Cellular biochemistry methods for investigating protein tyrosine phosphatases.

Abstract:

SIGNIFICANCE:The protein tyrosine phosphatases (PTPs) are a family of proteins that play critical roles in cellular signaling and influence many aspects of human health and disease. Although a wealth of information has been collected about PTPs since their discovery, many questions regarding their regulation and function still remain. CRITICAL ISSUES:Of particular importance are the elucidation of the biological substrates of individual PTPs and understanding of the chemical and biological basis for temporal and spatial resolution of PTP activity within a cell. RECENT ADVANCES:Drawing from recent advances in both biology and chemistry, innovative approaches have been developed to study the intracellular biochemistry and physiology of PTPs. We provide a summary of PTP-tailored techniques and approaches, emphasizing methodologies to study PTP activity within a cellular context. We first provide a discussion of methods for identifying PTP substrates, including substrate-trapping mutants and synthetic peptide libraries for substrate selectivity profiling. We next provide an overview of approaches for monitoring intracellular PTP activity, including a discussion of mechanistic-based probes, gel-based assays, substrates that can be used intracellularly, and assays tied to cell growth. Finally, we review approaches used for monitoring PTP oxidation, a key regulatory pathway for these enzymes, discussing the biotin switch method and variants of this approach, along with affinity trapping techniques and probes designed to detect PTP oxidation. FUTURE DIRECTIONS:Further development of approaches to investigate the intracellular PTP activity and functions will provide specific insight into their mechanisms of action and control of diverse signaling pathways.

journal_name

Antioxid Redox Signal

authors

Stanford SM,Ahmed V,Barrios AM,Bottini N

doi

10.1089/ars.2013.5731

subject

Has Abstract

pub_date

2014-05-10 00:00:00

pages

2160-78

issue

14

eissn

1523-0864

issn

1557-7716

journal_volume

20

pub_type

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