JP3066502B1 - Polypeptide - Google Patents

Abstract

The present invention provides a polypeptide having a phosphorylation consensus sequence and a method for simultaneous detection and screening of the activity of a Ser / Thr protein kinase using the same. [Solution] Lys Lys Arg Lys Ser Ser Leu Arg Arg Tr
p Polypeptide represented by Ser Pro Leu Thr ProArg Gln Met Ser Phe Asp Cys and its analogous polypeptides, and peptide conjugates of the polypeptide and polyamino acids.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a polypeptide and a peptide conjugate having a phosphorylation consensus sequence and a method for simultaneous detection and screening of Ser / Thr protein kinase activity using the same.

[0002]

2. Description of the Related Art In general, a stimulus transmitted from the outside of a cell to an intracellular cell activates a receptor or a channel protein on a cell membrane, and then is transmitted into the cell to cause calcium ions or cA.
Causes the production of second messengers such as MP.
Thereafter, activation and inactivation of various protein kinases occur, causing phosphorylation and dephosphorylation of the target protein (Cano, E., and Mahadevan, L. (19).
95) Parallel signal processing among mammalian MAPK
s. Trends Biochem. Sci. 20, 117-122). For example, Ser
It is known that the cascade of a series of protein kinases including MAPK, one of the / Thr protein kinases, is activated when the cell growth signal is turned on.

[0003] The properties and functions of the protein kinases discovered so far have been elucidated using specific peptide substrates in addition to the target protein substrates. For example, CaMKII specific substrates Autocamtide-2 and PKA
Kemptide, which is a specific substrate of E.g. However, as long as these specific substrates are used, there is a disadvantage that only the activity of a specific protein kinase can be detected. On the other hand, the in-gel phosphorylation method (Kameshita, I. an
d Fujisawa, H. (1989) A sensitive method for detect
ion of calmodulin-dependent protein kinase II acti
vity in sodium dodecyl sulfate-polyacrylamide gel.
Anal. Biochem. 183, 139-143; Kameshita, I. and F
ujisawa, H. (1996) Detection of protein kinase acti
vities toward oligopeptides in sodium dodecyl sulf
ate-polyacrylamide gel.Analyte. Biochem. 237, 198-20
3) is widely used as an indispensable technique for analyzing intracellular signal transduction mechanisms involved in protein phosphorylation, since the activity of various protein kinases can be easily detected together with their molecular weights at the level of crude extracts.

[0004] As a substrate used in the in-gel phosphorylation method, a protein substrate such as myelin basic protein has conventionally been the mainstream, but it has been recently improved (Kameshita, I. and Fujisawa, H. et al.
(1996) Detection of protein kinase activities towar
d oligopeptides in sodium dodecyl sulfate-polyacry
Various peptide substrates can also be used by coupling a peptide serving as a substrate with an amino acid polymer such as polylysine by lamide gel. Anal. Biochem. 237, 198-203).

[0005] However, the problem that the in-gel phosphorylation method can only detect the activity of a specific protein kinase capable of using them as a substrate as long as conventional protein / peptide substrates are used has been solved. Absent.

[0006]

SUMMARY OF THE INVENTION The present invention provides a polypeptide and a peptide conjugate having a phosphorylation consensus sequence and a method for simultaneous detection and screening of the activity of Ser / Thr protein kinase using the same. Aim.

[0007]

Means for Solving the Problems The present inventors have created peptide substrates that are recognized by several typical Ser / Thr protein kinases at the same time and applied them to the in-gel phosphorylation method. It has been found that it becomes possible to simultaneously detect the activity of the Ser / Thr protein kinase.

According to this method, Se having various substrate specificities
r / Thr protein kinase can be easily and simultaneously detected at the level of crude extract, and new Ser / Th with unknown substrate specificity
r It is also effective in screening for protein kinases. Ser / Thr protein kinases (CaMKII, calmodulin-dependent kinase IV for this purpose)
(CaMKIV), PKA, protein kinase C (PKC), characterized by having a phosphorylation consensus sequence of five sites recognized by MAPK), a peptide (Multid
e) and five of the five phosphorylation consensus sequences of Multide, each of which has five phosphorylation consensus sequences, each of which has five phosphorylation consensus sequences (Multide (5S), Multide (6S), Mude
ltide (11S), Multide (14T), Multide (19S)) were created. To apply these to the in-gel phosphorylation method,
Each peptide conjugate was prepared by binding to (Lys) or poly (Glu, Lys, Tyr).

