Phosphorylation of Receptor-Mimic Tyrosine Peptide on Surface Plasmon Resonance Sensor Chip and Its Interaction with Downstream Proteins

CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 36, Issue 10, October 2008 Online English edition of the Chinese language journal

Cite this article as: Chin J Anal Chem, 2008, 36(10), 1327–1332.

RESEARCH PAPER

Phosphorylation of Receptor-Mimic Tyrosine Peptide on Surface Plasmon Resonance Sensor Chip and Its Interaction with Downstream Proteins LI Xue-Ling1,2, HUANG Ming-Hui2, CAO Hui-Min1, CHEN Yao2, ZHAO Jian-Long1,*, YANG Meng-Su2,* 1 2

Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, Shanghai 200050, China Biotech and Health Center, Shenzhen Key Laboratory of Biotech Research, City University of Hong Kong, Shenzhen 518057, China

Abstract:

Insulin-like growth factor type-I receptor (IGF-1R) signal pathway is critical in several physiological activities and some

pathogenesis. IGF-1R derived nonphosphorylated peptide LYASVNPEY was immobilized on the surface of CM5 sensor chip specialized for surface plasmon resonance (SPR) biosensor. The peptide surface was treated with cell lysates containing or not containing IGF-1R kinase. The results show that using SPR biosensor PY20 can discriminate the different levels of peptide tyrosine phosphorylation (PTP) caused by different incubation times and different lysates. The effect of the PTP on peptide-protein interaction was further studied. The results demonstrated that only the phosphorylated peptide can interact with its downstream insulin receptor substrate-1 (IRS-1). The binding affinity (KA) and the association and dissociation kinetics (ka and kd) of the phosphorylated peptide with IRS-1 were measured by SPR biosensor, with the results of KA = (2.02 ± 0.08) × 108 M–1, ka = (2.30 ± 0.15) u 106 M–1 s–1 and kd = (1.14 ± 0.13) × 10–2 s–1, respectively. This new phosphorylation method of peptide that simulates cell receptor has the characteristics of real-time, speediness, label-free, and convenience for detection, which is instrumental for investigations on receptor phosphorylation signal pathways and drug screening against these signal pathways. Key Words:

Surface plasmon resonance biosensor; Insulin-like growth factor type-I receptor kinase; Peptide tyrosine

phosphorylation; Insulin receptor substrate-1; Kinetics and affinity

1

Introduction

Every active protein kinase phosphorylates a distinct set of substrates by chemically adding phosphate groups to the serine, threonine, or tyrosine etc. of the substrates in a regulated manner, which usually results in a functional change of the target protein (substrate) by changing enzyme activity, cellular location, or association with other proteins. Phoshorylation plays a critical role in the regulation of cell signaling, metabolism, and growth. The genes of tyrosine kinases in vivo are considerably fewer compared to that of serine and theorine kinases[1]. However, studies have shown

that these tyrosine kinases regulate cell signal transduction, cell proliferation, differentiation, apoptosis and neurite growth, etc. Insulin-like growth factor-1 receptor (IGF-1R) is an evolutionarily advanced receptor in tyrosine kinase receptor family. Owing to its high homology with insulin receptor (IR), IGF-1R, like IR, is categorized into insulin receptor subfamily[2]. IGF-1R is involved in cell transformation, proliferation, anti-apoptosis, and cancer cell malignant phenotype maintenance[3]. IGF-1R is composed of two extracellular Į chains and two transmembrane ȕ chains held together by disulfide bonds[3]. Binding of insulin-like growth

Received 21 April 2008; accepted 30 May 2008 * Corresponding author. ZHAO Jian-Long: E-mail: [email protected]; Tel: 86-21-62511070-5709; Fax: 86-21-62511070-8714; YANG Meng-Su: E-mail: [email protected]; Tel: 852-27887797; Fax: 852-27887406 This work was supported by the grants from Major Basic Research Program of the Science and Technology Commission Foundation of Shanghai of China (No. 04JC14081) and the Major State Basic Research Development Program of China (No. 2005CB724305). Copyright © 2008, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved.

