EP1218404A1 - Motif de liaison d'un recepteur - Google Patents

Motif de liaison d'un recepteur

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Publication number
EP1218404A1
EP1218404A1 EP00963808A EP00963808A EP1218404A1 EP 1218404 A1 EP1218404 A1 EP 1218404A1 EP 00963808 A EP00963808 A EP 00963808A EP 00963808 A EP00963808 A EP 00963808A EP 1218404 A1 EP1218404 A1 EP 1218404A1
Authority
EP
European Patent Office
Prior art keywords
receptor
protein
precursor
binding
binding motif
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00963808A
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German (de)
English (en)
Other versions
EP1218404A4 (fr
Inventor
Mark Andrew Guthridge
Frank Charles Stomski
Angel Francisco Lopez
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Berndt Michael C
Medvet Science Pty Ltd
Original Assignee
Berndt Michael C
Medvet Science Pty Ltd
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Publication date
Priority claimed from AUPQ2875A external-priority patent/AUPQ287599A0/en
Priority claimed from AUPQ8733A external-priority patent/AUPQ873300A0/en
Application filed by Berndt Michael C, Medvet Science Pty Ltd filed Critical Berndt Michael C
Publication of EP1218404A1 publication Critical patent/EP1218404A1/fr
Publication of EP1218404A4 publication Critical patent/EP1218404A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a binding motif of a receptor.
  • the binding motif is a cytoplasmic domain associated with stimulation of receptor mediated activities.
  • the present invention further contemplates methods of using the motif in particular for mediating activities of receptors.
  • Proteins including human granulocyte-macrophage colony-stimulating factor (GM- CSF), interleukin-3 (IL-3), and IL-5 are capable of stimulating normal and transformed hematopoietic cells.
  • the initiating event for signal transduction is the binding of the protein to their surface receptors.
  • These receptors may be composed of subunits such as the ⁇ chain and a common ⁇ chain ( ⁇ c ). Engagement of ⁇ c by the binding of the cytoplasmic protein to surface receptors results in the stimulation of cell survival, proliferation, and differentiation and mature cell effector function in the appropriate lineage, a fact that emphasises the major signaling role played by ⁇ c in mediating receptor induced biological activities.
  • tyrosine phosphorylation of ⁇ c One of the first events in activation of receptors and in the initiation of the signaling cascade is tyrosine phosphorylation of ⁇ c .
  • This is a common theme among receptor signaling subunits and can be seen in homodimeric receptors such as the erythropoietin (EPO) receptor, thrombopoietin (TPO) receptor, and granulocyte colony-stimulating factor (G-CSF) receptor as well as in heterodimeric receptors such as in the IL-6 and IL-2 receptors, and in the GM-CSF, IL-3, and IL- 5 receptor systems.
  • EPO erythropoietin
  • TPO thrombopoietin
  • G-CSF granulocyte colony-stimulating factor
  • Tyrosine phosphorylation of receptor signaling subunits appeared to be a critical step in the creation of docking sites for the association of signaling molecules. Despite the perceived importance of tyrosine phosphorylation of receptors, it is becoming apparent that signaling can proceed in its absence. This is demonstrated in the EPO and TPO receptors, in which the substitution of all tyrosines failed to abolish their activities.
  • the 14-3-3 family of proteins is one such protein, which consists of 7 different isoforms and is expressed ubiquitously from yeast to humans.
  • the ability of 14-3- 3 to bind to a number of motifs in a wide range of signaling molecules suggests that 14-3-3 proteins may participate in a number of cell signaling pathways that may include mitogenesis, transformation and survival.
  • 14-3-3 has been shown to bind a number of signaling molecules, it has been more difficult to determine how or where 14-3-3 can regulate signaling events directly or indirectly, or whether 14-3-3 is implicated at all.
  • an object of the present invention is to overcome some of the problems of the prior art and to understand how proteins can express their biological activities and to use this information to manipulate cellular functions.
  • a binding motif of a receptor capable of binding a cytoplasmic protein, said binding motif comprising an amino acid sequence wherein at least one amino acid is serine/threonine.
  • the serine/threonine residue corresponds to a serine residue at position 585 of the common ⁇ c according to Figure 1.
  • binding motif comprises an amino acid sequence including the sequence
  • the second serine/threonine of the motif is an indicator of the cytoplasmic protein binding motif.
  • the motif as a whole is involved in the cytoplasmic binding and requires the serine/threonine residue along with flanking amino acids.
  • the motif includes flanking amino acid sequences which may improve the binding of a cytoplasmic protein to the binding motif. More preferably the flanking amino acids are selected from R and X-P (wherein X is any amino acid such that the flanking amino acids individually or co-operatively contribute to the binding motif for binding to a cytoplasmic protein.
  • amino acid sequence of the binding motif includes the sequence:
  • a binding motif of a receptor capable of binding a cytoplasmic protein, said binding motif comprising an amino acid sequence, a functional equivalent or analogue thereof and wherein at least two (2) of the amino acids are serine.
  • the receptor may be any receptor that is capable of binding to an extracellular molecule or protein and which mediates its function through the binding of a cytoplasmic molecule or protein such as 14-3-3 protein, or any cytoplasmic molecule or protein capable of binding a further signaling molecule which activates a cascade of events leading to cell signaling pathways and biological functions such as mitogenesis, proliferation, transformation, differentiation and cell survival, or any other cytoplasmic molecule or protein which does not signal.
  • This molecule may act as an antagonist. More preferably, the cytoplasmic protein is 14-3-3.
  • the receptor is selected from the group including:
  • IL-6R-beta IL6 human interleukin-6 receptor beta chain precursor
  • LEPR human leptin receptor precursor LEP-R
  • OB RECEPTOR OB-R
  • TNR2 human tumor necrosis factor receptor 2 precursor tumor necrosis factor
  • TRK3 human receptor protein-tyrosine kinase TKT precursor (EC 2.7.1.112)
  • FGR1 human basic fibroblast growth factor receptor 1 precursor (BFGF-R)
  • PTPM human protein-tyrosine phosphatase mu precursor (EC 3.1.3.48) (R-PTP-MU).
  • R-PTP-MU PTPM human protein-tyrosine phosphatase mu precursor
  • PGDS human alpha platelet-derived growth factor receptor precursor (EC 2.7.1.112)
  • FGR4 human fibroblast growth factor receptor 4 precursor (EC 2.7.1.112)
  • FGR2 human fibroblast growth factor receptor 2 precursor FGFR-2)
  • MANR human macrophage mannose receptor precursor.
  • LRP2 human low-density lipoprotein receptor-related protein 2 precursor (megalin)
  • IDD human integral membrane protein dgcr2/idd precursor (KIAA0163)
  • AMFR human autocrine motility factor receptor precursor (AMF receptor) (9P78) (19) ACH5 human neuronal acetylcholine receptor protein, alpha-5 chain precursor.
  • KKIT human stem cell growth factor receptor (proto-oncogene tyrosine- protein kinase kit) (C-KIT) (CD117 antigen)
  • TPOR human thrombopoietin receptor precusor (TPO-R) (myeloproliferative leukemia protein (C-MPL). (TPOR or MPL).
  • TPO-R thrombopoietin receptor precusor
  • C-MPL myeloproliferative leukemia protein
  • TPOR mouse thrombopoietin receptor precursor (TPO-R) (myeloproliferative leukemia protein) (C-MPL). (TPOR or MPL).
  • TPO-R thrombopoietin receptor precursor
  • C-MPL myeloproliferative leukemia protein
  • the binding motif of the receptor is capable of interacting with a cytoplasmic molecule or protein such as 14-3-3 protein, or any cytoplasmic molecule or protein capable of binding a further signaling molecule which activates a cascade of events leading to cell signaling pathways and biological functions such as mitogenesis, proliferation, transformation, differentiation and cell survival, or any other cytoplasmic molecule or protein which does not signal.
  • This molecule may act as an antagonist.
  • the cytoplasmic protein is 14-3-3.
  • the binding motif of a receptor has an amino acid sequence selected from the group including: (i) HSRSLP
  • a phosphorylated binding motif of a receptor capable of binding a cytoplasmic protein comprising an amino acid sequence wherein at least one amino acid is serine.
  • the serine residue corresponds to a serine residue at position 585 of the common ⁇ c according to Figure 1.
  • a phosphorylated binding motif of a receptor capable of binding a cytoplasmic protein, said binding motif comprising an amino acid sequence, a functional equivalent or analogue thereof and wherein at least two (2) of the amino acids are serine and wherein at least one serine residue of the motif is phosphorylated.
  • a phophorylated binding motif of a GM-CSF/IL-3/IL-5 receptor capable of binding a cytoplasmic protein, said binding motif comprising an amino acid sequence of at least 582 HSRSLP 587 of the GM- CSF/IL-3/IL-5 receptor or a functional equivalent or analogue thereof wherein at least Ser 585 is phosphorylated.
  • a binding motif of a GM-CSF/IL- 3/IL-5 receptor capable of binding a cytoplasmic protein, said binding motif comprising an amino acid sequence of at least 582 HSRSLP 587 of the GM-CSF/IL- 3/IL-5 receptor or a functional equivalent or analogue thereof wherein at least Ser 585 is phosphorylated.
  • a method of binding a cytoplasmic protein to a receptor comprising:
  • the serine residue corresponds to a serine residue at position 585 of the common ⁇ c according to Figure 1.
