Genetically Encoded Bioluminescent Indicator for ERK2 Dimer in Living Cells

Abstract

In this study, a genetically encoded bioluminescent indicator for ERK2 dimer was developed with the split Renilla luciferase complementation method, in which the formation of ERK2 dimer induces a spontaneous emission of bioluminescence in living cells. In response to extracellular stimuli, such as epidermal growth factor (EGF) or 17beta-estradiol (E2), extracellular signal-regulated kinase 2 (ERK2) is phosphorylated by its upstream kinase MEK, and also phosphorylates its substrates in various regions of the cell, including the nucleus. Phosphorylated ERK2 is led to form its dimer, thereby transporting itself into the nucleus. We demonstrated with the indicator that stimulation with EGF or E2 induces the formation of ERK2 dimer in living MCF-7 cells. The dynamics of this dimer formation was examined and discussed.

Split Renilla Luciferase Protein Fragment-assisted Complementation (SRL-PFAC) to Characterize Hsp90-Cdc37 Complex and Identify Critical Residues in Protein/Protein Interactions

“…SRL-PFAC imaging has been validated to monitor protein/ protein interactions, including the heterodimerization between MyoD/Id (27), the interacting location of Tyr-941/Src homology 2 domain (41), the interaction of each Hsp90 isoform (␣/␤) with p23 (29), homodimeric formation of herpes simplex virus type 1-thymidine kinase (42), and dimerization of ERK2 (43). Here, we optimized the SRL-PFAC system to study full-length human Hsp90/human Cdc37 interaction, and we applied this system to identify critical residues for human Hsp90/Cdc37 interactions in living cells.…”

Bioluminescent Indicators for Ca2+ Based on Split Renilla Luciferase Complementation in Living Cells

“…[9][10][11] The mutation of cysteine 124 in Renilla luciferase increaseed its enzymatic activity, and its luminescence was 4.1 ¥ 10 fold higher than that with wild hRL (Table 1). The luminescence spectrum of this mutant C124A was similar to that of wild-type Renilla luciferase (Fig.…”

“…CaM, C124A, hRLn and hRLc were prepared as described previously. [9][10][11][12] Cell culture and transfection MCF-7 cells were cultured in MEM supplemented with 10% heat-inactivated fatal calf serum, 1 mM sodium pyruvate, 100 mM MEM non-essential amino-acid solution, 100 unit/mL penicillin and 100 mg/mL streptomycin. Cells were maintained in 5% CO2 at 37˚C.…”

“…The Ca 2+ -induced interaction between CaM and M13 leads to complementation of the N-and C-terminal halves of split Renilla luciferase in living cells. 9,10 This intramolecular interaction results in the spontaneous and simultaneous emission of bioluminescence from split Renilla luciferase domains with coelenterazine as a substrate.…”

Monomeric and Dimeric Models of ERK2 in Conjunction with Studies on Cellular Localization, Nuclear Translocation, and In Vitro Analysis

“…Dimerization upon phosphorylation was also studied in vivo using a bioluminescence indicator (Kaihara and Umezawa, 2008). Genetically encoded tandem array of ERK2 were linked in the N-and C-termini with the N-and C-terminal halves of split Renilla luciferase.…”