor of osteoclastic bone resorption within a highly conserved pentapeptide region of parathyroid hormone-related protein; PTHrP[107-111].
We have recently shown that a carboxyl-terminal sequence of parathyroid hormone-related protein, PTHrP[107-139], is a potent direct inhibitor of osteoclastic bone resorption. We now report that this bone resorption inhibitory activity, which we have called osteostatin, is entirely contained within the highly conserved pentapeptide PTHrP[107-111]. Our results indicate that processing at residue 106 and a free amino terminus is required for full activity of the peptide. The retroinverted peptide is considerably less potent than the parent peptide. The retention of full biological activity in such a short fragment was unexpected. This data provides the basis for the development of further analogs with potential therapeutic use in disorders associated with increased osteoclastic bone resorption.
Amino Acid Sequence↗
Mutagenesis of the pseudosubstrate site of protein kinase C leads to activation.
Protein kinase C is maintained in an inactive state by the action of an inhibitory region within the effector binding domain of the kinase. It has been suggested that a short stretch of amino acids (pseudosubstrate site) mediates this inhibition by binding to the active site and preventing substrate interaction [House, C. and Kemp, B. E. (1987) Science 238, 1726-1728]. A mutated version of protein kinase C-alpha containing a glutamic acid for alanine substitution in this region has been analysed for biochemical properties and biological function. Consistent with the importance of this pseudosubstrate site in regulating kinase activity, this altered protein has a significantly increased effector-independent kinase activity relative to wild-type protein kinase C-alpha and shows increased sensitivity to activation by proteolysis. The increased activity of this protein in the intact cell was confirmed by its ability to stimulate expression from a phorbol-ester-inducible reporter construct in a transient transfection system. Expression of a mutant kinase with the pseudosubstrate sequence deleted causes greater induction in this transient expression system, consistent with this kinase being independent of effectors and thus constitutively active.
Animals↗
The smooth muscle 132 kDa cyclic GMP-dependent protein kinase substrate is not myosin light chain kinase or caldesmon.
Atrial natriuretic peptide (ANP) stimulates the phosphorylation of three cyclic GMP-dependent protein kinase substrate proteins of 225, 132, and 11 kDa (P225, P132 and P11 respectively) in the particulate fraction of cultured rat aortic smooth muscle cells [Sarcevic, Brookes, Martin, Kemp & Robinson (1989) J. Biol. Chem. 264, 20648-20654]. Vrolix, Raeymaekers, Wuytack, Hofmann & Casteels [(1988) Biochem. J. 255, 855-863] have reported the presence of a 130 kDa cyclic GMP-dependent protein kinase substrate protein in the membrane fraction of pig aorta or stomach, and suggested that it may be myosin light chain kinase (MLCK). The aim of the present study was to determine whether P132 from rat aorta was MLCK or caldesmon. Although P132 co-migrates with purified chicken gizzard MLCK on SDS/polyacrylamide gels, it is distinct from rat aortic MLCK. Partially purified MLCK from rat aorta migrated as a 145 kDa protein on SDS/polyacrylamide gels. Immunoblotting the partially purified rat aortic MLCK with antibody to bovine tracheal MLCK identified rat aortic MLCK (145 kDa) and a corresponding 145 kDa protein in the particulate fraction of cultured rat aortic smooth muscle cells, but did not detect the 132 kDa protein. Phosphopeptide maps of purified rat aortic MLCK prepared by digestion with Staphylococcus aureus V8 protease were distinct from those of P132. P132 was not caldesmon, since antibodies to caldesmon cross-reacted with 136 and 76 kDa proteins in the particulate fraction of rat aortic cells, but not with P132. Furthermore, caldesmon was partially extracted from the particulate into the soluble fraction by heating at 90 degrees C, whereas P132 was not. These results demonstrate that the ANP-responsive cyclic GMP-dependent protein kinase substrate of 132 kDa from rat aortic smooth muscle cells is not MLCK or caldesmon.
Animals↗
A rapid whole-blood immunoassay system.
