WO2009087196A1 - Procédé pour la mesure de l'activité de plk4 - Google Patents

Procédé pour la mesure de l'activité de plk4 Download PDF

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WO2009087196A1
WO2009087196A1 PCT/EP2009/050179 EP2009050179W WO2009087196A1 WO 2009087196 A1 WO2009087196 A1 WO 2009087196A1 EP 2009050179 W EP2009050179 W EP 2009050179W WO 2009087196 A1 WO2009087196 A1 WO 2009087196A1
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plk4
autophosphorylation
activity
phosphorylation
antibody
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Thierry Andre Regis Grand-Perret
Nele Vloemans
Frederik Tack
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Janssen Pharmaceutica NV
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Janssen Pharmaceutica NV
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • 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
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes

Definitions

  • the present invention relates to a novel method for measuring PLK4 activity and/or PLK4 activation status, especially in cells and tissue samples.
  • Polo-like kinases are key enzymes that control mitotic entry of proliferating cells and regulate many aspects of mitosis necessary for successful cytokinesis, including centrosome duplication and maturation; DNA damage checkpoint activation; bipolar spindle formation; Golgi fragmentation and assembly; and chromosome segregation (Barr, F. A. et al., Nat. Rev. MoI. Cell Biol. 2004, 5, 429-441).
  • PLKs are found in organisms as diverse as yeast and human and contain two conserved domains, the N-terminal catalytic kinase domain and a C-terminal region composed of the so- called polo-boxes.
  • PLK 4 differs not only in structure, compared to the other PLKs it has only one polo-box, but also in the distribution of PLK4 mRNA in adults that is restricted to certain tissues such as testes and thymus (Karn, T. et al., Oncol. Rep. 1997, 4, 505-510; Fode, C. et al., Proc. Natl. Acad. Sci.
  • PLK2 was recently found to be a novel p53 target gene and RNAi silencing of PLK2 leads to mitotic catastrophe in taxol-exposed cells (Burns, TF., et al., MoI Cell Biol. 23, 2003, 5556-5571).
  • PLK3 it was found that it induces cell cycle arrest and apoptosis through perturbation of microtubule integrity (Wang, Q., et al., MoI Cell Biol.
  • PLK4 was shown to be transcriptionally repressed by p53 and induces apoptosis upon RNAi silencing (Li, J., et al., Neoplasia 7, 2005, 312-323). PLK4 was also found to be required for centriole duplication and flagella development. The absence of centrioles, and hence basal bodies, compromises the meiotic divisions and the formation of sperm axonemes (Bettencourt-Dias M., et al., Current Biology 15, 2005, 2199-2207).
  • a primary aim of the present invention comprises providing an easy and reliable method to measure PLK4 activity and/or PLK4 activation status, especially in cells and tissue samples.
  • the present invention provides a method for determining the activity of PLK4 comprising the steps of:
  • the activity of PLK4 is determined using an immunostaining procedure with an antibody reagent that is specific to the S 305 autophosphorylation site on PLK4.
  • the immunostaining procedure is immunofluorescent detection of autophosphorylated PLK4, using for example cultured cells in a flask or plate, or cell smears from tissue samples, biopsies or needle aspirates.
  • the immunostaining procedure is immunohistochemical detection of autophosphorylated PLK4, using for example cell smears from tissue samples, biopsies or needle aspirates, or tissue sections that have been fixed to preserve the tissue structure, e.g. by freezing, or by paraformaldehyde fixation and paraffin embedding.
  • Standard methods for cell or tissue fixation, binding of antibody reagents, and labeling or staining can be employed in these immunostaining procedures (e.g. see Using Antibodies, A Laboratory Manual, edited by Harlow, E. and Lane, D., 1999, Cold Spring Harbor Laboratory Press (e.g. ISBN 0-87969-544-7), particularly chapters 5 and 6 on staining cells and tissues).
  • the step of determining the level of autophosphorylation of S305 of PLK4 in said sample can be performed for example by using a phosphorylated S305-specific antibody, preferably fluorescently or radioactively labeled, or for example by using a sandwich ELISA assay in which a first antibody reagent is specific to PLK4 protein and a second antibody reagent is specific to the S305 autophosphorylation site on a phosphorylated S305-specific antibody, preferably fluorescently or radioactively labeled, or for example by using a sandwich ELISA assay in which a first antibody reagent is specific to PLK4 protein and a second antibody reagent is specific to the S305 autophosphorylation site on
  • S305 antibodies can be used for the measurement of PLK4 phosphorylation level in circulating blood cells such as Leukocytes or circulating tumor cells as a surrogate marker for the activity inhibitors aimed at reducing PLK4 activity.
  • FACS Fluorescence Activated Cell sorting
  • S 305 of PLK4 is determined using a sandwich ELISA assay in which a first antibody reagent is specific to PLK4 protein and a second antibody reagent is specific to the S 305 autophosphorylation sites on PLK4.
