STUDYING THE INTERACTION BETWEEN PROTEIN PHOSPHATASE 1 (PP1) AND ITS BINDING PROTEINS BY RESONANCE ENERGY TRANSFER APPROACHES
Document Type
Presentation
Start Date
22-10-2010 3:00 PM
End Date
22-10-2010 4:30 PM
Description
Protein-protein interactions are critical for many cellular processes, including synaptic plasticity, a cellular model for learning, memory and other experience dependent brain functions. For protein phosphatases-1 (PP1 ), its binding proteins function either to inhibit its enzymatic activities (inhibitors) or target it to distinct micro-compartments in cells to increase its substrates specificity (targeting molecules). Because both PP1 activity and its targeting have been shown to be critical for modulating the communication between neurons, being able to elucidate how neural activity dynamically modulates the interaction between PP1 and its binding proteins is critical for our understanding of the underlying molecular mechanisms of synaptic plasticity.
Resonance energy transfer (RET)-based approaches enable the study of the interaction between proteins in living cells, providing spatial and/or temporal resolution, and thus the opportunity of studying the dynamics of those interactions. Our lab has successfully established Fluorescence Resonance Energy Transfer (FRET) assay to study PP1 interactions with one of its inhibitors, inhibitor-2 (12), and one of its targeting proteins, neurabin (Nrb), in living cells. Basically we could detect robust FRET between CFP-I2 and YFP-PP1 as well as between CFP-Nrb(1-490) and YFP-PP1 in HEK 293 cells. Our present work aimed to develop Bioluminescence Resonance Energy Transfer (BRET) as an alternative assay to study the interaction between PP1 and 12 or Nrb. We have observed robust increase in BRET ratio between Rluc-I2 and YFP-PP1 in HEK293 cells. The increased BRET ratio was blocked by expression of non-luciferase or non-fluorescence tagged 12 and PP1, which suggested the specific binding between PP1 and 12. Furthermore, BRET ratio was decreased when there was a single point mutation H125Q in PP1, indicating the importance of this site in PP1 binding to 12. Our future goals are to study the dynamics of PP1 interaction with 12 and Nrb in neurons. in response to synaptic activity. The results obtained will provide insights in therapeutic intervention in diseases in which synaptic plasticity went awry, like mental retardation and Alzheimer's disease.
Supported by NIH & NSF
Recommended Citation
Yang, Hongtian; Siddoway, Ben; Hou, Hallong; and Xia, Hugh, "STUDYING THE INTERACTION BETWEEN PROTEIN PHOSPHATASE 1 (PP1) AND ITS BINDING PROTEINS BY RESONANCE ENERGY TRANSFER APPROACHES" (2010). Dr. Joseph M. Moerschbaecher, III Graduate Research Day. 27.
https://digitalscholar.lsuhsc.edu/grad_rs/2010/poster2/27
STUDYING THE INTERACTION BETWEEN PROTEIN PHOSPHATASE 1 (PP1) AND ITS BINDING PROTEINS BY RESONANCE ENERGY TRANSFER APPROACHES
Protein-protein interactions are critical for many cellular processes, including synaptic plasticity, a cellular model for learning, memory and other experience dependent brain functions. For protein phosphatases-1 (PP1 ), its binding proteins function either to inhibit its enzymatic activities (inhibitors) or target it to distinct micro-compartments in cells to increase its substrates specificity (targeting molecules). Because both PP1 activity and its targeting have been shown to be critical for modulating the communication between neurons, being able to elucidate how neural activity dynamically modulates the interaction between PP1 and its binding proteins is critical for our understanding of the underlying molecular mechanisms of synaptic plasticity.
Resonance energy transfer (RET)-based approaches enable the study of the interaction between proteins in living cells, providing spatial and/or temporal resolution, and thus the opportunity of studying the dynamics of those interactions. Our lab has successfully established Fluorescence Resonance Energy Transfer (FRET) assay to study PP1 interactions with one of its inhibitors, inhibitor-2 (12), and one of its targeting proteins, neurabin (Nrb), in living cells. Basically we could detect robust FRET between CFP-I2 and YFP-PP1 as well as between CFP-Nrb(1-490) and YFP-PP1 in HEK 293 cells. Our present work aimed to develop Bioluminescence Resonance Energy Transfer (BRET) as an alternative assay to study the interaction between PP1 and 12 or Nrb. We have observed robust increase in BRET ratio between Rluc-I2 and YFP-PP1 in HEK293 cells. The increased BRET ratio was blocked by expression of non-luciferase or non-fluorescence tagged 12 and PP1, which suggested the specific binding between PP1 and 12. Furthermore, BRET ratio was decreased when there was a single point mutation H125Q in PP1, indicating the importance of this site in PP1 binding to 12. Our future goals are to study the dynamics of PP1 interaction with 12 and Nrb in neurons. in response to synaptic activity. The results obtained will provide insights in therapeutic intervention in diseases in which synaptic plasticity went awry, like mental retardation and Alzheimer's disease.
Supported by NIH & NSF
Comments
See abstract book page 71