WO1999057305A1 - Method for screening for substances which are activators or inhibitors of protein kinase b - Google Patents
Method for screening for substances which are activators or inhibitors of protein kinase b Download PDFInfo
- Publication number
- WO1999057305A1 WO1999057305A1 PCT/SE1999/000609 SE9900609W WO9957305A1 WO 1999057305 A1 WO1999057305 A1 WO 1999057305A1 SE 9900609 W SE9900609 W SE 9900609W WO 9957305 A1 WO9957305 A1 WO 9957305A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- xaa
- amino acid
- preferably chosen
- sequence
- pkb
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/91—Transferases (2.)
- G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- G01N2333/91205—Phosphotransferases in general
- G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems
Definitions
- the present invention relates to method for screening for substances which are activators or inhibitors of Protein kinase B (PKB) and can be used as a kinase substrate for PKB by the use of peptides comprising a specific sequence which do not include a large hydrophobic residue at the C-terminal end.
- PKB Protein kinase B
- peptides comprising a specific sequence which do not include a large hydrophobic residue at the C-terminal end.
- These peptides can be used in assays measuring the activity of PKB, in screening for substances which are activators or inhibitors of gene transcriptional regulation of forkhead proteins through the catalytic activities of PKB and for discrimination between the effects of compounds which mediate insulin action through transcription from those which modulate activity of enzymes involved in metabolism by phosphorylation.
- the substrate peptide sequence comprises the sequence 1 ArgXaaArgXaaXaaSerXaa or sequence 2, ArgXaaArgXaaXaaThrXaa, in which Xaa in position 2 is any amino acid, preferably chosen from Pro and Gly, Xaa in positions 4 and 5 are any amino acid, preferably chosen from Thr and Ser and Xaa in position 7 is any amino acid, preferably chosen from Asn, Gin, Thr, Ser and with the proviso that the sequence does not include a large hydrophobic residue directly C-terminal to the phophorylation site.
- insulin receptor substrate A key downstream protein in insulin signalling is phosphoinositide 3-kinase (PI3K) which catalyses the production of the second messenger phosphatidylinositol 3,4,5-trisphosphate.
- PKB appears to be a key intermediary in the regulation of glucose utilisation and control of protein synthesis by insulin (Cross et al. 1995); (Cohen et al. 1997); (Peak et al. 1998); (Gingras et al. 1998)).
- GSK3 glycogen synthase kinase 3
- PKB has been shown to phosphorylate and activate phosphofructo kinase-2 (Deprezet al.
- a third likely substrate for PKB is the type 3B cyclic AMP phosphodiesterase (Wijkander et al. 1998), which in insulin-responsive tissues is activated by phosphorylation, leading to the inactivation of adrenergic-stimulated processes.
- Figure la Cos-7 transfected with HA-PKB. Phosphorylation of Crosstide (black bar) compared with a peptide according to the invention (grey bar)
- Figure lb Inactive rPKB ⁇ activated by incubation with IGF-1 stimulated muscle cell lysate. Phosphorylation of Crosstide (black bar) compared with a peptide according to the invention (grey bar)
- PKB Protein kinase B
- the peptides can be used for discrimination between the effects of compounds which mediate insulin action through transcriptional regulation from those compounds which modulate activity of enzymes involved in metabolism by phosphorylation. Such a discrimination has not been possible earlier. 4
- the invention relates to the use of a peptide sequence comprising the sequences
- ArgXaaArgXaaXaaSerXaa sequence 1, or ArgXaaArgXaaXaaThrXaa, sequence 2.
- the sequence can be included as a part of any peptide or protein provided that the sequence is accessible to the targeting enzyme PKB.
- the peptide is preferably SerThrPheArgProArgThrSerSerAsnAla, sequence 14.
- amino acids Asp, Glu, Lys and Arg are charged and could possibly have an influence on phosphorylation.
- large or strongly hydrophobic residue is for example meant Phe, Leu, He, Trp and
- Gene transcriptional regulation involves activation or repression of the enzymes and other components involved in metabolism, for example the repression of PEPCK
- FKHRL1, AFX and AF6q21 (Accession number AF032885, AF032886, X939996 and
- peptides as defined in the claims can be used in the search for new substrates for PKB as templates for sequence searches and/or for primers in techniques of molecular biology such as PCR.
- Example 1 The isoform of PKB was immunoprecipitated from lysates of Cos-7 cells (approx. lmg of protein) transfected with bovine PKB ⁇ which contained an N-terminal haemagglutinin (HA) tag. After washing, phosphorylation of the peptides RPRTSSF (black bar in Figure la) and STFRPRTSSNA (grey bar in Figure la) by PKB was measured in parallel assays by incubation in the presence of 33 P-labelled ATP. Comparison of results from the two peptides showed that within a 30 minute assay, equal amounts of peptide were converted to the phosphorylated form at a rate of 0.5pmol per minute under the assay conditions employed. 5
- the example suggest that the peptide of the invention is useful for determining PKB activity of other isoforms than the ⁇ -form.
