Tnk1/Kos1 knockout mice develop spontaneous tumors Sarasija Hoare, Kishalay Hoare, Mary K. Reinhard,...
-
Upload
steven-harrell -
Category
Documents
-
view
214 -
download
0
Transcript of Tnk1/Kos1 knockout mice develop spontaneous tumors Sarasija Hoare, Kishalay Hoare, Mary K. Reinhard,...
Tnk1/Kos1 knockout mice develop spontaneous tumors
Sarasija Hoare, Kishalay Hoare, Mary K. Reinhard, Young J. Lee, S. Paul Oh, and W. Stratford May
University of Florida
Cancer Research 68:8723-8732
November 2008
Tnk1/Kos1
Tnk1 is a non-receptor tyrosine kinase
Negatively regulates Ras activation through phosphorylation of Grb2
Do you predict this to be an oncogene or a TSG?
Trans Am Clin Climatol Assoc. 121: 281
Tnk1/Kos1
Alternative splicingIf the exon-intron junction at the end of exon 8 isn’t used, a polyA signal is encounteredEncodes Kos1 (or Tnk1b)
Fig. S2
Image adapted from www.nobelprize.org
Matches partsof Tnk1/Kos1
neor allows the cells to resist the drugs in the neomycin family (like G418)
HSV-tk is the thymidine kinase gene from herpes simplex virus
Making Tnk1/Kos1-/- mice
Some mosaics will be Tnk1/Kos1+/- in their germ cells.
Mate together for Tnk1/Kos1- /-
Image adapted from www.nobelprize.org
Recombinant ‘knockout’ cells injected into blastocysts of C57BL/6 mice
Making Tnk1/Kos1-/- mice
Making Tnk1/Kos1-/- miceDetermine mouse genotype by Southern blotting
Collect genomic DNA from tailDigest with BamHISeparate by gel electrophoresisBlot with “3’ probe”
Wt allele is 7.8kbDeletion allele is 4.2kb
Fig. 1AB
Making Tnk1/Kos1-/- mice
Fig. 1CD
Western blots
Liver Cell Lysates Mouse Embryo Fibroblasts (MEFs)
Only Kos1 (45kD) seen on western blots. No Tnk1 (72kD)
Phenotype of Tnk1/Kos1-/- mice
One tumor histology exampleStained with H&E (hematoxylin and eosin)Hepatocellular carcinoma at 40x or 400x
Fig. 2NO
Kos1 Inhibition of Ras
Are these tumors due to excessive activation of Ras?
How can we determine if Ras is activated or not in vivo?
We know that Ras-GTP binds Raf but Ras-GDP does not bind Raf
Measure how much Ras can bind Raf
Isolate the Ras Binding Domain (RBD) of Raf
Fuse to another protein (GST, glutathione-S-transferase), for easy manipulation
Raf-RBD
Created a GST (Glutathione-S-Transferase) fusion protein
Making a fusion gene: GST stop codon removedfirst codon of Raf-RBD cDNA is just after the last codon of GST
Recombinant Plasmid
Kos1 Inhibition of Ras
Immobilize Raf-RBD by creating a GST fusion proteinGlutathione-S-Transferase binds reduced glutathione (GSH)
Express protein in E. coli
Since GST binds to glutathione, the fusion protein willbind to insoluble beads with glutathione on the surface
GST
Raf-RBD
N
C
Kos1 Inhibition of Ras
Transform recombinant plasmid into E. coli host.
Ptac promoter is derived from the lac promoter
can be strongly induced with IPTG (structurally similar to lactose)
Lyse E. coli.
Purify GST-Raf-RBD on column of reduced glutathione (GSH)-beads.
Wash away other E. coli proteins.
GST
Raf-RBD
Kos1 Inhibition of Ras
Add mouse liver cell lysate (includes Ras-GDP and Ras-GTP)
Only Ras-GTP sticks. Ras-GDP (and all other proteins) are washed away.
Wash.
Elute proteins by denaturation.
Western blot for Ras.
GST
Raf-RBD
Kos1 Inhibition of Ras
Mouse liver cell lysate
Pulled down with GST-Raf-RBD
Ras western blot
Quantified
Fig. 3A
Tnk1/Kos1 Status:
Kos1 Inhibition of Ras
Does lack of Tnk1/Kos1 affect activation of Ras by EGF?
MEFs grown on a plate.
Serum-starved 24 hours
Treated with EGF for 5 min.
Pull-down with GST-Raf-RBD
Western blot for Ras
Fig. 3B
Kos1 Inhibition of Ras
Does lack of Tnk1/Kos1 affect activation of Ras by EGF?
Can also answer this question by looking downstream, at Erk phosphorylation.
Phospho-specific antibody
Normal tissue, tumor or MEFs
Fig. S4
Kos1 Inhibition of Ras
…AUGGAUUAUAAAGACCAUGAUGAUUAUAAAGACCAUGAUGAUUAUAAAGACCAUGAUAUGCUU…
N-Met-Asp-Tyr-Lys-Asp-His-Asp-Asp-Tyr-Lys-Asp-His-Asp-Asp-Tyr-Lys-Asp-His-Asp-Met-Leu-…
HA Epitopes
Start of Kos1
Kos13xHAN-
-C
Kos1
3xHA
Does lack of Tnk1/Kos1 affect activation of Ras by EGF?
Unmodified NIH3T3 cellsOverexpress Kos1
Kos1 on a plasmid with strong promoterHA epitope tagalso a CN = catalytically null allele Lys148 Ala blocks ATP binding
Kos1 Inhibition of Ras
Unmodified NIH3T3 cells. Overexpress Kos1 (or controls). Series of western blots
Fig. S3
HA western blot
Kos1 Inhibition of Ras
Unmodified NIH3T3 cells. Overexpress Kos1 (or controls). Series of western blots
Fig. S3
HA western blot
GST-Raf-RBD pull-down
Ras western blot
Phospho-Erk western blot
Erk western blot
Kos1 Inhibition of Ras
Kos1 Inhibition of Ras
Does Kos1 inhibit Ras?
