Phosphorylation is one of the most important mechanisms of signal transduction in eukaryotic cells, yet many aspects of it are poorly understood. The physiological targets and regulatory mechanisms of most kinases and phosphatases are not known; neither are the spatial and temporal distributions of phosphorylation activity in living cells, either at rest or in response to external stimuli. Achievements in these areas and others have been limited by the lack of general methods for detecting phosphorylation activity in living cells. There is a pressing need for indicators that can report with high spatial and temporal resolution the phosphorylation activities of specific kinases and phosphatases in vivo. We propose a broadly adaptable method for monitoring the phosphorylation state of specific tyrosine kinase and phosphatase targets in individual living mammalian cells. This method will be nondestructive and have spatial and temporal resolution comparable to that of Ca +2 imaging. We will engineer a transfectable chimeric tyrosine kinase and/or phosphatase substrate, incorporating proteins whose fluorescence properties change significantly as a function of phosphorylation state. To develop and optimize our indicator, we will take a library approach, using both fluorescence-activated cell sorting (FACS) and microtiter plate-based assays to screen for constructs with the highest sensitivity to phosphorylation.
| Ting, A Y; Kain, K H; Klemke, R L et al. (2001) Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells. Proc Natl Acad Sci U S A 98:15003-8 |
