KR20040009726A - Fluorescence-Labeled Peptide for ILK1 Protein Kinase Assay and Non-Radioactive ILK1 Protein Kinase Assay Using the Same - Google Patents

Abstract

PURPOSE: A fluorescence-labeled peptide for protein kinase ILK1 assay and a non-radioactive protein kinase ILK1 assay using the same are provided, thereby easily, rapidly, accurately and stably determining the activity of purified or unpurified protein kinase ILK1 without using radioactive isotope. CONSTITUTION: A peptide for protein kinase ILK1 assay is characterized by having the amino acid sequence of SEQ ID NO: 13, wherein the peptide is selected from the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 8; and the peptide is labeled with fluorescence. A method for determining activity of non-radioactive protein kinase ILK1 comprises the steps of: labeling a peptide for protein kinase ILK1 assay with fluorescence; reacting the fluorescence-labeled peptide with protein kinase ILK1; subjecting the reacted fluorescence-labeled peptide to electrophoresis to separate phosphorylated and unphosphorylated peptides; and determining the quantity of phosphorylated peptide.

Description

Fluorescence-Labeled Peptide for ILK1 Protein Kinase Assay and Non-Radioactive ILK1 Protein Kinase Assay Using the Same}

The present invention relates to a fluorescently labeled peptide for measuring ILK1 (integrin linked kinase 1) protein kinase activity and a non-radioactive ILK1 protein kinase activity assay using the same. More specifically, the present invention reacts fluorescently labeled peptides and electrofluorescent peptides with ILK1 protein kinase to measure the activity of protein kinase, electrophoreses at their isoelectric point, and then the amount of phosphorylated peptide is determined. By measuring, the present invention relates to a method for measuring the activity of ILK1 protein kinase.

Phoshpothidylinositol 3 kinase (PI3K) related signaling pathways are activated by protein-protein interactions as several receptors, growth factors, cytokines, and peripheral factors activate their receptors and signal transduction into the cell. Under the direct control of PI3K, ILK1 is a serine / threonine protein kinase, a serine / threonine of the hydrophobic pocket of a protein kinase belonging to the cyclic AMP / cyclic GMP dependent and protein kinase C related protein kinase (AGC) family containing Akt protein kinase. Phosphorylates residues and activates these protein kinase. Therefore, the ILK1 is assumed to play an important mediator in the process of overlapping each other and transmitting a signal from the cell membrane to the nucleus. In particular, ILK1 protein kinase, which is involved in the survival and death of cells and plays a pivotal role in regulating the action of serine / threonine protein kinase Akt protein kinase, is also receiving attention. It is the subject of intensive research.

However, conventionally, by measuring the degree to which 32P of the radioisotope gamma 32P ATP is transferred to a protein substrate or a peptide according to a phosphorylation reaction, the activity of various kinase including ILK1 protein kinase was measured. It was necessary to purify enzymes for the activity measurement of these enzymes in cells coexisting with different types of phosphatase, and it was difficult to measure them quickly, and it was difficult to deal with radioisotopes and potential for radiation exposure of experimenter and people around. The risk of being unavoidable was unavoidable.

For these reasons, there is a constant need to develop methods for measuring the activity of purified or unpurified ILK1 protein kinase without the use of radioisotopes in a simple, rapid, accurate and safe manner.

Accordingly, the present inventors have made efforts to develop a method for measuring the activity of purified, as well as unpurified ILK1 protein kinase without the use of radioisotopes. By measuring the activity of ILK1 protein kinase using, it was confirmed that it is possible to measure the activity of purified or unpurified ILK1 protein kinase without using a radioisotope, thereby completing the present invention.

1 is a photoelectrophoresis by conventional ILK1 protein kinase activity assay using radioisotopes.

Figure 2 is an electrophoresis picture showing the activity according to the amount (A) and reaction time (B) of the ILK1 protein kinase for the peptide of SEQ ID NO: 1.

Figure 3 is an electrophoresis picture showing the specificity (A) of the ILK1 protein kinase to the peptide of SEQ ID NO: 1 and the specificity (B) of the peptide of SEQ ID NO: 1 for various protein kinase.

Figure 4 is an electrophoresis picture of measuring the activity of ILK1 protein kinase in cell lysate after treatment with PI3 / Akt protein kinase activity inhibitor in human melanoma cell line, using the peptide of SEQ ID NO: 1 as a substrate.

