Download Searching for Binding Partners for the Novel PHKG1 Variant, PhKγ

SEARCHING FOR BINDING PARTNERS FOR THE
NOVEL PHKG1 VARIANT,
PhK-γ181
KISHORE POLIREDDY,
Western Kentucky University.
Kinases? What do they do?
Kinases are enzymes which transfer a
phosphate group from ATP to a specific
substrate, this process is known as
Phosphorylation.
PO3-2
kinases
ATP
ADP
Structure of Phosphorylase kinase
Alpha,β regulatory subunits
Gamma is catalytic subunit
Delta subunit is intrinsic molecule
calmodulin
Importance of glycogen
 Glycogen is the major carbohydrate reserve,
composed of linked glucose molecules stored
within the liver and muscle.
PhK is the rate limiting enzyme of the
glycogenolysis.
Role of PhK in glycogen breakdown
Alternate processing of PHKG1
 PHKG1-mRNA codes for 387 amino acids gamma
subunit
 Alternate processing of PHKG1-mRNA will code for
181 amino acid gamma subunit.
Calmodulin-binding (domain-N)
catalytic domain-PHKG1
PHKG1
PHKG1-human
387 aa
Calmodulin-binding (domain-C).
Truncated form, PhKγ-181, retains the kinase
activity and its activity is influenced by the
Phosphorylation of PKC.
Aims of the present study
To identify the potential binding partners for
γ-181 by using Lex-A based yeast two hybrid
system.
Current Study
Presently, we are trying to produce a LexA- γ181 fusion protein by using the vector pLex-A
(bait) and the vector pB42 (prey) to search for
binding partners within a brain cDNA library.
Interaction between γ-181 and the brain cDNA library proteins will be monitored using
Lac-Z and LEU2 as reporter genes.
Future Work
Cloning of γ-181 in to pLex-A vector
Identify the clones which shows positive
interaction between γ-181, c-DNA library
proteins
Library plasmids isolated from the clones
that show positive results on the yeast two
hybrid screen will be sequenced to identify
novel binding partners for γ-181.
Preliminary results
PCR amplification of γ-181
1
2
3
1. Marker
2. PCR amplified product1
3. PCR amplified product 2
Restriction digestion of vector
11
2
3
4
5
1. Marker
2. Uncut pLexA vector
3. Restriction digestion pLexA vector with EcoR1.
4. Restriction digestion pLexA vector with BamH1
5. Restriction digestion pLexA vector with EcoR1 &
BamH1
Ligation of restriction digested γ-181 in to
predigested pLex-A vector.
Restriction digested γ-181
Restriction digested vector
Ligation
γ-181
pLex-A vector
Acknowledgements
• Dr. Nancy A Rice
• Dr. B. Sharma
KBRIN
References
1. Three-Dimensional Structure of Phosphorylase Kinase at 22A
Resolution and Its Complex with Glycogen Phosphorylase b.
Structure, Vol. 10, 33–41, January, 2002, Elsevier Science Ltd.
2. The Fractal Structure of Glycogen: A Clever Solution to
Optimize Cell Metabolism. Biophysical Journal Volume 77
September 1999 1327–1332.
3. The crystal structure of a phosphorylase kinase peptide
substrate complex: kinase substrate recognition. The EMBO
Journal Vol.16 No.22 pp.6646–6658, 1997.
4. A Eukaryotic Transcriptional Activator Bearing the DNA
Specificity of a Prokaryotic Repressor. Cell, Vol. 43, 729–736,
December, 1985.
Thank you