The present invention relates to the following Multide, five types of Multid
e For analogous peptides and peptide conjugates.

[0010] The present invention also relates to a method for simultaneous detection and screening of Ser / Thr protein kinase activity using the peptide conjugate. Item 1. Polypeptide shown below (Multide) Lys Lys Arg Lys Ser Ser Leu Arg Arg Trp Ser Pro Leu Thr Pro Arg Gln Met Ser Phe Asp Cys Item 2. Polypeptide shown below (Multide (5S)) Lys Lys Arg Lys Ser Ala Leu Arg Arg Trp Ala Pro Leu Ala Pro Arg Gln Met Ala Phe Asp Cys Item 3. Polypeptide shown below (Multide (6S)) Lys Lys Arg Lys Ala Ser Leu Arg Arg Trp Ala Pro Leu Ala Pro Arg Gln Met Ala Phe Asp Cys Item 4. Polypeptide shown below (Multide (11S)) Lys Lys Arg Lys Ala Ala Leu Arg Arg Trp Ser Pro Leu Ala Pro Arg Gln Met Ala Phe Asp Cys Item 5. Polypeptide shown below (Multide (14T)) Lys Lys Arg Lys Ala Ala Leu Arg Arg Trp Ala Pro Leu Thr Pro Arg Gln Met Ala Phe Asp Cys Item 6. Polypeptide shown below (Multide (19S)) Lys Lys Arg Lys Ala Ala Leu Arg Arg Trp Ala Pro Leu Ala Pro Arg Gln Met Ser Phe Asp Cys Item 7. Item 7. A peptide conjugate comprising the polypeptide according to any one of Items 1 to 6 and an amino acid polymer. Item 8. Amino acid polymer is poly Lys or poly (Glu,
8. The peptide conjugate according to item 7, which is Lys, Tyr). Item 9. Item 9. At least two types of Ser / Thr protein kinases by the in-gel phosphorylation method, wherein the polypeptide according to any one of items 1 to 6 or the peptide conjugate according to claim 7 or 8 is used. Simultaneous measurement of activity. Item 10. A method for screening a novel Ser / Thr protein kinase by the in-gel phosphorylation method, wherein the polypeptide according to any one of items 1 to 6 or the peptide conjugate according to claim 7 or 8 is used.

