Dr. Julianne Grose, Department of Microbiology & Molecular Biology

Evaluation of academic objectives

PAS kinase is a newly discovered member of the nutrient sensing kinases that regulates glucose homeostasis in mice and yeast. The aim of our 2009 MEG “The Function of Yeast PAS kinase” was to characterize the role of a known PAS kinase substrate, Ugp1, as well as to identify novel PAS kinase substrates. We have made significant progress on each of these aims, which has facilitated the preparation of publications, presentations and abstracts. In addition, the findings described below have opened up further research as evidenced by our 2010 and 2011 MEG proposals as well as a 2009 NIH grant proposal. Thus, our 2009 MEG funding has furthered undergraduate education by training undergraduates in basic scientific theory and practice while allowing them to contribute to medically relevant scientific research within a strong mentoring environment. Six undergraduate and one graduate student were dedicated to this project and presented their results at scientific meetings as well as in research articles that are in preparation.

Description of the results/findings of the project

The aim of our 2009 MEG “The Function of Yeast PAS kinase” was to characterize the role of a known PAS kinase substrate, Ugp1, as well as to identify novel PAS kinase substrates. Specifically, we have identified novel Ugp1 constructs from selection in yeast that will allow characterization of phosphoUgp1, a known substrate of PAS kinase involved in glucose partitioning. In addition, the 2009 funding allowed for the construction of several yeast two- hybrid PAS kinase constructs that have been used to identify novel putative PAS kinase substrates.

Results of Aim 1

The essential enzyme Ugp1 is the only known cellular source of UDP-glucose and the only known bone fide substrate of PAS kinase. UDP-glucose is utilized as a glucose donor for many diverse processes in the cell including protein glycosylation, glycogen storage, and cell wall constituents. The Ugp1 protein is phosphorylated by PAS kinase in response to cell integrity stress or respiratory growth conditions and acts to direct UDP-glucose consumption for the production of cellular wall constituents at the expense of glycogen accumulation. That is, phosphorylation affects the utilization of the product of Ugp1 (UDP-glucose), rather than its enzymatic activity. This redirection of UDP-glucose utilization is accompanied by a tractable change in the conformation of Ugp1 which may facilitate localization, protein binding partners, or both. In our 2009 attempts to characterize the function of phosphoUgp1 were were unable to tag Ugp1 for protein purification or localization studies (data not shown). That is, any fusion constructs (GFP, Myc-tag, TAP-tag, His-tag) inserted at either the N- or C-terminus disrupted phosphorylation by PAS kinase. We, therefore, introduced a genetic screen to select for mutations in Ugp1 which trap it in the phosphorylated state. We were able to successfully isolate independent mutants with this phenotype.

Ugp1 mutants that appear to be trapped in the phosphorylated state are selected for using a temperature sensitive mutation in the tor2 gene of yeast. The tor2(ts) can be suppressed by overexpressing PAS kinase; this suppression is dependent on phoshorylation of Ugp1 (Mike Hall, personal communication). Thus, mutations that trap Ugp1 in a phosphorylated conformation may rescue growth of the tor2(ts) mutant. To produce the desired mutant, undergraduate Kevin Wiest created a mutant library of Ugp1 mutants using TAQ polymerase mediated PCR, which has a mutation error rate of 1 mutation in 1000 bp (Ugp1 is approximately 2 Kb). He then transformed the PCR products and digested expression plasmid into competent yeast cells, relying on the high recombination rate of yeast to produce the desired clones in vivo.

After selecting from approximately 153,500 colonies that have received the Ugp1 construct from yeast transformations, three colonies were successfully isolated that have been shown to be a result of a mutation in the Ugp1 gene on the expression plasmid (Figure 2). These constructs are currently being sequenced and validated by ion-exchange chromatography, which is capable of detecting the conformational change in Ugp1 induced upon phosphorylation. These constructs are currently being used to make Ugp1-GFP and Ugp1-Myc fusion proteins to see if Ugp1 is located in the cell wall after it is phosphorylated, and to identify proteins that copurify with phosphoUgp1. These associated proteins will allow us to discover the molecular mechanisms of PAS kinase in regulating the biosynthesis of the cell wall constituents.

Results of Aim 2

The goal of Aim2 was to identify novel PAS kinase substrates. Protein kinase subsrates are notoriously difficult to identify due to the transient nature of the interaction, the abundance of protein kinases, and the general level expression of protein kinases. The yeast 2- hybrid offers many advantages over conventional methods for identifying protein kinase substrates due to the increased sensitivity of the system. A yeast 2-hybrid system for identifying PAS kinase substrates (described in Aim 2 of our 2009 MEG proposal) was constructed by graduate student Chris Johnson and undergraduate Steve Sowas. The system was varied in 2009 by undergraduates Steve Sowas and Katie Harris who verified the full length yeast 2-hybrid PAS kinase constructs as functional and made several PAS kinase mutants. Mutations in the kinase domain that render PAS kinase “inactive” may increase PAS kinase/substrate interaction by inhibiting the phosphorylation event that triggers substrate release. Thus, we constructed a D1230A PAS kinase inactive mutant that is currently being used to identify PAS kinase substates in the yeast 2-hybrid screen. The D1230A amino acid substitution was constructed based on a recent finding that Asp166 of the model protein kinase PKA is the terminal proton acceptor during phosphate transfer. Thus, mutation of this conserved aspartic acid may hinder substrate release by inhibiting phosphate transfer without destroying the ATP and substrate binding motifs. The PAS kinase and PKA kinase domains are highly similar and the D1230A PAS kinase substitution completely abolishes the ability of PAS kinase to rescue toxicity due to Ugp1 overexpression (Figure 4, Grose, JH and Sowa, S, unpublished results). These mutations have been moved into the yeast two- hybrid constructs though site-directed mutagenesis, and are currently being used in our yeast 2-hybrid screen.