LI Xue-Ling et al. / Chinese Journal of Analytical Chemistry, 2008, 36(10): 1327–1332

factor-1 (IGF-1) with IGF-1R induces the conformational change and autophosphorylation of the receptor, which leads to the recruitment and phosphoylation of its intracellular substrates proteins, such as insulin receptor substrate-1 (IRS-1) and Src homology collagen protein (SHC), etc. These phosphorylated substrate proteins bind their downstream proteins, such as PI3-K and Grb-2 etc, thus activating the PI3-K/AKT and MAK signal transduction ways, which increase cell proliferation and prevent apoptosis[4,5]. Aberrant IGF-1R signaling has been implicated in cell malignant transformation, and the enzyme has emerged as a target for anticancer drug design. Previous research has demonstrated that Y950 and the motif NPXpY of IGF-1R are important in IGF-1R binding with its downstream protein IRS-1 and the binding is regulated by the phosphorylation and dephosphorylation of Y950[6–8]. Surface plasmon resonance (SPR) biosensor is widely used to observe the interactions of proteins because of its real-time, speediness, label-free, and convenience for detection. This technique has been applied to study the interactions of receptor-ligand, phosphorylated peptide-downstream proteins[9–12], and the phosphorylation detections of peptide and protein substrate[13–15], etc. However, these studies either

1 2 3 4 5

need synthesizing phosphorylated peptides, or need purifying specific kinases, and therefore, there are problems of high experimental cost, unsteadiness, and dephosphorylation of the synthesized phosphorylated peptide, and difficulty in direct immobilizing highly electronegative phosphorylated peptide on carboxymethylated dextran (CMD) chip surface. In this study, by direct immobilizing the unphosphorylated peptide via amino coupling and then phosphorylating the peptide on sensor surface by activated cell lysates (Fig.1 A–E), a novel method was developed for the research on protein phosphorylation events. The results have demonstrated that the phosphorylated peptide interacts with IGF-1R downstream protein, IRS-1[6–8]. The method avoids the disadvantages of high cost and poor producibility in the study of phosphoylated proteins. It lays foundation for the practical application of SPR biosensor technique as a research instrument of disease treatment drugs.

2

Experimental

2.1

Instruments and reagents

BiacoreTM X biosensor and CM5 (Carboxymethylated

Treat the peptide on the sensor chip surface with cell lysates in the kinase reaction buffer

6

LYASVNEPY O

a HN

Regeneration

7 6

Phosphorylated peptide interaction with IRS-1

Antibody detection

P

(B)

Regeneration

(A)

O

8 b c

(E) (C)

(D)

Fig.1 Scheme of the peptide surface phosphorylation, detection and interaction with IRS-1 A, scheme of peptide immobilization on CM5 sensor chip via amino-coupling; B, phosphorylation of peptide on solid surface; C, detection of tyrosine phosphorylation; D, measurement of peptide-protein interactions. 1, peptide; 2, carboxymethylated dextran gel; 3, gold film; 4, chrome cladding; 5, glass substrate; 6, tyrosine phosphate group; 7, anti-phosphotyrosine antibody (anti-pTyr, PY20); 8, IRS-1; Inset (E) is the enlarged square marked in (C); a of (E) is the linker, 6-aminohexanoyl (NH2(CH2)6CO-, Ahx-), of peptide (LYASVNPEY-NH2); b, CM5 sensor chip surface; c, a part of chip surface to be enlarged