  • a method of activating cellular activities said method including:
  • cytoplasmic protein is associated with activation of cellular activities.
  • a functional equivalent or analogue thereof regulating the activation of phosphoryaltion of a binding motif of a receptor as described above, a functional equivalent or analogue thereof; and subjecting the binding motif to a cytoplasmic protein to bind the cytoplasmic protein to the binding motif; and activating a cell signaling pathway by interacting the bound cytoplasmic protein with a signaling molecule involved in the pathway.
  • Figure 1 shows the amino acid sequence of the common ⁇ chain ( ⁇ c ).
  • Figure 2 shows the amino acid sequence of the Stem Cell Growth Factor Receptor (Proto-Oncogene Tyrosine- Protein Kinase Kit) (C-KIT) (CD 117 Antigen)
  • FIG 3 shows the amino acid sequence of the Human Thrombopoietin Receptor Precursor (TPO-R) (Myeloproliferative Leukaemia Protein) (C-MPL).
  • TPO-R Human Thrombopoietin Receptor Precursor
  • C-MPL Myeloproliferative Leukaemia Protein
  • FIG 4 shows the amino acid sequence of the Mouse Thrombopoietin Receptor Precursor (TPO-R). (Myeloproliferative Leukaemia Protein) (C-MPL).
  • TPO-R Mouse Thrombopoietin Receptor Precursor
  • C-MPL Myeloproliferative Leukaemia Protein
  • FIG. 5 shows that Human ⁇ c associates with 14-3-3 ⁇ and this association is mediated by the 544-626 region of ⁇ c .
  • HEK 293T cells were either mock transfected (mock), transfected with wild type ⁇ c (wt) or ⁇ c containing C terminal deletions. Lysates were prepared from transfected cells and either immunoprecipitated with anti-14-3-3 ⁇ antibody (A) or precipitated with either 14-3- 3-GST sepharose (B) or GST-sepharose (C). All proteins were separated on 7.5% SDS-PAGE under reducing conditions before Western blotting with anti- ⁇ c antibody (MAb 1C1).
  • Figure 6 shows that 14-3-3 ⁇ specifically binds the HSRSLP motif of the ⁇ c cytoplasmic domain.
  • HEK 293 cells were used either untransfected (UT), or transfected with wild type ⁇ c (wt), or with ⁇ c containing the sequence 581 PHSRSLP 587 mutated to 581 GEFAAAA 587 or with ⁇ c containing the sequence 820 RSKPSSP 826 mutated to 820 EFAAAAA 826 .
  • Lysates were made and immunoprecipitations were performed using GST-14-3-3-sepharose. The presence of ⁇ c was determined by Western blotting with an anti- ⁇ c antibody (MAb 1C1).
  • MAb 1C1 anti- ⁇ c antibody
  • Figure 7 shows that Inhibition of ⁇ c association with 14-3-3 ⁇ by phosphorylated but not by unphosphorylated ⁇ c and raf-1 peptides.
  • Lysates of HEK293T cells transfected with ⁇ c were immunoprecipitated with GST-14-3-3-sepharose in the absence or in the presence of chemically-synthesised peptides (100uM) containing a ⁇ c sequence (CLGPPHSRSLPDILG) or a Raf-1 sequence (CLSQRQRSTSTPNVHM).
  • CGSQRQRSTSTPNVHM chemically-synthesised peptides
  • the relevant phosphorylated serine is underlined.
  • the experiment was performed on 7.5% SDS-PAGE under reducing conditions.
  • the presence of ⁇ c in the precipitation experiment was determined by Western blotting with anti- ⁇ c antibody (MAb 1C1).
  • Figure 8 shows that Specific inhibition of ⁇ c association with 14-3-3 ⁇ by a phosphorylated peptide encompassing the 579-592 region of ⁇ c .
  • Lysates of HEK293T cells transfected with ⁇ c were precipitated with GST-14-3-3-sepharose in the absence or in the presence of various concentrations of chemically- synthesised peptides.
  • Two ⁇ c peptide sequences were used, CLGPPHSRSLPDILG either non phosphorylated (A) or serine 585 phosphorylated (B), and CPLSLRSKPSPGPGP either non phosphorylated (C) or serine phosphorylated (D).
  • the appropriate phosphorylated serine in each peptide is underlined.
  • the experiment was performed on 7.5% SDS-PAGE under reducing conditions.
  • the presence of ⁇ c in the immunoprecipitates was determined by Western blotting with anti- ⁇ c antibody (MAb 1C1).
  • Figure 9 shows that Binding of 125 l-labeled 14-3-3z to synthetic peptides corresponding to the 14-3-3 binding region of ⁇ c .
  • Microtiter wells were coated with synthetic peptides CLGPPHSRSLPDILG either non phosphorylated or phosphorylated on the second serine (underlined).
  • Various concentrations of 5 l- labeled recombinant 14-3-3z protein were added to microtiter wells and incubated at 22°C for 2 h. [Insert: Scatchard analyses of 14-3-3 interaction with the serine- phosphorylated peptide].
  • Figure 10 shows that 585 Ser in ⁇ c is phosphorylated in vivo by GM-CSF.
  • the anti-phospho- 585 Ser ⁇ c antibody specifically recognises the phosphorylated CLGPPHSRSLDILG peptide. Dot blots were prepared on nitrocellulose filters of either the non phosphorylated or the serine phosphorylated CLGPPHSRSLPDILG peptide and the scrambled peptide CLPLSGPDSHIRGPL before probing with anti-phospho- 585 Ser ⁇ c antibody.
  • B The serine phosphorylated
  • CLGPPHSRSLPDILG peptide specifically inhibits the binding of anti-phospho- 585 Ser ⁇ c antibody to ⁇ c .
  • Lysates of HEK293T cells transfected with wild type ⁇ c were immunoprecipitated with anti- ⁇ c antibody (MAb 8E4). Immunoprecipitates were run on 7.5% SDS-PAGE under reducing conditions.
  • Anti-phospho- 585 Ser ⁇ c antibody was then pre-incubated with either medium (none), 100 fold molar excess of the serine phosphorylated (1) or non phosphorylated (2) CLGPPHSRSLPDILG peptide, or the scrambled peptide CLPLSGPDSHIRGPL (3).
  • FIG 11 shows Phosphorylation of Ser585 of ⁇ c in response to IL-3 and the recruitment of 14-3-3.
  • CTL-EN cells expressing IL-3Ra and either wt ⁇ c or ⁇ c HSRSLP- EFAAAA were washed, starved for 4 hours in DMEM containing 0.1% FCS and then stimulated with 50ng/ml IL-3 for the indicated times. Cells were then lysed and the ⁇ c immunoprecipitated (7x10 7 cells/IP) with the 1C1 anti- ⁇ c specific mAb.
  • Immunoprecipitates were divided and 80% or 20% were separately run on SDS-PAGE and transferred to nitrocellulose filters, (a) 80% of the immunoprecipitates were probed with the anti-phospho-Ser585 ⁇ c antibody, the filters stripped, and reprobed with the 1C1 anti- ⁇ c mAb. 20% or the immunoprecipitates were probed with the 4G10 anti-phosphotyrosine antibody, (b) Regulation of 14-3-3 association with ⁇ c following IL-3 stimulation. CTL-EN cells were starved and stimulated as above, and ⁇ c was immunoprecipitated with the 1C1 mAb coupled to Sepharose beads.
  • Immunoprecipitates were boiled in a non-reducing sample buffer and subjected to SDS-PAGE on a non-reducing gel. Immunoblot analysis was performed with anti-14-3-3 antibodies and the filter stripped and re-probed with the 1C1 antibody. These results were typical of 3 experiments.
  • Figure 12 shows PKA phosphorylates Ser585 of ⁇ c .
  • Figure 13 shows 14-3-3 binding to ⁇ c in response to IL-3 stimulation is required for the recruitment and activation of PI 3-kinase.
  • CTL-EN cells expressing IL-3Ra and either wt ⁇ c or ⁇ c HSRSLP ⁇ EFAAAA were starved for 12 hours in DMEM containing 0.5% FCS and stimulated with 50ng/ml IL-3 for the indicated times. Cells were then lysed and phospho-tyrosine containing proteins were immunoprecipitated (2x10 7 cells/IP) using the 4G10 antibody.
  • Immunoprecipitates were washed in 1x PI 3-kinase buffer (20mM HEPES, pH 7.5, 5mM MgCI 2 , 1mM EGTA) and PI 3-kinase activity was analysed in a lipid kinase assay using phosphatidyl inositol and g 32 PATP as substrates as described in the "Materials and Methods". Autoradiogram of TLC plate is shown with 32 P-labelled phosphatidyl inositols (PIP) and the origin indicated. These results were typical of 3 experiments, (b) CTL-EN cells were also examined for the association of ⁇ c with p85.
  • CTL-EN cells expressing either wt ⁇ c or ⁇ c HSRSLP ⁇ EFAAAA were starved for 12 hours in DMEM containing 0.5% FCS and then stimulated with 50ng/ml IL-3 for the indicated times.
  • Cells were then lysed and the ⁇ c immunoprecipitated (2x10 7 cells/IP) with the 1C1 anti- ⁇ c specific mAb.
  • Immunoprecipitates were then subjected to SDS-PAGE, transferred to nitrocellulose and immunoblotted with anti-p85 (1 mg/ml) followed by detection with HRP-conjugated anti-rabbit IgG antibody and ECL.
