This web page was produced as an assignment for Genetics 677, and undergraduate course at UW-Madison.
Conclusion
Below is a copy of my final project which shows the expected results I believe I would see. File only in Powerpoint.
final_presentation.pptx | |
File Size: | 5071 kb |
File Type: | pptx |
The goal of this experiment was to determine what role the phosphorylation of KCNQ1 plays in producing the symptoms associated in Long QT Syndrome. I began by researching information on KCNQ1 and its association with Long QT Syndrome. I found a lot of information that proved that KCNQ1 was indeed the main cause of the syndrome but not a lot as to what was actually going wrong to cause the problems. But I did discover that KCNQ1 did play a critical role in keeping the heart beating and that the protein was highly conserved throughout many mammalian species such as mouse, rat, dog, and cow. This allowed me to choose the best organism for my experiment. In this case I choose the mouse as it is the easiest to keep and has a shorter life span to allow for more research to be done in a shorter amount of time.
Further investigation into KCNQ1 protein domains and ontology terms allowed it to be narrowed down to its cellular role, functioning as a membrane transport protein transferring ions into and out of the cell. I found that the protein contained two domains: an Ion_trans domain which does the transporting across the membrane and a KCNQ_channel domain which after further research appears to be big location for phosphorylation sites as well as binding domains. This finding is what led me into the next part of my experiment to see what may be phosphorylating KCNQ1 and if there may be a problem with it.
A search through the STRING network database led me to three different proteins that showed signs of being phosphorylating kinases or involved in such a process: AKAP9, PRKAR2A, and PRKAR2B (the last two form a dimer to activate a protein of interest). PRKAR2A/B turned out to be average run of the mill phosphorylating kinases but what was interesting was that they were found within the cytoplasm and not in the membrane where KCNQ1 is located. This is what led me to AKAP9.
AKAP9 turned out to be a scaffold protein that brought phospho kinases to KCNQ1 to allow them to activate KCNQ1 to maintain its transporting. I found that there is an almost hidden domain (they were difficult to find through Pfam and SMART) that is RII binding domain. This binding domain is what the PRKAR2A/B complex binds to as AKAP9 presents the complex to KCNQ1.
To begin the experiment I wanted to see if my hypothesis that AKAP9 does in fact act as a taxi cab for the PRKAR2A/B complex. The first thing that I looked at was to see where all four proteins localized within the cell. I found that the KCNQ1 proteins localized within the cell membranes while the PRKAR2A/B complex was mostly found within the cytoplasm. The interesting part was that AKAP9 was found in both areas of the cell suggesting that it does indeed spend time in the cytoplasm as well as the cell membrane. This data was backed up by looking at Gene ontology terms found through AmiGo.
Next I wanted to see if changes in the RII binding domain in AKAP9 would affect protein binding and in turn phosphorylation of KCNQ1. To do this I created two mouse populations one wild type and the other a RII binding domain knockout. After lysing the cells the AKAP9/KCNQ1 protein complex was put onto a microarray plate and PRKAR2A/B complexes with fluorophored cAMP attached was allowed to wash over the plate. The spots with wild type AKAP9 showed phosphorylated KCNQ1 while the knock out did not.
Lastly, to give a lead in to further research I did DNA sequencing of patients with known Long QT Syndrome that had unknown mutations and upon sequencing and aligning their sequences looked to see if there were mutations within their AKAP9 DNA sequence that could lead to further experiments in the role of AKAP9 with KCNQ1 and Long QT Syndrome.
In conclusion, the data I have found throughout all my research on KCNQ1 and Long QT Syndrome shows that even though there is a lot known about the syndrome further research is still needed on the overall KCNQ1 protein networks, and see how those relationships may affect the symptoms observed in patients with Long QT Syndrome.
Further investigation into KCNQ1 protein domains and ontology terms allowed it to be narrowed down to its cellular role, functioning as a membrane transport protein transferring ions into and out of the cell. I found that the protein contained two domains: an Ion_trans domain which does the transporting across the membrane and a KCNQ_channel domain which after further research appears to be big location for phosphorylation sites as well as binding domains. This finding is what led me into the next part of my experiment to see what may be phosphorylating KCNQ1 and if there may be a problem with it.
A search through the STRING network database led me to three different proteins that showed signs of being phosphorylating kinases or involved in such a process: AKAP9, PRKAR2A, and PRKAR2B (the last two form a dimer to activate a protein of interest). PRKAR2A/B turned out to be average run of the mill phosphorylating kinases but what was interesting was that they were found within the cytoplasm and not in the membrane where KCNQ1 is located. This is what led me to AKAP9.
AKAP9 turned out to be a scaffold protein that brought phospho kinases to KCNQ1 to allow them to activate KCNQ1 to maintain its transporting. I found that there is an almost hidden domain (they were difficult to find through Pfam and SMART) that is RII binding domain. This binding domain is what the PRKAR2A/B complex binds to as AKAP9 presents the complex to KCNQ1.
To begin the experiment I wanted to see if my hypothesis that AKAP9 does in fact act as a taxi cab for the PRKAR2A/B complex. The first thing that I looked at was to see where all four proteins localized within the cell. I found that the KCNQ1 proteins localized within the cell membranes while the PRKAR2A/B complex was mostly found within the cytoplasm. The interesting part was that AKAP9 was found in both areas of the cell suggesting that it does indeed spend time in the cytoplasm as well as the cell membrane. This data was backed up by looking at Gene ontology terms found through AmiGo.
Next I wanted to see if changes in the RII binding domain in AKAP9 would affect protein binding and in turn phosphorylation of KCNQ1. To do this I created two mouse populations one wild type and the other a RII binding domain knockout. After lysing the cells the AKAP9/KCNQ1 protein complex was put onto a microarray plate and PRKAR2A/B complexes with fluorophored cAMP attached was allowed to wash over the plate. The spots with wild type AKAP9 showed phosphorylated KCNQ1 while the knock out did not.
Lastly, to give a lead in to further research I did DNA sequencing of patients with known Long QT Syndrome that had unknown mutations and upon sequencing and aligning their sequences looked to see if there were mutations within their AKAP9 DNA sequence that could lead to further experiments in the role of AKAP9 with KCNQ1 and Long QT Syndrome.
In conclusion, the data I have found throughout all my research on KCNQ1 and Long QT Syndrome shows that even though there is a lot known about the syndrome further research is still needed on the overall KCNQ1 protein networks, and see how those relationships may affect the symptoms observed in patients with Long QT Syndrome.
Future Directions
1. Is the mutation that is found in the RII binding site causing a problem in localization of the PRKAR2A/B complex?
2. Does the same interactions of proteins occur in all cells known to have these proteins?
3. Is there a mutation in the PRKAR2A/B complex?
2. Does the same interactions of proteins occur in all cells known to have these proteins?
3. Is there a mutation in the PRKAR2A/B complex?