Protein Tyrosine Phosphatase Assays

Overview

Journal Curr Protoc Immunol

Specialty Allergy & Immunology

Date 2011 Apr 5

PMID 21462163

Citations 24

Authors

Ulrike Lorenz

Affiliations

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Abstract

Tyrosine phosphorylation and dephosphorylation of proteins play a critical role in many processes of the immune system, from early development to fully differentiated effector function. Since the opposing actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) determine the steady-state level of tyrosine phosphorylation on a given protein, it is often important for mechanistic studies to determine the specific activities of PTKs and PTPs. PTPs are defined by their enzymatic activity that catalyzes the dephosphorylation of phosphotyrosine residues. This unit focuses on methods to determine the enzymatic activity of PTPs. While there are many varieties of PTP assays, the focus in this unit is on immune complex PTP assays, which do not require elaborate biochemical purifications and are commonly used to test the activities of specific PTPs in the immune system.

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References

Gallagher S . Immunoblot detection. Curr Protoc Protein Sci. 2008; Chapter 10:Unit 10.10. DOI: 10.1002/0471140864.ps1010s04. View

Tiganis T, Bennett A . Protein tyrosine phosphatase function: the substrate perspective. Biochem J. 2007; 402(1):1-15. PMC: 1783993. DOI: 10.1042/BJ20061548. View

Alonso A, Sasin J, Bottini N, Friedberg I, Friedberg I, Osterman A . Protein tyrosine phosphatases in the human genome. Cell. 2004; 117(6):699-711. DOI: 10.1016/j.cell.2004.05.018. View

Sozio M, Mathis M, Young J, Walchli S, Pitcher L, Wrage P . PTPH1 is a predominant protein-tyrosine phosphatase capable of interacting with and dephosphorylating the T cell receptor zeta subunit. J Biol Chem. 2003; 279(9):7760-9. DOI: 10.1074/jbc.M309994200. View

Pao L, Badour K, Siminovitch K, Neel B . Nonreceptor protein-tyrosine phosphatases in immune cell signaling. Annu Rev Immunol. 2007; 25:473-523. DOI: 10.1146/annurev.immunol.23.021704.115647. View

Kilgore N, Carter J, Lorenz U, Evavold B . Cutting edge: dependence of TCR antagonism on Src homology 2 domain-containing protein tyrosine phosphatase activity. J Immunol. 2003; 170(10):4891-5. DOI: 10.4049/jimmunol.170.10.4891. View

Hermiston M, Xu Z, Weiss A . CD45: a critical regulator of signaling thresholds in immune cells. Annu Rev Immunol. 2002; 21:107-37. DOI: 10.1146/annurev.immunol.21.120601.140946. View

Markova B, Gulati P, Herrlich P, Bohmer F . Investigation of protein-tyrosine phosphatases by in-gel assays. Methods. 2004; 35(1):22-7. DOI: 10.1016/j.ymeth.2004.07.004. View

Van Veldhoven P, Mannaerts G . Inorganic and organic phosphate measurements in the nanomolar range. Anal Biochem. 1987; 161(1):45-8. DOI: 10.1016/0003-2697(87)90649-x. View

10.

Echan L, Speicher D . Protein detection in gels using fixation. Curr Protoc Protein Sci. 2008; Chapter 10:10.5.1-10.5.18. PMC: 5647780. DOI: 10.1002/0471140864.ps1005s29. View

11.

Montalibet J, Skorey K, Kennedy B . Protein tyrosine phosphatase: enzymatic assays. Methods. 2004; 35(1):2-8. DOI: 10.1016/j.ymeth.2004.07.002. View

12.

Zhang Z . Protein-tyrosine phosphatases: biological function, structural characteristics, and mechanism of catalysis. Crit Rev Biochem Mol Biol. 1998; 33(1):1-52. DOI: 10.1080/10409239891204161. View

13.

Holmes N . CD45: all is not yet crystal clear. Immunology. 2006; 117(2):145-55. PMC: 1782222. DOI: 10.1111/j.1365-2567.2005.02265.x. View

14.

Carter S, Karl D . Inorganic phosphate assay with malachite green: an improvement and evaluation. J Biochem Biophys Methods. 1982; 7(1):7-13. DOI: 10.1016/0165-022x(82)90031-8. View

15.

Huntington N, Tarlinton D . CD45: direct and indirect government of immune regulation. Immunol Lett. 2004; 94(3):167-74. DOI: 10.1016/j.imlet.2004.05.011. View