  • Figure 14 shows 14-3-3 binding to ⁇ c is required for Akt activation but not STAT5, ERK or JNK activation
  • CTL-EN cells expressing IL-3Ra and either wt ⁇ c or ⁇ c HSRSLP ⁇ EFAAAA were starved for 12 hours in DMEM containing 0.5% FCS and then stimulated with 50ng/ml IL-3 for the indicated times.
  • Cells were lysed, whole cell lysates were subjected to SDS-PAGE and transferred to a nitrocellulose filter.
  • the immunoprecipitates were then washed in 1x JNK buffer (10mM MgCI 2 , 10mM Tris-HCI ph 7.4) and the JNK activity was then analysed in an in vitro kinase assay using g 32 PATP and 1mg GST-jun1-79.
  • Kinase assays were subjected to SDS-PAGE and transferred to a nitrocellulose filter. An autoradiogram of the filters is shown.
  • Figure 15 shows 14-3-3 binding to ⁇ c in response to IL-3 regulates cellular viability
  • CTL-EN cells expressing IL-3Ra and either wt ⁇ c or ⁇ c HSRSLP ⁇ EFAAAA were washed and then plated in duplicate at 2.5x10 5 cells/ml in DMEM containing 0.1% FCS and either 10ng/ml IL-3, 10 ng/ml IL-2 or 10ng/ml IL-3 and 5 mM LY294002. Viable cells were counted using the trypan blue exclusion method after 4 days. The results shown are typical of 3 experiments.
  • Figure 16 shows 14-3-3 binding to ⁇ c is not required for cell cycle progression
  • a) CTL-EN cells expressing IL-3Ra and either wt ⁇ c or ⁇ .HSRSLP ⁇ EFAAAA were washed and then plated at 2.5x10 5 cells/ml in DMEM containing 0.1% FCS and starved for 24 hours. Cells were then stimulated for a further 24 hours with 50 ng/ml IL-3 in the same medium after which the cells were harvested by centrifugation, fixed in ethanol and stained with propidium iodide. Cell cycle distribution was then analysed by flow cytometry.
  • CTL-EN cells were washed, starved and stimulated with IL-3 for 2 hours as described above.
  • Total RNA was extracted and subjected to Northern blot analysis. Filters were probed sequentially with 32 P-labelled cDNAs for c-myc and 18SrRNA. Signals were detected by autoradiography.
  • Figure 17 shows 14-3-3 binding to ⁇ c in response to IL-3 stimulation promotes cellular survival by suppressing apoptosis.
  • CTL-EN cells expressing IL-3Ra and either wt ⁇ c or ⁇ c HSRSLP ⁇ EFAAAA were washed and then plated at 5x10 5 cells/ml in DMEM containing 0.1% FCS and either 10ng/ml IL-3 or 10 ng/ml IL-2 for 18 hours. Cells were then harvested by centrifugation and stained with annexin V according to the manufacturers instructions. Annexin V positive cells were then analysed by flow cytometry.
  • Figure 18 shows a proposed model for the regulation of survival by IL-3.
  • Binding of IL-3 to the IL-3 receptor composed of an ⁇ -chain ( ⁇ ) and a ⁇ -Chain ( ⁇ c ), results in receptor oligomerization (only one ⁇ -chain and one ⁇ -chain are shown for simplicity), increased tyrosine phosphorylation and also increased Ser585 phosphorylation (S).
  • Ser585 phosphorylation of ⁇ c allows recruitment of 14-3-3, which in tun recruits PI 3-kinase (PI 3-K) either directly, or indirectly through an additional dapto molecule.
  • the recruitment of PI 3-kinase and the production of phosphatidyl inositols (PIP) couples the activated receptor to down-stream signalling molecules such as Akt and promotes cellular survival.
  • Figure 19 shows 585 Ser in ⁇ c of acute myeloid leukaemia (AML) is constitutively phosphorylated.
  • CTL-EN cells expressing IL-3R ⁇ and ⁇ c were either untreated or stimulated with IL-3 (2ng/ml) for 1 or 5 minutes.
  • AML cells expressing GM- CSFR ⁇ and ⁇ c were either untreated or stimulated with GM-CSF (2ng/ml) for 5 minutes.
  • Lysates of either CTL-EN cells or AML cells were immunoprecipitated with anti- ⁇ c antibody (MAb 8E4) and the immunoprecipitates run on 10% SDS- PAGE under reducing conditions.
  • MAb 8E4 anti- ⁇ c antibody
  • Figure 21 shows PKA phosphorylates Ser585 of ⁇ c .
  • A The ability of either PKA or CKII to phosphorylate purified His ⁇ c 445-881 was examined in an in vitro kinase assay using ⁇ 32 P-ATP.
  • B The ability of PKA and CKII to phosphorylate Ser585 of His ⁇ c 445-881 was examined by immunoblot analysis using the anti-phospho- Ser585 ⁇ c antibody.
  • C Determination of the K m and the V max for PKA phosphorylation of the ⁇ c peptide.
  • D Pharmacologic regulation of PKA activity and its effect on ⁇ c Ser585 phosphorylation.
  • Figure 22 shows 14-3-3 binding to ⁇ c in response to IL-3 stimulation is required for the recruitment and activation of PI 3-kinase.
  • A Analysis of PI 3-kinase activity.
  • B CTL-EN cells were also examined for the association of ⁇ c with p85.
  • C The association of recombinant His ⁇ c 445-881 with p85 and 14-3-3.
  • Figure 23 shows 14-3-3 binding to ⁇ c is required for Akt activation but not STAT5, ERK, JNK or JAK2 activation.
  • A Phosphorylation of Akt, STAT5 and ERK2.
  • B Activation of JNK activity.
  • C Tyrosine phosphorylation of JAK2.
  • Figure 24 shows 14-3-3 binding to ⁇ c in response to IL-3 stimulation promotes cellular survival by suppressing apoptosis.
  • A Cellular viability. Shown is the viability of CTL-EN cells expressing wt ⁇ c in the presence of IL-2 (•) or IL-3 ( ⁇ ) and the viability of CTL-EN cells expressing ⁇ c HSRSLP ⁇ EFAAAA in the presence of IL-2 (T) or IL-3 (A).
  • Figure 27 shows Ser585 phosphorylation and PI 3-K signalling is constitutive in primary human AML cells.
  • a binding motif of a receptor capable of binding a cytoplasmic protein, said binding motif comprising an amino acid sequence wherein at least one amino acid is serine/threonine.
  • the serine/threonine residue corresponds to a serine residue at position 585 of the common ⁇ c according to Figure 1.
  • a binding motif of a receptor capable of binding a cytoplasmic protein, said binding motif comprising an amino acid sequence, a functional equivalent or analogue thereof and wherein at least two (2) of the amino acids are serine.
  • binding motif comprises an amino acid sequence including the sequence:
  • the second serine/threonine of the motif is an indicator of the cytoplasmic protein binding motif.
  • the motif as a whole is involved in the cytoplasmic binding and requires the serine/threonine residue along with flanking amino acids.
  • the motif includes flanking amino acid sequences which may improve the binding of a cytoplasmic protein to the binding motif. More preferably the flanking amino acids are selected from R and X-P (wherein X is any amino acid such that the flanking amino acids individually or co-operatively contribute to the binding motif for binding to a cytoplasmic protein.
  • amino acid sequence of the binding motif includes the sequence:
  • motif as used herein in relation to the receptor, means a distinctive portion of the receptor but is not intended to include the whole receptor.
  • the receptor may be any receptor that is capable of binding to an extracellular molecule or protein and which mediates its function through the binding of a cytoplasmic molecule or protein such as 14-3-3 protein, or any cytoplasmic molecule or protein capable of binding a further signaling molecule which activates a cascade of events leading to cell signaling pathways and biological functions such as mitogenesis, proliferation, transformation, differentiation and cell survival, or any other cytoplasmic molecule or protein which does not signal.
  • This molecule may act as an antagonist. More preferably, the cytoplasmic protein is 14-3-3.
  • Signaling molecules may be molecules involved in cellular pathways such as those pathways involved in proliferation, survival or differentiation. Examples of such pathways may include the JAK/STAT pathway, the ras/MAP kinase pathway or the PI-3-Kinase pathway. All pathways may be involved directly or indirectly with these functions.
  • the receptor is selected from the group including:
  • IL-6R-beta IL6 human interleukin-6 receptor beta chain precursor
  • LEPR human leptin receptor precursor LEP-R
  • OB RECEPTOR OB-R
  • TNR2 human tumor necrosis factor receptor 2 precursor tumor necrosis factor
  • TRK3 human receptor protein-tyrosine kinase TKT precursor (EC 2.7.1.112)
  • FGR1 human basic fibroblast growth factor receptor 1 precursor (BFGF-R)
  • PTPM human protein-tyrosine phosphatase mu precursor (EC 3.1.3.48) (R-PTP-MU).
  • R-PTP-MU PTPM human protein-tyrosine phosphatase mu precursor
  • PDGS human alpha platelet-derived growth factor receptor precursor (EC 2.7.1.112)
  • FGR4 human fibroblast growth factor receptor 4 precursor (FGFR-4) (EC 2.7.1.112)
  • FGR2 human fibroblast growth factor receptor 2 precursor (FGFR-2) (EC 2.7.1.112)
  • LRP2 human low-density lipoprotein receptor-related protein 2 precursor (megalin)
  • IDD human integral membrane protein dgcr2/idd precursor (KIAA0163)
  • AMFR human autocrine motility factor receptor precursor AMF receptor
  • gp78 ACH5 human neuronal acetylcholine receptor protein, alpha-5 chain precursor.