Figure 5 is an electrophoresis picture of the activity of ILK1 protein kinase in the cell lysate after the protein kinase activator was treated to a human melanoma cell line, using the peptide of SEQ ID NO: 1 as a substrate.

Phosphorylated Substrate Proteins and Consensus Sequences (motif) by ILK1 Candidate proteins ILK phosphorylation site Akt 1,2,3 473 S; PHFPQFSYAS Peptide of SEQ ID NO: 1 p70 S6K 389 T; QVFLGFTYVA Peptide of SEQ ID NO: 2 PKC delta 664 S; SAFAGFSFVN Peptide of SEQ ID NO: 3 SGK 1,2,3 422 S; EAFLGFSYAP Peptide of SEQ ID NO: 4 MAPKKK5 / ASK1 998 S; IKYDPFSFKT Peptide of SEQ ID NO: 5 PDK1 147 T; FVYLYFTFQD Peptide of SEQ ID NO: 6 Integrin beta 1 704 T; DCWFVFTYSV Peptide of SEQ ID NO: 7 MSK 1,2 376 S; KLFQGYSFVA Peptide of SEQ ID NO: 8 Consensus xxΦxxΦT / SΦXX Peptide of SEQ ID NO: 13

The inventors of the present invention searched through the protein database (http://www.ncbi.nlm.nih.gov/entrez/) for the amino acid sequence conserved in the hydrophobic pocket of AGC, and as a result SEQ ID NO: 13 Was found to exist (Table 1). The Akt protein kinase 3 (Akt3) belonging to the AGC family was searched by entrez to find that the peptide of SEQ ID NO: 1 corresponding to the conserved sequence was present at the 473 th amino acid serine. The mutation of the 473 th serine of Akt 3 with alanine was used to verify whether the wild type Akt 3 was phosphorylated by ILK. Therefore, the ILK1 protein was synthesized using the fluorescent peptide of SEQ ID NO. 1 synthesized to complete the method for measuring the activity of ILK1 protein kinase simply, quickly, accurately and safely, as in PDK1, Akt kinase and MAPK kinase kinase 3. The activity of kinase was measured (FIG. 2). However, the phosphorylation of this peptide was not phosphorylated by other protein kinase (FIG. 3). The results show that this conserved sequence is required to phosphorylate the substrate of ILK1. Based on the results of the protein database search and the results of analysis of the conventional ILK1 protein kinase activity assay using gamma 32P ATP, the invention of the fluorescent label peptide for measuring the ILK1 protein kinase activity was completed as follows (FIG. 4). ).

Fluorescent label peptide for measuring ILK1 protein kinase activity of the present invention is represented by the peptide of SEQ ID NO: 13. ILK1 kinase shows a reaction specificity to the peptide of SEQ ID NO: 13, and does not react at all with a peptide in which the seventh serine or threonine of the peptide of SEQ ID NO: 13 is substituted with another 18 amino acids (FIG. 4). Thus, these peptides act as highly selective substrates for ILK1 protein kinase, allowing accurate activity measurements even with partially purified enzymes or unpurified cell lysates. As the above peptides, peptides of SEQ ID NOS: 1 to 8 which exist in nature, preferably in Table 1, may be mentioned.

In addition, the non-radioactive ILK1 protein kinase activity assay of the present invention comprises the steps of fluorescently labeling the peptide; Reacting the fluorescently labeled electric peptide with ILK1 protein kinase; Electrophoresis at the isoelectric point after completion of the reaction to separate the phosphorylated peptide from the non-phosphorylated peptide; And measuring the amount of phosphorylated peptide.

Hereinafter, the non-radioactive ILK1 protein kinase activity assay described above will be described in more detail.

