1. SEARCHING FOR BINDING PARTNERS FOR THE NOVEL PHKG1 VARIANT, PhK-γ KISHORE POLIREDDY, Western Kentucky University.

2. 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.
kinases PO3-2
ATP ADP

3. Structure of Phosphorylase kinase
Alpha,β regulatory subunits
Gamma is catalytic subunit
Delta subunit is intrinsic molecule calmodulin

4. 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.

5. Role of PhK in glycogen breakdown

6. 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.

7. Truncated form, PhKγ-181, retains the kinase activity and its activity is influenced by the Phosphorylation of PKC.

8. Aims of the present study
To identify the potential binding partners for γ-181 by using Lex-A based yeast two hybrid system.

10. Current Study
Presently, we are trying to produce a LexA- γ-181 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 c-DNA library proteins will be monitored using Lac-Z and LEU2 as reporter genes.

11. 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.

12. Preliminary results PCR amplification of γ-181
Marker
PCR amplified product1
PCR amplified product 2

13. Restriction digestion of vector
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

14. Ligation of restriction digested γ-181 in to predigested pLex-A vector.
Restriction digested vector
Ligation
γ-181
pLex-A vector

15. Acknowledgements
Dr. Nancy A Rice
Dr. B. Sharma
KBRIN

16. 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 – The crystal structure of a phosphorylase kinase peptide substrate complex: kinase substrate recognition. The EMBO Journal Vol.16 No.22 pp.6646–6658, A Eukaryotic Transcriptional Activator Bearing the DNA Specificity of a Prokaryotic Repressor. Cell, Vol. 43, 729–736, December, 1985.

17. Thank you