LI Xue-Ling et al. / Chinese Journal of Analytical Chemistry, 2008, 36(10): 1327–1332

dextran 5) sensor chips were purchased from Amersham Pharmacia (Uppsala, Sweden). High speed centrifuge (Centrifuge 5810R) and super speed centrifuge (CS150GXL) were purchased from Eppendorf company (Hamburg, Germany) and Hitachi Ltd. (Tokyo, Japan), respectively. HBS-EP buffer (10 mM Hepes, 150 mM NaCl, 3.4 mM EDTA, 0.05% P20, pH 7.4), and the amine coupling kit containing N-ethyl-N’-(3-diethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), and ethanolamine hydrochloride were supplied by Amersham Pharmacia (Uppsala, Sweden). A peptide derived from human IGF-1R protein (position Y950 in hIGF-1R) Ahx-LYASVNPEY-NH2 (Ahx, 6-aminohexanoic acid; -NH2, C-terminal amidation) was synthesized by GL Biochem Ltd (China), > 96% purity by HPLC and of the appropriate molecular weight (1167) by mass spectrometry. Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Invitrogen (Carlsbad, CA, USA). ATP was purchased from Shanghai Sangon Biological Engineering Technology & Services Co. Ltd. (China). Antibody against phosphotyrosine (PY20) was obtained from Southern Biotechnology Associates, Inc. (Birmingham, AL, USA). Protease inhibitor cocktail tablets were obtained from Roche Applied Science Company (Basel, Switzerland). IRS-1 was from Upstate Biotechnology Inc. (Lake Placid, NY, USA). All other chemicals used were of analytical grade, and doubly distilled deionized water (ddH2O) was used in all experiments. All solutions that flowed through Biacore instrument were filtered with 0.22 Pm nylon filter membrane after preparation, and degassed before use. 2.2

Cell culture and cell lysates preparation

NIH-3T3 cell line overexpressing IGF-1R was a gift from Professor Derek LeRoith (National Institute of Diabetes and Digestive and Kidney diseases, NIH, Bethesda, MA, USA). The cell culture, treatment, and cell lysates preparation were detailed in republished literature[16]. 2.3

Peptide chip preparation

The Ahx-LYASVNPEY-NH2 peptide elongated with a 6-aminohexanoic acid (Ahx) spacer was covalently coupled to a CM5 chip using EDC/NHS chemistry. Specifically, equal volumes of 0.1 M NHS and 0.4 M EDC were mixed just before use and 35 Pl of the mixture was injected over the CM5 sensor chip to activate the carboxyl groups of CMD hydrogel on the sensor surface. 50 Pl peptide of 1 mM in 10 mM sodium acetate (pH 4.8) containing 0.5% DMSO as solvent was injected over F1 flow cell with F2 flow cell surface as control, followed by 50 Pl ethanolamine hydrochloride of 1M to quench the residual NHS esters. The above immobilization procedure was carried out at 25 ºC and

at a constant flow rate of 5 ȝl min–1 in HBS-EP buffer. HBS-EP buffer flowed over the sensor chip. After the baseline was stable, the immobilized peptide sensor chip was undocked for the following use. Several peptide chips were prepared using the above method. 75 Pl lysate of IGF-1R overexpressing cell with IGF-1 stimulation (RS), 60 Pl 5 × kinase buffer (Tris-HCl of 100 mM, pH 7.4, MgCl2 of 50 mM, MnCl2 of 5 mM, 5 mM dithiothreitol), 0.6 Pl ATP of 100 mM, 1.5 Pl Na3VO4 of 200 mM, and ddH2O were finally mixed well to a volume of 250 Pl, which was prepared on ice before use. 50 Pl kinase solution above mentioned was dropped on the sensor chip surface immobilized with peptide, and incubated for 5, 15, 30, 45, and 60 min, respectively at 25 °C. This process was carried out outside the Biacore X instrument. Meanwhile, sensor chips without peptides and sensor chips with immobilized peptide treated with kinase buffer only were used as control. After incubation, the above sensor chip surfaces were washed thoroughly with HBS-EP for the following use. Peptide surfaces treated with lysates of IGF-1R overexpressing cell without IGF-1 stimulation (RNS) and control cells (C), respectively, were also prepared. 2.4