First, a fluorescent material such as fluorescein is conjugated to the peptide to fluoresce, and then phosphorylation occurs on the fluorescent peptide by reacting ILK1 protein kinase with a fluorescently labeled peptide in the presence of ATP. Since unphosphorylated peptides are not charged at their neutral pH (isoelectric point), they do not migrate in the electric field, and phosphorylated peptides carry a -divalent charge, which moves to the anode when the electric field is applied. Using this difference, agarose gel electrophoresis is performed to separate both peptides. Phosphorylated peptide can be quantitated by analyzing agarose gel using densitometry, or by fluorescence measurement after separating phosphorylated peptide from agarose gel. Can be detected within 2 hours. On the other hand, since the substrate peptide of the present invention exhibits a substrate specificity that does not react with other kinase present in the sample as described in the following examples, even partially purified enzymes or unpurified cell lysates can be used. Compared to the method for measuring kinase activity using isotopes, there is little noise or background, and the potential risk of using isotopes can be avoided.

Through the following examples will be described in more detail the present invention. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Comparative Example 1 Synthesis of Fluorescent Labeled Peptide for Measurement of ILK1 Protein Phosphorase Activity

In the presence of Gamma 32P ATP, ILK1 protein kinase was reacted with SGK1 S189A in which the substrate protein wild-type SGK1 (SGK1 WT) and SGK1 protein serine at position 189 were replaced with alanine and then treated with or without alkaline dekinase. After electrophoresis, X-ray pictures were taken (see FIG. 1). In the upper part of FIG. 1, the used substrate proteins (SGK1 WT, SGK1 S189A) are displayed, and the alkaline dephosphatase treated (+) and the untreated (-) are indicated, and the negative control (negative control) The substrate protein reacted without adding ILK1 or adding ATP containing 32P, respectively. In the arrow to the right, ILK1 protein kinase comprising 32P is labeled ILK1 and Akt 3 WT or Akt3 S473A is labeled Akt3. As shown in FIG. 1, as a result of electrophoresis by conventional ILK1 protein kinase activity assay using radioisotopes, wild type Akt 3 (Akt 3 WT) was phosphorylated by ILK1 protein kinase, but 473 serine of Akt 3 was phosphorylated. Since the mutant protein (Akt3 S473A) substituted with alanine was not phosphorylated, it was confirmed that the ILK1 protein kinase phosphorylates itself with serine 473 of Akt3, and using this amino acid sequence information, the Akt3 hydrophobic pocket (fluorescerin The peptide labeled peptide of SEQ ID NO: 1) was synthesized.

Example 1 activity measurement according to the amount and reaction time of the ILK1 protein kinase

In order to confirm the sensitivity of the ILK1 protein kinase activity assay using the fluorescently labeled substrate peptide (peptide of SEQ ID NO: 1), the activity according to the amount (A) and reaction time (B) of the ILK1 protein kinase was as follows. Measurement (see Fig. 2): A peptide of SEQ ID NO: 1 was prepared by attaching isothiocyanate fluorescein (FITC) to the amino terminus as a substrate of ILK1 protein kinase. 5 μl of an electrofluorescein conjugated fluorescent peptide was reacted with 20 μl of protein kinase with 5 μl of differentially-treated cell lysate or 5, 10 and 20 ng of activated ILK1 protein kinase. Solution (20 mM HEPES, pH 7.2, 10 mM MgCl2, 10 mM MnCl2, 1 mM dithiothreitol, 0.2 mM EGTA, 20 mM ATP, 1 mg phosphatidylserine, ILK1 protein kinase activator, 30 to 30 minutes) After the reaction, the reaction was stopped by heating at 95 for 10 minutes The reaction was electrophoresed at 0.8 V agarose gel, TBE buffer (pH 8.0) for 15 minutes at 100V voltage, and the phosphorylated peptide was not phosphorylated. The electrophoretic gel was placed on a UV-spectrometer and photographed, the phosphorylated peptide band was cut, eluted with a predetermined amount (1 ml) of distilled water for 30 minutes, quantified at an optical density of 488 mm, and used. The conversion ratio of the phosphorylated product to the amount of was calculated as black arrowheads in Figure 2. As shown in Figure 2, an increase in the amount of ILK1 protein kinase and a certain amount of ILK1 Increasing the reaction time of the protein kinase can increase the activity of ILK1 protein kinase, and can detect the activity of ILK1 protein kinase up to about 5ng.

