Assessment of Protein-tyrosine Phosphatase 1B Substrate Specificity Using “Inverse Alanine Scanning”

Abstract

An "inverse alanine scanning" peptide library approach has been developed to assess the substrate specificity of protein-tyrosine phosphatases (PTPases). In this method each Ala moiety in the parent peptide, AcAAAApYAAAA-NH 2 , is separately and sequentially replaced by the 19 non-Ala amino acids to generate a library of 153 well defined peptides. The relatively small number of peptides allows the acquisition of explicit kinetic data for all library members, thereby furnishing information about the contribution of individual amino acids with respect to substrate properties. The approach was applied to protein-tyrosine phosphatase 1B (PTP1B) as a first example, and the highly potent peptide substrate Ac-ELEFpYMDYE-NH 2 (k cat /K m 2.2 ؎ 0.05 ؋ 10 7 M ؊1 s ؊1 ) has been identified. More importantly, several heretofore unknown features of the substrate specificity of PTP1B were revealed. This includes the ability of PTP1B to accommodate acidic, aromatic, and hydrophobic residues at the ؊1 position, a strong nonpreference for Lys and Arg residues in any position, and the first evidence that residues well beyond the ؉1 position contribute to substrate efficacy.

Substrate Specificity of Lymphoid-specific Tyrosine Phosphatase (Lyp) and Identification of Src Kinase-associated Protein of 55 kDa Homolog (SKAP-HOM) as a Lyp Substrate

“…A more systematic and thorough approach, such as the use of peptide libraries, is needed for the determination of substrate specificity for individual PTPs. Indeed, combinatorial peptide libraries have been useful in the determination of optimal amino acid sequence for PTP recognition (18,(32)(33)(34)(35)(36)(37)(38).…”

“…This method allows the acquisition of explicit kinetic data for all library members and enables the assessment of the contribution of individual amino acids to PTP substrate recognition based on the actual enzymatic activity of the PTP against its putative peptide substrates in solution. The Lyp-catalyzed dephosphorylation of each individual Tyr(P) peptide within the library was monitored by the increase in tyrosine fluorescence (18,39) at pH 7.0 and 25°C. The k cat /K m value, a measure of substrate specificity, was directly calculated from the reaction progress curve for each peptide (see "Experimental Procedures").…”

“…To identify novel Lyp substrates in an unbiased manner, we employed an "inverse alanine scanning" strategy (18) to profile the sequence specificity of Lyp. Briefly, each Ala residue in the parent peptide, Ac-AAAApYAAAA-NH 2 , is separately and sequentially replaced by the 19 non-Ala amino acids to generate a library of 153 well defined phosphopeptides (Fig.…”

“…The Lyp-catalyzed hydrolysis of Tyr(P)-containing peptides was continuously monitored at 305 nm for the increase in tyrosine fluorescence with excitation at 280 nm (18 Peptide Synthesis and Characterization-The synthesis and characterization of the inverse alanine phosphopeptide library were described previously (18). The Lyp consensus peptides (Ac-YGEEpYDDLY-NH 2 and Ac-YGYEpYDDEY-NH 2 ) and the peptide 71 DGEEpYDDPF 79 derived from SKAP-HOM were prepared using standard solid phase Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry.…”

“…Identification and characterization of novel Lyp substrates will help define the chain of molecular events coupling Lyp dysfunction to diseases. In the current study, we sought to determine Lyp substrate specificity using an "inverse alanine scanning" peptide library approach (18). The obtained consensus peptide corresponds to a stretch of amino acid sequence in the integrinsignaling adaptor SKAP-HOM (19,20), which is a homolog of Src kinase-associated protein of 55 kDa (SKAP-55) (21).…”