Download Summer 2011 Proposal for UNCA Undergraduate Research

BIOLOGY
Elucidation of G12-HSP90 Protein Interactions Necessary for a G12
Dependent Cell Growth Pathway
Abstract
Heterotrimeric guanine nucleotide binding proteins (G proteins) are important cell
signaling proteins that mediate a wide variety of cellular responses via signal transduction
across the plasma membrane. They do so by interacting with a number of downstream
target proteins to drive various signaling pathways. The G protein G12 governs
pathways regulating cell growth, proliferation, and shape, and has been implicated in
tumorigenesis and metastasis. My research will focus primarily on the mechanisms and
evolutionary origin of protein-protein interactions between G12 and one of its
downstream targets, Heat Shock Protein of 90kDa (HSP90), as part of the cell growth
related Serum Response Factor (SRF) pathway. Prior screenings of various G12 point
mutants against HSP90 have revealed several key amino acid residues that appear
important for this protein-protein interaction to occur. Interestingly, substitution of these
residues with the corresponding residues of the fruit fly homolog of G12 abolishes its
ability to stimulate SRF. My research will consist of two phases. First, I will compare the
amino acid sequences of mammalian G12 to its evolutionary homologs across a broad
range of vertebrates and invertebrates, and catalog variations at residues critical for
HSP90 interaction. In the second phase, I will engineer mutants of mouse G12 that
substitute the residues found to vary in the G12 homologs, and test these for HSP90
binding and SRF activation to determine the precise structural features that conferred
upon G12 the ability to stimulate this cellular growth pathway. In addition, I will
examine variation in HSP90 across a variety of taxa to identify residues for mutation;
these studies should facilitate mapping of G12 binding motifs within HSP90.
Background
G proteins have been studied extensively in order to elucidate their numerous
functions in cell signaling and response. Heterotrimeric G proteins are comprised of 2
functional signaling units: an  subunit and a  heterodimer. When inactive, the subunits
are bound together as a heterotrimer at the inner face of the plasma membrane, bound to a
G protein coupled receptor (GPCR). Ligand binding at the extracellular face of the GPCR
stimulates the G subunit to release a bound guanosine diphosphate (GDP) molecule,
allowing for its replacement with a guanosine triphosphate (GTP). This transition causes
a conformational shift in the  subunit, dissociating it from the  subunit. From there,
the  subunit interacts with a number of downstream proteins to promote various cellular
responses (Suzuki et al., 2009).
The G12 subunit is ubiquitously expressed in mammalian tissues. Functions
specific to it include promoting cell growth and mitosis through a variety of signaling
pathways. Due to the nature of its function, G12 has the potential to promote malignant,
uncontrolled cell growth and metastasis, classifying it as a proto-oncoprotein. Indeed,
analysis of cDNA libraries collected from sarcoma tissue samples has implicated G12
as a strong transforming oncoprotein (Chan et al., 1993). Importantly, G12 need only be
over-expressed, not mutated, in order to potentially drive cancerous proliferation through
these pathways (Meigs and Lyakhovich, in review). Fromm et al. (1997) demonstrated
G12 stimulation of this SRF pathway to promote cell growth, and potentially cancer cell
progression. A better understanding of the protein-protein interactions of these pathways
may prove important in discovering ways to combat cancer. Previous research done by
Dr. Meigs and colleagues, screening G12 point mutants against a number of its
downstream targets, has elucidated several regions of G12 important for interaction. My
research will focus on the evolutionary emergence of a G12 dependent cell growth
pathway, mediated through its downstream target protein Heat Shock Protein of
90kDaltons (HSP90).
HSP90 is primarily understood to be a molecular chaperone protein, maintaining
the functional shape of signal transduction proteins including G proteins (Suzuki et al.,
2009). Vaiskunaite et al. (2001) reported that a G12 driven SRF pathway is dependent
on the function of HSP90, however, other G proteins, namely RhoA and G13, are not.
In evolutionary terms, this pathway has been conserved in spite of alternative SRFspecific growth pathways. The amino acid sequence of G12 contains key differences
that distinguish it from other G proteins. Dr. Meigs has engineered G12 point mutants
that replace native amino acids with those found at identical structural positions in other,
“ancestral” G protein subfamilies. Assays designed to examine these G12 mutants for
HSP90 binding have identified several single amino acid substitutions that disrupt this
interaction. Dr. Meigs’s lab also examined the fruit fly version of G12, a protein termed
Concertina, for its function in several aspects of cell biology. A surprising finding was
that Concertina, despite harboring many properties similar to G12, lacks the ability to
drive the SRF pathway in cells. Furthermore, introduction of two single amino acids from
Concertina into G12 caused a full loss of its ability to stimulate SRF (T. Meigs,
personal communication).
