Recently, we found that an alkoxide-bridged dinuclear zinc(II) complex of 1,3-bis[bis(pyridin-2-ylmethyl) amino]propan-2-olate acts as a novel phosphate capture molecule under physiological conditions. The dizinc(II) complex forms a stable 1:1 complex with a phosphate monoester dianion in aqueous solutions. The X-ray crystal analysis of the 1:1 dizinc(II) complex with a p-nitrophenyl phosphate dianion disclosed that each phosphate oxygen anion binds to a zinc(II) at the fifth coordination site and that the two zinc(II) ions are separated by a distance of 3.6 Å. Thus, the dizinc(II) complex having a vacancy on the two zinc(II) ions is suitable for the access of a phosphate monoester dianion. In an aqueous solution, the dizinc(II) complex strongly binds to the phenyl phosphate dianion (Kd=2.5×10-8 M) at a neutral pH. The anion selectivity indexes against SO42-, CH3COO-, Cl-, and the diphenyl phosphate monoanion at 25°C are 5.2×103, 1.6×104, 8.0×105, and >2×106, respectively. In addition, the 1:1 formation of the dizinc(II) complex and an inorganic phosphate dianion (HOPO32-) was clearly detected by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) based on a characteristic mass shift and a total-charge change of the phosphate (from -2 to +1). This finding introduced a new procedure for the analysis of phosphorylated compounds using the dizinc(II) complex as an MS probe. Therefore, we termed the dinuclear metal complex "Phos-tag" as a phosphate-binding tag molecule.
Phosphorylation is a major post-translational modification widely used in regulation of many cellular processes. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase activated by activation subunit p35. Cdk5-p35 regulates various neuronal activities such as neuronal migration, synaptic activity, and cell death. The kinase activity of Cdk5 is regulated by proteolysis of p35: proteasomal degradation causes downregulation of Cdk5 whereas cleavage of p35 by calpain causes overactivation of Cdk5. Phosphorylation of p35 determines the proteolytic pathway. We have previously identified Ser8 and Thr138 as major phosphorylation sites using metabolic labeling of cultured cells followed by 2D-phosphopeptide mapping and phospho-specific antibodies. However, these approaches cannot determine the extent of p35 phosphorylation in vivo. Here we report the use of Phos-tag SDS-PAGE to reveal the in vivo phosphorylation states of p35. Using Phos-tag acrylamide, electrophoretic mobility of phosphorylated p35 was delayed because it is trapped at Phos-tag sites. We constructed phosphorylation-dependent banding profiles of p35 and Ala substitution mutants at phosphorylation sites co-expressed with Cdk5 in COS-7 cells. Using the standard banding profiles, we assigned respective bands of endogenous p35 with combinations of phosphorylation states, and quantified Ser8, Ser91 and Thr138 phosphorylation. This is the first quantitative and site-specific measurements of phosphorylation of p35, demonstrating the usefulness of Phos-tag SDS-PAGE for analysis of phosphorylation states of in vivo proteins.
Activation of myosin by phosphorylation has been implicated in regulation of smooth muscle contractions. The ability to analyze myosin phosphorylation is crucial for better understanding of the regulatory mechanisms of smooth muscle contraction. In myosin phosphorylation analysis, it is important to quantify the ratio between phosphorylated and unphosphorylated myosin because those can have opposite effects on contractions. For this reason, urea/glycerol-PAGE and 2-D electrophoresis in combination with Coomassie blue staining or Western blotting are commonly used to separate and quantify myosin based on its phosphorylated state. However, those conventional techniques are not sensitive enough to analyze minute smooth muscles, such as terminal resistant arterioles, partly because of a poor separation in electrophoresis with a small sample. Recently we overcame this problem by introducing a phos-tag technique, and achieved picogram sensitivity. In this article, the sensitive phosphorylation quantification method and application of phos-tag electrophoresis to smooth muscle physiology research are reviewed.
Two-dimensional electrophoresis using Phos-tag affinity electrophoresis enabled us to sensitively detect and separate the phosphorylated forms of protein. When we used 2-DE with isoelectric focusing using IPG gel strip in the first dimension and Phos-tag affinity electrophoresis in the second dimension, the nuclear fraction contained more than 20 spots of endogenous heterogeneous nuclear ribonucleoprotein K (hnRNP K) on the 2-DE map. Mass spectrometric analysis indicated that these multiple forms of hnRNP K were produced mainly by alternative splicing of the single hnRNP K gene and phosphorylation of Ser116 and/or Ser284. By applying this two-dimensional electrophoresis map of hnRNP K, we indicated that the multiple forms were differentially modulated in response to external stimulation.
Enrichment of phosphopeptides is an important process for mass spectrometric analysis. Here, we used the enrichment method using the biphasic Phos-tag/C18 tip, which consists of Phos-tag agarose beads overlaid on the C18 disk filter in a micropipet tip. In the cell lysates of GIST882 cells, 3187 phosphopeptides (1688 unique phosphopeptides) were identified by nano LC-MS/MS analysis coupled with phosphopeptide enrichment using this method. This method was comparable to another phosphopeptide enrichment method using Titansphere Phos-TiO Kit in numbers of peptides detected. A total number of unique phosphopeptides detected by the two methods was 3202, and the overlap between phosphopeptides enriched by two methods was 36%. Therefore, it is concluded that Phos-tag agarose method is an alternative method for identification of phosphoproteins.