Which figure makes the strongest argument?Why so much redundancy?
Fig. S4
Fig. 3AB
Fig. S3
Kos1 Phosphorylates Grb2Kos1 is a kinase; does it inhibit Ras activity by phosphorylation?
in vitro kinase assay
Recombinant Grb2 protein
Mammalian cells transfected with plasmid expressing GFP-Kos1 or GFP-Kos1(CN)
GFP-Kos1 was purified by immunoprecipitation (IP)
A
Immunoprecipitation (IP)
Lyse cells.Add anti-GFP antibody
Add Protein A attached to an insoluble bead
Wash away unattached molecules
Kos1 Phosphorylates Grb2
in vitro kinase assay
Kinase: GFP-Kos1Substrate: Grb2 proteinSubstrate: 32P-g-ATP
Separate proteins by electrophoresisExpose gel to film.Radioactivity will expose the film
Kos1 Phosphorylates Grb2
in vitro kinase assay
Kinase: GFP-Kos1Substrate: Grb2 proteinSubstrate: 32P-g-ATP
Separate proteins by electrophoresisExpose gel to film.Radioactivity will expose the film
Kos1 Phosphorylates Grb2
Fig. S6
Another way to detect the phosphorylated products.
Nonradioactive ATP
After allowing the kinase to work,western blot with an anti-phosphotyrosine antibody
Kos1 Phosphorylates Grb2
Fig. S6
Kos1 Phosphorylates Grb2
Fig. 4B
That was in vitro. Does Kos1 phosphorylate Grb2 in vivo?
Cells transfected with Flag-tagged Kos1 (wt or CN)
IP Sos1
Grb2 also co-immunoprecipitates(coIPs)
Series of western blots
Conclusion?
Kos1 Phosphorylates Grb2
Fig. 4C
That was in cell culture. Does Kos1 phosphorylate Grb2 in a living animal?
Livers from knockout miceIP Grb2. Detect phosphorylation with anti-phosphotyrosine antibody western blot
Kos1 Phosphorylates Grb2
Fig. 4D
Hypothesis: Phosphorylation of Grb2 blocks it’s association with Sos1
coIP experiment. Liver cell lysate. IP with Sos1 antibody. Western blot for Grb2.
Is the hypothesis supported or refuted?
Epigenetic Silencing of Tnk1/Kos127% (14/52) of the heterozygous mice developed tumors.
If Kos1 is a tumor suppressor protein, why do these tumors develop?
Hypothesis #1: HaploinsufficiencyTwo functional Tnk1/Kos1 genes are needed to suppress tumors
Hypothesis #2: Somatic mutationThe wildtype allele has been mutated
Hypothesis #3: Epigenetic silencingThe wildtype allele has been silenced
Epigenetic Silencing of Tnk1/Kos1
from
non
-tum
or ti
ssue
Fig. 5A
Testing Hypothesis #1: Haploinsufficiency
GTP-Ras assay on mice
What data would support this hypothesis? GTP-Ras level similar to non-tumor tissue
What data would refute this hypothesis? GTP-Ras level similar to Tnk1-/-
Epigenetic Silencing of Tnk1/Kos1
Fig. 5C
Testing Hypothesis #1: Haploinsufficiency
Also measured Kos1 levels by western blotting
Epigenetic Silencing of Tnk1/Kos1Testing Hypothesis #2: Somatic mutation
Not directly tested. How would you test it?
Epigenetic Silencing of Tnk1/Kos1Testing Hypothesis #3: Epigenetic silencing
Methylation of CpG dinucleotides near promoters leads to silencing
Epigenetic Silencing of Tnk1/Kos1Testing Hypothesis #3: Epigenetic silencing
Purify genomic DNA from tumors
Modify DNA with Sodium Bisulfite
Unmethylated cytosines become Uracils
Methylated cytosines are protected
Epigenetic Silencing of Tnk1/Kos1Testing Hypothesis #3: Epigenetic silencing
Designed PCR primers for 136 bp of Tnk1/Kos1 promoter
Primer: 5’GAAAACGAAAAAAACAACTACGAA3’
Target Site: GAAAACGAAAAAAACAACTACGAACTTTTGCTTTTTTTGTTGATGCTT
if not methylated, primers won’t bind well!GAAAAUGAAAAAAACAACTAUGAACTTTTGUTTTTTTTGTTGATGUTT
-CH
3
-CH3
-CH
3
-CH3
Epigenetic Silencing of Tnk1/Kos1
Testing Hypothesis #3: Epigenetic silencing
Methylation-Specific PCR
Fig. 5B
Aden
ocar
cino
ma
Nor
mal
Liv
er T
issu
e, T
nk1+/
+
Hep
atoc
ellu
lar c
arci
nom
a
Hep
atom
a
Lym
phom
a
Lym
phom
a
Lym
phom
a
Major Conclusions
• Loss of Tnk1/Kos1 increases tumor frequency (Table 1)
• Kos1 inhibits Ras activity (Fig. 3)
• Kos1 phosphorylates Grb2, inhibiting its association with Sos1 (Fig. 4)
• Tnk1/Kos1 promoter can be methylated (Fig. 5)
Major New Techniques for us
• Knockout mouse
• GST fusion proteins
• Epitope tagging
• IP/coIP
• in vitro kinase assay
• Phosphorylation-specific western blot
• Methylation-specific PCR