  • KKIT human stem cell growth factor receptor (proto-oncogene tyrosine- protein kinase kit) (C-KIT) (CD117 antigen)
  • C-KIT stem cell growth factor receptor
  • TPOR human thrombopoietin receptor precusor (TPO-R) (myeloproliferative leukemia protein (C-MPL).
  • C-MPL myeloproliferative leukemia protein
  • MPL thrombopoietin receptor precursor
  • TPOR or MPL thrombopoietin receptor precursor (TPO-R) (myeloproliferative leukemia protein)
  • the binding motif of the receptor is capable of interacting with a cytoplasmic molecule or protein, or any cytoplasmic molecule or protein capable of binding a further signaling molecule which activates a cascade of events leading to cell signaling pathways and biological functions such as mitogenesis, proliferation, transformation, differentiation and cell survival, or any other cytoplasmic molecule or protein which does not signal.
  • This molecule may act as an antagonist. More preferably, the cytoplasmic protein is 14-3-3.
  • the 14-3-3 protein is a family of proteins which consists of 7 different isoforms and is expressed ubiquitously from yeast to humans. The ability of 14-3-3 to bind to a number of motifs in a wide range of signaling molecules suggests that 14-3-3 proteins may participate in a number of cell signaling pathways that may include mitogenesis, transformation and survival. Although 14-3-3 has been shown to bind a number of signaling molecules, it has been more difficult to determine how 14-3-3 can regulate signaling events.
  • binding motif of a receptor has an amino acid sequence selected from the group including:
  • the binding capacity of the motif may be analysed by any binding studies or experiments available to the skilled addressee. Such experiments may include measuring the binding ability of a designated cytoplasmic protein to the binding motif. For instance electrophoretic mobility shift assays (EMSA or band shift assays) or foot print assays or pull down experiments are available to measure specific binding.
  • ESA electrophoretic mobility shift assays
  • band shift assays foot print assays or pull down experiments are available to measure specific binding.
  • the binding motif can be identified by the presence of a, serine residue preferably in an amino acid sequence as described above, and the ability to bind a designated cytoplasmic protein.
  • the designated cytoplasmic protein may be 14-3-3 protein, or any cytoplasmic protein capable of binding a further signaling molecule which activates a cascade of events leading to cell signaling pathways and biological functions such as mitogenesis, proliferation, transformation, differentiation and cell survival. More preferably, the cytoplasmic protein is 14-3- 3.
  • the receptor is the GM-CSF/IL-3/IL-5 receptor which includes the common beta chain ( ⁇ c ). It is found that the cytokines GM-CSF, IL-3 and IL-5 signal their actions through the surface receptor via the ⁇ c .
  • the binding motif comprises a sequence which includes amino acids HSRSLP corresponding to amino acids 582 to 587 of the common ⁇ c according to Figure 1 a functional equivalent or analogue thereof.
  • the common ⁇ chain ( ⁇ c ) is a component of a cytokine receptor and is part of a signaling subunit of the receptor. It is thought that the cytokine signals its functions through the ⁇ c , initiating events which cascade and culminate in an identifiable biological function such as cell survival, proliferation, differentiation and mature cell effector functions.
  • the present invention is not limited to motifs of the ⁇ c but includes motifs of receptors having similar sequences to the ⁇ c and including a serine/threonine residue.
  • the region or motif comprising amino acids 582 to 587 of the common ⁇ c may include 582 HSRSLP 587 which preferably interacts with a cytoplasmic protein selected from the group including 14-3-3 protein, WW-domain of the prolyl isomerase, Pin1 , and the ubiquitin ligase, NEDD4 or any cytoplasmic protein capable of binding a further signaling molecule which activates a cascade of events leading to cell signaling pathways and biological functions such as mitogenesis, proliferation, transformation, differentiation and cell survival.
  • a cytoplasmic protein selected from the group including 14-3-3 protein, WW-domain of the prolyl isomerase, Pin1 , and the ubiquitin ligase, NEDD4 or any cytoplasmic protein capable of binding a further signaling molecule which activates a cascade of events leading to cell signaling pathways and biological functions such as mitogenesis, proliferation, transformation, differentiation and cell survival.
  • a cytoplasmic protein selected from the group including 14-3-3 protein, WW
  • C-Kit Stem Cell Growth Factor Receptor
  • C-KIT Proto-Oncogene Tyrosine- Protein Kinase Kit
  • CD 117 Antigen preferably including amino acids 863 to 869 according to Figure 2 or amino acid residues 965 to 969 according to Figure 2 or a functional equivalent or analogue thereof.
  • TPO-R Thrombopoietin Receptor Precursor
  • C-MPL Myeloproliferative Leukemia Protein
  • TPOR or MPL preferably including amino acids 573 to 579 according to Figure 3 or a functional equivalent or analogue thereof.
  • TPO-R Thrombopoietin Receptor Precursor
  • C-MPL Myeloproliferative Leukemia Protein
  • TPOR or MPL preferably including amino acids 564 to 570 according to Figure 4 or a functional equivalent or analogue thereof.
  • IL6B HUMAN interleukin-6 receptor beta chain precursor IL-6R-BETA
  • amino acids 735- 739 having the sequence SSSRP or a functional equivalent or analogue thereof
  • LEPR HUMAN leptin receptor precursor (LEP-R) (OB receptor) (OB-R)
  • OB receptor OB receptor
  • amino acids 991- 995 having the sequence SNSKP or a functional equivalent or analogue thereof.
  • TNR2 HUMAN tumor necrosis factor receptor 2 precursor preferably including amino acids 368- 372 having the sequence SDSSP or a functional equivalent or analogue thereof.
  • VGR1 HUMAN vascular endothelial growth factor receptor 1 precursor preferably including amino acids 1197- 1201 having the sequence SISAP or a functional equivalent or analogue thereof.
  • TRK3 HUMAN receptor protein-tyrosine kinase TKT precursor EC 2.7.1.112
  • amino acids 444- 448 having the sequence SLSLP or a functional equivalent or analogue thereof.
  • Q01974 protein-tyrosine kinase transmembrane receptor ROR2 precursor preferably including amino acids 435- 439, having the sequence SASTP or a functional equivalent or analogue thereof.
  • FGR1 HUMAN basic fibroblast growth factor receptor 1 precursor BFGF-R
  • BFGF-R basic fibroblast growth factor receptor 1 precursor
  • Q15426 protein-tyrosine phosphatase, receptor-type, H precursor (EC 3.1.3.48), preferably including amino acids 1082- 1086, having the sequence SNSQP or a functional equivalent or analogue thereof.
  • PTPM HUMAN protein-tyrosine phosphatase MU precursor (EC 3.1.3.48) (R- PTP-MU), preferably including amino acids 818- 822, 833- 837, 1082- 1086 having the sequences SVSSP, STSVP, SKSPP or functional equivalents or analogues thereof.
  • PGDS HUMAN alpha platelet-derived growth factor receptor precursor (EC 2.7.1.112), preferably including amino acids 616- 620 having the sequence SRSQP or a functional equivalent or analogue thereof.
  • FGR4 HUMAN fibroblast growth factor receptor 4 precursor (FGFR-4) (EC 2.7.1.112), preferably including amino acids 439- 443, 791- 795 having the sequences SSSGP, SSSFP or functional equivalents or analogues thereof.
  • FGR2 HUMAN fibroblast growth factor receptor 2 precursor (FGFR-2) (EC 2.7.1.112), preferably including amino acids 780- 784 having the sequence SPSYP or a functional equivalent or analogue thereof.
  • Q 13635 patched protein homolog (PTC), preferably including amino acids 1290- 1294 having the sequence SGSLP or a functional equivalent or analogue thereof.
  • MANR HUMAN macrophage mannose receptor precursor preferably including amino acids 1432- 1436 having the sequence SQSSP or a functional equivalent or analogue thereof.
  • LRP2 HUMAN low-density lipoprotein receptor-related protein 2 precursor preferably including amino acids 4616- 4620 having the sequence SPSLP or a functional equivalent or analogue thereof.
  • IDD HUMAN integral membrane protein DGCR2/IDD precursor preferably including amino acids 526- 530 having the sequence SGSTP or a functional equivalent or analogue thereof.
  • AMFR HUMAN autocrine motility factor receptor precursor (AMF receptor) (GP78), preferably including amino acids 203- 207 having the sequence SVSPP or a functional equivalent or analogue thereof.
  • ACH5 HUMAN neuronal acetylcholine receptor protein alpha-5 chain precursor, preferably including amino acids 382-386 having the sequence SGSGP or a functional equivalent or analogue thereof.
  • a phosphorylated binding motif of a receptor capable of binding a cytoplasmic protein, said binding motif comprising an amino acid sequence wherein at least one amino acid is serine/threonine and wherein the serine residue is phosphorylated.
  • the serine residue corresponds to a serine residue at position 585 of the common ⁇ c according to Figure 1.
  • a phosphorylated binding motif of a receptor capable of binding a cytoplasmic protein, said binding motif comprising an amino acid sequence, a functional equivalent or analogue thereof and wherein at least two (2) of the amino acids are serine and wherein at least one serine residue of the motif is phosphorylated.
