Enhanced kinase translocation reporters for simultaneous real-time measurement of PKA, ERK, and calcium

Figures

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  Figure 1. The bNLS in PKA-KTR is too weak to direct proteins to the nucleus.A, representative fluorescence micrographs of HEK293 cells expressing the (red) PKA-KTR1/mCherry, (blue) H2B/mTagBFP2, and (green) mNeonGreen, grown in the absence of Fsk (−FSK) or after 30 min exposure to 30 μM forskolin (+FSK). Bar represents 50 μM. B, bar graph showing C/N ratios of PKA-KTR1/mCherry (gray) prior to the addition of Fsk or (pink) 30 min after the addition of Fsk. C, representative fluorescence micrographs of HEK293 cells expressing the (red) PKA-KTR1/tdCherry, (blue) H2B/mTagBFP2, and (green) mNeonGreen, grown in the absence of Fsk (−FSK) or after 30 min exposure to 30 μM forskolin (+FSK). Bar represents 50 μM. D, bar graph showing C/N ratios of PKA-KTR1/tdCherry (gray) prior to the addition of Fsk or (pink) 30 min after the addition of Fsk. All data in this figure are from a minimum of three independent biological replicates, the data for all bar graphs came from the analysis of independent groups of cells, the error bars reflect the SEM, and p values were calculated by two-tailed t test.

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  Figure 2. Tuning NLS strength and KTR size improves PKA-KTR sensitivity and dynamic range.A, amino acid sequences of PKA-KTR sensor domains, showing the kinase docking site, bNLS, PKA phosphorylation sites (in pink, with asterisk), and NES, with predicted NLS strength scores listed to the right. Bar represents 50 μm. B, representative fluorescence microscopy images of HEK293 cells expressing the (red) ePKA-KTR1.1, 1.2, 1.3, 1.4, and 1.5 proteins, (blue) H2B/mTagBFP2, and (green) mNeonGreen, grown in the absence of Fsk (−FSK) or after 30 min exposure to 30 μM forskolin (+FSK). C, bar graph showing C/N ratios of ePKA-KTR1.1, 1.2, 1.3, 1.4, and 1.5/tdCherry proteins (gray) prior to the addition of Fsk or (pink) 30 min after the addition of Fsk. D, C/N ratios of ePKA-KTR1.1, 1.2, 1.3, 1.4, and 1.5/tdCherry (gray) prior to the addition of leptomycin B or (green) after 30 min incubation in leptomycin B. E, bar graphs comparing the C/N ratios of ePKA-KTR1.2/mCherry (pink) and ePKA-KTR1.2/tdCherry (purple) following 30 min incubation in 0, 1, 3, 10, or 30 μM Fsk, or 5 nM leptomycin B (LMB). F, bar graphs comparing the C/N ratios of (pink) ePKA-KTR1.4/mCherry and (purple) ePKA-KTR1.4/tdCherry…

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  Figure 3. Tuning the NLS of ERK-KTR.A, amino acid sequence alignments of ERK-KTR sensor domains, showing the kinase docking site, bNLS, (pink, asterisk) PKA phosphorylation sites, and NES, with predicted NLS strength scores listed to the right. Bar represents 50 μm. B, representative live-cell fluorescence images of HEK293 cells expressing H2B/mTagBFP2, mNeonGreen, and variants of ERK-KTR/tdCherry proteins. −EGF refers to fluorescence images taken prior to the addition of EGF, while +EGF refers to fluorescence micrographs taken 15 min after the addition of 10 nM EGF. Images are from the same cells observed prior to and after addition of EGF. C, bar graph showing C/N ratios of eERK-KTR1.1, 1.2, and 1.3/tdCherry1, following 15 min incubation in 0, 0.3, 3, or 10 nM EGF. D, C/N ratios of eERK-KTR1.2/mScarlet (gold) or eERK-KTR1.2/mScarlet/BCR (brown), after 15 min incubation in 0, 0.1, 0.3, 1, or 10 μM Fsk. The data for these bar graphs came from the analysis of independent groups of cells. All data in this figure are from a minimum of three independent biological replicates, the data for all bar graphs came from the analysis of independent groups of cells, the error bars reflect the SEM, and…