Detection of peptide tyrosine phosphorylation levels

The above sensor chips that were treated with three kinds of cell lysates were respectively docked into the SPR instrument. Then the peptide surface was washed three times for 1 min with HCl of 10 mM to remove the nonspecifically absorbed cell lysates. After the baseline was stable, anti-pTyr antibody (PY20) of various concentrations was injected over the peptide surface for 5 min to decide the optimal PY20 concentration for tyrosine phosphorylation detection. Between two cycles of PY20 injections, HCl of 100 mM was flowed over the sensor chip surface for 1 min to regenerate the peptide surface. The above procedure was performed at 25 °C with a flow rate of 10 Pl min–1. The results demonstrated that the optimal PY20 concentration is 3.57 Pg ml–1 (as shown in Fig.2). Phosphorylation levels of surface peptide were compared, which were respectively prepared with different cell lysates for different time spans as mentioned above. 2.5

Effect of peptide phosphorylation on peptide-protein interaction

The surface phosphoylated peptide was prepared with RS for 30 min as detailed above. 50 Pl IRS-1 (concentrations were 2.5–80 nM in HBS-EP) was injected over the peptide surface for 300 s, followed by a 180-s wash with running buffer. After subtracting the sensorgram from the control channel, the binding sensorgram of IRS-1 to the

LI Xue-Ling et al. / Chinese Journal of Analytical Chemistry, 2008, 36(10): 1327–1332

IGF-1R-mimic peptide was obtained. The sensor surface was then regenerated by one pulse of injection with 5 Pl HCl of 100 mM to dissociate the binding IRS-1. After the baseline was steady, the regenerated surface was used for another cycle of binding experiment (Fig.1).

3

Result and discussion

3.1

Determination of optimal PY20 concentrations

Various concentrations of PY20 were used to detect the tyrosine phosphorylation level of the peptide that was treated for 60 min with RS. Figure 2 shows that with the increase of PY20, PY20-tyrosinephosphoylated peptide binding increased accordingly. However, when the PY20 reached 3.57 Pg ml–1, the binding level did not change significantly (Fig.2). To obtain the obvious interaction sensorgrams and meanwhile not to waste antibody, we chose 3.57 Pg ml–1 as the detection concentration of PY20. The following experimental results also indicate that this concentration can well discriminate the different of phosphorylation levels of peptide. 3.2

Tyrosine phosphorylation level detection by PY20

After sensor chip surface without peptide was incubated with cell lysates for 60 min and then washed and desorbed thoroughly, almost no tyrosine phosphorylation was detected,

Fig.2

which demonstrates that almost no tyrosine phosphorylated proteins in cell lystates are nonspecifically absorbed to the sensor chip surface after severe wash. Therefore, the incubation of the cell lystates with the peptide surface will not affect the following peptide phosphorylation detection as shown in Fig.3A. With the increase of incubation time, bindings of 3.57 Pg ml–1 PY20 increased accordingly. The results demonstrates that SPR biosensor based tyrosine phosphorylation detection by 3.57 Pg ml–1 PY20 can discriminate well the change of phosphorylation levels caused by different incubation periods. At the incubation time points of 15, 30, 45 and 60 min, binding response units of antiphosphotyrosine antibody (PY20) plotted against incubation time presents a sigmoid curve as shown in Fig.3B. One possible explanation is that at the beginning of incubation, the activities of protein tyrosine kinases (PTKs) in cell lysates were relatively low; after 15 min incubation, the PTKs in cell lysates were activated by the effectors of Mg2+, Mn2+ and ATP, etc. in PTKs buffer, probably through PTKs conformational change or autophosphorylation; after 45 min incubation, because of the depletion of ATP and increase of peptide phosphorylation level, the activities of PTKs decreased, and meanwhile, the protein tyrosine phosphatase in cell lysates began to function; consequently, the phosphorylation rate decreased greatly, while the phosphorylation level increased slowly, and finally stopped increasing.