A new immunoassay system has been developed which allows the detection of circulating antigens, antibodies or drugs in whole blood without specialized personnel or equipment. This is achieved by the use of bispecific reagents, which comprise specific antibodies or antigens that are coupled to a non-agglutinating antierythrocyte antibody. Within two minutes, these reagents cause specific agglutination of a patient's own red cells in samples that contain the relevant analyte. Levels of low molecular weight haptens also can be measured by the use of an indirect, agglutination-inhibition assay. This simple immunoassay method would fulfil the needs of many physicians and Third-World countries and also has mass-screening and veterinary applications.
AIDS Serodiagnosis↗
Comparison of the effects of amino-terminal synthetic parathyroid hormone-related peptide (PTHrP) of malignancy and parathyroid hormone on resorption of cultured fetal rat long bones.
We have compared the effects of of various synthetic amino-terminal forms of human parathyroid hormone-related peptide (PTHrP) of malignancy with synthetic parathyroid hormone (PTH) on the resorptive responses of fetal rat long bones in organ culture. PTH and PTHrP increased 45Ca release at concentrations of 0.1-25 nM. PTHrP (1-40) and bovine PTH (1-34) were more potent than human PTH (1-34) and PTHrP (1-34). However, the slopes of the dose-response curves and the maximal resorptive effects were similar. There was a marked decrease in the potency of amino-terminal PTHrP peptides as the length was decreased. PTHrP (1-29) and PTHrP (1-25) were inactive at 120 nM. Further comparison of bPTH (1-34) and PTHrP (1-34) showed that both could induce bone resorption after a brief (6 hours) exposure and that the response to PTHrP (1-34) was qualitatively similar to that of bPTH (1-34) with respect to enhancement by ACTH and inhibition by calcitonin and glucocorticoids. Hydroxyurea and indomethacin did not block the resorptive response to either agonist. Cyclic AMP production in response to PTHrP (1-34) and (1-40) was similar to that for bPTH (1-34) in ROS 17/2.8 cells. The cyclic AMP (cAMP) response was much smaller in fetal rat long bones and calvariae, and bPTH was more potent than PTHrP. These studies confirm that PTHrP is quantitatively similar in its effects on bone resorption to PTH and are consistent with the two agents acting on the same receptor.
Animals↗
Protein kinase C pseudosubstrate prototope: structure-function relationships.
A structure-function study of the protein kinase C (PK-C) pseudosubstrate sequence (R19FARK-GALRQKNV31) has been undertaken. The role of specific residues was investigated using an alanine substitution scan. Arg-22 was the most important determinant in the inhibitor sequence, since substitution of this residue by alanine gave a 600-fold increase in the IC50 value to 81 +/- 9 microM. Substitutions of other basic residue also increased the IC50, 5-, 11- and 24-fold for the Ala-19, Ala-23 and Ala-27 substitutions, respectively. The importance of basic residues in determining the potency of the pseudosubstrate peptide reflects the requirements for these residues in peptide substrate phosphorylation. The residues Gly-24, Leu-26 and Gln-28 were also important for pseudosubstrate inhibitor potency. The large difference in the IC50 value for the [A22]PK-C(19-31) peptide makes it a valuable control in studies employing the pseudosubstrate peptide to explore functional roles of PK-C.
Amino Acid Sequence↗
Active tyrosine phosphatase in immunoprecipitates of multiple isoforms of Ly-5.
Using tumour cell lines expressing specific isoforms of murine Ly-5 (molecular weights of 180,000, 200,000 and 240,000) we find that all forms of Ly-5 and immuno-affinity purified forms of Ly-5 contain tyrosyl phosphatase activity. These results demonstrate that these isoforms of Ly-5 belong to the same family of functional receptor-linked tyrosine phosphatases as the human leukocyte common antigen. CD45.
Animals↗
Protein kinase recognition sequence motifs.
Protein kinases play a crucial role in the regulation of many cellular processes. They alter the functions of their target proteins by phosphorylating specific serine, threonine and tyrosine residues. Identification of phosphorylation site sequences and studies with corresponding model peptides have provided clues to how these important enzymes recognize their substrate proteins. This knowledge has made it possible to identify potential sites of phosphorylation in newly sequenced proteins as well as to construct specific model substrates and inhibitors.
