Download Figure 2 - York College of Pennsylvania

km23: a Novel Protein in the TGF Signaling Pathway
Nicole Dague
Department of Biological Sciences, York College of Pennsylvania
ABSTRACT
Cell Culture:
293T cells were chosen for experimentation. This particular
cell line is epithelial and was derived from the human
kidney. The cells were designed to over-express the Tumor
(T) antigen, which in turn would enhance expression of an
encoded protein (km23). Cell media was DMEM + 10%
FBS.
Figure 1
CONCLUSION
TGF
TGF
TGF
RI
INTRODUCTION
METHODS
RI
An innovative method for screening expression
libraries for TGF interacting proteins was developed by
Dr. Kathleen M. Mulder at the Pennsylvania State
University College of Medicine. The screening method
isolated a novel TGF receptor-interacting protein named
km23. Research was conducted to determine if km23
was present with TGF intracellularly and if km23 was
phosphorylated as a means of initiating signaling.
Western blots served as controls for each experiment.
Experimentation confirmed that km23 was present with
TGF intracellularly and that km23 was phosphorylated
by TGF. Further experimentation is necessary to
determine km23’s exact role in the TGF signaling
pathway.
P
2. RII causes RI to bind
3. RI phosphorylated,
1. TGFß binds
signal is initiated
to RII
Nucleus
Verify km23 present with TGF ß intracellularly:
Figure 1: Signal transduction through TGF ß-receptor phosphorylation and activation.
Model adapted from Yue and Mulder (2001).
•293T cells transiently transfected with tagged receptors
or km23 using LipofectAMINE PlusTM. Product procedures
Figure
2
were followed.
•Cells were labeled with 125I- TGF for 4hr at 4°C.
km23-Flag
•DSS cross-linking agent added for 15 min.
RI-myc
•Lysates immunoprecipitated (IP’d) with anti-flag M2 Ab.
RII-HA
unlabelled TGF
•Visualized with SDS-PAGE and autoradiography
•Western blotting confirmed the expression of receptors
and km23 in relevant lanes
•Repeat experimentation confirmed results.
Cancer is one of the top afflictions of Americans
today. In the year 2002, 1,284,900 new cancer cases
are expected to be diagnosed (American Cancer Society
2002). Transforming growth factor (TGF) plays
an essential role in the development of cancers and has
become a popular target for research. TGF functions as
a natural potent growth inhibitor of solid tumor formation
of a number of cell types. Past research has
demonstrated that TGF initiates cell signaling by
Determine if km23 is phosphorylated by RI/RII TGF ß
phosphorylating intracellular proteins. TGF is known to receptor complex:
interact with two receptors on the cell surface, TGF
•293T cells were transiently transfected with tagged
Receptor I (RI) and TGF Receptor II (RII). (See Figure
receptors or km23
1) Known molecular weights for the receptors have been
•In vivo phosphorylation assays conducted (Methods
published: RI ranges from 65-70kDa and RII from 85from Yue and Mulder 1999). 3h labeling period with
110kDa (Yue and Mulder 2001).
[32Pi], and IP’s with anti-flag Ab.
An alteration in the expression of TGF or in the
•Western blotting confirmed expression in relevant lanes
proteins of its signaling pathways has been associated
•Results were indicative of three experiments.
with human diseases (Yue and Mulder 2001). Since
TGF has been demonstrated to participate in signaling
RESULTS
pathways that have been implicated in human
pathologies, understanding it’s vital role in the
km23
was
present
with
TGF
ß
intracellularly
(Figure
2)
development of cancer may be the key to finding a cure.
