PAS Kinase Interactions

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Steven Sowa and Dr. Julianne Grose, Microbiology

Goal

To discover the substrates of PAS kinase.

Introduction

Signal transduction pathways are critical to the function of a cell. Correct signals help the cell to manage its resources effectively while improper or inactive signals contribute to cancer and many other human diseases. One protein involved in signal transduction is PAS kinase.

Researchers have discovered that PAS kinase influences obesity and type II diabetes in mammals. PAS-kinase-deficient mice are “nearly completely protect[ed] from the deleterious effects of a high fat diet including obesity and insulin resistance [i.e., diabetes]” (Hao et al 2007). However, researchers still do not understand the molecular mechanism in mice that enacts this change.

Our aim was to identify the substrates of PAS kinase, so that we could study the function of these substrates. We experimented with two system by which we could determine the substrates of PAS kinase. First, using coimmunoprecipitation to purify PAS kinase from the cell with its substrates. Second, designing a yeast-2 hybrid system to identify kinase-substrate interactions. We ended up largely using the yeast 2-hybrid screen to identify PAS kinase substrates.

Results

Initially, we tried to use coimmunoprecipitation to discover PAS kinase substrates. We knew that if we coimmunoprecipitate PAS kinase from the cell with its substrate, we could identify the substrate using mass spectrometry. The interactions of PAS kinase with its substrates are expected to be transient, so we tried to create mutants of PAS kinase that increased its affinity for its substrates. We used a paper on Protein Kinase A to help us target specific amino acid residues for mutation in PAS kinase (Taylor et al 2004). With this paper as background, we created a mutant of PAS kinase that had amino acid 1230 changed from an aspartic acid to a serine (D1230S) and it showed signs of efficacy. However, but we didn’t progress to coimmunoprecipitation with this construct because we decided the yeast 2-hybrid system would give us better results.

Therefore, we switched our focus to the yeast 2-hybird system. This system is much more sensitive than coimmunoprepitation and generally produces overlapping, but often differing results. The other advantage to the yeast 2-hybrid system is that it provides in vivo evidence of kinase-substrate interactions, which is much stronger evidence of true protein interactions than the in vitro results of coimmunoprecipitation. The yeast 2-hybrid system takes some time to construct and calibrate. We made several constructs of PAS kinase to use in performing the screen. We able to transfer the D1230S mutation from our coimmunoprecipitation experiment to our yeast 2-hybrid experiment and we created three other new constructs. We also amplified the three genomic libraries to use in our screen. We spent six months calibrating the conditions of the screen. We determined the right concentration of antibiotic to use in our plates, optimized our yeast transformation efficiency, and tested the functionally of our PAS kinase constructs. We started screening the yeast genomic library with our first construct and found five putative substrates that we are in process of sequencing so we can identify these substrates. I trained a new undergraduate, Jordan Mackey, who is helping me with this project. We expect to start screening the genomic libraries with other constructs in the coming months. While this project has been moving slowly, our work looks promising.

Academic Outcomes

I gave a poster presentation on my progress at an American Society for Microbiology meeting on April 10, 2010. I will also give poster presentation on my research on Feb 18, 2011 at an undergraduate research conference at Weber State University. We currently gathering data for a publication on PAS kinase substrates that will be published as soon as we finish our screen and characterizing the substrates.

Conclusion

In spite of a number of setbacks and disappointments, I have learned much and I continue to enjoy contributing to my project. I was able to spend 20-30 hours a week in my lab working on my project and learning from Dr. Grose. I was able to give a poster presentation and quarterly presentations at lab meetings. I trained an undergraduate and we made significant progress on calibrating the yeast 2-hybrid system and screening through our yeast genomic libraries. We struggled at times to control contamination and obtain conclusive results. In spite of these challenges, we still identified several putative substrates and are in the process of sequencing these hits so we can isolate and characterize these substrates. I feel very confident that these efforts will ultimately lead to a publication in a good scientific journal. I am very grateful for the opportunity I have to do this research and receive ORCA grant funding for it.

Sources

  • Hao HX, Cardon CM, Swiatek W, Cooksey RC, Smith TL, Wilde J, Boudina S, Abel ED, McClain DA, Rutter J. 2007. PAS kinase is required for normal cellular energy balance. Proc. Natl. Acad. Sci. U.S.A.; 104(39): 15466-15471.
  • Taylor, S.S., et al. 2004. PKA: A portrait of protein kinase dynamics. Biochim Biophys Acta, 1697(1-2): 259-269.