Amino Acid Sequence↗
Regulatory and structural motifs of chicken gizzard myosin light chain kinase.
The amino acid sequence for chicken smooth muscle myosin light chain kinase (smMLCK) was deduced from a full-length cDNA. This has allowed definition of both the complete sequence of the inactive 64-kDa proteolytic fragment, which contains the pseudosubstrate autoregulatory sequence, and of the active 61-kDa Ca2+/calmodulin-independent fragment, which lacks the autoregulatory domain. Comparison of the two sequences shows that the autoregulatory domain extends from Asn-780 to Arg-808. The peptide Leu-774 to Ser-787 does not inhibit smMLCK, whereas peptides of similar or shorter length from the pseudosubstrate region (Ser-787 to Val-807) are potent inhibitors. These data define the autoregulatory region as being contained within and probably identical to the pseudosubstrate domain. The catalytic and regulatory regions are flanked by several copies of 100-amino acid segments containing one of two consensus motifs. These motifs are absent from mammalian skeletal muscle MLCK or from Dictyostelium discoideum MLCK but are present in the Caenorhabditis elegans unc-22 gene product and the titin molecule of skeletal muscle myofibrils. These results indicate that the amino acid sequence of smMLCK encodes multiple functional motifs in addition to the catalytic domain.
Amino Acid Sequence↗
Use of a conserved immunodominant epitope of HIV surface glycoprotein gp41 in the detection of early antibodies.
An enzyme immunoassay (EIA) utilizing a synthetic peptide analogue of HIV gp41 (amino acids 579-599, RILAVERYLKDQQLLGIWGCS) as antigen was compared with two commercial assays (Genetic Systems, Abbott ENVACORE) for the ability to detect antibodies in the early stages of infection. Two panels, consisting of 96 sera from 15 people and 140 sera from 44 people seroconverting to HIV, were examined. In the first group the synthetic peptide assay (gp41 EIA) detected antibodies before the Genetic Systems EIA in seven out of 15 people and concurrently in the remaining eight. With the second panel the Abbott ENVACORE assay detected antibodies before the gp41 EIA in two out of 44 people while the gp41 EIA detected antibodies first in six out of 44. In the remaining 36 people antibodies were detected simultaneously by the two tests. The gp41 EIA usually detected anti-HIV antibodies before or concurrently with the two commercial assays examined suggesting that the epitope cluster represented by this peptide is recognized early in infection.
AIDS Serodiagnosis↗
A monoclonal antibody raised against an undecapeptide sequence from human transforming growth factor alpha recognizes a hexapeptide epitope in mitotic centrosomes.
A monoclonal antibody (mAbIIa138) raised against the second-loop undecapeptide (residues [21-31]) of human transforming growth factor alpha, known to be involved in binding to its cell receptor, was found to define a hexapeptide epitope (RFLVQE, residues [22-27]). In enzyme-linked immunosorbent assay testing mAbIIa138 did not react with either native or reduced human transforming growth factor alpha, indicating that conformational restraints in this protein interfered with the antigenicity of the cognate sequence. Despite its failure to react with human transforming growth factor alpha, this monoclonal antibody proved very interesting when used to immunostain mammalian cells. mAbIIa138 was found to bind with great specificity to the centrosomes of late-interphase and mitotic cells. The staining of the centrosomes was most intense when the centrosomes were active in organizing the mitotic spindle and faded during telophase as the spindle was disaggregated. The epitope that mAbIIa138 recognizes is thus in a centrosomal protein, denoted CSP alpha, involved in some aspect of mitotic spindle organization or function. Analysis of the antigenic stringency of the epitope, using an amino acid replacement set, showed that 25 individual single amino acid replacements were possible without significant loss of antigenicity. Data bank searches for proteins of possible relevance were unsuccessful, so CSP alpha is an as yet unsequenced protein, specific to the centrosome.
