tnk1/kos1 knockout mice develop spontaneous tumors

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

Adapted from Molecular Cell Biology by Lodish et al.

(ES cells)

Making Tnk1/Kos1-/- mice

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



neor addedHSV-tk lost


Fig. 1A


Sites for restriction enzymes


Fig. 1A

Image adapted from Molecular Cell Biology by Lodish et al.

(heterozygous)

Some mosaics will be Tnk1/Kos1+/- in their germ cells.

Mate together for Tnk1/Kos1- /-


Recombinant ‘knockout’ cells injected into blastocysts of C57BL/6 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


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-/- mice27% (14/52) of the heterozygous mice developed tumors. 72% epithelial

Phenotype of Tnk1/Kos1-/- mice43% (12/28) of the homozygous mice developed tumors. 75% lymphoid

Phenotype of Tnk1/Kos1-/- mice

One tumor histology exampleStained with H&E (hematoxylin and eosin)Hepatocellular carcinoma at 40x or 400x

Fig. 2NO

Phenotype of Tnk1/Kos1-/- mice

Trans Am Clin Climatol Assoc. 121: 281

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


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


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


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


Mouse liver cell lysate

Pulled down with GST-Raf-RBD

Ras western blot

Quantified

Fig. 3A

Tnk1/Kos1 Status:


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



Can also answer this question by looking downstream, at Erk phosphorylation.

Phospho-specific antibody

Normal tissue, tumor or MEFs

Fig. S4


…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


Unmodified NIH3T3 cellsOverexpress Kos1

Kos1 on a plasmid with strong promoterHA epitope tagalso a CN = catalytically null allele Lys148 Ala blocks ATP binding


Unmodified NIH3T3 cells. Overexpress Kos1 (or controls). Series of western blots

Fig. S3

HA western blot


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



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


Kinase: GFP-Kos1Substrate: Grb2 proteinSubstrate: 32P-g-ATP

Separate proteins by electrophoresisExpose gel to film.Radioactivity will expose the film



Kinase: GFP-Kos1Substrate: Grb2 proteinSubstrate: 32P-g-ATP

Separate proteins by electrophoresisExpose gel to film.Radioactivity will expose the film


Fig. S6

and a few controls


Fig. S6

Kos1 also phosporylates itself!


Fig. S6

Another way to detect the phosphorylated products.

Nonradioactive ATP

After allowing the kinase to work,western blot with an anti-phosphotyrosine antibody


Fig. S6


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?


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


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-/-


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


Purify genomic DNA from tumors

Modify DNA with Sodium Bisulfite

Unmethylated cytosines become Uracils

Methylated cytosines are protected


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


Testing Hypothesis #3: Epigenetic silencing

Methylation-Specific PCR

Fig. 5B

Aden

ocar

cino

ma

Nor

mal

Liv

er T

issue

, Tnk

1+/+

Hepa

toce

llula

r car

cino

ma

Hepa

tom

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

tnk1/kos1 knockout mice develop spontaneous tumors

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