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Use of intein-mediated phosphoprotein arrays to study substrate specificity of protein phosphatases
 
Samvel Kochinyan, Luo Sun, Inca Ghosh, Tanya Barshevsky, Jie Xu, and Ming-Qun Xu
New England Biolabs, Inc., Ipswich, MA, USA
BioTechniques, Vol. 42, No. 1, January 2007, pp. 63–69
Full Text (PDF)
Abstract

Synthetic peptides incorporating various chemical moieties, for example, phosphate groups, are convenient tools for investigating protein modification enzymes, such as protein phosphatases (PPs). However, short peptides are sometimes poor substrates, and their binding to commonly used matrices is unpredictable and variable. In general, protein substrates for PPs are superior for enzymatic assays, binding to various matrices, and Western blot analysis. The preparation and characterization of phosphoproteins, however, can be difficult and technically demanding. In this study, the intein-mediated protein ligation (IPL) technique was used to readily generate phosphorylated protein substrates by ligating a synthetic phosphopeptide to an intein-generated carrier protein (CP) possessing a carboxyl-terminal thioester with a one-to-one stoichiometry. The ligated phosphoprotein (LPP) substrate was treated with a PP and subsequently subjected to array or Western blot analysis with a phosphospecific antibody. This approach is highly effective in producing arrays of protein substrates containing phosphorylated amino acid residues and has been applied for screening of PPs with specificity toward phosphorylated tyrosine, serine, or threonine residues, resulting in an approximately 240-fold increase in sensitivity in dot blot analysis compared with the use of synthetic peptides. The IPL technique overcomes the disadvantages of current methods and is a versatile system for the facile production of protein substrates containing well-defined structural motifs for the study of protein modification enzymes.

Introduction

Protein phosphorylation and dephosphorylation are predominant events in signal transduction pathways. Characterization of the specificities of protein kinases (PKs) and protein phosphatases (PPs) is pivotal for the understanding of molecular mechanisms and the determination of drug targets related to numerous diseases (1). Despite a rapidly growing appreciation of the important function of PPs in signal transduction, it is still a tremendous challenge for investigators to understand how PPs distinguish between the diversity of phosphoproteins that they encounter within the living cells. In vitro production of protein substrates for PP analysis relies on the phosphorylation of purified proteins by a PK in the presence of ATP, a reaction that is often inefficient (2,3,4). Although 32P-labeled proteins are widely used in assays of PP activity due to the high sensitivity they afford, the preparation and characterization of 32P-labeled substrates requires a considerable expenditure of time and effort and depends on the availability of the appropriate PK. Many proteins can also present various problems in handling, particularly with respect to solubility. Thus, it is rather difficult to obtain a sufficient quantity of phosphoprotein to use on a routine basis as a phosphatase substrate. Synthetic peptide substrates have become convenient tools for studying enzyme specificities since they are chemically well-defined entities that can be acquired in a highly purified form and in a large quantity (5,6). In addition, chemical synthesis allows for the systematic alteration of the sequence and the length of the substrate peptide, which could aid in the study of substrate specificity, as well as in the optimization of an enzyme-specific substrate. However, it should be noted that in comparison to protein substrates, short phosphopeptides are sometimes not optimal enzyme substrates (2,7). Enzyme-linked immunosorbent assays (ELISAs) and arrays are commonly used for peptide-based assays, whereas Western blot analysis is not suitable for small peptides. Direct synthesis of peptides on cellulose membranes (SPOT) allows for screening of large arrays of peptide substrates (8,9). However, SPOT-based assays become less sensitive and nonquantitative when the amount of peptide is saturated. In ELISA, short peptides often exhibit variable binding to polystyrene, thereby causing inconsistency in signal detection. Furthermore, arraying peptides on commonly used membranes, such as low-cost nitrocellulose, is usually hindered due to low and variable binding that results in poor sensitivity and ambiguity.

We have previously demonstrated production of peptide arrays on nitrocellulose by the ligation of synthetic peptide substrates to an inteingenerated carrier protein (CP) (10). This technique, termed intein-mediated peptide array (IPA), takes advantage of the catalytic activity of an engineered intein to generate a reactive thioester at the C terminus of a CP. A peptide possessing an N-terminal cysteine residue is linked to the C terminus of the CP via a native peptide bond using intein-mediated protein ligation (IPL) (11). This method circumvents the problem associated with variable binding by small peptide substrates to various matrices, leading to successful applications for antibody characterization and epitope scanning, with an increase in sensitivity of up to 104- fold compared with the use of peptide antigens (10).

In this report, we have constructed a diverse array of ligated phosphoprotein (LPP) substrates containing a phosphotyrosine, phosphoserine, or phosphothreonine residue. Synthetic phosphopeptides based on the phosphorylation sites of various PKs were covalently linked to the C terminus of a CP. Three types of PPs, phosphotyrosine-specific PP (PTP), phosphoserine/threonine-specific PP (PSP), and dual specificity PP (DSP), have been used for the investigation of substrate specificity by dot blot and Western blot analysis.

Materials and Methods

Peptide Synthesis

All peptides (Table 1) were synthesized with an N-terminal cysteine and purified by high-performance liquid chromatography (HPLC; New England Biolabs, Ipswich, MA, USA). The phosphopeptides based on the phosphorylation sites of various PKs were described previously (10). The peptides were dissolved in 5 mM Tris-HCl, pH 7.5, 50 M NaCl at a final concentration of 5 mM. Phosphoserine containing peptide PB1-pSer was derived from mouse Bcl2-antagonist of cell death (BAD) (12). Phosphotyrosine-containing peptide Cdc2-pTyr15 was derived from a phosphorylation site of human cyclindependent kinase (13).

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