  • the first antibody reagent is adsorbed onto a surface (e.g. a plate or dish, e.g. a 96-well plate), a cell extract is prepared from the sample of cells to be tested such that the PLK4 protein is solubilized (e.g.
  • the cell extract is treated if necessary to ensure that any agents used to solubilize PLK4 will not affect antibody binding to PLK4 (e.g. by dilution, addition of detergents such as Triton X-100), PLK4 protein in the extract is adsorbed onto the surface by contacting with the first antibody, and the phosphorylation level of the adsorbed PLK4 is quantitated by contacting with a labeled second antibody reagent that is specific to the S305 autophosphorylation sites on PLK4 .
  • a protein solubilizing agent such as SDS or PROTEOEXTRACT®
  • ELISA methods are particularly advantageous where a rapid assay of PLK4 activity is required, or where large numbers of sample have to be analyzed, e.g. in a high- throughput compound screen.
  • ELISA methods are well known to those of skill in the art, e.g. see International Patent Publication No. WO 95/14930, or Using Antibodies, A Laboratory Manual, edited by Harlow, E. and Lane, D., 1999, Cold Spring Harbor Laboratory Press, (e.g. ISBN 0- 87969-544-7).
  • a dot blot assay may be used for the determination of the level of autophosphorylated PLK4. Accordingly, the latter embodiment provides a method for determining the activity of PLK4 comprising the step of adsorbing the PLK4 protein onto a membrane (e.g. a hydrophobic membrane, nitrocellulose, nylon), and contacting with a labeled antibody reagent that is specific to the S 305 autophosphorylation site on PLK4.
  • a membrane e.g. a hydrophobic membrane, nitrocellulose, nylon
  • the level of phosphorylation of PLK4 is determined by electrophoretic separation of the proteins in the sample and immunoblot analysis using an antibody reagent specific to the S 305 autophosphorylation site on PLK4.
  • electrophoretic separation of the proteins in the sample is achieved by SDS- PAGE.
  • Figure 1 describes the identification of PLK4 autophosphorylation sites within the N- terminus of the kinase:
  • A A diagram showing the position of the kinase domain (KD), crypto Polobox (CPB), Polobox (PB) and PEST sequences (numbered 1, 2 and 3) within PLK4.
  • KD kinase domain
  • CPB crypto Polobox
  • PB Polobox
  • PEST sequences numbered 1, 2 and 3
  • Figure 2 shows multiple autophosphorylation sites exist within PLK4:
  • A A diagram showing the PLK4 fragments that were fused to SUMO, expressed and purified from bacteria.
  • B SUMO-PLK4 fragments were used as substrates in in vitro kinase assays using SUM0-PLK4 1-285. Autoradiography demonstrated that all of the fragments were phosphorylated to varying extent with the exception of fragment 888-971 where no phosphorylation could be detected. It should be noted that fragment 365-639 migrated at a similar rate as SUM0-PLK4 1-
  • Serine residue 305 appeared to be the major autophosphorylation site within PLK4.
  • the plus sign indicates a positive control carried out using a 12 amino acid peptide from RAF containing serine 338 closely matching the consensus phosphorylation sequence of PLK4.
  • Figure 4 shows that serine 305 of PLK4 is autophosphorylated in vitro and in vivo.
  • Lysates were prepared from HCTl 16 cells that had been transiently transfected with EGFP-PLK4 or -PLK4 K41M in conjuction with control or PLK4 siRNA and western blotted with anti-PLK4 CPB and anti-PLK4 pS305 antibodies.
  • the anti-PLK4 pS305 antibody was found to recognize specifically the active form of the kinase in vivo.
  • Figure 5 describes an amino acid sequence of PLK4. However, sequences containing polymorphisms and synonyms are included (e.g. Uniprot PLK4_HUMAN 000444)
  • PLK4 itself is its own substrate and multiple autophosphorylation sites within the protein are identified. It is shown that autophosphorylation of S305 can be used as a marker of PLK4 activity.
  • All SUMO fusion plasmids were created by polymerase chain reaction using the full length PLK4 as a template and cloned into a modified version of pSUMO (Invitrogen ® ). Fusion proteins were expressed in BL21 (DE3) cells (Invitrogen ® ) and purified with HIS-Select HF Nickel affinity gel (Sigma c ). Site directed mutagenesis was performed using a Quickchange XL mutagenesis kit (Strategene ® ) according to manufacturers protocol. Protein expression, purification condition and all primers sequences are further illustrated below.
  • HCTl 16 colorectal carcinoma cells were cultured in McCoys 5 A media (Gibco c ) and Hams F12 media (Gibco ® ) supplemented with 1% L-Glutamine (Gibco ® ), 1% gentamycin and 10% fetal calf serum (Gibco c ) in a humidified incubator (37°C, 5% CO 2 ).
  • HeLa centrin-1 GFP cells were cultered in DMEM supplemented with 10% FCS, 1 % penicillin/streptomycin and 1 % L-glutamine.
  • PLK4 plasmids and/or siRNAs were transfected with Lipofectamine2000 (Invitrogen ® ).
  • PLK4 specific (5' -AAGGACCTTATTCACCAGTTA-S ') and Mismatch (Ambion ® , 4611) siRNA were used at 5OnM.
  • PLK4 specific (5' -AAGGACCTTATTCACCAGTTA-S ') and Mismatch (Ambion ® , 4611) siRNA were used at 5OnM.
  • cells were lysed in triple detergent buffer.