  • binding motif comprises an amino acid sequence including the sequence:
  • the second serine/threonine motif is an indicator of the cytoplasmic protein binding motif.
  • the motif as a whole is involved in the cytoplasmic binding and requires the serine/threonine residue along with flanking amino acids.
  • the motif includes flanking amino acid sequences which may improve the binding of a cytoplasmic protein to the binding motif. More preferably the flanking amino acids are selected from R and X-P (wherein X is any amino acid) such that the flanking amino acids individually or co-operatively contribute to the binding motif for binding to a cytoplasmic protein.
  • amino acid sequence of the binding motif includes the sequence:
  • the motif must have at least one serine residue Preferably there are two.
  • the second serine residue from the 5' end of the motif is phosphorylated.
  • the receptor is selected from the group of receptors described above and the amino acid sequence is any one of the binding motifs described above.
  • a binding motif of a GM-CSF/IL- 3/IL-5 receptor capable of binding a cytoplasmic protein, said binding motif comprising an amino acid sequence including the sequence 582 HSRSLP 587 of the GM-CSF/IL-3/IL-5 receptor or a functional equivalent or analogue thereof wherein at least Ser 585 is capable of being phosphorylated.
  • a phophorylated binding motif of a GM-CSF/IL-3/IL-5 receptor capable of binding a cytoplasmic protein, said binding motif comprising an amino acid sequence including the sequence 582 HSRSLP 587 of the GM-CSF/IL-3/IL-5 receptor or a functional equivalent or analogue thereof wherein at least Ser 585 is phosphorylated.
  • the binding motif binds to the cytoplasmic protein 14-3-3.
  • receptors as described above can be phosphorylated and induced to bind a cytoplasmic protein such as 14-3-3 by phosphorylation preferably of the second serine residue from the 5' end of the motif.
  • the serine residue corresponds to a serine residue at position 585 of the common ⁇ c according to Figure 1.
  • the binding motif is any one of the motifs described above.
  • phosphorylation of the binding motif is caused by the binding of a triggering molecule to its corresponding receptor.
  • Triggering molecules may be cytokines which bind to cytokine receptors.
  • the receptor is a heterodimeric receptor. More preferably it is a GM-CSF/IL-5/IL-3 receptor bound by a GM-CSF, IL-5 or IL-3 cytokine.
  • Other triggering molecules may be the corresponding triggering molecule that binds to any one of the receptors listed above.
  • binding motif is as described above.
  • any triggering molecule which binds to a receptor or receptor signaling system may be capable of causing phosphorylation of the binding motif.
  • the triggering molecule is a cytokine which binds to a homodimeric or heterodimeric receptor prior to phosphorylation of the binding motif.
  • the cytokine binds to a heterodimeric cytokine receptor.
  • the heterodimeric cytokine receptor may comprise two (or three) subunits which subserve distinct and specialised functions. These subunits include the major ligand binding subunit ( ⁇ subunit) and the signaling subunit which may comprise a ⁇ or ⁇ subunit.
  • the signaling subunit may recognise several cytokines on the ⁇ subunit which can then tranduce signals from the cytokines into the cell.
  • the common beta chain ( ⁇ c ) is found in the receptor signalling systems of cytokines including granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and interleukin-5 (IL-5).
  • the binding motif comprises an amino acid sequence including 582 HSRSLP 587 on the ⁇ c . More preferably, this sequence is phosphorylated upon binding of GM-CSF, IL-3 or IL-5 to the receptor. More preferably Ser 585 is phosphorylated particularly when GM- CSF, IL-3 or IL-5 bind to their corresponding receptors.
  • other triggering molecules can cause phosphorylation of an equivalent region of the receptor. For instance, the molecule which binds to the receptor (as listed above) may cause phosphorylation to the binding motif.
  • a method of binding a cytoplasmic protein to a receptor comprising:
  • the phosphorylation of the binding motif may improve the binding of a cytoplasmic protein to the binding motif so that when the cytoplasmic protein is reacted with the motif, or equivalent thereof, binding may occur to bring other cytoplasmic proteins or signaling molecules into close proximity to the receptor.
  • Phosphorylation may occur by any means which transfers a phosphoryl (phosphate) group to the cytoplasmic binding motif.
  • the cytoplasmic protein may be 14-3-3 protein, or any cytoplasmic protein capable of binding a further signaling molecule which activates a cascade of events leading to cell signaling pathways and biological functions such as mitogenesis, proliferation, transformation, differentiation, cell survival, chemotaxis, motility, enhanced phagocytosis, bacterial killing, superoxide production and cytotoxicity.
  • a method of activating cellular activities said method including:
  • cytoplasmic protein regulating the activation of phosphorylation of a binding motif of a receptor as described above, a functional equivalent or analogue thereof; and subjecting the binding motif to a cytoplasmic protein wherein said cytoplasmic protein is associated with cellular activities.
  • the cytoplasmic protein is 14-3-3 protein, or any cytoplasmic protein capable of binding a further signaling molecule which activates a cascade of events leading to cell signaling pathways and biological functions such as mitogenesis, proliferation, transformation, differentiation cell survival, chemotaxis, motility, enhanced phagocytosis, bacterial killing, superoxide production and cytotoxicity.
  • the cytoplasmic binding protein is 14-3-3.
  • the 14-3-3 molecule binds not only to the cytoplasmic binding motif (as found by the applicants) but has the ability to bind to a wide range of signaling molecules and to participate in a number of cell signaling pathways resulting in mitogenesis, transformation, differentiation cell survival, chemotaxis, motility, enhanced phagocytosis, bacterial killing, superoxide production and cytotoxicity.
  • 14-3-3 or an equivalent binds to the binding motif of the receptor, its ubiquitous nature can bind cytoplasmic proteins involved in signaling pathways which activate these pathways. For instance, not being limited by theory and by example, it has now been found by the Applicants that cytokines such as GM- CSF, IL-3 or IL-5, will bind to the ⁇ c of the receptor.
  • the binding motif of the receptor is then phosphorylated and preferably phosphorylates the 585 Ser or equivalent residue.
  • 14-3-3 can bind to the phosphorylated motif thereby positioning the 14-3-3 close to the receptor for further binding of cytoplasmic proteins involved in cell signaling (signaling molecules) for cellular activities such as proliferation, differentiation, cell survival, chemotaxis, motility, enhanced phagocytosis, bacterial killing, superoxide production and cytotoxicity.
  • cytoplasmic proteins involved in cell signaling signaling molecules
  • signaling molecules for cellular activities such as proliferation, differentiation, cell survival, chemotaxis, motility, enhanced phagocytosis, bacterial killing, superoxide production and cytotoxicity.
  • the cytoplasmic protein is 14-3-3 protein, or any cytoplasmic protein capable of binding a further signaling molecule which activates a cascade of events leading to cell signaling pathways and biological functions such as mitogenesis, proliferation, transformation, differentiation, cell survival, chemotaxis, motility, enhanced phagocytosis, bacterial killing, superoxide production and cytotoxicity.
  • the cytoplasmic binding protein is 14-3-3.
  • a molecule that binds to a phospho-serine bound 14-3-3 molecule such that a pathway is coupled to the motif or equivalent unit in a receptor and brought into close proximity to downstream signaling proteins at, or near, the cell membrane.
  • Cellular activities may include cell survival, proliferation, transformation, differentiation, mitogenesis, chemotaxis, motility, enhanced phagocytosis, bacterial killing, superoxide production and cytotoxicity.
  • Figure 18 shows proposed model for the regulation of survival by IL-3. Binding of IL-3 to the IL-3 receptor, composed of a ligand specific ⁇ -chain ( ⁇ ) and a common ⁇ -chain ( ⁇ c ), results in receptor oligomerization (only one ⁇ chain and one ⁇ -chain are shown for simplicity).
  • ⁇ ligand specific ⁇ -chain
  • ⁇ c common ⁇ -chain
  • Receptor oligomerization results in the activation of tyrosine kinases which results in tyrosine phosphorylation of ⁇ c (P-Y) and the recruitment of SH2- and PTB-binding proteins (not shown).
  • activation of PKA results in the phosphorylation of Ser585 of ⁇ c (S-P).
  • Ser585 phosphorylation allows the recruitment of 14-3-3, which in turn recruits PI 3-kinase (PI 3-K) either directly through the p110 or p85 subunits or indirectly through an additional adaptor molecule(s).
  • Phosphorylation of the motif may be regulated by any means which results in inhibition or activation of the phosphorylation of the cytoplasmic protein binding motif particularly the Ser 585 residue.
  • the phosphorylation is induced by a triggering molecule such as a cytokine selected from the group including GM- CSF, IL-3 or IL-5. More preferably for cell survival, it is induced by IL-3.
  • the ⁇ c is phosphorylated. More preferably the binding motif comprising the amino acid sequence HSRSLP is phosphorylated. More preferably the second serine from the 5' end is phosphorylated. In ⁇ c , this correlates to 585 Ser.
  • Regulation of cell survival may include enhancing or reducing cell survival or even causing cell death. This may be achieved by enhancing or inhibiting any of the steps described above. For instance enchancing phosphorylation of the binding motif may enhance survival. Alternatively, inhibiting phosphorylation may inhibit cell survival.
  • a method of inhibiting cell survival including inhibiting the binding of a cytoplasmic protein to a binding motif of a receptor as desc ⁇ bed above.
  • the cytoplasmic protein is 14-3-3.