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  Figure 4. Kinetics of ePKA-KTR1.2 and eERK-KTR1.2 responses.A, representative fluorescence micrographs of ePKA-KTR1.2/tdCherry-expressing, ER/mTagBFP2–expressing HEK293 cells at t = 3 min, t = 23 min, and t = 43 min, with cells exposed to Fsk alone at t = 3 min, Fsk at t = 3 min, and H89 at t = 23 min, or Fsk at t = 3 min and EGF at t = 23 min. Bar represents 50 μm. B, plot of ePKA-KTR1.2/tdCherry C/N ratios at every minute in cells exposed to (orange) Fsk only, (green) Fsk followed by H89, and (purple) Fsk followed by EGF. Lines at top denote times of chemical addition; arrows denote the times at which images in (A) were taken. C, plot of ePKA-KTR1.2/tdCherry C/N ratios every minute, in cells exposed to (orange) EGF alone, (green) EGF then VX11e, or (purple) EGF then Fsk. D, double plot of (red) ePKA-KTR1.2/tdCherry C/N ratios and (black) anti-phosphoPKA antibody staining intensity. In these experiments, the cells were incubated for 10 min in 0, 0.3, 1, 3, 10, and 30 μM Fsk, after which the cells were fixed, permeabilized, processed for immunofluorescence microscopy, and imaged, with phospho-PKA levels and C/N ratios calculated from the digital image files. E, representative fluorescence…

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  Figure 5. Simultaneous measurement of PKA activity, ERK activity, and calcium.A, representative fluorescence images of HEK293 cells co-expressing ePKA-KTR1.2/tdTomato, eERK-KTR1.2/emiRFP670, and ER/mTagBFP2 exposed to Fsk at t = 3 min, then EGF at t = 23 min, then H89 at t = 50 min, then VX11e at t = 75 min, with images collected at t = 3, 23, 43, 68, and 93 min. Bar represents 50 μm. B, plot of C/N ratios of (red) ePKA-KTR1.2/tdTomato and (orange) eERK-KTR1.2/emiRFP670 every 60 s, showing their responses to Fsk, then EGF, then H89, then VX11e. Lines at the top denote the times of chemical addition, and arrows at the bottom denote the times at which the images of (A) were collected. C, representative fluorescence images of HEK293 cells co-expressing ePKA-KTR1.2/tdTomato, eERK-KTR1.2/emiRFP670, and ER/mTagBFP2 exposed to Fsk at t = 3 min, then EGF at t = 23 min, then H89 at t = 50 min, then VX11e at t = 75 min. Bar represents 50 μm. D, plot of C/N ratios of (red) ePKA-KTR1.2/tdTomato and (orange) eERK-KTR1.2/emiRFP670 every 60 s, showing their responses to EGF, then Fsk, then VX11e, then H89. E–J, plots of (green) GCaMP8 fluorescence intensity, (red) ePKA-KTR1.2/tdTomato C/N ratio, and…

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  Figure 6. Superior performance of ePKA-KTR1.4 and eERK-KTR1.2 in DRG neurons.A, graph of C/N ratios of (pink) ePKA-KTR1.2, (purple) ePKA-KTR1.4, and (green) PKA-KTR3 in DRG neurons, at baseline, in response to Fsk, and in response to subsequent addition of H89. The p values for the changes in PKA-KTR3 C/N ratio were 0.07 and 0.06, respectively. B, plot of C/N ratios of ePKA-KTR1.4/tdTomato collected every minute in response to (orange) Fsk alone, (green) Fsk followed later by the addition of H89, or (purple) Fsk followed later by the addition of PMA. Lines at the top denote the times of chemical addition. C, graph of C/N ratios of (green) ERK-KTR1 and (orange) eERK-KTR1.2 in DRG neurons at baseline, in response to PMA, and in response to subsequent addition of H89. The p values for the changes in ERK-KTR1 C/N ratio were 0.08 and 0.3, respectively. D, plot of C/N ratios of eERK-KTR1.2/emiRFP670 collected every minute in response to (orange) PMA alone, (green) PMA then VX11e, or (purple) PMA then VX11e. Lines at the top denote the times of chemical addition. E, representative fluorescence images of DRG co-expressing ePKA-KTR1.4/tdTomato, eERK-KTR1.2/emiRFP670, and ER/mTagBFP2, exposed…