  • Primary antibodies were obtained from the following sources: humanized anti-ninein single chain antibody (Bornens laboratory), anti-gamma tubulin monoclonal antibody (Sigma ® ), anti-C-Napl (BD Biosciences ® ), anti-PLK4 antibody (Abeam ® ), anti- GAPDH (Abeam ® ).
  • HRP- secondary antibodies were obtained from Santa Cruz c .
  • Alexa488 and Alexa680 secondary antibodies were obtained from Molecular Probes ® . Cy3, Cy5, AMCA secondary antibodies were obtained from Jackson Laboratories ® .
  • a proprietary peptide library containing putative phosphorylation sites in PLK4 was prepared by Jerini c using their proprietary microscale technology.
  • the peptides, corresponding locations in PLK4 and mutations of each peptide scanned are displayed in Table I.
  • the peptides were subjected to a kinase reaction using purified SUMO- PLK4]_ 2 85 and processed as described above.
  • HeLa centrin-1 GFP cells growing on fibronectin/collagen-coated coverslips were washed once with PBS and fixed with methanol at -20 0 C for 20 minutes. Coverslips were sequentially washed with PBS and antibody blocking buffer: PBS containing 1% bovine serum albumin fraction V (Sigma ® ) and 0.5% Triton X-100 (Sigma ® ) prior to adding primary antibodies diluted in antibody blocking buffer. Cells were stained with primary antibodies for 1 hour at ambient temperature and washed extensively with antibody blocking buffer before incubating with secondary antibodies, diluted in the same buffer, for 30 minutes at ambient temperature.
  • the coverslips were washed with antibody blocking buffer, DNA stained with a 0.2 ⁇ g/ml solution of 4',6-diamidino-2- phenylindole dihydrochloride (DAPI) (Sigma ® ), and sequentially washed with antibody dilution buffer and distilled water. After allowing to air-dry, the coverslips were mounted onto glass slides using Mowiol mounting medium. Images were captured on a Leica DMRA2 microscope, fitted with a CoolSNAP camera (Princeton Instruments c ), using a IOOX 1.4 N.A. objective lens (Leica c ) and Metamorph software (Universal Imaging ® ). Processing of images was carried out using Metamorph software.
  • DAPI 4',6-diamidino-2- phenylindole dihydrochloride
  • BL21 (DE3) competent cells (Invitrogen ® ) were transformed with the recombinant DNA according to manufacturer's protocol. Following culture upon agar plates containing Kanamycin and 1 % glucose, a single colony was resuspended in 2YT media containing Kanamycin and 0.1% glucose for 3 hours. ImI of the pre-culture was then incubated in 400ml auto-induction medium (Novagen ® ) without glucose at 37° for 3 hours to reach an OD (600nm) of 0.6. At this point the temperature was reduced to 23°C for another 16 hours. The samples were spun down at 3500rpm for 25 minutes at
  • the HIS beads were spun down at 5000 rpm for 5 minutes, washed twice with 2OmM Tris pH8, 30OmM NaCl, 1OmM Imidazole pH8, 0.2% NP40 , 20% glycerol and 0.007% ⁇ mercapto-ethanol for lOminutes under agitating conditions at 4°C. Finally the proteins were eluted from the beads with 2OmM Tris pH8, 30OmM NaCl, 50OmM Imidazole pH8, 0.2% NP40 , 20% glycerol and 0.007% ⁇ mercapto-ethanol for 15 minutes under agitating conditions at 4°C and collecting the supernatant after centrifugation at 5000rpm for 5minutes at 4°C.
  • the primers used for sited directed mutagenesis were:
  • S305A FW S'-CCAGTACCAGTATAAGTGGTGCGTTATTTGACAAAAGAAGAC-S' ;
  • S305A RV 5'-GTCTTCTTTTGTCAAATAACGCACCACTTATACTGGTACTGG-S' ;
  • S305D FW 5'-CCAGTACCAGTATAAGTGGTGATTTATTTGACAAAAGAAGAC-S'
  • S305D RV 5'-GTCTTCTTTTGTCAAATAAATCACCACTTATACTGGTACTGG-S' .
  • Inter-molecular phosphorylation ofPLK4 detectable in various regions of the protein To determine if autophosphorylation occurred elsewhere in the protein, a series of
  • PLK4]_ 2 85 A significant phosphorylation was observed in all fragments apart from PLK4 8 o3-889 and PLK4 8 88-970 where weak and no phosphorylation was observed, respectively.
  • PLK4 3 65-63 9 had a similar migration distance as PLK4i_ 2 85, therefore masking the phosphorylation signal.
  • SyproRuby staining confirmed that similar molarities of substrate protein were used in the kinase reaction ( Figure 2B). These results indicated that multiple sites at various regions of the protein can be phosphorylated by PLK4.
  • S305 is a major autophosphorylation site in PLK4
  • mutant peptides with alanine substitutions were also examined in order to unambiguously determine the phosphorylation site (see Table I for a complete list of scanned peptides).
  • Four peptides contained serine/threonine residues that were phosphorylated by PLK4i_ 2 85 ( Figure 3B). The strongest phosphorylation was observed on peptide 20 containing S305. This peptide contains another serine at residue 303, however, when this was mutated to alanine the phosphorylation of the peptide still occurred (peptide 22).
  • PLK4 and PLK4 K41M were detected with the CPB antibody, however only active PLK4 was detected with the pS305 antibody indicating that this residue is exclusively phosphorylated by PLK4 in cells.
  • Upon co-transfection of a PLK4 specific siRNA complete ablation of total PLK4 and PLK4 K41M was observed and, more importantly, resulted a loss of signal upon detection with the pS305 antibody.

