In vitro studies of binding interactions that regulate the function of Noxa in leukemia cells
UROP Investigation Proposal
Mentor: Dr. Ameeta Kelekar
Background: The ability for tumor cells to avoid programmed cell death (PCD) or apoptosis can play a
significant role in their resistance to conventional chemotherapeutic treatments and often leads to an
unfavorable prognosis (Kasibhatla and Tseng, 2003). Apoptosis alone is a highly regulated process that
recruits the use of many different proteins. One such class of these proteins is the Bcl-2 family of
apoptosis (programmed cell death) regulators. The Bcl-2 family comprises pro-death (pro-apoptotic) and
pro-survival (anti-apoptotic) proteins that are mainly responsible for regulating the mitochondrial or
intrinsic apoptosis pathway. Interestingly, overexpression of the pro-survival Bcl-2 proteins is often
observed in hematopoietic cancers and underlies the resistance to the cell death and unfavorable
pathogenesis that is characteristic of malignant cells (Lessene, Peter, and Czabotar, 2008).
Among the many Bcl-2 proteins, one of interest is Noxa. Noxa is a pro-apoptotic Bcl-2 family
member that functions by binding to the pro-survival protein Mcl-1L. The interaction promotes
apoptosis by inactivating Mcl-1L and targeting it for degradation. In most epithelial cells and cancers
Noxa is expressed only in response to a death stimulus and its promoter is the target of both tumor
suppressors as well as oncogenes that function as transcription factors, such as p53, HIF-1 and myc
(Ploner, Kofler, and Villunger, 2009). Recently, Dr. Ameeta Kelekar’s group observed steady
expression of Noxa across various human hematopoietic (blood) cancers (Lowman et al, 2010) that
suggested the protein could be modified to mask its death-promoting properties. Bioinformatic analysis
of Noxa in a hematopoietic cancer setting revealed a serine site at the 13th residue of the protein (Ser13)
that could be a likely target for post-translational modification via phosphorylation, specifically by
action of the cyclin dependent kinase 5 (Cdk5) (Lowman et al, 2010). Further analysis revealed that the
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phosphorylation was a novel regulatory switch that converted Noxa from a pro-death to a pro-survival
protein allowing the cancer cells to avoid apoptosis. The authors also discovered that this regulatory
mechanism was glucose dependent; in that in the presence of glucose, Noxa was phosphorylated and
became a pro-survival protein while in the absence of glucose, Noxa lost the modification and became a
promoter of death.
These findings raised a number of interesting questions. While it was evident that nonphosphorylated Noxa was able to bind to Mcl-1L and promote entry of cells into the apoptosis pathway
(Lowman et al, 2010) it was unclear how phosphorylated Noxa imparted protection. Notably, the
investigators were able to successfully pull down Noxa in its native form from leukemia cell extracts in
the absence, but not in the presence, of glucose. This observation suggested that Noxa phosphorylation
results in a conformational change in the protein that alters its interaction properties or its function or
both. Preliminary studies suggested that the modified protein was unable to interact with Mcl-1L, but
this needs to be further investigated (Lowman et al, 2010). Unpublished biophysical analyses of Noxa
and phospho-Noxa revealed profound differences in the structure of each protein. It is therefore posited
that conformational protein modifications conferred upon Noxa subsequent to glucose enhanced
phosphorylation results in the alteration of the Noxa protein that limits its apoptotic potential. Another
key question raised by the inaccessibility of phosphorylated Noxa to immuoprecipitation antibodies
(Lowman et al), led to the discovery that the modified protein was encompassed in a multi-protein
particle in proliferating, non-apoptotic cells (manuscript in preparation). Proteomic analysis has revealed
that one of the components of this particle is PHLPP2 (PH Domain and Leucine Rich Repeat Protein
Phosphatase-2). A phosphatase is a protein that is responsible for reversing phosphorylation in that it
catalyzes the removal of the phosphate group. Early studies indicate that Noxa is a target of PHLPP2
activity. Since phosphatases are known to be tightly regulated tumor suppressors often found localized
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in proximity to their targets, these data suggested that in the absence of glucose, PHLPP2 becomes
activated and dephosphorylates Noxa, converting the protein to its pro-death form.
