
	1999 Summer Scholars Program

Previous: Laurel Griggs

Next: Farley C. Johnson

Using a Two-Hybrid System to Identify Proteins that Interact with Glut4-interacting Kinase and Akt

By Glenda-Gayle Haskin
Mentors: Dr. Mike M. Mueckler, Haru Murata
Department of Cell Biology, Washington University School of Medicine, St. Louis, MO

Glucose is a critical nutrient in the human body. It is the main source of energy and provides nutrition for living cells. Proteins known as glucose transporters (GluT) are responsible for bringing glucose inside cells, and are the primary focus of the Mueckler lab. As of now, five glucose transporters have been identified. They are, in order of discovery, GluT1, GluT2, GluT3, GluT4, and GluT5. GluT4 is the isoform that is regulated by insulin. When GluT4 fails to mediate the entrance of glucose into cells in the presence of insulin, a disease results known as Type II Diabetes.

My project focused on GluT4. It has been found that this particular glucose transporter, when not in the presence of insulin, remains sequestered inside the cell in unique storage compartments. After exposure to insulin, it moves to the surface of the cell to carry glucose across the cell membrane. The details of this shuttle to and from the surface of the cell are still cloudy. The protein Akt is known to have some role in the movement of GluT4, and a kinase that interacts directly with GluT4 was recently found in the Mueckler lab (tentatively termed GluT4-interacting kinase). My objective was to find proteins that interacted with Akt and the GluT4-interacting kinase.

The chosen method to do this was the Two-Hybrid System. In this system, the protein of interest is fused to the DNA binding domain (DBD) of a transcription factor, and a library of proteins is fused to the transcription activation domain (TAD) of a transcription factor. Then, if the two proteins interact, the proximity of the DBD and TAD will constitute the activity of a functioning transcription factor. When this occurs in specially engineered yeast the reaction transcribes a reporter gene, b -galactosidase, which generates a blue product in the presence of the chromogenic substrate X-gal.

During my six-week period, I prepared the Akt and GluT4-interacting Kinase "bait" hybrids. These procedures called for the construction of recombinant DNA. A plasmid, pBTM116, and DNA encoding the "bait" proteins were digested with BamHI and EcoRI. The Akt and GluT4-interacting kinase were then ligated with the plasmid and transformed into a strain of E. coli called DH5a. After a selection process using ampicillin and a restriction digest analysis of the DNA, the recombinant plasmids were transformed into yeast. At this point, the "bait" constructs were tested to ensure that they did not transactivate the reporter gene on their own.

The next procedure in this project would be to initiate the actual screening. If the X- gal screening identifies any positive yeast clones, the DNA of the interacting protein in the Library hybrid would be isolated and sequenced to reveal its identity.

Previous: Laurel Griggs

Scholars Index

Next: Farley C. Johnson

This page was last updated on Fri, Jun 16, 2000 at 2:16:01 PM by Tom Elgin with Userland Frontier.