- peptide substrates for use in PKB phosphorylation assays were refined by investigating the ability of recombinant activated PKB to phosphorylate various different peptides ( Figures 2a and b).
- peptides (lOO ⁇ M) were incubated with 0.2 ⁇ g activated recombinant PKB (purchased from UBI) in the presence of 1 O ⁇ M 33 P-ATP and the extent of phosphorylation was compared with that of the peptide sequence RPRTSSF, previously used as a substrate for PKB (Alessi et al, 1996).
- Figure 2a shows that addition of up to three neutral or small hydrophobic amino acids to either end of the sequence PKB is thought to phosphorylate had no marked effect on the extent of phosphorylation of each respective peptide.
- peptides KKRNRTLTK (a peptide often used to measure the activity of a kinase related to PKB, p70 S6 kinase) and APRPRVETSQ (derived from pyruvate dehydrogenase kinase 1) were phosphorylated to less than one quarter the extent of GRPRTSSF by PKB.
- APRPRVETSQ derived from pyruvate dehydrogenase kinase 1
- the compounds found from the claimed screening are anticipated also to be used against long term complications resulting from insulin resistance, such as vascular dysfunction, loss of neuronal cells and ⁇ -cells in pancreas. These compounds are not possible to find by using the modulators as described in WO 97/22360. 7
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Enzymes And Modification Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU42982/99A AU754508B2 (en) | 1998-04-30 | 1999-04-16 | Method for screening for substances which are activators or inhibitors of Protein kinase B |
JP2000547256A JP2002514389A (en) | 1998-04-30 | 1999-04-16 | Screening method for substances that act as activators or inhibitors of protein kinase B |
EP99948555A EP1090140A1 (en) | 1998-04-30 | 1999-04-16 | Method for screening for substances which are activators or inhibitors of protein kinase b |
CA002327540A CA2327540A1 (en) | 1998-04-30 | 1999-04-16 | Method for screening for substances which are activators or inhibitors of protein kinase b |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
SE9801530-8 | 1998-04-30 | ||
SE9801530A SE9801530D0 (en) | 1998-04-30 | 1998-04-30 | Method of screening |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1999057305A1 true WO1999057305A1 (en) | 1999-11-11 |
Family
ID=20411158
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/SE1999/000609 WO1999057305A1 (en) | 1998-04-30 | 1999-04-16 | Method for screening for substances which are activators or inhibitors of protein kinase b |
Country Status (6)
Country | Link |
---|---|
EP (1) | EP1090140A1 (en) |
JP (1) | JP2002514389A (en) |
AU (1) | AU754508B2 (en) |
CA (1) | CA2327540A1 (en) |
SE (1) | SE9801530D0 (en) |
WO (1) | WO1999057305A1 (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006023879A1 (en) * | 2004-08-20 | 2006-03-02 | Board Of Regents, The University Of Texas System | Screening of agents for activity against ischemic myocardial insults |
US7491702B2 (en) | 2001-04-18 | 2009-02-17 | The Open University | Polypeptides related to amyloid precursor protein, pharmaceutical compositions thereof, and methods of treatment using the same |
US7622446B2 (en) | 2001-04-18 | 2009-11-24 | The Open University | Polypeptides, derivatives and uses thereof |
US8158586B2 (en) | 2005-04-11 | 2012-04-17 | Pharmagap Inc. | Inhibitors of protein kinases and uses thereof |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1995018823A2 (en) * | 1994-01-07 | 1995-07-13 | Beth Israel Hospital | Substrate specificity of protein kinases |
WO1997018303A1 (en) * | 1995-11-16 | 1997-05-22 | Novartis Ag | Rac-proteine kinase as therapeutic agent or in diagnostics |
WO1997022360A2 (en) * | 1995-12-20 | 1997-06-26 | Medical Research Council | Control of protein synthesis, and screening method for agents |
-
1998
- 1998-04-30 SE SE9801530A patent/SE9801530D0/en unknown
-
1999
- 1999-04-16 AU AU42982/99A patent/AU754508B2/en not_active Ceased
- 1999-04-16 WO PCT/SE1999/000609 patent/WO1999057305A1/en not_active Application Discontinuation
- 1999-04-16 CA CA002327540A patent/CA2327540A1/en not_active Abandoned
- 1999-04-16 JP JP2000547256A patent/JP2002514389A/en not_active Withdrawn
- 1999-04-16 EP EP99948555A patent/EP1090140A1/en not_active Withdrawn
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1995018823A2 (en) * | 1994-01-07 | 1995-07-13 | Beth Israel Hospital | Substrate specificity of protein kinases |
WO1997018303A1 (en) * | 1995-11-16 | 1997-05-22 | Novartis Ag | Rac-proteine kinase as therapeutic agent or in diagnostics |
WO1997022360A2 (en) * | 1995-12-20 | 1997-06-26 | Medical Research Council | Control of protein synthesis, and screening method for agents |
Non-Patent Citations (1)
Title |
---|