[0011]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Method (i) [γ- ³² P] ATP (5000 Ci / mmol) used in the invention was purchased from Amersham. Poly (Lys) (average molecular weight 87,000), poly (Glu, Lys,
Tyr) (average molecular weight 23000), Kemptide (LRRASLG) and myelin
The basic protein used was manufactured by Sigma. Recombinant mo
use p42MAPK and Recombinant human MEK1 are Upstate Biote
Chnology was used. K252a, a general protein kinase inhibitor, was purchased from Calbiochem. Mult
ide, 5 kinds of Multide-like peptides (Multide (5S), Multide
de (6S), Multide (11S), Multide (14T), Multide (19S))
And Syntide-2 (PLARTLSVAGLPGKK) were synthesized by fmoc peptide synthesis using a peptide solid phase synthesizer PSSM8 manufactured by Shimadzu Corporation. The synthesized peptide was purified by high performance liquid chromatography using reverse phase column chromatography (ODS-80Tm, manufactured by Tosoh Corporation). The molecular weight of the peptide was determined using a fast atom bombardment-mass spectrometry mass spectrometer. Poly (Lys) or p
oly (Glu, Lys, Tyr) was reacted with the synthesized peptide to form a peptide conjugate (Kameshita, I. an
d Fujisawa, H. (1996) Detection of protein kinase
activities toward oligopeptides in sodium dodecyl
Sulfate-polyacrylamide gel.Analyte. Biochem. 237, 19
8-203). Calmodulin was purified from rat testis. C
aMKII was purified from rat cerebral cortex. PKA (catalytic subunit) was purified from bovine heart. (ii) Colo201 (non-adhere) which is a cancer cell derived from human colon
nt colon adenocarcinoma cell line) to RPMI1640 10%
The cells were cultured in a medium supplemented with fetal calf serum, penicillin and streptomycin, and cells reacted with K252a (300 nM) for 30 minutes and unreacted cells were prepared. Cells are incubated at 0 ° C in extraction buffer (20 m
M Tris-HCl (pH 7.4), 20 mM NaCl, 1 mM sodium orthov
anadate, 10 mM NaF, 5 mM sodium pyrophosphate, 0.5
mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 0.2
μM okadaic acid, 10 μg / ml leupeptin, 10 μg / ml a
ntipain, 10 μg / ml chymostatin, 10 μg / ml pepstati
n). Thereafter, the mixture was passed through a 25-gauge injection needle five times, and crushed four times for 30 seconds using a cell disrupter (Olympus UC-100-D). The crushed product was centrifuged at 15000 g for 10 minutes, and the supernatant was used as a cell extract. (iii) Recombinant mouse p42MAPK (1 μg) is 50 μl of the following reaction mixture (50 mM Tris-HCl (pH 7.4), 10 mM MgCl ₂ , 2
mM EGTA, 2 mM dithiothreitol, 50 μM ATP, 80ng rec
It was activated by reacting with ombinant human MEK1) for 30 minutes at 30 ° C. The reaction was stopped by adding 10 mM EDTA or SDS buffer. (iv) The activity of each protein kinase is 30 ° C [γ- ³²
[ ³² P] per minute to synthetic peptide in the presence of [P] ATP
Phosphoric acid uptake was determined by the method of Roskoski using phosphocellulose paper (Roskoski, R. (1983) Assay of prot.
It was calculated by measuring with ein kinases. Methods in Enzymology 99, 3-6). The composition of the reaction solution for measuring the activity of each protein kinase is as follows. PKA: 40 mM HEPES-NaOH (pH 8.0), 5 mM Mg (CH ₃ COO) ₂ ,
0.1 mM EGTA, 2 mM dithiothreitol, 50 μM [γ- ³² P] A
TP (200-500 cpm / pmol), 0.01 μg PKA, 20 μM oligopep
tide CaMKII; 40 mM HEPES-NaOH (pH 8.0), 5 mM Mg (CH ₃ COO) ₂ ,
0.1 mM EGTA, 2 mM dithiothreitol, 50 μM [γ- ³² P]
ATP (200-500 cpm / pmol), 0.2 mM CaCl ₂ , 1 μM calmodu
lin, 0.01 μg CaMKII, 20 μM oligopeptide (v) In-gel phosphorylation was performed as follows. Polyacrylamide gel electrophoresis was performed using the Laemmli method (Laemml
i, U. (1970) Cleavage of structural proteins during
the assembly of the head of bacteriophage T4. Nat
ure 227, 680-685). A slab gel was prepared using 10% acrylamide separation gel and 3% stacking gel. 20 μM peptide conjugate (20 μg / ml poly
(Lys) or 20 μg / ml poly (Glu, Lys, Tyr) was made to react with the peptide. ) Was used as a substrate to perform an in-gel phosphorylation method using a cell extract (10 μg) of Colo201 cells. When looking at the autophosphorylation of protein kinase, the phosphorylation in gel was performed in the absence of peptide conjugate. Phosphorylation reaction, 25 degrees, 2 hours, 2.5 ml
Reaction solution (50 mM Tris-HCl (pH 7.4), 10 mM MgCl ₂ , 2 mM
EGTA, 2 mM dithiothreitol, 50 μM [γ- ³² P] ATP (10
μCi / ml) and minigel (8.5 × 4.5 × 0.1 cm). When detecting CaMKII, 0.2 mM CaCl ₂ and 1 mM
μM calmodulin was added to the reaction solution.

[0012]

According to the present invention, Multide is a peptide having 22 amino acids bonded, which has five phosphorylation consensus sequences recognized by Ser / Thr protein kinase. Among the five phosphorylation consensus sequences of Multide, five kinds of Multide-like peptides each having a phosphorylation consensus sequence (Multide (5S), Multide (6S),
Multide (11S), Multide (14T), Multide (19S)) and Multide
Can be used in combination with the in-gel phosphorylation method to achieve calmodulin-dependent kinase II (CaMKI
I), cyclic AMP-dependent kinase (PKA), mitogen-act
The intracellular activity of Ser / Thr protein kinase such as ivated protein kinase (MAPK) can be detected at once and simultaneously. In addition, this method enables screening of novel Ser / Thr protein kinases.