Example 2 : Specificity of ILK1 Enzyme for Fluorescently Labeled Peptide of SEQ ID NO: 1

To confirm the substrate specificity of the phosphorylation reaction by ILK1 protein kinase, Akt3 hydrophobic pocket peptide (SEQ ID NO: 1) Akt3 T loop peptide (SEQ ID NO: 9), Rac1 peptide (SEQ ID NO: 10), MEKK3 Pepetide (peptide of SEQ ID NO: 11), IRS-2 pepetide (peptide of SEQ ID NO: 12) were synthesized, and fluorescein was conjugated to fluoresce, and reacted with ILK1 protein kinase in the same manner as in Example 1 above. Was performed. As a result, it was observed that ILK1 caused phosphorylation only in response to Akt3 peptide (peptide of SEQ ID NO: 1) (FIG. 3A). In addition, using the peptide of SEQ ID NO: 1 as a substrate, the reaction was performed in the same manner as in Example 1 with 20 ng of other protein kinases (PKC, GSK3, PDK1, Akt1, Akt 3 SGK1, MEKK3,) instead of ILK1 protein kinase. Was performed. As shown in FIG. 3B, the peptides of Rac1 and IRS-1 did not react with ILK1, and the ILK peptide did not react at all with other protein kinase enzymes.

Example 3 Changes in the Activity of ILK1 Protein Kinase According to the Treatment Concentration of PI3K / Akt Protein Kinase Inhibitor

To confirm that the PI3K / Akt signaling activator (LY29002) was tested for the effect on the ILK1 protein kinase by a method using fluorescently labeled peptides, the following experiment was performed: This inhibitor was treated in human melanoma cell lines. Next, the activity of ILK1 protein kinase in cell lysate was measured. As a substrate, the peptide of SEQ ID NO: 1 of Table 1 was used, and the reaction was carried out in the same manner as in Example 1, using 5 µl of sorted-treated cell lysate which was not purified. ). Phosphorylated peptides in FIG. 4 are indicated by black arrowheads. As shown in Figure 4, it was confirmed that the phosphorylation of the ILK1 substrate peptide decreases in proportion to the treatment concentration of the PI 3 / Akt protein kinase inhibitor. This shows that ILK activity is affected by the PI3K / Akt signaling system.

Example 4 Changes in the Activity of ILK1 Protein Phosphorylase According to the Treatment Concentration of Protein Kinase C Activator

In order to confirm that the change in the enzyme activity regulated according to the treatment concentration of the protein kinase C (okadaic acid) was examined by the method using a fluorescent substrate, the following experiment was performed: The protein kinase activator was activated in human black. After treatment with the species cell line, the activity of ILK1 protein kinase in cell lysate was measured. The peptide used as the substrate was used as the peptide of SEQ ID NO: 1 in Table 1, and the reaction was carried out in the same manner as in Example 1 using 5 µl of the separated-treated cell lysate which was not purified. 5). In FIG. 5, phosphorylated peptides are indicated by black arrowheads. As shown in FIG. 5, irrespective of the treatment concentration of the protein kinase activator, the phosphorylation of the ILK substrate peptide was not changed.

As described in detail above, the present invention provides a method for measuring the activity of ILK1 protein kinase, simply, quickly, accurately and safely using a fluorescently labeled peptide. According to the method for measuring the activity of the ILK1 protein kinase of the present invention, it is simple, rapid, accurate and safe to measure the activity of purified and unpurified ILK1 protein kinase without using radioisotopes. It is possible to contribute greatly to the study of the role of ILK1 protein kinase in the field of cell signaling and the screening of ILK activation / inhibitor.

Claims (5)

Peptide for measuring ILK1 protein kinase activity represented by the peptide of SEQ ID NO: 13 The peptide for measuring ILK1 protein kinase activity according to claim 1, which is one selected from the group consisting of peptides of amino acid sequence numbers 1-8. The peptide for measuring ILK1 protein kinase activity according to claim 1, wherein the peptide is fluorescently labeled. Fluorescently labeling the peptide for measuring ILK1 protein kinase activity of claim 1; Reacting the electrofluorescent peptide with ILK1 protein kinase; Electrophoresis of the reacted electrofluorescent peptide at its isoelectric point to separate the phosphorylated peptide from the non-phosphorylated peptide; And, non-radioactive ILK1 protein kinase activity assay comprising the step of measuring the amount of phosphorylated peptide. The non-radioactive ILK1 protein kinase activity assay according to claim 4, wherein the electrical peptide is one selected from the group consisting of the peptides of Table 1.