Research Interests
Cyclic Nucleotide Dependent Protein Kinase–Binding Proteins in Bovine Cardiac Tissues
Rationale
for Project:
Approximately 5 million Americans are afflicted with
some form of heart failure or cardiac disease at an estimated cost of 40 billion
dollars annually (Olson and Molkentin, 1999). The major symptom associated
with heart failure or cardiac disease is the inability of the heart to circulate
oxygenated blood as well as it should. This
decrease in pumping efficiency is often caused by blockages in coronary arteries
(arteries that supply blood to the heart itself), high blood pressure, or a
defect in the cardiac walls or valves. The inability of the heart muscle to adequately deliver
oxygenated blood to peripheral tissues leads to fatigue, weakness, and shortness
of breath in the patient making everyday tasks a tiring chore thus decreasing
the quality of life for said victims. In most instances, heart failure and heart disease can be
managed by administration of medications but patients continue to lead a
lifestyle of reduced activity (American Heart Association, 2000).
Even though much is known about the causes and
effects of heart failure and disease, the underlying molecular mechanisms have
yet to be elucidated. We must
therefore, through scientific laboratory research, develop an understanding of
the biochemistry of molecules in the cardiac tissue. Knowledge of how these molecules interact with each other is
of utmost importance to the subsequent development of novel pharmaceuticals to
treat and prevent heart failure and cardiac disease. Progress has recently been made on this basic medical
research front with the discovery that a pair of enzymes known as the cyclic
nucleotide-dependent protein kinases are intrinsically involved with maintenance
of normal cardiac function.
The two known cyclic nucleotide-dependent protein
kinases in mammals are the cyclic AMP-dependent protein kinase (PKA) and the
cyclic GMP-dependent protein kinase (PKG).
These two enzymes are closely related in that they have similar amino
acid sequences and similar functions in the cell (Francis and Corbin, 1994).
In response to various cellular signals, each kinase is responsible for
the transfer of a phosphate group from adenosine triphosphate (ATP) to a set of
target proteins in a process termed phosphorylation.
It has recently been discovered that PKA is responsible for the
phosphorylation of the major cardiac proteins, phospholamban and troponin (Sulakhe
and Vo, 1995, Ardelt et al, 1998). Olson and Molkentin (1999) have hypothesized that PKA
phosphorylation of phospholamban and troponin are vitally important in the
development of cardiac hypertrophy which often accompanies heart failure and
cardiac disease.
In separate studies, a novel paradigm for the control
or regulation of protein kinases is gaining acceptance.
This novel paradigm is that kinases can have other proteins bound to them
for the purpose of regulating the activity of the kinase.
For example, in the brain, PKA is bound to and consequently regulated by
another protein named AKAP (A-kinase anchoring protein)
(Skalhegg and Tasken, 2000). Protein
kinases other than PKA and PKG have been shown to complex with a different class
of enzymes called phosphatases (Westphal et
al, 1998). It is highly
probable that either PKA or PKG forms a complex in cardiac tissues with other
protein molecules and that said complex is responsible for the control and
regulation of PKA or PKG in cardiac tissues.
The hypothesis of my research is that cyclic nucleotide-dependent protein kinases in cardiac tissues do indeed complex with other proteins (binding proteins). Identification of these binding proteins would be a major advancement toward the understanding of the regulation of PKA and PKG in normal and heart disease conditions. Therefore, a major goal of my research is to isolate and identify proteins in cardiac tissues that bind to and regulate the activity of PKA and PKG through the use of standardized biochemical techniques.
Description
of Project Methods:
Isolation and identification of cyclic nucleotide-dependent protein
kinase binding proteins are done using bovine (cow) hearts as the model system.
The major advantage to using the cow heart is the ease of procuring the
quantity of hearts needed to begin a protein purification project such as this.
Typically four hearts are obtained from a regional slaughterhouse for one
attempt to purify any PKA- or PKG-binding proteins.
The hearts are dissected into small pieces and homogenized in 10 liters
of cold buffer solution to produce a crude cellular extract as has been
previously performed for bovine lungs (Reed et
al, 1996).
A variety of chromatographic techniques are employed to attempt to
isolate the binding proteins. Chromatography
techniques typically use a gel-like matrix that has specific molecules adhered
to it. When the extract is passed
over the matrix, only proteins of interest in the extract adhere to the
molecules of the matrix. The
proteins that remain bound to the matrix can then be eluted off the matrix by
changing the conditions in the buffer solution.
This procedure thus provides a measure of purification for specific
proteins in the extract.
One type of chromatography used has cyclic AMP
molecules adhered to the matrix (Reed et
al, 1997). Both PKA and PKG
will adhere to this matrix as would any proteins specifically bound to PKA or
PKG. Theoretically, all other
proteins would pass on through this matrix without binding to it.