[0013]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in more detail with reference to embodiments. Example 1 Multide (KKRKSSLRRWSPLTPRQMSFDC) is characterized by having five phosphorylation consensus sequences recognized by five Ser / Thr protein kinases (CaMKII, CaMKIV, PKA, PKC, MAPK), 22 amino acids Is a peptide bound. Multide 5 for analysis of Multide phosphorylation sites
Among the phosphorylation consensus sequences at five sites, five types of Multide-like peptides each having a phosphorylation consensus sequence (Multide (5S), Multide (6S), Multide (11S), Multide
de (14T) and Multide (19S)) were synthesized (FIG. 1). Multide and
In order to examine the degree of phosphorylation of five kinds of Multide-like peptides by PKA and CaMKII, Kemptide (LRRASLG) and Syntide-2 (P
LARTLSVAGLPGKK) was simultaneously used to examine phosphorylation (in vitro kinase assay) by PKA and CaMKII (Table 1).

[0014]

[Table 1]

Although Multide and Multide (6S) were strongly phosphorylated by PKA, Multide (5S), Multide (11S),
(19S) was weakly phosphorylated. Multide
(14T) was not phosphorylated at all by PKA. on the other hand,
Multide and Multide (19S) were phosphorylated by CaMKII, but to a lesser extent than Syntide-2.

The peptide may be poly (Lys) or poly (Gl
u, Lys, Tyr) have been shown not only to be retained in gels but also to be good substrates for protein kinases (Kameshita, I. and Fujisa
wa, H. (1996) Detection of protein kinase activities
toward oligopeptides in sodium dodecyl sulfate-po
lyacrylamide gel. Anal. Biochem. 237, 198-203; Kam
eshita, I., Ishida, A. and Fujisawa, H. (1997) A new
peptide conjugate as a highly specificsubstrate f
or MAP kinase. J. Biochem., 122, 168-172). Therefore
Multide and 5 Multide-like peptides (Multide (5S), M
ultide (6S), Multide (11S), Multide (14T), Multide (19
(S)) was linked to poly (Lys) to prepare a product (Multide-poly (Lys)), which was used for the in-gel phosphorylation method. Multide-poly (L
In-gel phosphorylation was performed using PKA as a substrate (ys) (FIG. 2A). As a result, the catalytic subunit of PKA (41 kD
A strong phosphorylation product was observed at the position of the molecular weight corresponding to a). Multide the (6S) -poly (Lys) from the even stronger phosphorylation when the substrate was observed, it was found that phosphorylation by PKA is mainly Ser ^6. In case of CaMKII, Mult
Only when ide-poly (Lys) and Multide (19S) -poly (Lys) were used as substrates, two phosphorylation products were found at the positions corresponding to the α subunit (50 kDa) and β subunit (60 kDa). (Figure 2, B). In the case of MAPK, a phosphorylation reaction in a gel was performed using MAPK activated by MEK. Mu
Strong phosphorylation was observed in ltide-poly (Lys) and Multide (14T) -poly (Lys). These results are for Pro-Xaa- Ser / Thr -Pro
Is a phosphorylation consensus sequence of MAPK.

From the above results, Multide and five types of Multide
It was considered that the use of a similar peptide by the in-gel phosphorylation method could be used for screening for a novel protein kinase in cells. Colo20, a human colon-derived cancer cell
Treatment of 1 with K252a, a protein kinase inhibitor
It is known that the cell morphology changes rapidly after 30 minutes (Mohri, T., Kameshita, I., Suzuki, S., Hioki, K.,
Tokunaga, R. andTaketani, S. (1998) Rapid adhesio
n and spread of non-adherent colon cancer Colo201
cells induced by the protein kinase inhibitors, K2
52a and KT5720 and suppression of the adhesion by
the immunosuppressants FK506 andcyclosporin A. Cel
l Struct. Funct. 23, 255-264). In other words, since the inhibition of the protein kinase causes a morphological change of the present cells, the change in the protein kinase before and after the morphological change was determined. So Multide and 5 kinds of Multide
In-gel phosphorylation was performed using a similar peptide (FIG. 3). When poly (Lys) was used as the polymer support, the phosphorylation band of PKA was too strong, so p
oly (Glu, Lys, Tyr) was used as the polymer support.
When Multide-poly (Glu, Lys, Tyr) was used as a substrate, the phosphorylation bands of 29 kDa, 50 kDa, and 90 kDa were reduced after K252a treatment. On the other hand, 60 kDa (doublet), 76 kD
a, 150 kDa band increased. The 150 kDa band is Mul
A weak band was observed when tide (5S) -poly (Glu, Lys, Tyr) was used as a substrate, but the band of 60 kDa (doublet) was
tide (5S) -poly (Glu, Lys, Tyr) and Multide (6S) -poly (Gl
u, Lys, Tyr) were also observed when both were used as substrates. These results indicate that the two protein kinases have different substrate specificities. On the other hand, the band of 76 kDa is observed even when the substrate is not present.
It was found that autophosphorylation was occurring. The 90 kDa band reduced after K252a treatment was Multide-poly (Glu, Ly
s, Tyr) and Multide (6S) -poly (Glu, Lys, Tyr) were both used as substrates, indicating that they had the same substrate specificity as PKA. 50 kDa band is Mu
ltide-poly (Glu, Lys, Tyr), Multide (5S) -poly (Glu, L
ys, Tyr), Multide (6S) -poly (Glu, Lys, Tyr), Multide
A band was also observed when (19S) -poly (Glu, Lys, Tyr) was used as a substrate.