Hypothesis and Specific Aims: Based on work described earlier, it is hypothesized that Noxa will
interact with Mcl-1L only when it is unphosphorylated whereas the phosphorylated form of Noxa will
interact with the phosphatase PHLPP2, but fail to interact with Mcl-1L. To test this hypothesis the
following two specific aims are proposed:
Aim 1: Purify His epitope-tagged recombinant Mcl-1L protein and analyze the interactions of
full length non-phosphorylated and Ser13 phosphorylated Noxa peptides with the purified Mcl1L using in vitro binding assays, immunoprecipitation and Western blotting.
Aim 2: Explore interactions between PHLPP2 and Noxa in vitro. Members of the Kelekar lab
have been able to successfully co-purify Noxa and PHLPP2 from leukemia cell extracts,
however, details of direct interactions between Noxa and PHLPP2 await analysis. FLAG
epitope-tagged purified recombinant PHLPP2 protein will be purified and its interactions with
non-phospho- and phospho-Noxa will be analyzed using in vitro binding assays, co immunoprecipitation and Western blotting.
Experimental Plan: To complete this investigation, the student will use a variety of experimental tools
common in protein interaction research. In separate experiments, his-tagged Mcl-1L and FLAG-tagged
PHLPP2 plasmids will be transfected into a bacteria and mammalian host culture, respectively. Briefly,
Mcl-1L and PHPP2 will then be purified by the appropriate purification technique (e.g. FLAG
purification) and binding studies between the purified proteins and de-novo synthesized Noxa and
phospho-Noxa will be performed. Using monoclonal antibodies targeting Mcl-1L or PHLPP2, coimmunoprecipitations will then be used to isolate the protein complexes containing Mcl-1L or PHLPP2
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and Western blotting probing for Noxa and phospho-Noxa will examine whether or not the two forms of
Noxa interact with Mcl-1L and PHLPP2.
Expected Outcome: Firstly, it is expected that the hypothesis presented will be supported. Dr. Kelekar
is one of the leading experts on Noxa and her group is responsible for the remarkable amount of
preliminary work done on Noxa (described earlier) and utilized in the development of the presented
hypothesis. But there is always the chance that the hypothesis will be refuted and PHLPP2 and Noxa do
not bind or phospho-Noxa and Mcl-1L interact. Despite the amount of circumstantial preliminary work
done it can never be fully predicted what the exact result of the investigation will be; as such, the
investigators are prepared for all the possible outcomes. As with all scientific investigation, problems are
expected to arise. To prepare for these often unavoidable encounters, the student will thoroughly review
the current experimental protocols set in place and research alternative methods to adopt, if necessary, to
be able to continue on with the investigation in a timely manner. A poster and abstract presentation will
certainly result from this work as well as being included in a future publication on the topic coming out
of the Kelekar lab.
Significance of research: Defective apoptosis presents a troubling hurdle to overcome in the treatment
of cancer. In cancer, the apoptosis pathway goes awry allowing the cancer cells to bypass cell death and
resist the effects of conventional chemotherapy treatment resulting in unfavorable prognosis.
Understanding the mechanisms of regulating apoptosis is critical in expanding the current chemotherapy
repertoire that includes drugs that are effective and overcome the cancer’s drug resistance. The results of
this UROP investigation will add a tremendous amount of knowledge on the regulation of apoptosis via
modulating the pro – death activity of Noxa. This project will also explore the potential of developing
the Noxa/PHLPP2 axis into a novel chemotherapeutic resource.
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References
Kasibhatla, S., Tseng, B. Why target apoptosis in cancer treatment? Mol. Cancer. Ther., 2: 573 –
580, 2003.
Lessene, G., Czabotar, P., Colman, P. Bcl – 2 family antagonists for cancer therapy. Nature
Reviews Drug Discovery, 7: 989 – 1000, 2008.
Xazmin H. Lowman, Maureen A. McDonnell, Ashley Kosloske, Oludare A. Odumade, Christopher
Jenness, Christine B. Karim, Ronald Jemmerson, Ameeta Kelekar. The proapoptotic function of
noxa in human leukemia cells is regulated by the kinase cdk5 and by glucose. Molecular Cell,
40: 823 – 833.
Ploner, C., Kofler, R., Villunger, A. Noxa: at the tip of the balance between life and death.
Oncogene, 27: S84 – S92, 2009.
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