| Title: | Mapping dynamic protein interactions in MAP kinase signaling using live-cell fluorescence fluctuation spectroscopy and imaging |
| Author(s): | Slaughter BD; Schwartz JW; Li R; |
| Address: | "Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, MO 64110, USA" |
| ISSN/ISBN: | 1091-6490 (Electronic) 0027-8424 (Print) 0027-8424 (Linking) |
| Abstract: | "Fluorescence correlation spectroscopy (FCS), fluorescence cross-correlation spectroscopy (FCCS), and photon counting histograms (PCH) are fluctuation methods that emerged recently as potentially useful tools for obtaining parameters of molecular dynamics, interactions, and oligomerization in vivo. Here, we report the successful implementation of FCS, FCCS, and PCH in live yeast cells using fluorescent protein-tagged proteins expressed from their native chromosomal loci, examining cytosolic dynamics and interactions among components of the mitogen activated protein kinase (MAPK) cascade, a widely occurring signaling motif, in response to mating pheromone. FCS analysis detailed the diffusion characteristics and mobile concentrations of MAPK proteins. FCCS analysis using EGFP and mCherry-tagged protein pairs observed the interactions of Ste7 (MAPK kinase) with the MAPKs, Fus3 or Kss1, and of the scaffold protein, Ste5, with Ste7 and Ste11 (MAPK kinase kinase) in the cytosol, providing in vivo constants of their binding equilibrium. The interaction of Ste5 with Fus3 in the cytosol was below the limit of detection, suggesting a weak interaction, if it exists, with K(d) >400-500 nM. Using PCH, we show that cytosolic Ste5 were mostly monomers. Artificial dimerization of Ste5, as confirmed by PCH, using a dimerizing tag, stimulated the interaction between Ste5 and Fus3. Native Ste5 was found to bind Fus3 preferentially at the cortex in pheromone-treated cells, as detected by fluorescence resonance energy transfer (FRET). These results provide a quantitative spatial map of MAPK complexes in vivo and directly support the model that membrane association and regulation of the Ste5 scaffold are critical steps in MAPK activation" |
| Keywords: | "Fungal Proteins/genetics/*metabolism Mitogen-Activated Protein Kinases/genetics/*metabolism Protein Interaction Mapping/*methods Recombinant Fusion Proteins/genetics/metabolism Signal Transduction Spectrometry, Fluorescence/*methods Yeasts/metabolism;" |
| Notes: | "MedlineSlaughter, Brian D Schwartz, Joel W Li, Rong eng F32 GM077923/GM/NIGMS NIH HHS/ R01 GM057063/GM/NIGMS NIH HHS/ F32 GM077923-01A1/GM/NIGMS NIH HHS/ GM057063/GM/NIGMS NIH HHS/ Research Support, N.I.H., Extramural 2007/12/14 Proc Natl Acad Sci U S A. 2007 Dec 18; 104(51):20320-5. doi: 10.1073/pnas.0710336105. Epub 2007 Dec 12" |