From the above results, 22 amino acids, which are characterized by having five phosphorylation consensus sequences recognized by Ser / Thr protein kinase (CaMKII, CaMKIV, PKA, PKC, MAPK) Multide peptide and Mul
Among the five phosphorylation consensus sequences of tide, five types of Multide having phosphorylation consensus sequences one by one
Similar peptides (Multide (5S), Multide (6S), Multide (11
(S), Multide (14T), Multide (19S)) as a peptide conjugate can be applied to the in-gel phosphorylation method to simultaneously detect the activity of several Ser / Thr protein kinases I understood. This method is also useful for screening novel Ser / Thr protein kinases with unknown substrate specificity.

[0019]

[Sequence List] <110> Koji Kajimura: Director General, Agency of Industrial Science and Technology <120> Polypeptides and peptide-conjugates (130> 113MS0235 <160> 6 <210> 1 <211> 22 <212> PRT <213 > Artificial Sequence <400> 1 Lys Lys Arg Lys Ser Ser Leu Arg Arg Trp Ser Pro Leu Thr Pro Arg 1 5 10 15 Gln Met Ser Phe Asp Cys 20 22 <210> 2 <211> 22 <212> PRT <213> Artificial Sequence <400> 2 Lys Lys Arg Lys Ser Ala Leu Arg Arg Trp Ala Pro Leu Ala Pro Arg 1 5 10 15 Gln Met Ala Phe Asp Cys 20 22 <210> 3 <211> 22 <212> PRT <213> Artificial Sequence <400> 3 Lys Lys Arg Lys Ala Ser Leu Arg Arg Trp Ala Pro Leu Ala Pro Arg 1 5 10 15 Gln Met Ala Phe Asp Cys 20 22 <210> 4 <211> 22 <212> PRT <213> Artificial Sequence <400> 4 Lys Lys Arg Lys Ala Ala Leu Arg Arg Trp Ser Pro Leu Ala Pro Arg 1 5 10 15 Gln Met Ala Phe Asp Cys 20 22 <210> 5 <211> 22 <212> PRT <213> Artificial Sequence < 400> 5 Lys Lys Arg Lys Ala Ala Leu Arg Arg Trp Ala Pro Leu Thr Pro Arg 1 5 10 15 Gln Met Ala Phe Asp Cys 20 22 <210> 6 <211> 22 <212> PRT <213> Artificial Sequence <400> 6 Lys Lys Arg Lys Ala Ala Leu Arg Arg Trp Ala Pro Leu Ala Pro Arg 1 5 10 15 Gln Met Ser Phe Asp Cys 20 22

[Brief description of the drawings]

FIG. 1: Multide and five Multide-like peptides (Mult
ide (5S), Multide (6S), Multide (11S), Multide (14T), M
2 shows the amino acid sequence of C. ultide (19S)) and its phosphorylation site. The underlined part of the sequence in FIG. 1 indicates the phosphorylation site of each protein kinase.

FIG. 2: Multide by PKA, CaMKII and MAPK and 5 kinds of Multide-like peptides (Multide (5S), Multide (6S), Mude
1 shows phosphorylation in gel of ltide (11S), Multide (14T) and Multide (19S)). Purified preparations of PKA (20 ng) (A), CaMKII (250 ng) (B) and activated MAPK-GST (20 ng) (C) were prepared at 20 μg / ml poly (Lys) (lane 1), 20 μM Multide poly (Lys) (lane 2), 20 μM Multide (5S) -poly (Lys) (lane 3), 20 μM
Multide (6S) -poly (Lys) (lane 4), 20 μM Multide (11
(S) -poly (Lys) (lane 5), 20 μM Multide (14T) -poly (Ly
s) (lane 6) and SDS-polyacrylamide gel electrophoresis using 20 μM Multide (19S) -poly (Lys) (lane 7) as a substrate.

