Fluorescent indicators for imaging protein phosphorylation in single living cells

Sato, Moritoshi; Ozawa, Toshio; Inukai, Kouichi; Asano, Tomohiko; Umezawa, Yoshio

doi:10.1038/nbt0302-287

Cited by 263 publications

(130 citation statements)

References 46 publications

Supporting

Mentioning

130

Contrasting

Order By: Relevance

“…1a. Upon phosphorylation of the substrate sequence by activated endogenous Src, the adjacent SH2 domain binds with this phosphorylated sequence, and FRET is induced between the two fluorescent units (10,11). Phosphorylation-dependent FRET responses of the indicator are thus observed as a decrease in the fluorescence emission ratio of two GFP mutants.…”

Section: Fluorescence Indicators Visualize Rapid Src Signalingmentioning

confidence: 99%

Epidermal Growth Factor Directs Sex-specific Steroid Signaling through Src Activation

Hitosugi¹,

Sasaki²,

Sato

et al. 2007

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Estrogens and androgens exert many biological effects that do not require interactions of their receptors with chromosomal DNA. However, it has been a long-standing question how the sex steroid receptors provoke signal transduction outside the nucleus. Here we have shown that epidermal growth factor (EGF) directs sex-specific steroid signaling through Src activation. We have revealed that estrogen (E2)-induced Src activation takes place in, not only plasma, but also endomembranes. This was found ascribed to the existence of EGF and the occurrence of EGF receptor (EGFR)-involved endocytosis of estrogen receptor together with Src. EGFR, estrogen receptor, and Src were found to form a complex upon E2 stimulation. The cell growth of breast cancer-derived MCF-7 cells was found to remarkably increase through the above EGF-involved estrogen-signaling process. In contrast, the androgen 5␣-dihydrotestosterone-induced Src activation occurs only in the plasma membrane free from the interaction of EGFR with androgen receptor, irrespective of EGF. The cell growth occurred only moderately as a result. The spatial difference in Src activation between E2 and 5␣-dihydrotestosterone may be responsible for the different extent of observed cell growth.Besides traditional genomic pathways of sex steroid receptors in the nucleus, the extranuclear non-genomic pathways of these receptors are being revealed to strongly relate to many biological consequences, including vascular protection and cell proliferation (1-3). These non-genomic pathways are rapidly mediated through several critical protein kinases. A non-receptor protein tyrosine kinase, Src, is known to be activated immediately after a steroid stimulation (4, 5). This activated Src phosphorylates a wide variety of substrate proteins (such as Shc), which finally induce gene transcription (6, 7). In previous studies, the rapid activation of Src and other kinases by steroids has never been demonstrated in living cells.To answer the question about how the Src activity is nongenomically regulated by steroid receptors in single living cells, we developed a fluorescent indicator for Src kinase activity, and named it Srcus. This indicator can monitor substrate phosphorylation by activated endogenous Src as a fluorescent resonance energy transfer (FRET) 5 response in single living cells. Based on the fluorescence imaging with the present fluorescent indicators, we demonstrated that E2-induced Src activation takes place in not only plasma but also endomembranes. This was found ascribed to the existence of epidermal growth factor (EGF) and the occurrence of EGF receptor (EGFR)-involved endocytosis of estrogen receptor (ER) together with Src. EGFR, ER, and Src were found to form a complex upon E2 stimulation. The cell growth of breast cancer-derived MCF-7 cells was found to remarkably increase through the above EGF-involved estrogen-signaling process. In contrast to estrogen-activated Src signaling, the male steroid hormone 5␣-dihydrotestosterone (DHT) was found to activate Src only in th...

show abstract

Section: Fluorescence Indicators Visualize Rapid Src Signalingmentioning

confidence: 99%

Epidermal Growth Factor Directs Sex-specific Steroid Signaling through Src Activation

Hitosugi¹,

Sasaki²,

Sato

et al. 2007

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Activation of OB-R Monitored by BRET-Energy transferbased techniques such as fluorescence and bioluminescence resonance energy transfer have been successfully used to monitor membrane receptor activation in living cells (25,26,31,32). Here, we assessed the effect of leptin stimulation on basal BRET signals in intact cells expressing either OB-R s or OB-R l .…”

Section: Functional Expression Of Ob-r Fusion Proteins-c-terminalmentioning

confidence: 99%

Activation of the Leptin Receptor by a Ligand-induced Conformational Change of Constitutive Receptor Dimers

Couturier¹,

Jockers

2003

Journal of Biological Chemistry

116

113

View full text Add to dashboard Cite

Binding of leptin to the leptin receptor is crucial for body weight and bone mass regulation in mammals. Leptin receptors were shown to exist as dimers, but the role of dimerization in receptor activation remains unknown. Using a quantitative Bioluminescence Resonance Energy Transfer approach, we show here in living cells that ϳ60% of the leptin receptor exists as constitutive dimers at physiological expression levels in the absence of leptin. No further increase in leptin receptor dimerization was detected in the presence of leptin. Importantly, in cells expressing the short leptin receptor isoform, leptin promoted a robust enhancement of energy transfer signals that reflect specific conformational changes of pre-existing leptin receptor dimers and that may be used as read-out in screening assays for leptin receptor ligands. Both leptin receptor dimerization and the leptin-induced energy transfer were Janus kinase 2-independent. Taken together, our data support a receptor activation model based on ligand-induced conformational changes rather than ligand-induced dimerization.

show abstract

“…These results demonstrate that the YFP/CFP emission ratio of Chimera reflects the activation of endogenous SFK at membrane ruffles. DISCUSSION GFP-based FRET technology is increasingly being used to visualize intracellular signaling in living cells (21,(25)(26)(27)(28)(29)(30). With this technique we have examined the interaction between Csk (a negative regulatory kinase for SFK) and Cbp (a transmembrane protein that can recruit Csk when phosphorylated by SFK).…”

Section: Monitoring Of Sfk Activation With a Single-molecule Fretmentioning

confidence: 99%

Mechanism of Csk-mediated Down-regulation of Src Family Tyrosine Kinases in Epidermal Growth Factor Signaling

Matsuoka

Nada

Okada

2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

Fluorescent indicators for imaging protein phosphorylation in single living cells

Cited by 263 publications

References 46 publications

Epidermal Growth Factor Directs Sex-specific Steroid Signaling through Src Activation

Epidermal Growth Factor Directs Sex-specific Steroid Signaling through Src Activation

Activation of the Leptin Receptor by a Ligand-induced Conformational Change of Constitutive Receptor Dimers

Mechanism of Csk-mediated Down-regulation of Src Family Tyrosine Kinases in Epidermal Growth Factor Signaling

Contact Info

Product

Resources

About