Summer Undergraduate Research Program - Fred Hutchinson ...

Regula'on of Cdk2 Ac'vity by Posi've Feedback Control
Madeline M. Schu. 1 , Bridget T. Hughes2 , Bruce E. Clurman2 1Whitman College, Walla Walla , WA; 2Fred Hutchinson Cancer Research Center, Sea.le, WA
Gamma Irradia'on Does Not Induce
Cdc25a Degrada'on
Results
Abstract
2011 Best Poster Award
Figure 6. The indicated cell
lines were exposed to 10 Gy
of gamma irradiaKon and
harvested at the specified
Kme points. Whole cell
extracts were
immunoblo.ed for Cdc25a,
Wee1, Cdk2 and p21. Cell
lysates were
immunoprecipitated with
Cdk2 anKbody and
immunoblo.ed for
phospho-­‐Y15 Cdk1/2 and
Cdk2.
Cdk2 Inhibitory Phosphoryla'on is Reduced in
Cell Lines with Increased Cdk2 Ac'vity
Figure 3. The indicated cell lines were either grown asynchronously or treated with HU. Cell lysates were
immunoprecipitated with Cdk2 anKbody and immunoblo.ed for phospho-­‐Y15 Cdk1/2 and Cdk2.
Growth Curve of Cdk2AF Cell Lines
The cell cycle is a very Kghtly regulated process and the fidelity of cell division is crucial
to the prevenKon of tumorigenesis and cancer growth. The key cell-­‐cycle regulators are cyclin-­‐
dependent kinases (Cdks), protein kinases which, upon binding their regulatory molecule cyclins,
become acKvated and phosphorylate target proteins o^en involved in cell cycle regulaKon. In
addiKon to cyclin binding, acKvity of Cdks is also controlled by both acKvaKng and inhibitory
phosphorylaKons. To be fully acKve, Cdks must be phosphorylated on T160 by Cdk acKvaKng
kinase (CAK) and the inhibitory phosphorylaKons on T14 and Y15 must be removed by the Cdc25
phosphatase. It is known that Cdk1 can regulate its own acKvity by inhibiKng Wee1 (the Y15
kinase) and acKvaKng the Cdc25c phosphatase. This posiKve feedback control by Cdk1 is, in part,
responsible for the switch-­‐like mechanism of Cdk1 acKvaKon at the start of mitosis. It has been
suggested that Cdk2, the G1/S-­‐phase Cdk, can also feed back to regulate its own acKvity by a
similar mechanism, however it has not been formally shown in vivo. To invesKgate whether Cdk2
can regulate its own inhibitory phosphorylaKon in vivo, we used a geneKc approach and
generated cell lines that contain a mutaKon in T14 and Y15 (Cdk2AF) that prevents inhibitory
phosphorylaKon. Using immunoprecipitaKon and immunoblo_ng to assay Y15 phosphorylaKon,
we show that Y15 inhibitory phosphorylaKon is reduced in Cdk2AF/+ cell lines compared to a
Cdk2+/-­‐ cell line (both of which have one WT Cdk2 allele). This suggests that the hyperacKve
Cdk2AF can regulate Y15 phosphorylaKon on the WT copy of Cdk2. The differences in Y15
phosphorylaKon between these cell lines is not due to a difference in Wee1 or Cdc25a protein
levels, and shRNA-­‐mediated knockdown of Wee1 failed to rescue the difference in Y15
phosphorylaKon implying a Wee1-­‐independent mechanism. These data support the hypothesis
that Cdk2 regulates its own inhibitory phosphorylaKon by a posiKve feedback mechanism and
future experiments will test if changes in Cdc25a acKvity or an unknown mechanism contributes
to this posiKve feedback control. These findings provide further insight into regulaKon of Cdk2
acKvity during the cell cycle and may provide addiKonal therapeuKc targets for cancers
overexpressing the Cdk binding partners, cyclins A and E.
Doubling Time:
WT 20 Hrs
+/Neo 20.5 Hrs
+/AF 21.1 Hrs
AF/AF 23.8 Hrs
Analysis of Wee1 and Cdc25a Protein
Levels in Cdk2AF Cell Lines
149
B
A
B
Figure 7. These four cell lines were seeded at 88,000 cells per well in a 12-­‐well plate
on day zero in triplicate. Cell number was counted daily over a four day period.
Cdk Inhibitory Phosphoryla'on
Conclusions/Future Direc'ons
C
C
Cdk Cdk cyclin Cdk
cyclin
cyclin Cdk cyclin
P
CAK Wee1 P
P P
Cdc25
Ac've Inac've
The data support the hypothesis that Cdk2 regulates its own inhibitory
phosphorylaKon by a posiKve feedback mechanism.
Figure 4. (A) The indicated cell lines were either grown asynchronously or treated with HU. Cell lysates were
immunoprecipitated with Cdk1 anKbody and immunoblo.ed for phospho-­‐Y15 Cdk1/2 and Cdk1. (B) The indicated cell
lines were either grown asynchronously or treated with HU. Whole cell extracts were immunoblo.ed for Wee1 or (C)
Cdc25a. (B) and (C) are from separate experiments.
Figure 1. Cyclin-­‐Cdk acKvity is regulated by both acKvaKng and inhibitory phosphorylaKons. CAK
phosphorylates T160 which is required for Cdk acKvaKon. Wee1 phosphorylates T14 and Y15 which
inhibits Cdk2. These phosphorylaKons must be removed by the Cdc25 phosphatase for the complex
to become acKve.
It is not the difference in Wee1 or Cdc25a expression across cell lines that is causing
the difference in Y15 phosphorylaKon.
It is possible that the observed difference in Y15 phosphorylaKon is a result of
changes in Cdc25a or Wee1 acKvity, not protein levels. AlternaKvely, an unknown
mechanism could be controlling this process.
Methods
In the future, the difference in Cdc25a and Wee1 acKvity will be measured using a
Cdc25a phosphatase assay and a Wee1 kinase assay, respecKvely.
Knockdown of Wee1 Does Not Rescue
Differences in Y15 Phosphoryla'on
AF
Cdk2
AF
Cdk2
AF
Cdk2
P
Cdk2
Cdk2
P
Cdk2
Cdk2
P
Cdk2
P
Acknowledgements
This work was funded by Grant R01 CA102742. Bridget T. Hughes is a recipient of an
American Cancer Society Postdoctoral Fellowship. The Summer Undergraduate
Research Program is supported in parts by the Cancer Center Support Grant (CCSG)
CURE Supplement: 5 P30 CA015704-­‐37S1 and FHCRC AdministraKon. Special thanks
to the Clurman Lab members for all their help and support!
Figure 5. The indicated cell lines were infected with a virus containing a non-­‐silencing control shRNA or Wee1
shRNA and treated with HU. Cell lysates were immunoprecipitated with Cdk2 anKbody and immunoblo.ed
for phospho-­‐Y15 Cdk1/2 and Cdk2. Whole cell extracts were immunoblo.ed for Cdk2 and Wee1.
WT +/Neo +/ AF AF/AF
Figure 2: Diagram of cell lines used in these experiments. The cell lines +/AF and AF/AF were generated using standard
AAV gene targeKng; the +/Neo cell line was an intermediate in this process.
Cells were cultured in DMEM with 10% FBS and HU treatment (2mM) was done for
~17hrs.
ImmunoprecipitaKon and immunoblo_ng were done using standard techniques.
shRNA mediated knockdown of Wee1 was performed using a lenKviral shRNA
construct obtained from Open Biosystems.