FIG. 3 Colo201 cells (K252a-treated (+) and untreated (-))
2 shows the detection of a novel protein kinase using the in-gel phosphorylation method and Multide and Multide-like peptides. Colo201 cells were treated with K252a for 30 minutes, washed with cold PBS, and collected by centrifugation. Crude extract (10 μg) derived from K252a-treated cells (+) or untreated cells (−) was 20 μg / ml poly (Glu, Lys, Tyr) (A), 20 μM Multide poly (Glu, Lys, Tyr)
(B), 20 μM Multide (5S) -poly (Glu, Lys, Tyr) (C), 20 μM
Multide (6S) -poly (Glu, Lys, Tyr) (D), 20 μM Multide (11
(S) -poly (Glu, Lys, Tyr) (E), 20 μM Multide (14T) -poly (G
lu, Lys, Tyr) (F), 20 μM Multide (19S) -poly (Glu, Lys, Ty
r) SDS containing (G) as a substrate or without substrate (H)
Polyacrylamide gel electrophoresis was performed. "△" on the right side of the gel indicates increased protein kinase activity after K252a treatment, and "▼" on the left side of gel indicates decreased protein kinase activity after K252a treatment.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Atsuhiko Ishida, Asahikawa City, Hokkaido Asahikawa Medical University (72) Person Hitoshi Fujisawa Nishi Kagura 4-5, Asahikawa City, Hokkaido Asahikawa Medical University (56) References Biochem. (1999), 126, p. 991-995 J.P. Biol. Chem. (1991), 226 (33), p. 22159-22163 J.C. Biol. Chem. (1991), 226 (23), p. 15180-15184 FEBS Lett. (1999 Aug.), 456 (2), p. 249-252 Anal. Biochem. (1996), 237 (2), p. 198-203 Biochem. (Tokyo) (1997), 122 (1), p. 168-172 J.C. Biochem. (1990), 265 (32), p. 19728-19735 (58) Fields investigated (Int. Cl. ⁷ , DB name) C07K 14/00 C12Q 1/48 Geneseq / FASTA / DDBJ Medline (STN) REGISTRY (STN)

Claims (10)

(57) [Claims] 1. Polypeptide (Multide) Lys Lys Arg Lys Ser Ser Leu Arg Arg Trp Ser Pro Leu Thr Pro Arg Gln Met Ser Phe Asp Cys 2. The polypeptide shown below (Multide (5
S)) Lys Lys Arg Lys Ser Ala Leu Arg Arg Trp Ala Pro Leu Ala Pro Arg Gln Met Ala Phe Asp Cys 3. The polypeptide shown below (Multide (6
S)) Lys Lys Arg Lys Ala Ser Leu Arg Arg Trp Ala Pro Leu Ala Pro Arg Gln Met Ala Phe Asp Cys 4. The polypeptide shown below (Multide (11
S)) Lys Lys Arg Lys Ala Ala Leu Arg Arg Trp Ser Pro Leu Ala Pro Arg Gln Met Ala Phe Asp Cys 5. A polypeptide shown below (Multide (14
T)) Lys Lys Arg Lys Ala Ala Leu Arg Arg Trp Ala Pro Leu Thr Pro Arg Gln Met Ala Phe Asp Cys 6. A polypeptide shown below (Multide (19
S)) Lys Lys Arg Lys Ala Ala Leu Arg Arg Trp Ala Pro Leu Ala Pro Arg Gln Met Ser Phe Asp Cys 7. A peptide conjugate comprising the polypeptide according to claim 1 and an amino acid polymer. 8. The amino acid polymer is poly-Lys or poly (G
lu, Lys, Tyr). 9. A method according to claim 1, wherein at least two types of Ser / S are prepared by using the polypeptide according to any one of claims 1 to 6 or the peptide conjugate according to claim 7 or 8. Simultaneous measurement of Thr protein kinase activity. 10. A novel Ser / Thr protein phosphor by an in-gel phosphorylation method, wherein the polypeptide according to any one of claims 1 to 6 or the peptide conjugate according to claim 7 or 8 is used. An oxidase screening method.