Phos-tag serves as an analytical tool for selective enrichment of phosphorylated molecules. Recent technological advances regarding mass spectrometry-based quantitative proteomics, in combination with Phos-tag technology, have enabled us to describe the comprehensive status of phosphorylated signaling molecules in a time-resolved manner. Integrative analysis of phosphoproteome dynamics with transcriptome regulation in breast cancer cells uncovered core network hubs related to drug resistance at the system level.
In the unicellular eukaryote Colpoda cucullus, resting cyst formation (encystment), which is a cellular morphogenetic process, is mediated by intracellular signaling pathways, which are triggered by an inflow of Ca2+ due to cell-to-cell mechanical contact. The enhanced chemiluminescence detection (ECL) for phosphorylated proteins using biotinylated Phos-tag showed that the phosphorylation level in several proteins was enhanced in Ca2+-dependent manner prior to the beginning of the cyst formation (within 1 h after the onset of encystment induction). The cAMP enzyme immunoassay (EIA) showed that the intracellular cAMP concentration was elevated in encystment-induced cells. The encystment induction and the phosphorylation of some proteins are slightly promoted by the addition of membrane-permeable derivatives of cAMP or non-selective phosphodiesterase inhibitor, while they tend to be suppressed by the addition of an inhibitor of cAMP-dependent kinase (PKA). These results suggest that a Ca2+-activated signaling pathway involving cAMP/PKA-dependent protein phosphorylation may be responsible for the encystment induction of C. cucullus. Encystment-specific phosphorylated proteins were isolated by phosphate-affinity chromatography using Phos-tag agarose beads, and some of them were tentatively identified by mass spectrometry analysis.
Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are growth factor-like bioactive lipids having a phosphate monoester residue. Phos-tag can bind to them and be used both for purification and quantification. In a two-phase solvent system consisting of chloroform/methanol/water, addition of Phos-tag move LPA and S1P from a hydrophilic phase to a hydrophobic phase in the form of their Phos-tag complexes. Using this property, we developed a method for purification of LPA and S1P in biological materials by the phase separation technique. Advantages of use of Phos-tag for detection of LPA and S1P in MALDI-TOF MS are an increase in ionization efficiency and detection as a single-ion form. Homologues of LPA and S1P in natural samples can be quantified by MALDI-TOF MS by using internal standards.
We describe an improved Phos-tag SDS-PAGE (Zn2+-Phos-tag SDS-PAGE) using a dizinc(II) complex of Phos-tag acrylamide in conjunction with a neutral-pH gel system buffered with Bis-Tris hydrochloride (Bis-Tris-HCl) to detect shifts in the mobility of phosphorylated proteins. Our alternative technique (Mn2+-Phos-tag SDS-PAGE) using a polyacrylamide-bound Mn2+-Phos-tag and a conventional Laemmli's buffer system under alkaline pH conditions has limitations for separating certain phosphoproteins. The major improvements and utilities were demonstrated by visualizing novel up-shifted bands of commercially available pepsin, recombinant substrate protein tau treated in vitro with tyrosine kinases, and endogeneous β-catenin in whole cell lysates. Additionally, the Zn2+-Phos-tag SDS-PAGE gels showed better long-term stability than the Mn2+-Phos-tag SDS-PAGE gels. We can thus present a simple, convenient, and more reliable "in-house" gel system for phosphate-affinity SDS-PAGE.
We introduce a novel fluorescence resonance energy transfer (FRET) system for the detection of a phosphorylated molecule such as a phosphopeptide using a phosphate-binding tag molecule, Zn(II)-Phos-tag (1,3-bis[bis (pyridin-2-ylmethyl)amino]propan-2-olato dizinc(II) complex) attached with a 7-amino-4-methylcoumarin-3-acetic acid (AMCA). 5-Carboxyfluorescein (FAM)-labeled phosphopeptides and nonphosphopeptides were prepared as the target molecules for the FRET system. A set of FAM (a fluorescent acceptor, emission at 520 nm) and AMCA (a fluorescent donor, excitation at 345 nm) is frequently used for a FRET system. The AMCA-labeled Zn(II)-Phos-tag captured specifically the FAM-labeled phosphopeptide to form a stable 1:1 complex, resulting in efficient FRET. After the FAM-labeled phosphopeptide was dephosphorylated with alkaline phosphatase, the FRET disappeared. Using this FRET system, we demonstrated the detection of the time-dependent reversible phosphorylation of the FAM-labeled substrate peptide. The Phos-tag-based FRET system has the following major advantages: i) The real-time analysis of the reversible phosphorylation reaction is possible without multiple samplings, ii) the analysis requires a simple procedure just using two solutions of AMCA-labeled Phos-tag and a FAM-labeled compound, and iii) the system would be useful for the reliable and comprehensive phosphorylation assays for various phosphopeptides containing phosphoserine, phosphothreonine, or phosphotyrosine, in vitro. Thus, the principle of this system would be applied to high-throughput kinase/phosphatase profiling, measurement of enzyme activity, and determination of an activator or an inhibitor.