DARIO R. ALESSI ET AL.: "Molecular basis for the substrate specificity of protein kinase B; comparison with MAPKAP kinase-1 and p70 S6 kinase", FEBS LETTERS, vol. 399, 1996, pages 333 - 338 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7491702B2 (en) | 2001-04-18 | 2009-02-17 | The Open University | Polypeptides related to amyloid precursor protein, pharmaceutical compositions thereof, and methods of treatment using the same |
US7622446B2 (en) | 2001-04-18 | 2009-11-24 | The Open University | Polypeptides, derivatives and uses thereof |
WO2006023879A1 (en) * | 2004-08-20 | 2006-03-02 | Board Of Regents, The University Of Texas System | Screening of agents for activity against ischemic myocardial insults |
US7531318B2 (en) | 2004-08-20 | 2009-05-12 | Board Of Regents, The University Of Texas System | Screening of agents for activity against ischemic myocardial insults |
US8158586B2 (en) | 2005-04-11 | 2012-04-17 | Pharmagap Inc. | Inhibitors of protein kinases and uses thereof |
Also Published As
Publication number | Publication date |
---|---|
AU754508B2 (en) | 2002-11-21 |
JP2002514389A (en) | 2002-05-21 |
CA2327540A1 (en) | 1999-11-11 |
EP1090140A1 (en) | 2001-04-11 |
SE9801530D0 (en) | 1998-04-30 |
AU4298299A (en) | 1999-11-23 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Büther et al. | KIBRA is a novel substrate for protein kinase Cζ | |
Laine et al. | The protooncogene TCL1 is an Akt kinase coactivator | |
Zheng et al. | Activation of phosphoinositide 3-kinase activity by Cdc42Hs binding to p85. | |
Chen et al. | Association of focal adhesion kinase with its potential substrate phosphatidylinositol 3-kinase. | |
Aitken et al. | Specificity of 14-3-3 isoform dimer interactions and phosphorylation | |
CHEUNG et al. | Characterization of AMP-activated protein kinase γ-subunit isoforms and their role in AMP binding | |
FURLANETTO et al. | 14-3-3 Proteins interact with the insulin-like growth factor receptor but not the insulin receptor1 | |
Ching et al. | Cloning of three novel neuronal Cdk5 activator binding proteins | |
Asselin et al. | X-linked inhibitor of apoptosis protein activates the phosphatidylinositol 3-kinase/Akt pathway in rat granulosa cells during follicular development | |
Zhang et al. | The insulin receptor-related receptor. Tissue expression, ligand binding specificity, and signaling capabilities. | |
US6004757A (en) | Substrate specificity of a protein kinases | |
Ando et al. | Enhanced insulin-induced mitogenesis and mitogen-activated protein kinase activities in mutant insulin receptors with substitution of two COOH-terminal tyrosine autophosphorylation sites by phenylalanine. | |
US20070196883A1 (en) | Enzyme | |
AU726294B2 (en) | Ikk-alpha proteins, nucleic acids and methods | |
Polek et al. | The TNF receptor, RELT, binds SPAK and uses it to mediate p38 and JNK activation | |
Heierhorst et al. | Phosphorylation of myosin regulatory light chains by the molluscan twitchin kinase | |
US6207393B1 (en) | Inhibition of intracellular signal transduction by 14-3-3-binding peptides | |
US8604163B2 (en) | EEF2K assays for identifying compounds that inhibit EEF2K activity | |
WHEELER-JONES et al. | Identification of 14-3-3 proteins in human platelets: effects of synthetic peptides on protein kinase C activation | |
Grange-Midroit et al. | G protein-coupled receptor kinases, β-arrestin-2 and associated regulatory proteins in the human brain: postmortem changes, effect of age and subcellular distribution | |
Kojima et al. | Role of phosphatidylinositol-3 kinase and its association with Gab1 in thrombopoietin-mediated up-regulation of platelet function | |
AU754508B2 (en) | Method for screening for substances which are activators or inhibitors of Protein kinase B | |
Csehi et al. | Tumor necrosis factor (TNF) interferes with insulin signaling through the p55 TNF receptor death domain | |
Heierhorst et al. | Substrate specificity and inhibitor sensitivity of Ca2+/S100‐dependent twitchin kinases | |
Stephens et al. | A heterotrimeric GTPase-regulated isoform of PI3K and the regulation of its potential effectors |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AU CA JP NZ US |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
ENP | Entry into the national phase |
Ref document number: 2327540 Country of ref document: CA Ref country code: CA Ref document number: 2327540 Kind code of ref document: A Format of ref document f/p: F |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1999948555 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 42982/99 Country of ref document: AU |
|
WWP | Wipo information: published in national office |
Ref document number: 1999948555 Country of ref document: EP |
|
WWG | Wipo information: grant in national office |
Ref document number: 42982/99 Country of ref document: AU |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1999948555 Country of ref document: EP |