Transcript Phosphorylation of Pitx2 Elizabeth A. Hardardt, SOU Mentors

Phosphorylation of Pitx2
Elizabeth A. Hardardt, SOU
Drs. Theresa Filtz and Chrissa Kioussi, OSU,
Mentors
06/29/2009
Significance
Source:
http://www.benettontalk.com/obese.j
pg(top)
http://media.photobucket.com/image
/muscles/bidule-78/muscles-1.jpg
Significance, cont’d
• Muscle diseases
o
o
Axenfeld-Rieger syndrome
Lack of left/right asymmetry of the heart
• Development and regeneration of
muscle tissue
• Further understanding of human life
Sources:http://www.molvis.org/molvis/v
13/a108/v13a108f1.jpg (top);
http://www.landesbioscience.com/curie/
chapter/1865/
Muscle Development and Regeneration
• All cells contain the genome, regulation
determines which genes are expressed
o
occurs during any stage of gene expression
• Muscle development (myogenesis)
o
Is positively regulated by Pitx2
Source: http://missinglink.ucsf.edu/lm/IDS_101_histo_resource/nerves_muscle.html#118
Regulation of Gene Expression
• Transcription
Source:
https://eapbiofield.wikispaces.com/Transcription+and+Tran
slation+KW
Source:
http://serc.carleton.edu/microbelife/research_methods/ge
nomics/transcrip.html
Regulation of Gene Expression
• Post-translational modification
Source: http://www.thestandard.org.nz/wp-content/uploads/2003/01/phosphate.gif (top);
http://kinasephos.mbc.nctu.edu.tw/document.html
Hypothesis
Wnt and growth factor signal the phosphorylation
of Pitx2, switching its transcription activity from
repression to activation
C2C12 Culture
Stimulation/Extraction
• C2C12 cells
• With/without Calyculin, which induces
hyperphosphorylation
• Varying stages of development
• Total cell lysate
• Embryonic skeletal muscle cells
•
•
•
•
10% fetal bovine serum
10 mM LiCl
Varying incubation time
Immunoprecipitation
Methods
o
o
o
o
Stimulation
Immunoprecipitation or Total cell extraction
Protein assay (Lowry method)
Separation & detection (Western Blot)
Source:
http://thunder.biosci.umbc.edu/clas
ses/biol414/spring2007/index.php/
Co-immunoprecipitation
Results
E12.5
Limb
E12.5
Limb
E12.5
Limb
+Li+
E12.5
Limb
+Li+
PM
PM
+Li+
Experiment 1. Western Blot: overnight development, α-Pitx2 rabbit
polyclonal antibody and goat α-rabbit secondary antibody with ECL
detection
Results
PM PM
w/o w/
2D 2D 4D 4D
DM DM DM DM
w/o w/ w/ w/o
30 kDa
Experiment 2. Western Blot: 3 minute development, α-Pitx2 rabbit
polyclonal antibody and goat α-rabbit secondary antibody with ECL
detection
Results
35 kDa
25 kDa
PM PM 3h 3h 1d 1d 1d
w/o w/ DM DM DM DM DM
w/o w/ w/o w/ w/
Experiment 3. Western Blot: overnight development, α-Pitx2 rabbit
polyclonal antibody and goat α-rabbit secondary antibody with ECL
detection
Conclusion
•Phosphorylation may mark Pitx2 for
degradation, thus ending any of its transcription
activity, but more testing is needed to confirm
this result.
•Subsequent experiments should utilize
immunoprecipitation to remove interfering
substances.
Acknowledgements
Dr. Theresa Filtz
Dr. Chrissa Kioussi
Dr. Michelle Becker
Dr. Traci Hilton
Dr. Walter Vogel
Dr. Kevin Ahern
HHMI
Funding provided by the College of Pharmacy
Questions?