By: Chris CarrBy: Chris Carr

Background InformationBackground Information

Theileria parvaTheileria parva is an intracellular protozoan is an intracellular protozoan parasite that is transmitted by ticks and causes parasite that is transmitted by ticks and causes East Coast fever, a disorder of cattle in East East Coast fever, a disorder of cattle in East and Central Africa and Central Africa

They invade and immortalize bovine They invade and immortalize bovine lymphocyteslymphocytes

The major playersThe major players

C-MycC-Myc: a transcription factor that binds to specific sites in the : a transcription factor that binds to specific sites in the promoters of at least 1382 different genespromoters of at least 1382 different genes

JAK2/STAT3JAK2/STAT3: a signalling pathway that contributes to c-Myc : a signalling pathway that contributes to c-Myc transcriptiontranscription

Mcl-1Mcl-1: target gene of c-Myc: target gene of c-Myc CK2CK2: a factor that increases the stability of c-Myc: a factor that increases the stability of c-Myc BW720cBW720c: a drug that kills : a drug that kills TheileriaTheileria AG 490AG 490: a drug that inhibits JAK2 activity: a drug that inhibits JAK2 activity PARPPARP: poly(ADP-ribose) polymerase: poly(ADP-ribose) polymerase Z-VADZ-VAD: Caspase inhibitor: Caspase inhibitor

What on earth is this about?What on earth is this about?

TheileriaTheileria initiates a strong and sustained initiates a strong and sustained induction of c-Mycinduction of c-Myc

This is caused by intervening with both c-Myc This is caused by intervening with both c-Myc transcription and stabilitytranscription and stability

Infection then results in a c-Myc mediated Infection then results in a c-Myc mediated antiapoptotic responseantiapoptotic response

How is this possible?How is this possible?

Activation of CK2Activation of CK2 Activation lf NF-Activation lf NF-κκB signalling pathwayB signalling pathway Induction of anti-apoptotic proteinsInduction of anti-apoptotic proteins Decrease in the number of pro-apoptotic proteinsDecrease in the number of pro-apoptotic proteins Infected lymphocytes use autocrine loopsInfected lymphocytes use autocrine loops

-TNF autocrine loop-TNF autocrine loop NF-NF-κκB activationB activation

-GM-CSF autocrine loop-GM-CSF autocrine loopP13K activation

AP-1 induction

What are autocrine loops?What are autocrine loops?

A process of cell signaling whereby a cell both A process of cell signaling whereby a cell both releases and responds to a soluble factorreleases and responds to a soluble factor

The loops refer to the signal both originating The loops refer to the signal both originating and ending in the same locationand ending in the same location

Experiment 1: Theileria infection leads Experiment 1: Theileria infection leads to phosphorylation of STAT3, and to phosphorylation of STAT3, and

induction of c-Myc and Mcl-1 (part 1)induction of c-Myc and Mcl-1 (part 1) Theileria pursuade lymphocytes to proliferate Theileria pursuade lymphocytes to proliferate

uncontrollably via GM-CSF autocrine loop.uncontrollably via GM-CSF autocrine loop.

Does this proliferation occur in tandem with a signalling pathway?

Is the end result of this signalling pathway an increase in c-Myc levels?

YES

Infected

Non-

infected

Experiment 1: Part 2Experiment 1: Part 2

Are the phosphorylation of STAT3 and the induction Are the phosphorylation of STAT3 and the induction of c-Myc due directly to infection of Theileria?of c-Myc due directly to infection of Theileria?

Of Course

Note: Stat3 is a protein that is present consistently with or without infection. However, the phosphorylation of this protein is dramatically increased upon infection.

BW 720c was used to kill Theileria

Wait there’s moreWait there’s moreExperiment 1: Part 3Experiment 1: Part 3

Does STAT3 signalling to c-Myc involve the JAK2 Does STAT3 signalling to c-Myc involve the JAK2 kinase?kinase?

Indubitably

AG490 was used to inhibit JAK2

Over the course of the experiment, blocking JAK2 signalling resulted in a greater decrease in STAT3 phosphorylation than eliminating Theileria altogether

Experiment 2: Experiment 2: TheileriaTheileria dependent dependent post-translational stabilization of c-post-translational stabilization of c-

Myc involves CK2Myc involves CK2 Does parasite infection affect the stability of the c-Myc Does parasite infection affect the stability of the c-Myc

protein?protein?