PKA, PKG, and their binding proteins are eluted off the matrix by adding
cAMP to the buffer solution thus separating these proteins from the rest of the
extract proteins. A second type of
chromatography system takes advantage of the fact that both PKA and PKG are
negatively charged and will therefore adhere to a chromatography matrix that is
positively charged. Proteins that
specifically bind PKA and PKG would also be associated with this type of
chromatography matrix and could be eluted from it by increasing the ionic
strength of the buffer (Smith et al,
2000).
An alternative method of identifying proteins that bind to PKA or PKG is to use antibodies raised against PKA or PKG in experiments called immunoprecipitations. For this type of experiment, the extracts are incubated with the aforementioned antibodies. The antibodies are then collected by centrifugation based upon their characteristic adherence to the bacterial protein A that had been conjugated to a matrix bead. The collected antibodies can then be analyzed for the presence of binding proteins by a technique called electrophoresis. In this technique, proteins are separated from each other by an electrical field which pulls proteins through a matrix at a rate proportional to the size of the individual proteins. The separated proteins can be visualized by application of Coomassie Blue, an organic dye that specifically stains proteins. PKA and PKG-binding proteins that have been separated by electrophoresis are then to be extracted from the electrophoresis matrix in a highly purified state. By a process known as Edman degradation peptide sequencing, the identity of the protein/s may be determined.
At
the “heart” of any science is the objective to acquire more knowledge.
The main reason, therefore, to performing biochemical experiments is to
learn more about the functioning of an organism at the molecular level.
It is anticipated that conclusions drawn from these studies in the bovine
heart would increase our understanding of the functioning of both the normal
human heart and the diseased human heart since the hearts of these two organisms
are remarkably similar. The type of
research proposed here should be classified as basic medical research since the
direct application of this research is unclear at present.
The objective behind this basic medical research is to discover new
information that one day may be directly applied to the treatment of human heart
disease through the development of new drugs or other technologies.
Future
Goals of Project:
It has once been said that a good research project actually results in
the asking of many more questions than it answered.
It is the hope that this preliminary research project would ask more
questions than it answers. These
questions would then be the springboard for future research projects.
For example, what is the exact molecular nature of the interaction
between the kinases and their binding proteins?
How do the PKA- and PKG-binding proteins regulate the function of these
kinases in cardiac tissues? Is the
PKA- or PKG-binding protein complex affected by hormone influence?
Do these PKA- and PKG-binding proteins play similar roles of regulation
in other tissues where PKA and PKG are known to function?
The data collected from the successful completion of this research
project would be incorporated into a much larger research grant proposal aimed
at attracting extramural funding from such resources as the American Heart
Association (AHA) or the National Institutes of Health (NIH).
Follow-up studies to this project would be dictated by the results obtained from research of the project proposed here. It is conceivable that future studies might require the isolation of genes that encode these novel PKA- and PKG-binding proteins thus requiring techniques in molecular biology. Understanding of the regulation and function of a PKA- or PKG-binding protein complex may only be discovered through in vivo experiments involving heart cells (cardiomyocytes) grown in tissue culture. The hope is that future studies might provide opportunities for unique intramural collaborations with other scientists here at A.P.S.U. and other institutions.
American
Heart Association (2000) http://www.americanheart.org
Ardelt, P., Dorka, P., Jaquet, K., Heilmeyer, L.M., Jr.,
Kortkle, H., Korfer, R., and Notohamiprodjo, G.
(1998) Biol. Chem. 379,
341-347.
Francis,
S.H., and Corbin, J.D. (1994)
Annu. Rev. Physiol.
56, 237-272.
Olson,
E.N. and Molkentin, J.D. (1999)
Circulation Research, 84,
623-632.
Reed,
R.B., Sandberg, M., Jahnsen, T., Lohmann, S.M., Francis, S.H., and Corbin, J.D.
Reed,
R.B., Sandberg, M., Jahnsen, T., Lohmann, S.M., Francis, S.H., and Corbin, J.D.
Skalhegg,
B.S., and Tasken, K. (2000)
Front. Biosci.
5, D678-693.
Smith,
J.A., Reed, R.B., Francis, S.H., Grimes, K., and Corbin, J.D.
(2000) J.
Biol.
Sulakhe,
P.V., and Vo, X.T. (1995)
Mol. Cell Biochem.
149-150, 103-126.
Westphal,
R.S., Anderson, K.A., Means, A.R., and Wadzinski, B.E.
(1998) Science
280,
Biochemical Reagents Inventory
Other Projects: Aralia spinosa