  • the receptor is the GM-CSF/IL-5/IL-3 receptor and the triggering molecule is IL-3, although a phosphorylation event which phosphorylates 585 Ser may also trigger the binding of 14-3-3 to the motif.
  • the binding motif is as described above.
  • Antagonists that bind to the receptor motif in either the phosphorylated or unphosphorylated form may be useful to inhibit cell survival or activation.
  • Preferably antagonists may be useful to inhibit cell survival or activation by preventing phosphorylation preferably by preventing serine phosphorylation of the ⁇ c or equivalent thereby preventing the cytoplasmic protein binding to the binding motif.
  • they may prevent the interaction of a signaling molecule binding to a phosphoserine bound 14-3-3 or equivalent.
  • Prevention of phosphorylation of the ⁇ c or binding motif as described above may be by inhibition of the specific kinases involved in the phosphorylation of the serine/threonine residue or it may include mutation of the binding motif to prevent the cytoplasmic protein such as 14-3-3 from binding and activating cell cycle pathways.
  • Kinase inhibitors such as H89 which binds to PKA may be used.
  • cell permeable kinase inhibitors are used.
  • the signaling molecule is a PI-3- kinase involved in the PI-3-kinase pathway which leads to cell survival or cell activation.
  • Antagonists may include antibodies, small peptides, small molecules, peptide mimetics or any type of molecule known to those skilled in the art that are directed to the cytoplasmic binding motif so as to prevent attachment of cytoplasmic proteins such as 14-3-3 to a phosphoserine residue or a signaling molecule.
  • Antibodies may be generated in response to any of the binding motifs described above by methods known and available to the skilled addressee.
  • the antagonists as described may be useful as cancer therapeutics to prevent cell survival of cancer cells or cell activation such as myeloid cell activation and may be useful for preventing or treating leukaemia such as acute myeloid leukaemia (AML).
  • leukaemia such as acute myeloid leukaemia (AML).
  • Other uses of antagonists may be in prevention and treatment of inflammatory diseases.
  • Applicants have also found that the 585 Ser in ⁇ c of acute myeloid leukaemia (AML) is constitutively phosphorylated (see Figure 19). Hence by preventing phosphorylation of the 585 Ser, further binding of 14-3-3 to the binding motif of ⁇ c may be prevented thereby further preventing the binding of PI-3-kinase which interacts with the PI-3-kinase pathway to activate cell survival.
  • the functions may be selected from the group including chemotaxis, motility, enhanced phagocytosis, bacterial killing, superoxide production and cytoxicity. These functions may also contribute to inflammation including, but not limited to, asthma and rheumatoid arthritis.
  • a method of inhibiting cell activation including inhibiting the binding of a cytoplasmic protein to a binding motif of a receptor as described above.
  • this method of interaction may be a useful tool for a method of treating or preventing cell proliferative diseases such as AML or cancer.
  • Targeting may be by way of the use of antagonists as described above or by any means that prevents activation of cell cycles via the binding motif described in the present invention. Targeting may be by blocking or mutating the motif.
  • Techniques such as gene therapy may also be employed which targets or removes the binding motif.
  • a method of treating a cytokine mediated condition comprising:
  • the cytokine mediated condition is a condition which requires a cytokine to bind to its receptor to induce a cellular activity. By regulating the activator, cellular action may be activated to increase the phosphorylation or to decrease phosphorylation.
  • the cytokine mediated condition is a GMCSF/IL-5 mediated condition and the binding motif includes the amino acid sequence 582 HSR SLP 587 on the ⁇ c .
  • Substitution mutations of two sequences within the cytoplasmic domain of the human ⁇ c cDNA were constructed using oligonucleotide-directed mutagenesis (Altered-sites, Promega, Sydney, NSW, Australia) as described in Wood Cock, J.M. et al (1994) EMBO, 13, 5176. Both mutants were essentially poly-alanine substitutions. Mutagenesis oligonucleotides encoding non-alanine residues were included to facilitate restriction enzyme screening of mutant clones. The first motif was 58 HSRSLP 587 mutated to 582 EFAAAA 587 , and the second was 820 RSKPSSP 826 mutated to 820 EFAAAAA 826 .
  • the point mutant S585A was also constructed, however this mutant created a cryptic proteolytic site in ⁇ c and was not able to be used.
  • the mutations were confirmed by nucleotide sequencing and the mutant ⁇ c cDNAs suloned into the eukaryotic expression vector pcDNAI (Invitrogen, San Diego, CA).
  • the ⁇ c deletion mutant cDNAs were a kind gift of Dr A. Miyajima.
  • GM-CSF and IL-3 Recombinant human IL-3 and GM-CSF were produced in E.coli essentially as described in Barry, S.C. et al (1994) J. Biol chem, 269, 8488 and Hercus, T.R. et al (1994) Proc. Natl. Acad. Sci., 91 , 5838. Cytokine purity and quantitation was determined by HPLC analysis and Coomasie staining of SDS-PAGE separated proteins. The activity of the cytokines based on the ED 50 values in a TF-1 proliferation assay (Kitamura, T. et al (1989) J. Cell Physiol, 140, 323) was 0.03ng/ml for GM-CSF and 0.1 ng/ml for IL-3.
  • the specificity of the anti-phospho- 585 Ser ⁇ c antibody was verified by dot immunoblots against the CLGPPHSRSLPDILG peptide either non phosphorylated or serine phosphorylated form and a scrambled peptide LPLSGPDSHIRGPL. The corresponding phosphorylated serine in each peptide is underlined. Peptides were synthesised by Chiron Mimotopes, Melbourne, Australia. The anti-14-3-3z antibody was kindly provided by Dr A. Aitken.
  • M1 cell line expressing GM-CSF receptor alpha chain and ⁇ c wild type was maintained in RPMI-1640 supplemented with 10 % v/v FCS.
  • the M1 cell line was kindly provided by Dr N. Nicola. (e) Surface marker analysis by flow cytometry. Expression of receptors on transfected cells was verified by flow cytometry. Briefly, cells were incubated with the anti- ⁇ c MAb (1C1) (Stomski, F.C. et al (1996) Mol. Cell. Biol, 16, 3035) or anti- GM-CSFRa MAb (4H1) (Stomski, F.C.
  • ⁇ c peptide sequence CLGPPHSRSLPDILG either non phosphorylated or serine phosphorylated and a scrambled peptide CLPLSGPDSHIRGPL.
  • Raf1 peptides corresponding to the sequence CLSQRQRSTSTPNVHM were also used and were either non phosphorylated or serine phosphorylated. The corresponding phosphorylated serine in each peptide is underlined.
  • Peptides were synthesised by Chiron Mimotopes, Melbourne, Australia. The presence of ⁇ c in either the immunoprecipitation or precipitation experiment was determined by Western blotting with anti- ⁇ c antibody (MAb 1C1).
  • nitrocellulose membranes were blocked in a solution of PBS/0.05% (v/v) Tween 20 (PBT) containing 1% (w/v) blocking reagent 1096 176 (Boehringer Mannheim) and probed with anti- ⁇ c (1C1), anti-14-3-3z 33 or anti-phospho- 585 Ser ⁇ c followed by either anti-mouse or rabbit peroxidase conjugated antibodies.
  • Immunoreactive proteins were detected by chemiluminescence using the ECL kit (Amersham, Little Chalfont, U.K.) following the manufacturer's instructions.
  • microtiter well-bound radioactivity were estimated in a g-counter.
  • 14-3-3 is known to be a phospho-serine binding protein which interacts with the RSXSXP motif, where S is phosphorylated.
  • S is phosphorylated.
  • 585 Ser phosphorylation within the 582 HSRSLP 587 motif would be required for 14-3-3 association.
  • M1 myeloid leukaemic cells were stimulated with 2 ng/ml GM-CSF, ⁇ c was immunoprecipitated, and immunoprecipitates probed with the anti-phospho- 585 Ser antibody.
  • GM-CSF stimulation strongly upregulated 585 Ser phosphorylation of ⁇ c .
  • CTL-EN cells were tranduced with viral constructs expressing IL-3Ra and either wt ⁇ c , a substitution mutant that encompasses the 14-3-3 binding site ( ⁇ c HSRSLP ⁇ EFAAAA) or a point mutant in which Ser585 of the 14-3-3 binding motif was substituted for alanine ( ⁇ c S585A).
  • the ⁇ c S585A point was not able to be used in these studies as it appeared to introduce a cryptic proteolytic cleavage site.
  • Western blot analysis and flow cytometry indicated that this mutant was proteolysed and failed to be expressed on the cell surface (Stomski et al., 1999) Blood, 94, 1933-1942).
  • ⁇ c Ser585 phosphorylation in response to IL-3 stimulation was then examined in these cell lines using anti-phospho-Ser585 ⁇ c antibodies. These antibodies have been shown to specifically recognize the phosphorylated 14-3-3 binding motif HSRSLP (where S is phosphorylated) but not the non- phosphorylated motif.
  • CTL-EN cells expressing wt ⁇ c or ⁇ c HSRSLP ⁇ EFAAAA were starved for 4 hours in 0.1 % FCS and then stimulated with 50ng/ml of IL-3. The ⁇ c was immunoprecipitated and then subjected to immunoblot analysis using the anti-phospho-Ser585 ⁇ c antibodies.
  • Ser585 phosphorylation was upregulated in response to IL-3 stimulation in CTL-EN cells expressing wt while no signal was detected for the ⁇ c HSRSLP-»EFAAAA mutant control (Fig. 11a).