Determination of optimal PY20 detection concentration (A) binding responses of different concentrations of PY20 with tyrosine phosphorylated peptides on sensor chip surface and (B) plot of PY20 response units versus PY20 concentration

Fig.3 Sensorgrams of PY20 binding with peptide phosphorylated by RS for various time periods (A) and detections of peptide tyrosine phosphorylation catalyzed by different cell lysates (RS, RNS, and C) for different time periods, respectively (B) RS: lysate of IGF-1R overexpressing cell stimulated with IGF-1; RNS: lysate of IGF-1R overexpressing cell without IGF-1 stimulation; C: NIH 3T3 control cell lysate

LI Xue-Ling et al. / Chinese Journal of Analytical Chemistry, 2008, 36(10): 1327–1332

3.3

Effectors of peptide tyrosine phosphorylation

Figure 3B indicates that the level of the peptide tyrosine phosphorylation catalyzed by RS is higher than that catalyzed by RNS within 1 h, and the latter is higher than that caused by C, which expresses no or very low IGF-1R (P < 0.05). Since the main differences of the three lysates are the IGF-1R expression level and stimulation or no stimulation of IGF-1R, we propose that the different levels of tyrosine phosphorylation may be caused by the different amounts of IGF-1R and its different activity states, and that IGF-1R kinase in RS contributed more to the tyrosine phosphorylation. The peptide phosphorylation level (PPL) caused by RS greater than that by RNS in the period range of 5–45 min may be explained by that IGF-1 stimulation facilitated the fast activation of IGF-1R by Mg2+, Mn2+ and ATP, and that IGF-1 stimulation led to higher tyrosine kinase activity, while RNS needed an extra period of autophosphorylation and longer time of activation. After 1 h incubation, PPL caused by C is close to that by RS and RNS. The reason may be that most of the other nonspecific kinases in cell lysate were activated after a longer incubation. 3.4

Effect of tyrosine phosphorylation on interactions of peptide-downstream proteins

2.5, 5, 10, 20 and 40 nM of IRS-1 were respectively flowed over the phophorylated peptide surface for 300 s, followed by HBS-EP flowing over the surface for 150 s to naturally dissociate IRS-1. The sensorgram is shown in Fig.4. With the increase of IRS-1 concentration, the binding responses of IRS-1 and the phosphorylated IGF-1R mimic peptide increased accordingly (Fig.4A), while IRS-1 did not bind the unphosphorylated peptide. IRS-1 binding response units were plotted versus its concentration and then fitted with a single-site equilibrium binding model (Fig.4B) to generate affinity constant (KA). The dissociation rate (kd) was

determined by fitting the dissociation phase, and the association rate (ka) was estimated by multiplying the kd with KA. The obtained affinity constant (KA) is (2.02 ± 0.09) ×108 M–1 and the rate constants (ka and kd) are (2.30 ± 0.15) u106 M–1 s–1 and (1.14 ± 0.13) × 10–2 s–1, respectively (Table 1). Comparison with the measured KA = (1.37 ± 0.20) u 109 M–1, ka = (1.48 ± 0.11) × 106 M–1 s–1 , and kd = (1.08 ± 0.06) × 10–3 s–1of the interaction of phosphorylated IGF-1R with IRS-1 in our previous study[16] demonstrates that the affinity of IRS-1 and IGF-1R mimic peptide is 6.8-fold lower than that of IRS and IGF-1R, which mainly resulted from the different dissociation rates. It suggests that other IGF-1R sequence plays an important role in maintaining the IGF-1R conformation, which leads to slower dissociation of IRS from IGF-1R and faster binding that guarantees the effective signal transduction to downstream proteins. The high affinity of this phosphorylated peptide and IRS-1 proved that NPXpY motif plays a critical role in IGF-1R binding with IRS-1[6–9]. Using a similar method as above, the interaction of this tyrosine phosphorylated peptide (TPP) and PI3-K was also studied. The results show that the TPP does not bind PI3-K, which demonstrates that the binding site of PI3-K/IGF-1R is not at NPXpY site. This is consistent with the previous reports that the C-terminal of IGF-1R Y1113 site plays a critical role in IGF-1R/PI3-K interaction[17]. Besides the physiological phosphorylation site at its C-terminal tyrosine, IGF-1R derived peptide, LY-7AS-5VNPEY, has another two phosphorylation sites, the tyrosine at -7 site (Y-7) and the serine at -5 site (S-5). The latter two sites are very likely phosphorylated indiscriminatingly in vitro, and the effect of which on the peptide-IRS-1 interaction needs further study. However, considering the binding properties of the phosphorylated peptide with IRS-1, we conclude that the phosphorylated peptide can partly mimic IGF-1R, which can be used to study the IGF-1R interactions with IRS-1 and other intracellular downstream proteins, such as SHC and PI3-K, and determine the contribution of this specific site to these

