Fluorescent Indicators for Imaging Protein Phosphorylation in Single Living Cells
To study protein phosphorylation, investigators have used electrophoresis, immunocytochemistry, and in vitro kinase assays. However, these methods do not provide enough information about spatial and temporal dynamics of protein phosphorylation in each living cell. To overcome this limitation, we have developed genetically encoded novel fluorescent indicators and visualized signal transduction based on protein phosphorylation in living cells (Sato et al., 2002) (Fig. 1). Within the fluorescent indicator, a substrate domain for a protein kinase of interest is fused with a phosphorylation recognition domain via a flexible linker sequence. The tandem fusion unit consisting of the substrate domain, linker sequence, and phosphorylation recognition domain is sandwiched with two different color fluorescent proteins, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), which serve as the donor and acceptor fluorophores for fluorescence resonance energy transfer (FRET). As a result of phosphorylation of the substrate domain and subsequent binding of the phosphorylated substrate domain with the adjacent phosphorylation recognition domain, FRET is induced from CFP to YFP when CFP is excited at 440 ± 10nm. Upon activation of phosphatases, the phosphorylated substrate domain is dephosphorylated and the FRET signal is decreased. This FRET change is represented by the change in the fluorescence emission ratio of CFP at 480 ± 15 nm and YFP at 535 ± 12.5 nm, both of which were monitored continuously by a dual-emission fluorescence microscope. We named this indicator "phocus" (a fluorescent indicator for protein phosphorylation that can be custom-made). Until now, by using suitable substrates and phosphorylation recognition domains, we have developed a large number of phocus variants for several key protein kinases, such as a receptor tyrosine kinase, insulin receptor, and a serine/threonine protein kinase, Akt/PKB (Table I). In addition, these phocus variants were further tailored to visualize subcellular local activity of the respective protein kinases in living cells (Table I). For example, the phocus variant for Akt protein kinase was tethered to the cytoplasmic surface of mitochondria or Golgi membranes by connecting each appropriate sequence/domain. This membrane tethering prevented the free diffusion of the indicator and avoided the resulting loss of spatial information as to phosphorylation by the activated Akt. We thus found that the activated Akt is not in the cytosol but is localized at subcellular membranes, including Golgi and mitochondria membranes, when the cells were stimulated.
II. MATERIALS AND INSTRUMENTATION
ECFP (Cat. No. 6900-1) and EYFP (Cat. No. 6006-1) expression vectors are from Clonthech. Ham's F-12 medium (Cat. No. 21700), fetal bovine serum (Cat. No. 10099-141), and LipofectAMINE 2000 (Cat. No. 11668- 019) reagent are from Invitrogen. Other chemicals used are all of analytical reagent grade. Glass-bottom dishes are from Asahi Techno Glass (Cat. No. 3911-035).
Cells are observed with a 40× oil-immersion objective (Carl Zeiss) on a Axiovert 135 microscope (Carl Zeiss) with a cooled CCD camera MicroMAX (Roper Scientific Inc.) controlled by MetaFluor (Universal Imaging). An excitation filter (440AF21), a dichroic mirror (455DRLP), and emission filters for CFP (480AF30) and YFP (535AF26) are from Omega Optical.
Substrates for protein kinases and phosphatases often exhibit each unique localization, including mitochondria, Golgi, nucleus, and plasma membrane in living cells, which is thought to be critical for specific signal transduction in the respective intracellular loci (Hunter, 2000). Thus, we further tailored our phocuses to analyze the phosphorylation events in such particular locations in single living cells. Here we exemplify phocus for insulin receptor and that for Akt/PKB; the latter was named Aktus.
A. Phocus for Imaging Phosphorylation by Insulin Receptor
IRS-1 is one of the major substrates of insulin receptor. It contains a peckstrin-homology (PH) domain and a phosphotyrosine-binding (PTB) domain in its Nterminal end. The PH and PTB domains bind, respectively, with the phosphoinositides at the plasma membrane and with the juxtamembrane domain of insulin receptor, which is immediately tyrosine phosphorylated by insulin stimulation (Paganon et al., 1999). Thus, the concentration of IRS-1 is thought to be increased around the insulin receptor at the plasma membrane upon insulin stimulation. These PH and PTB domains were fused with phocus, named phocus- 2pp, to locate the phocus around the insulin receptor like the IRS-1 and to measure the local phosphorylation event there. When phocus-2pp was expressed in CHO-IR cells, fluorescence was observed throughout the cells (Fig. 2, time 0s). Upon insulin stimulation, the CFP/YFP emission ratio, which is expressed with pseudocolor, was decreased in the cytosol due to phosphorylation-induced FRET from CFP to YFP within phocus-2pp. Three hundred seconds after insulin stimulation, membrane ruffles, in which a large extent of phocus-2pp were accumulated, appeared around the plasma and disappeared in 1000s (Fig. 2). In these membrane ruffles, phocus-2pp has been found to colocalize with the insulin receptor accumulated there by insulin stimulation. Interestingly, in these membrane ruffles, the extent of phocus-2pp phosphorylation was visualized to be ~2-fold greater than that in the cytosol. This difference in phosphorylation levels between intracellular loci could be due to a different balance of kinase and phosphatase activities between intracellular loci. Phocus-2pp should contribute to reveal the biological significance of such a characteristic domain for tyrosine kinase signaling in the membrane ruffles, which were formed upon insulin stimulation, with high spatial and temporal resolution.
B. Aktus for Imaging Phosphorylation by Akt/PKB
Akt/PKB is a serine/threonine kinase that regulates a variety of cellular responses, such as cell proliferation, cell survival, and angiogenesis (Marte and Downward, 1997). To provide information on the spatial and temporal dynamics of the Akt activity in single living cells, we have developed a genetically encoded fluorescent indicator for Akt, named Aktus (Sasaki et al., 2003). Almost all Akt substrates are localized to subcellular regions. For example, eNOS (Fulton et al., 2001), which mediates a vasodilatory effect by nitric oxide production, is localized predominantly to the Golgi apparatus, whereas Bad (Chao and Korsmeyer, 1998), which is related to apoptosis promotion, is present in mitochondrial outer membranes. By fusing the Aktus with the respectively subcellular localization domains within the eNOS and Bad, eNOSAktus and Bad-Aktus, which are respectively localized to the Golgi apparatus and mitochondrial outer membrane, were developed as shown in Table I and compared with the cytosolic diffusible indicator Aktus. We have shown that in vascular endothelial cells, the Golgi-localized indicator, eNOS-Aktus, was phosphorylated upon stimulation with insulin and with 17β- estradiol, whereas the mitochondria-localized Bad-Aktus was phosphorylated by 17β-estradiol but not by insulin (Table II). However, the diffusible indicator Aktus was not phosphorylated efficiently upon both insulin or 17β-estradiol stimulation (Table II). From these results, it is suggested that the activated Akt is localized to subcellular compartments, including the Golgi apparatus and/or mitochondria, rather than diffusing in the cytosol, thereby efficiently phosphorylating its substrate proteins. Different observation with the mitochondria-localized indicator indicates that localization of the activated Akt to mitochondria is directed differently between insulin and 17β-estradiol via distinct mechanisms. The present indicators and their applications are thus expected to contribute to the studies of a whole range of dynamics of the activated Akt in living cells.
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