  • Tyrosine phosphorylation of ⁇ c was detected for both the wt ⁇ c and the ⁇ c HSRSLP-»EFAAAA mutant (Fig. 11a).
  • Co-immunoprecipitation experiments showed that increased ⁇ c Ser585 phosphorylation in response to IL-3 stimulation also resulted in increased 14-3-3 binding to ⁇ c (Fig. 11b). No association of 14-3- 3 with the ⁇ c HSRSLP ⁇ EFAAAA mutant control in response to IL-3.
  • CTL-EN cells expressing wt ⁇ c were starved for 12 hours in DMEM containing 0.5% FCS and then either left unstimulated (nil), stimulated with 50ng/ml IL-3, 50mM forskolin or 100m dibutyryl cAMP for 10 minutes.
  • Cells were lysed, ⁇ c was immunoprecipitated and subjected to immunoblot analysis with the anti-phospho-Ser585 antibody.
  • IL-3 stimulation resulted in increased Ser585 phosphorylation (Fig. 12c, IL-3).
  • agents that elevate intracellular levels of cAMP and activate PKA were also found to result in increased Ser585 phosphorylation of ⁇ c .
  • the results shown in Figure 12 would suggest that PKA is likely to phosphorylate Ser585 of ⁇ c in response to IL-3 stimulation.
  • GM-CSF and IL-3 are known to activate at least three pathways; the JAK STAT pathway, the ras/MAP kinase pathway and the PI 3-kinase pathway (Guthridge et al., 1998 skin cells 16, 301-313). These pathways are not necessarily mutually exclusive and may have substantial overlap.
  • the PI 3-kinase pathway has been implicated as having an important role in regulating cellular survival in a number of systems (Marte and Downward, (1997); Trends Biochem Sci 22, 355-358 cell, 88, 435-437) Franke et al., 1997) and, significantly, has been shown to bind 14-3-3 (Bonnefoy-Berard et al., (1995); Lui et al., 1996). We therefore examined the possibility that Ser585 of ⁇ c and 14- 3-3 maybe important in the regulation of the PI 3-kinase pathway.
  • CTL-EN cells expressing wt ⁇ c , or ⁇ c HSRSLP ⁇ EFAAAA were starved and stimulated with IL-3, tyrosine phosphorylated proteins were immunoprecipitated and the PI 3-kinase activity of the immunoprecipitates was determined in an in vitro kinase assay using phosphatidyl inositol and g 32 P-ATP as substrates.
  • IL-3 stimulation resulted in the association of p85 with wt ⁇ c which was maximal at 5 minutes and was decreased by 30 minutes (Fig. 13b).
  • the recruitment of p85 to the ⁇ c HSRSLP ⁇ EFAAAA mutant in response to IL-3 was virtually abolished (Fig. 13b).
  • SHP2 protein tyrosine phosphatase
  • his ⁇ c 445-881 expressed in bacteria is not phosphorylated on Ser585, however as shown in Figure 12a and 12b, we have found that cAMP-dependent protein kinase (PKA) can efficiently phosphorylate Ser585 of his ⁇ c 445-881 in vitro.
  • PKA cAMP-dependent protein kinase
  • his ⁇ c 445-881 coupled to Sepharose and phosphorylated on Ser585 by PKA was incubated with COS-7 cell lysates and examined for its ability to pull-down p85 by immunoblot analysis.
  • a number of peptides encompassing the 14-3-3 binding site in ⁇ c (listed in Fig. 3c) were examined for their ability to competitively inhibit the interaction of his ⁇ c 445-881 with p85 (Fig. 12c).
  • his ⁇ c 445-881 Sepharose was able to pull down p85 from the COS-7 cell extracts (Fig. 13d, lane 1) and the presence of scrambled (Fig. 13d, lane 2), Ser585Ala (Fig. 13d, lane 3), or non-phosphorylated-Ser585 (Fig. 13d, lane 4) control peptides had no apparent effect on this interaction.
  • the interaction of p85 with his ⁇ c 445-881 was markedly reduced in the presence of a phospho-Ser585 peptide (Fig. 13d, lane 5).
  • CTL- EN cells expressing either wt ⁇ c or the ⁇ c HSRSLP ⁇ EFAAAA receptors were starved and then stimulated with IL-3 and the activation of Akt (or protein kinase B), the signal transducer and activator of transription protein STAT5, and the extracellular-regulated kinases ERK1 and ERK 2 (or MAP kinase) was examined in whole cell lysates by Western blot using phospho-specific antibodies.
  • Akt is a known downstream target of PI 3-kinase whereas STAT5 and ERK are thought to be regulated by pathways distinct from the PI 3-kinase pathway.
  • CTL-EN cells expressing wt ⁇ c or the ⁇ c HSRSLP-»EFAAAA mutant induced STAT5 and ERK phosphorylation in response to IL-3 (Fig. 14a). While CTL-EN cells expressing wt ⁇ c demonstrated phosphorylation of Akt in response to IL-3, the phosphorylation of Akt was almost completely abolished in cells expressing the ⁇ c HSRSLP ⁇ EFAAAA mutant (Fig. 14a).
  • JNK c-jun N-terminal kinase
  • CTL-EN cells expressing the wt IL-3 receptor remain greater than 90% viable for up to 3-4 days under low serum conditions (0.1% FCS) in the presence of IL-3.
  • CTL-EN cells expressing either wt ⁇ c or ⁇ c HSRSLP- EFAAAA were plated out at 2.5x10 5 cells/ml in the presence of IL-3 or IL-2 in medium containing 0.1 % FCS and viable cells were counted after 4 days.
  • CTL-EN cells expressing wt ⁇ c remained greater than 90% viable for up to 3-4 days in the presence of either IL-3 or IL-2
  • cells expressing the ⁇ c HSRSLP ⁇ EFAAAA mutant showed a loss of viability in the presence of IL-3 with only 7% viable cells remaining after 4 days (Fig 15a).
  • Cells expressing the mutant receptor were able to maintain viability in the presence of IL-2.
  • CTL-EN cells expressing the wt ⁇ c lost viability in the presence of IL-3 and the PI 3-kinase inhibitor, LY294002, confirming that PI 3-kinase activity is essential for the survival these cells (Fig. 15a).
  • CTL-EN cells were starved in medium containing 0.1% FCS and no IL-3 for 24 hours and then stimulated with IL-3 for up to 24 hours. The cells were then fixed in ethanol and the cell cycle distribution was examined by prodidium iodide staining and flow cytometry. Under these starvation conditions, nearly 90% of CTL-EN cells accumulate in the G Q /G, phase of the cell cycle (Fig. 16a).
  • CTL-EN cells expressing either wt ⁇ c or the ⁇ c HSRSLP ⁇ EFAAAA mutant and synchronized in G ⁇ G were both able to re-enter the cell cycle in response to IL-3 indicating that Ser585 phosphorylation, and 14-3-3 binding are not essential for signals that promote cell cycle progression (Fig. 16a).
  • EXAMPLE 9 LOSS OF 14-3-3 BINDING TO ⁇ r RESULTS IN CELL DEATH BY APOPTOSIS.
  • Annexin V binding studies to determine if the loss in cell viability in CTL-EN cells expressing the ⁇ c HSRSLP ⁇ EFAAAA mutant was due to apoptosis.
  • CTL-EN cells expressing wt ⁇ c or ⁇ c HSRSLP ⁇ EFAAAA were washed and placed in medium containing 0.1% FCS and either IL-2 or IL-3 for 16 hours. The cells were then stained with annexin V and apoptotic cells were analysed by flow cytometry.
  • CTL-EN cells expressing wt ⁇ c or ⁇ c HSRSLP ⁇ EFAAAA showed negligible annexin V staining in the presence of IL-2 (Fig. 17).
  • CTL-EN cells expressing the ⁇ c HSRSLP ⁇ EFAAAA mutant showed increased annexin V staining compared to cells expressing the wt ⁇ c indicating that the defect in cell survival is due to increased apoptosis (Fig. 17).
  • 14-3-3 To address the functional significance of 14-3-3 binding to ⁇ c cell lines were generated expressing either wild type (wt) or mutant IL-3 receptors lacking the 14- 3-3 binding site. Constructs expressing the IL-3-specific ⁇ . chain (IL-3R ⁇ ..and either wt ⁇ c , ⁇ c HSRSLP- EFAAAA, ⁇ c RSL ⁇ AAA, ⁇ c S585A or ⁇ c S585G were introduced into the CTL-EN T-cell line.
  • IL-3R ⁇ ..and either wt ⁇ c , ⁇ c HSRSLP- EFAAAA, ⁇ c RSL ⁇ AAA, ⁇ c S585A or ⁇ c S585G were introduced into the CTL-EN T-cell line.
  • the ⁇ c was immunoprecipitated and then subjected to immunoblot analysis using the anti- phospho-Ser585 ⁇ c antibody.
  • Ser585 phosphorylation was upregulated in response to IL-3 stimulation in CTL-EN cells expressing wt ⁇ c while no signal was detected for either the ⁇ c HSRSLP ⁇ EFAAAA or the ⁇ c S585G mutant controls (Fig. 20A).
  • Co-immunoprecipitation experiments showed that increased ⁇ c Ser585 phosphorylation in response to IL-3 stimulation also resulted in increased 14-3-3 binding to ⁇ c (Fig. 20A).