Amino Acid Sequence↗
Atrial natriuretic peptide-dependent phosphorylation of smooth muscle cell particulate fraction proteins is mediated by cGMP-dependent protein kinase.
The atrial natriuretic peptide (ANP) stimulates cGMP production and protein phosphorylation in a particulate fraction of cultured rat aortic smooth muscle cells. Three proteins of 225, 132, and 11 kDa were specifically phosphorylated in response to ANP treatment, addition of cGMP (5 nM), or addition of purified cGMP-dependent protein kinase. The cAMP-dependent protein kinase inhibitor had no effect on the cGMP-stimulated phosphorylation of the three proteins but inhibited cAMP-dependent phosphorylation of a 17-kDa protein. These results demonstrate that the particulate cGMP-dependent protein kinase mediates the phosphorylation of the 225-, 132-, and 11-kDa proteins. The 11-kDa protein is phospholamban based on the characteristic shift in apparent Mr from 11,000 to 27,000 on heating at 37 degrees C rather than boiling prior to electrophoresis. ANP (1 microM) increased the cGMP concentration approximately 4-fold in the particulate fractions, from 4.3 to 17.7 nM, as well as the phosphorylation of the 225-, 132-, and 11-kDa proteins. In contrast, the biologically inactive form of ANP, carboxymethylated ANP (1 microM), did not stimulate phosphorylation of any proteins nor did the unrelated peptide hormone, angiotensin II (1 microM). These results demonstrate the presence of the cGMP-mediated ANP signal transduction pathway in a particulate fraction of smooth muscle cells and the specific phosphorylation of three proteins including phospholamban, which may be involved in ANP-dependent relaxation of smooth muscle.
Adenosine Triphosphate↗
Myosin light chain kinase structure function analysis using bacterial expression.
A 40-kDa fragment of chicken smooth muscle myosin light chain kinase was produced and partially purified from a bacterial expression system. This fragment exhibits calmodulin binding and substrate phosphorylation properties similar to those of the isolated chicken gizzard enzyme. A series of 3'-deletion mutants was prepared and used to produce proteins with the same NH2 terminus but with COOH termini varying over 180 amino acids. Results show that truncation of the enzyme at Ser-512 (based on the amino acid numbering system described for the partial cDNA clone by Guerriero, V., Jr., Russo, M. A., Olson, N. J., Putkey, J. A., and Means, A. R. (1986) Biochemistry 25, 8372-8381) does not alter calmodulin binding, calmodulin regulation, or enzymatic properties. Removal of an additional 5 residues from the COOH terminus completely inhibits calmodulin binding and results in an inactive kinase that can be fully activated by limited proteolysis. Site specific mutations within these 5 residues demonstrate that Gly-508 and Arg-509 are independently involved in calmodulin-dependent binding and activation of myosin light chain kinase. Truncation of the enzyme at residues within the protein kinase catalytic domain results in inactive protein that cannot be activated by proteolysis.
Animals↗
NMR study of a 34-residue N-terminal fragment of the parathyroid-hormone-related protein secreted during humoral hypercalcemia of malignancy.
The proton resonances of the biologically active peptide parathyroid-hormone-related protein (residues 1-34) were assigned using one-dimensional spin-decoupling techniques, two-dimensional correlated spectroscopy and by comparing the spectra of the peptides 1-20, 1-25, 1-29, 7-34 and 15-34. The conformation of 1-34 was determined using one- and two-dimensional nuclear Overhauser enhancement spectroscopy in the rotating frame. Amide proton temperature coefficients, vicinal coupling constants and circular dichroic spectra helped reveal a surprisingly compact structure with residues 3-9 forming alpha-helix, type-I beta-turns between residues 10-13 and 16-19 and several interactions between the N-terminal residues and the C-terminal residues. Of these latter, the strongest appeared to be between Asp-10 and Phe-22. One peptide surface in the deduced model presents multiple positive charges, while the opposite surface has a hydrophobic character possibly functioning to exclude water from the binding interface and enhancing the binding constant.
Amino Acid Sequence↗