The constructs described above were used in 2009/2010 yeast 2-hybrid screens that have lead to the identification of putative novel PAS kinase substrates (see 2010 interim report). The identification of new PAS kinase substrates is arguably the most important Aim of our lab because it would lead to a deeper understanding of the function of PAS kinase. Since many proteins and pathways are conserved between yeast and other eukaryotes, many of these substrates may be conserved, facilitating the understanding of metabolic regulation in mammals.

Evaluation of the mentoring environment

In our lab, students are encouraged to take ownership of their project and to contribute by offering new avenues to explore as well as to suggest alternative experimental techniques. We have weekly lab meetings where the students take turns presenting their findings, providing students with a broader view of science and aiding in the development of presentation skills. In addition, we have weekly project-specific subgroup meetings where we discuss data interpretation, experimental theory and protocols, and solutions to any current experimental difficulties. The students are arranged in hierarchies within these subgroups, those who have the most experience are involved in training the newer lab members. This hierarchy facilitates higher learning as students explain concepts behind their research to others and gradually acquire more independence and responsibility. I am available in the lab virtually every day performing experiments, and I have an open-door policy for the students in my lab. I also had the undergraduate students in my lab take a short course from our librarian Greg Nelson, in which they become familiar with the on-line molecular biology resources (such as NCBI Blast, Clustal W, Pub Med, etc.)

Participating Students

I began working at BYU in September 2008. Since then I have had the opportunity to work with 20 undergraduate and two graduate students in my laboratory thanks to the funding from ongoing MEG’s. Twelve students were trained specifically during the 2009 year. Six of these students have had the opportunity to present presentations or posters of their research at scientific meetings (ASM branch meeting). In addition, I was able to recruit one graduate student to the lab in 2009, Chris Johnson. The research described herein has facilitated our 2010 research, including an NIH grant proposal submitted in October of 2009, which barely missed the funding cutoff and is currently being resubmitted. In addition, we are in the process of preparing our first manuscript from my laboratory at BYU. The authors consist solely of undergraduate and graduate students from my lab. A list of all undergraduates and graduate students in my lab in 2009 is given below with students who have coauthored abstracts, posters or manuscripts in bold and those who have submitted an ORCA grant with an asterick.

Undergraduates trained in the Grose lab in 2009:

Christopher Bird*
Igor Baldow*
Michael Fry
Katie Harris**
David Herbert
Mark Herzog*
Kent Jarvis*
Jonathan Neubert*
Matthew Sheppard
Steve Sowa*
Christina Swenson*
Kevin Wiest*

Graduate students trained in the Grose lab in 2009:
Chris Johnson

Abstracts/Presentations resulting from 2009 undergraduate research in the Grose lab:

• Katie Harris, Steve Sowas, Christopher Johnson and Julianne H. Grose. A yeast two hybrid screen for novel PAS kinase substrates. Poster, 2010, Intermountain Branch ASM meeting.
• Kent Jarvis, Jonathan Neubert, Ivor Benjamin, Julianne H. Grose. Yeast as a model for studying cryABR120G cardiomyopathy. Poster, 2010, Intermountain Branch ASM meeting.
• Christopher Johnson, Nick Whipple and Julianne H. Grose. Identifying novel pathways involved in NAD biosynthesis. Poster, 2010, Intermountain Branch ASM meeting.
• Christina Swenson, Donald P. Breakwell and Julianne H. Grose. Mendelian Segregation of Alleles in Saccaharomyces cerevisiae. 2010 Presentation, ASMCUE, San Diego, CA

Description of how the 2009 budget was spent

$9,600 5 undergraduate students for 15 weeks, 8 hours per week at $8.25 per hour.
This pay was for months (spring/summer) in which these students will not be enrolled in the research for credit course 494R. The following undergraduates were paid to work in the Grose lab in the 2009 academic year: Christopher Bird, Michael Fry, David Herbert, Mark Herzog, Kent Jarvis, Jonathan Neubert, Steve Sowa and Kevin Weist.

$8,000 Research expenses include laboratory consumables, reagents and supplies for wet lab research.

• $800- DNA extraction and amplification and modification $1200- 32P-ATP for kinase enzymatic assays
• $2000- yeast and bacterial media, antibiotics and supplies $2000- protein purification supplies
• $1000- DNA sequencing and protein mass spectrometry
• $1000- gloves, pipettes, kim-wipes, parafilm, and other consumables

$400 Registration and poster fees for the 6 undergraduates to a local scientific conference (ASM branch meeting).

***The above funding was supplemented with start-up funds for the Grose lab