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PROFILING THE INTRINSIC SEQUENCE SPECIFICITY OF PROTEIN TYROSINE PHOSPHATASES

Selner, Nicholas
http://rave.ohiolink.edu/etdc/view?acc_num=osu1384269733

Abstract Details

2013, Doctor of Philosophy, Ohio State University, Chemistry.
Many signaling pathways are mediated by protein tyrosine phosphorylation. Regulation of protein tyrosine phosphorylation is balanced between protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Numerous studies have shown PTPs to exhibit site-specific dephosphosphorylation in vivo, although much is still unknown regarding the factors that determine the substrate specificity of PTPs. Our lab has developed a method to profile the sequence specificity of PTPs by using combinatorial peptide libraries with an enzyme-coupled assay to detect PTP activity. In this study, the PTP screening method was applied to nine PTPs (PTP1B, SHP1, VHR, TCPTP, SHP2, LMW-A, LMW-B, PTPH1, and PTPRC) which resolved several different aspects of PTP sequence specificity that was previously unknown. Many potential PTP substrates contain multiple pY motifs in close proximity. Previous studies have suggested that PTP1B specifically recognizes a tandem pY motif. As a result, the sequence specificity of PTP1B, SHP1, and VHR PTPs for multiple pY motifs were profiled and validated with solution-phase kinetics. Our results suggest that PTPs do not preferentially recognize specific multiple pY motifs, but prefer additional pY residues due to their role as an acidic residue. Low molecular weight protein-tyrosine phosphatases (LMW-PTPs) have been classified as the fourth class of PTPs due to their unique primary sequence. Although LMW-A and LMW-B are isoforms, studies have implicated them with having different substrate specificities. Therefore, the sequence specificity of LMW-A and LMW-B were determined and exhibited differences in both activity and protein recruitment. The sequence specificity of many PTPs have been studied in our lab (HePTP, PTP-PEST, SHP1, SHP2, PTP1B, TCPTP, PTPH1, PTPD2, PTPRB, PTPRC, PTPRD, PTPRO), which also demonstrated a similar preference for pY peptides rich in acidic residues (e.g., Asp and Glu) and disfavor positively charged sequences libraries. However, kinetic analyses of the 14 PTPs against acidic, neutral, and basic pY peptide substrates revealed that the PTPs have diverse levels of sequence selectivity and catalytic efficiency. PTPs also have vastly different intrinsic catalytic efficiencies (kcat/KM values against optimal substrates), which differ by >105-fold due to different kcat and/or KM values. Several PTPs have shown little positional preference for the acidic residues relative to the pY residue of a peptide substrate. The structural basis of this unusual substrate specificity was investigated by site-directed mutagenesis and X-ray crystallography. Mutation of Arg47 and Arg24 of PTP1B, which are located near the bound substrate, decreased the enzymatic activity of the pY peptides that contained acidic residues within the pY-6 to pY+5 region. A co-crystal structure of PTP1B bound with a nephrin pY1193 peptide confirmed the ability of Arg24 to engage in electrostatic interactions with acidic residues at the pY+1, pY+2, and likely other positions. These results suggest that Arg24, Arg47, and other positively charged amino acids near the PTP active site make long-range electrostatic interactions with all acidic residues on a bound pY substrate.
Dehua Pei (Advisor)
129 p.
Biochemistry; Chemistry
Combinatorial library, catalytic activity, kinetics, phosphotyrosine, phosphatase, PTP, substrate specificity

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Citations

  • Selner, N. (2013). PROFILING THE INTRINSIC SEQUENCE SPECIFICITY OF PROTEIN TYROSINE PHOSPHATASES [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1384269733

    APA Style (7th edition)

  • Selner, Nicholas. PROFILING THE INTRINSIC SEQUENCE SPECIFICITY OF PROTEIN TYROSINE PHOSPHATASES. 2013. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1384269733.

    MLA Style (8th edition)

  • Selner, Nicholas. "PROFILING THE INTRINSIC SEQUENCE SPECIFICITY OF PROTEIN TYROSINE PHOSPHATASES." Doctoral dissertation, Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1384269733

    Chicago Manual of Style (17th edition)

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