  • ⁇ c was immunoprecipitated from I 125 surface-labelled CTL-EN cells (to avoid detection of intracellular-only ⁇ c ) with either anti-phosphorSer585, anti- phosphotyrosine or anti- ⁇ c antibodies from both IL-3-stimulated (+IL-3) or non- stimulated (-IL-3) cells expressing wt ⁇ c or ⁇ c S585G receptors.
  • Immunoprecipitates were electrophoresed on a polyacrylamide gel and the amount of radiolabelled receptor immunoprecipitated was quantified using a phosphorlmager.
  • wt ⁇ c non- stimulated cells
  • ⁇ c as the receptor is not phosphorylated on either Ser585 or tyrosine
  • Fig. 20D tyrosine
  • Ser585 of ⁇ c lies within an 585 SXXD 588 motif which is a possible casein kinase II (CKII) consensus phosphorylation site and also within an 582 HSRS 585 motif which is a possible, but poor, cAMP-dependent protein kinase (PKA) consensus phosphorylation site (Kennely and Krebs, (1991)) (J. Biol. Chem. 266, 15,555- 15,558).
  • CKII casein kinase II
  • PKA cAMP-dependent protein kinase
  • CTL-EN cells expressing the wt ⁇ c or MO7e cells were either not stimulated (nil), stimulated for 10 minutes with IL-3 (IL-3), or pretreated with 30 ⁇ M H89 for 15 minutes prior to IL-3 stimulation for 10 minutes (H89 + IL-3). After various treatments, cells were lysed, ⁇ c was immunoprecipitated, and subjected to immunoblot analysis with the anti-phospho-Ser585 antibody.
  • GM-CSF and IL-3 are known to regulate at least three, not necessarily mutually exclusive pathways; the JAK/STAT, the ras/MAP kinase and the PI 3-kinase pathways.
  • JAK/STAT the JAK/STAT
  • ras/MAP kinase the ras/MAP kinase pathway
  • PI 3-kinase pathway The possibility that Ser585 of ⁇ c and 14-3-3 association may be important in the regulation of the PI 3-kinase pathway in response to IL-3 was exmained.
  • CTL-EN cells expressing IL-3R ⁇ and either the wt ⁇ c , the ⁇ c HSRSLP ⁇ EFAAAA mutant or the ⁇ c S585G mutant were factor-deprived, stimulated with IL-3, tyrosine phosphorylated proteins were immunoprecipitated, and the PI 3-kinase activity of the immunoprecipitates was determined in an in vitro kinase assay using phosphatidylinositol and ⁇ 32 P-ATP as substrates.
  • IL-3 stimulation of cells expressing wt ⁇ c resulted in the rapid and transient activation of PI 3-kinase activity which was blocked by the PI 3-kinase inhibitors Wortmannin and LY294002 (Fig.
  • CTL-EN cells expressing IL-3R ⁇ and either wt ⁇ c , ⁇ c HSRSLP ⁇ EFAAAA or ⁇ c S585G were stimulated with IL-3, ⁇ c was immunoprecipitated and the immunoprecipitates were examined for associated p85 subunit by immunoblot analysis.
  • IL-3 stimulation resulted in the recruitment of p85 to wt ⁇ c whereas no recruitment of p85 to the ⁇ c HSRSLP ⁇ EFAAAA or the ⁇ c S585G mutants was observed (Fig. 22B).
  • CTL-EN cells expressing either wt ⁇ c or ⁇ c HSRSLP ⁇ EFAAAA were plated out at 5x10 5 cells/ml in the presence of 10 ng/ml IL-3/0.1% FCS and viable cells were counted over 3 days.
  • IL-3 was able to promote viability of CTL-EN cells expressing wt ⁇ c for up to 3 days
  • cells expressing the ⁇ c HSRSLP ⁇ EFAAAA mutant showed a loss of viability in the presence of IL-3 with only 18% viable cells remaining after 3 days (Fig 24A).
  • the viability of CTL- EN cells expressing wt ⁇ c or ⁇ c HSRSLP ⁇ EFAAAA were maintained in the presence of the IL-2 control cytokine.
  • CTL-EN cells expressing the ⁇ c HSRSLP ⁇ EFAAAA in response to IL-3 were also reflected in loss of cellular metabolic activity as determined by the MTS reduction assay.
  • CTL-EN cells expressing wt ⁇ c maintain appreciable levels of metabolic activity in the presence of 10ng/ml IL-3 ( ⁇ ) and 0.1% FCS for up to 3 days but lost metabolic activity in the absence of IL-3 (T)(Fig. 24B).
  • CTL-EN cells expressing the ⁇ c HSRSLP ⁇ EFAAAA mutant receptor lost metabolic activity in the presence of 10ng/ml IL-3 (Fig. 24B, A).
  • CTL-EN cells expressing either the wt ⁇ c or the ⁇ c HSRSLP ⁇ EFAAAA mutant were plated out as described above in 20 ng/ml IL- 2, no factor (NF) or 50 ng/ml IL-3. Cells were harvested 48 hours later, the DNA extracted and subjected to agarose gel electrophoresis and ethidium bromide staining.
  • DNA from CTL-EN cells expressing either wt ⁇ c or the ⁇ c HSRSLP ⁇ EFAAAA mutant exhibited the characteristic laddering typical of apoptotic cells in the absence of factor (NF) while no significant DNA laddering was observed in the presence of IL-2 (Fig. 24C).
  • CTL-EN cells expressing wt ⁇ c and plated in IL-3 showed no significant DNA laddering, however, DNA from CTL- EN cells expressing the ⁇ c HSRSLP ⁇ EFAAAA mutant which were plated in IL-3 showed a clear DNA ladder typical of apoptotic cells.
  • Cells were plated out in 20 ng/ml IL-2, no factor or 50 ng/ml IL-3, stained with annexin V and propidium iodide after 30 hours and analysed by flow cytometry.
  • CTL-EN cells expressing the wt ⁇ c or the ⁇ c HSRSLP ⁇ EFAAAA mutant were examined in fixed and permeablized cells by propidium iodide staining and flow cytometry.
  • CTL-EN cells growing asynchronously in the presence of IL-2 have approximately 37% of cells in G Q /G, phase of the cell cycle (Fig. 25A, asynchronous).
  • AML cells were starved for 12h and then stimulated with GM-CSF for 5 minutes. Where indicated cells were pre-incubated in the PKA inhibitor, H89 (10 ⁇ M), for 60 min. prior to GM-CSF stimulation. Cells were then lysed.
  • B 25% of the lysates were subjected to anti-phosphotyrosine immuno-precipitation and PI 3-K assays were performed.

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Abstract

La présente invention concerne un motif de liaison d'un récepteur. Ce motif de liaison est notamment un domaine cytoplasmique associé à la stimulation des activités induites par le récepteur. L'invention concerne également des procédés d'utilisation dudit motif, en particulier pour déclencher les activités des récepteurs. Dans un aspect, l'invention porte sur un motif de liaison d'un récepteur pouvant se lier à une protéine cytoplasmique, ledit motif comprenant une séquence d'acides aminés dans laquelle au moins un acide aminé est la sérine/thréonine et, de préférence, deux sérines indiquent le motif de liaison cytoplasmique. Ce motif de liaison comprend de préférence une séquence d'acides aminé contenant la séquence : -S-X-S/T-, dans laquelle X représente n'importe quel acide aminé.
EP00963808A 1999-09-15 2000-09-15 Motif de liaison d'un recepteur Withdrawn EP1218404A4 (fr)

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AUPQ287599 1999-09-15
AUPQ2875A AUPQ287599A0 (en) 1999-09-15 1999-09-15 A binding motif of a receptor
AUPQ873300 2000-07-12
AUPQ8733A AUPQ873300A0 (en) 2000-07-12 2000-07-12 A binding motif of a receptor (2)
PCT/AU2000/001118 WO2001019847A1 (fr) 1999-09-15 2000-09-15 Motif de liaison d'un recepteur

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US6670150B1 (en) * 2001-05-10 2003-12-30 Applera Corporation Isolated human receptor proteins, nucleic acid molecules encoding human receptor proteins, and uses thereof
US20140047572A1 (en) * 2012-08-13 2014-02-13 University Of Rochester Thrombopoietin mimetics for the treatment of radiation or chemical induced bone marrow injury

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WO1992001788A1 (fr) * 1990-07-18 1992-02-06 Schering Corporation Chaine beta du recepteur de gm-csf human
US5677144A (en) * 1993-11-16 1997-10-14 Ullrich; Axel Recombinant DNA encoding CCK 2, a receptor tyrosine kinase
WO1997048728A1 (fr) * 1996-06-20 1997-12-24 Koster, Henk, Wilhelmus Peptides derives de l'il-6 et du recepteur de l'il-6 presentant une activite agoniste ou antagoniste vis-a-vis de l'il-6

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WO1992001788A1 (fr) * 1990-07-18 1992-02-06 Schering Corporation Chaine beta du recepteur de gm-csf human
US5677144A (en) * 1993-11-16 1997-10-14 Ullrich; Axel Recombinant DNA encoding CCK 2, a receptor tyrosine kinase
WO1997048728A1 (fr) * 1996-06-20 1997-12-24 Koster, Henk, Wilhelmus Peptides derives de l'il-6 et du recepteur de l'il-6 presentant une activite agoniste ou antagoniste vis-a-vis de l'il-6

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