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. 2006 Mar;26(6):2065–2079. doi: 10.1128/MCB.26.6.2065-2079.2006

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Copyright © 2006, American Society for Microbiology

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FIG. 5. — Sorbitol-induced micro-FRET changes for MEKK2/MEK5 and MEK5/ERK5 interactions. (A) CFP-WT MEK5 and YFP-WT MEKK2 were coexpressed in COS7 cells. After 4 h of serum starvation, cells were stimulated with 200 mM sorbitol. Images were captured at the indicated time points after sorbitol stimulation. YFP, CFP, and FRET^c images are shown in the three left columns. The FRET^c signals between CFP-MEK5 and YFP-⁴⁷K-A MEKK2 and between CFP-³¹¹S³¹⁵T-AV MEK5 and YFP-MEKK2 are presented in the middle two columns. The FRET^c images between CFP-MEK5 and YFP-MEKK2 in the presence of Xpress-ERK5 and CFP-³¹¹S³¹⁵T-DD MEK5 and YFP-MEKK2 are shown in the right two columns. (B) Time course of FRET^c between CFP-WT MEK5 and YFP-WT ERK5 in COS7 cells stimulated with 200 mM sorbitol (left three columns). The middle two columns represent FRET^c time courses for CFP-PB1 domain-deleted MEK5 (ΔPB1 MEK5) and YFP-ERK5 and for CFP-³¹¹S³¹⁵T-AV MEK5 and YFP-ERK5. The FRET^c images between CFP-MEK5 and YFP-ERK5 in the presence of HA-MEKK2 and CFP-³¹¹S³¹⁵T-DD MEK5 and YFP-ERK5 are shown in the right two columns. (C, D, and E) Analysis of the changes in FRET^c/CFP between CFP-MEK5 and YFP-MEKK2 after sorbitol stimulation. FRET^c/CFP between CFP-MEK5 and YFP-MEKK2 (C), CFP-MEK5 and YFP-⁴⁷K-A MEKK2 (D), and CFP-³¹¹S³¹⁵T-AV MEK5 (designated ^311.315ST-AV MEK5 in figure) and YFP-MEKK2 (E) was evaluated. The value of FRET^c divided by CFP (FRET^c/CFP) ± the SD for multiple images (n = 5 to 7) was used for statistical analysis of multiple experiments. The results shown are in each case representative of three replicate experiments. The asterisk in panel C indicates statistical significance (P < 0.01) between the cells at time zero and at 1 min as determined by paired t test. (C to E, inset) Anti-MEKK2 antibody blot of WT or ⁴⁷K-A MEKK2 expressed in COS7 cells harvested at the indicated times after sorbitol stimulation showing the MEKK2 gel shift that correlates with MEKK2 activation. (F to H) Data analyses of the FRET^c/CFP kinetics between CFP-MEK5 and YFP-ERK5 after sorbitol stimulation. FRET between CFP-MEK5 and YFP-ERK5 (F), CFP-ΔPB1 MEK5 and YFP-ERK5 (G), and CFP-³¹¹S³¹⁵T-AV MEK5 and YFP-ERK5 (H) was calculated as FRET^c/CFP ± the SD for multiple images (n = 6). The results shown are in each case representative of three replicate experiments. The asterisk in panel F indicates statistical significance (P < 0.01) versus the cells at time zero as determined by paired t test. (F to H, inset) Anti-phospho-ERK5 and anti-ERK5 antibody blots at the indicated time point after sorbitol stimulation.

FIG. 5. — Sorbitol-induced micro-FRET changes for MEKK2/MEK5 and MEK5/ERK5 interactions. (A) CFP-WT MEK5 and YFP-WT MEKK2 were coexpressed in COS7 cells. After 4 h of serum starvation, cells were stimulated with 200 mM sorbitol. Images were captured at the indicated time points after sorbitol stimulation. YFP, CFP, and FRET^c images are shown in the three left columns. The FRET^c signals between CFP-MEK5 and YFP-⁴⁷K-A MEKK2 and between CFP-³¹¹S³¹⁵T-AV MEK5 and YFP-MEKK2 are presented in the middle two columns. The FRET^c images between CFP-MEK5 and YFP-MEKK2 in the presence of Xpress-ERK5 and CFP-³¹¹S³¹⁵T-DD MEK5 and YFP-MEKK2 are shown in the right two columns. (B) Time course of FRET^c between CFP-WT MEK5 and YFP-WT ERK5 in COS7 cells stimulated with 200 mM sorbitol (left three columns). The middle two columns represent FRET^c time courses for CFP-PB1 domain-deleted MEK5 (ΔPB1 MEK5) and YFP-ERK5 and for CFP-³¹¹S³¹⁵T-AV MEK5 and YFP-ERK5. The FRET^c images between CFP-MEK5 and YFP-ERK5 in the presence of HA-MEKK2 and CFP-³¹¹S³¹⁵T-DD MEK5 and YFP-ERK5 are shown in the right two columns. (C, D, and E) Analysis of the changes in FRET^c/CFP between CFP-MEK5 and YFP-MEKK2 after sorbitol stimulation. FRET^c/CFP between CFP-MEK5 and YFP-MEKK2 (C), CFP-MEK5 and YFP-⁴⁷K-A MEKK2 (D), and CFP-³¹¹S³¹⁵T-AV MEK5 (designated ^311.315ST-AV MEK5 in figure) and YFP-MEKK2 (E) was evaluated. The value of FRET^c divided by CFP (FRET^c/CFP) ± the SD for multiple images (n = 5 to 7) was used for statistical analysis of multiple experiments. The results shown are in each case representative of three replicate experiments. The asterisk in panel C indicates statistical significance (P < 0.01) between the cells at time zero and at 1 min as determined by paired t test. (C to E, inset) Anti-MEKK2 antibody blot of WT or ⁴⁷K-A MEKK2 expressed in COS7 cells harvested at the indicated times after sorbitol stimulation showing the MEKK2 gel shift that correlates with MEKK2 activation. (F to H) Data analyses of the FRET^c/CFP kinetics between CFP-MEK5 and YFP-ERK5 after sorbitol stimulation. FRET between CFP-MEK5 and YFP-ERK5 (F), CFP-ΔPB1 MEK5 and YFP-ERK5 (G), and CFP-³¹¹S³¹⁵T-AV MEK5 and YFP-ERK5 (H) was calculated as FRET^c/CFP ± the SD for multiple images (n = 6). The results shown are in each case representative of three replicate experiments. The asterisk in panel F indicates statistical significance (P < 0.01) versus the cells at time zero as determined by paired t test. (F to H, inset) Anti-phospho-ERK5 and anti-ERK5 antibody blots at the indicated time point after sorbitol stimulation.