Dr. Mulder’s method for screening expression libraries
•Top panel: total cell lysates confirm position of RI and
has isolated a novel protein involved in the TGF RII (lane 2). Banding noted in Lane 5 indicates km23
signaling pathway, named km23. Initial experimentation
present with RI/RII complex.
on km23 determined it to be a 96 amino acid protein,
encoded by a 291 base pair reading frame with a
•Lower panels: Western blots for Flag, myc, and HA
predicted molecular weight of 10.6667kD. Western
confirmed expression in relevant lanes
blotting determined actual molecular weight to be 11kD.
km23 was phosphorylated by the RI/RII TGF ß receptor
km23 was believed to play a role in the TGF
complex (Figure 3)
pathway and the discovery of its role became the goal of
•Top panel: Expression of km23, RI or RII alone did not
this project. Since previous research had demonstrated
result in phosphorylation (lanes 2,3,4). km23 not
that TGF phosphorylates intracellular proteins as a
phosphorylated when expressed in cell (lane 2).
mechanism for initially signaling events, a key
Phosphorylation of km23 with RI/RII receptor complex
component to our research was to determine if km23
(banding in lane 5).
was phosphorylated by TGF. As a component in the
pathway, km23 would have the potential to serve as a
•Lower panel: Western blot analysis confirmed
therapeutic and, or diagnostic target in the battle against
expression of km23-flag.
cancers.
-
+
+
+
-
-
+
-
+
+
+
-
+
+
+
+
RII
RI
1
2
3
cell
lysates
4
5
FURTHER RESEARCH
6
Flag IP
Western Blots for
Experimental Controls:
Flag Ab:
km23
1
2
3
4
5
6
myc Ab:
RI
1
2
3
4
5
RII
1
2
3
4
5
6
Figure 2: km23 is present with the RI/RII immunocomplexes intracellularly.
Figure 3
+ - - - - - + - - + - - + - + +
- - - + + +
km23
1
2
3
IP Ab:
4
5
6
Flag
Flag Western Blot
Experimental Control:
km23
1
2
3
4
5
6
Figure 3: km23 is phosphorylated by the RI/RII complex.
Further research has been conducted since the time of
initial experimentation. Additional research has
concluded that once bound to the receptor complex,
km23 can mediate at least one signaling event after
TGF activation and that km23 is also associated with
the motor protein dynein. The eventual goal is to
determine if km23 can serve as a therapeutic or
diagnostic target in the battle against cancer.
6
HA Ab:
Empty Vector
km23-Flag
RI-V5
RII-HA
Experimentation supported hypothesis that km23
was present with TGF intracellularly. (See Figure 2)
Banding noted in lanes where km23 was complexed with
both TGF receptors (see Lane 5). Unlabelled TGF
served as a control and no banding was expected (see
Lane 6). Separate western blots confirmed the presence
of each component (km23, RI, or RII) by blotting for
their specific tags. (See lower panels).
Further experimentation supported that km23 was
phosphorylated by TGF. (See Figure 3). Banding was
noted in lane 5, when km23 was complexed with the
RI/RII complex. Western blots confirmed the presence
of km23.
Data supported the hypothesis that km23
was present with TGF intracellularly and that
km23’s phosphorylation by TGF suggests that
km23 is part of a signaling pathway.
LITERATURE CITED
Derynck, R. and Feng, X. 1997. TGF-β receptor signaling.
Biochimica et Biophysica Acta 1333: F105-F150.
Hill, C.S. 1996. Signaling to the Nucleus by Members of the
Transforming Growth Factor- β (TGF β) Superfamily.
Cell Signaling 8: 533-544.
Tang, Q., Staub, C.M., Gao, G., Qunyan, J., Aurigemma, R.,
Mulder, K.M. 2002. A novel TGFß receptorinteracting protein that is also a light chain of the motor
protein dynein. Molecular Biology of the Cell 13: In press.
Yue, J. and Mulder, K.M. 2001. Transforming growth factor-β
signal transduction in epithelial cells.
Pharmacology & Therapeutics 91: 1-34.
ACKNOWLEDGEMENTS:
Dr. Kathleen M. Mulder, Professor
Department of Pharmacology
Pennsylvania State College of Medicine
Cory Staub, Senior Research Technician
Qian Tang, Research Technician
Dr. Jeffery Thompson, Faculty Advisor