Fig.4 Real-time sensorgrams of IRS-1 interaction with IGF-1R derived peptide (A) and the curve fitted with one-site equilibrium binding model (B) Table 1 Comparison of binding kinetics and affinity of IRS-1 and IGF-1R derived phosphorylated peptide with those of IGF-1R/IRS-1 Binding IRS-1/peptide IRS-1/IGF-1R

ka (M–1 s–1)

kd (s–1)

KA (M–1)

(2.30 ± 0.15) u 106 (1.48 ± 0.11) × 106

(1.14 ± 0.13) ×10–2 (1.08 ± 0.06) ×10–3

(2.02 ± 0.09) × 108 (1.37 ± 0.20) u 109

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interactions. We propose that the recognition sites and the interface of IGF-1R and its substrate proteins can be studied by designing rational receptor derived peptides. 3.5

Significance of SPR biosensor based tyrosine protein phosphorylation events study

The tertiary structures of insulin and insulin-like growth factor-1 (IGF-1) are very similar. Insulin and IGF-1 can competitively bind each other’s receptors, which are both receptor protein tyrosine kinases. 83% of the receptors’ intracellular kinase domains are homologous and their signal pathways are very similar. The study of insulin signal pathway is very important for screening drugs against type I and type II diabetes and obesity. Meanwhile, IGF-1 is mainly involved in proliferation of normal cells and carcinogenesis. In the cells of some kinds of tumors, IGF-1R is overexpressed or over activated, which enhances DNA synthesis and cell proliferation and prevents apoptosis. Recent studies have shown that IGF-1 plays an important role in treatment of type II diabetes, acute or chronic nephritis, osteoporosis, and metabolic diseases. Therefore, it will be a new scheme for cancer treatment to specifically inhibit IGF-1R kinase autophosphorylation, its association with IRS-1, SHC, and PI3-K, and their phosphorylation by IGF-1R, while not affecting the associations of insulin receptor with these three proteins and their phosphorylation involved in insulin pathway; in other words, inhibit cancer cell proliferation while not leading to any side effects of diabetes, etc. Screening out activators or inhibitors respectively targeting insulin receptor and IGF-1R will provide more choices for treatment of diabetes and cancer. SPR biosensor was used to observe the interactions of proteins. Since most recombinant proteins can be obtained, the binding order and the complex formation process of a wide range of signal proteins can be real-time observed. Therefore, SPR biosensor has prominent advantages in screening drug analogs targeting cell surface receptors and in qualitatively and quantitatively examining the interactions of proteins. By immobilizing a peptide that mimics a protein, or immobilizing a protein itself, then phosphorylating and dephosphorylating the immobilized peptide or protein in turn by kinases and phosphatases, we can also study the effects of the phosphorylation and dephosphorylation on the interactions of

the peptide or protein and its down or up stream proteins. This method is real-time, label-free, detection-convenient, etc., and therefore, great amounts of data can be obtained in short time. This is instrumental to study the signal pathways involving protein phosphorylation and dephosphorylation and to screen drugs targeting these pathways.

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