BW 720c was used to kill Theileria

Cyclohexamide was used to inhibit the transcription of c-Myc

In B-cells containing Theileria, c-Myc half-life was significantly prolonged

Yes

Experiment 2: continuedExperiment 2: continued

The presence of live Theileria parasites leads to a CK2-The presence of live Theileria parasites leads to a CK2-mediated increase in the stability of c-Mycmediated increase in the stability of c-Myc

Apigenin was used as a CK2 inhibitor

Experiment 3: Theileria-induced Experiment 3: Theileria-induced transcriptional activation of c-Myc is transcriptional activation of c-Myc is

mediated in part by GMC-SF via activation of mediated in part by GMC-SF via activation of STAT3STAT3

Does Does TheileriaTheileria induce high c-Myc levels by induce high c-Myc levels by transcriptional regulation?transcriptional regulation?

Theileria induces a four-fold increase in c-Myc driven luciferase activity

Yup

Experiment 3: ContinuedExperiment 3: Continued

C-Myc transactivation is dependent on live parasites

C-Myc binds to the luciferase gene which results in luciferase activty

Experiment 3: Yes, there is moreExperiment 3: Yes, there is more

Addition of recombinant GM-CSF to infected cells increased endogenous c-Myc transactivation

Experiment 3: further continued Experiment 3: further continued

Luciferase activity is decreased because there are an increased number of binding sites for c-Myc to attach to.

A promotor containing 4 E-box elements were introduced upstream from the luciferae gene.

Thus, target gene transcription is reduced.

Experiment 3: Wait, there is moreExperiment 3: Wait, there is more

The co-transfection of different mutants significantly lowered c-Myc driven luciferase activity

Therefor, transcriptional induction of c-Myc clearly involves JAK2 and STAT3

cont

rol

Kin

ase

dead

JA

K2

Experiment 4: Theileria-Experiment 4: Theileria-transformed B cell survival is c-Myc transformed B cell survival is c-Myc

dependentdependent How significant is the contribution of c-Myc to the How significant is the contribution of c-Myc to the

survival of survival of TheileriaTheileria-infected B cells?-infected B cells?

C-Myc transcription was interfered with by the addition of antisense oligonucleotides

Very Significant

Non

-sen

se

Ant

i-se

nse

Experiment 4: continuedExperiment 4: continued

Cell death correlated to a loss of c-Myc levels, Mcl-1 expression, and PARP cleavage

Thus, Mcl-1 can be directly attributed to c-Myc activation

Experiment 5: Inhibition of JAK2 Experiment 5: Inhibition of JAK2 results in caspase-dependent apoptosis of results in caspase-dependent apoptosis of

Thjeileria- transformed B cellsThjeileria- transformed B cells

AG 490 induced apaptosis is caspase dependent

Experiment 5: continuedExperiment 5: continued

LETD-AFC is a caspase 8 substrate

DEVD-AFC is a general caspase substrate

There is no LETD activity which means caspase 8 is not a part of this mechanism of cell death

Experiment 5: Can you handle it?Experiment 5: Can you handle it?

In correlation with caspase 9 activation, there is PARP cleavage (fig 5c)

There is also Annexin-V positivity and nuclear fragmentation (fig 5d)

Therefore, the inhibition of the JAK2 signalling pathway leads to B-cell apoptosis

Experiment 6: Ectopic expression of c-Experiment 6: Ectopic expression of c-Myc rescues Theileria infected B cells Myc rescues Theileria infected B cells

from AG 490 mediated apoptosisfrom AG 490 mediated apoptosis

Apoptosis results when AG 490 blocks JAK2 activity, however, the addition of ectopic c-Myc reverts cell death.

Experiment 6: continuedExperiment 6: continued

Ectopic expression of CMV-cMyc augments c-Myc levels, but does not effect STAT3 or its phosphorylation status

Results: Yes, the experiments are Results: Yes, the experiments are finally overfinally over

Theileria infection leads to increased levels of c-Myc

The life of the c-Myc transcription factor is prolonged due to CK2-mediated phosphorylation

A JAK 2/STAT3 signalling pathway also contributes to increased c-Myc transcription

This anti-apoptotic process can be inhibited at several junctures (Apigenin, BW 720c, AG 490)

Further studiesFurther studies

Other cytokines may be secreted that activate Other cytokines may be secreted that activate JAK2 and c-Myc by yet-to-be described JAK2 and c-Myc by yet-to-be described autocrine loopsautocrine loops

How do P13K activation and AP-1 induction How do P13K activation and AP-1 induction lead to the phosphorylation of STAT3lead to the phosphorylation of STAT3

The EndThe End

Now you may start clapping