In my initial proteomic comparisons of G12 across numerous taxa, I uncovered
interesting variations at several of these key amino acid positions in the G12 homologs
of the frogs Xenopus laevis and Xenopus tropicalis. These species exhibit the Concertinaspecific amino acid residues similar to Dr. Meigs’s engineered point mutants, yet retain
many of the same characteristics as mouse G12. Further investigation led to the
discovery that these frog species exhibited a divergent sequence for HSP90 as well.
Methods
My primary focus will be to create fusion proteins of HSP90 and the Xenopus
variant and screen them against G12 point mutants engineered to harbor key residues
found in G12 homologues. Specifically, I will be interacting a constitutively active
mutant of G12—continuously bound to GTP—termed G12QL against the target protein
in a series of “pulldown” assays. A fusion of the target protein HSP90 to the protein
Glutathione-S-Transferase (GST) will be bound to heavy sepharose beads coated in
glutathione. HSP90-GST fusion proteins will be achieved via transformation of bacterial
cells with plasmid vectors engineered to encode the HSP90-GST fusion. HSP90 protein
is quite large at 90kDa, making fusion of the whole protein difficult and impractical.
Previous work in Dr. Meigs’s lab has shown that G12 binds at the C-terminus of
HSP90. Therefore a GST fusion of that specific HSP90 region should be sufficient for
performing these experiments.
The immobilized HSP90-GST fusion proteins will be mixed with cell lysates
containing the G12QL point mutants. Any interaction with HSP90 will effectively coprecipitate the G12 proteins along with the sepharose beads. Subsequently, gel
electrophoresis and G12-specific immunoblotting will be used to detect these
interactions.
Conclusion
The purpose of this research is to better understand the mechanism of interaction
between G12 and HSP90 as part of a potentially cancerous cell growth pathway.
Understanding specifically how these interactions occur should improve our ability to
design and discover new methods for the treatment of various types of cancer associated
with these pathways.
References
Suzuki N, Hajicek N, Kozasa T. (2009) Regulation and physiological functions of
G12/13-mediated signaling pathways. Neurosignals 17: 55-70.
Chan A.M., Fleming T.P., McGovern E.S., Chedid M., Miki T, Aaronson S.A. (1993)
Expression cDNA cloning of a transforming gene encoding the wild-type G12 gene
product. Mol. Cell. Biol. 13: 762-768.
Fromm C., Coso O., Montaner S., Xu N., Gutkind J.S. (1997) The small GTP-binding
protein Rho links G protein coupled receptors and G12 to the serum response element
and to cellular transformation. Proc. Natl. Acad. Sci. USA 94: 10098-10103.
Meigs T., Lyakovich A. G protein alpha 12. In review; Springer Publishing.
Vaiskunate R., Kozasa T., Voyno-Yasenetskaya T.A. (2001) Interaction between the G
subunit of the heterotrimeric G12 protein and Hsp90 is required for G12 signaling. J.
Biol. Chem. 276: 46088-46093.
Budget and Justification
Student stipend for 8 weeks of full-time summer research
$ 1500.00
Supplies/Reagents for Cell Growth, DNA Mutagenesis, and Protein Analysis
Acrylamide solution
Gel-loading micropipet tips
$ 57.60
$ 54.23
Falcon tubes, sterile (for bacterial transformations)
$ 109.46
Anti-G{alpha}12 polyclonal antibody
$ 259.00
Wizard PCR/gel cleanup kit
$ 129.60
T4 DNA ligase
$ 34.58
JM109 competent bacterial cells
$ 222.76
Benchmark Protein Standards
$ 133.00
_____________________________________________________________
TOTAL:
$ 2500.23
Time Period
6/6/2011 – 7/6/2011: Structural comparisons of G12 and its homologs using BLAST
program. Polymerase Chain Reaction based engineering of constructs encoding HSP90GST fusion proteins, and verification by DNA sequencing. Transformation of bacterial
cells to express the protein constructs.
7/7/2011 – 7/30/2011: Bacterial expression and affinity purification of protein products.
Interaction assays between HSP90-GST fusions and G12. Computer-assisted
quantification of protein binding.
Publication Outlet
This work will be submitted to the UNC-Asheville Journal and also presented at the
Undergraduate Symposium on Research and Creativity. Also, it is our anticipation that
data from this research ultimately will comprise part of a manuscript Dr. Meigs will
submit for publication in a peer-reviewed journal.