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Abstract

La présente invention porte sur un procédé pour déterminer l'activité de PLK4, consistant à : se procurer un échantillon devant être testé en ce qui concerne l'activité de PLK4, déterminer le niveau d'autophosphorylation de S305 de PLK4 dans ledit échantillon, et déterminer l'activité d'activité de PLK4 dans l'échantillon de cellules, le niveau d'autophosphorylation de S305 étant directement corrélé au niveau d'activité de PLK4.
PCT/EP2009/050179 2008-01-10 2009-01-08 Procédé pour la mesure de l'activité de plk4 Ceased WO2009087196A1 (fr)

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EP08150157.9 2008-01-10

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007146436A2 (fr) * 2006-06-14 2007-12-21 Vertex Pharmaceuticals Incorporated Structure cristalline de la kinase plk3 (polo-like kinase 3) et de ses poches de liaison

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007146436A2 (fr) * 2006-06-14 2007-12-21 Vertex Pharmaceuticals Incorporated Structure cristalline de la kinase plk3 (polo-like kinase 3) et de ses poches de liaison

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JANG, Y-J., ET AL: "Phosphorylation of Threonine 210 and the Role of Serine 137 in the regulation of Mammalian Polo-like Kinase", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 277, no. 46, 15 November 2002 (2002-11-15), pages 44115 - 44120, XP002483097 *
LEUNG ET AL: "Determination of the Plk4/Sak consensus phosphorylation motif using peptide spots arrays", FEBS LETTERS, ELSEVIER, AMSTERDAM, NL, vol. 581, no. 1, 23 December 2006 (2006-12-23), pages 77 - 83, XP005815647, ISSN: 0014-5793 *

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