Pilot Research Project Ten
Comparison of protein-protein interactions of Grb7 and s-SHIP within the P13 kinase signaling pathway of stem cells.
Co-Principal Investigators
Dr. Larry Rohrschneider, Full Member, Basic Sciences – FHCRC
Dr. Barbara Lyons, Assistant Professor, Chemistry and Biochemistry – NMSU
This pilot project will investigate functional interactions between two signaling proteins common to the PI3-Kinase signaling pathway from organisms as diverse as C. elegans and man. The Grb7 (MIG10-family) protein has been studied in the Lyons laboratory while the s-SHIP product has been a focus of research in the Rohrschneider laboratory. Grb7 is a downstream messenger protein important in PI3-kinase signaling, while s-SHIP is important in down-regulation of PI3-kinase signaling pathways. Each protein may have a role in mammary tumor formation or maintenance, yet little, or nothing, is known about their interactions in mammary tumors or their stem cells. Recently, work in the Rohrschneider lab has identified and isolated normal mammary tissue stem cells from a transgenic mouse model. The work on this pilot project will combine expertise from both labs to investigate how each protein may function in mammary tissue stem cells. The proposed molecular analyses of signaling protein interactions in mammary stem cells has not previously been possible. This project represents a novel combined approach derived from the distinct interests of the Lyons and Rohrschneider laboratories.
The specific aims of this pilot project are to:
- Aim 1: Screen for stem cell proteins interacting with Grb7 and s-SHIP. We will prepare a cDNA library from mammary stem cells isolated by flow cytometry, and use the yeast and mammalian hybrid screen methods (with and without an included tyrosine kinase activity) to identify interacting stem cell proteins.
- Aim 2: Verify interactions in stem cells using immunoprecipitation, co-localization, with and without overexpression of exogenous proteins.
- Aim 3: Demonstrate functional importance of interactions using a) siRNA to knockdown host proteins, b) expression of dominant-negative Grb7 or s-SHIP proteins, c) transplantation of mammary tissue stem cells (treated as in a or b above), into mammary fat pads for regrowth of a new mammary gland.
For More Information
To learn more about the research talking place in Dr. Rohrschneider’s lab, visit: www.fhcrc.org/science/labs/rohrschneider/index.html
Information about Dr. Lyons' research can be found at: www.chemistry.nmsu.edu/lyons.html
Faculty and students interested in learning more about this pilot project may contact Dr. Larry Rohrschneider at lrohrsch@fhcrc.org or Dr. Barbara Lyons at blyons@nmsu.edu.
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