Human 70-kDa SHP-1L Differs from 68-kDa SHP-1 in Its C-terminal Structure and Catalytic Activity

Chao-Lan Yu,Yong-Jiu Jin,Steven J Burakoff

doi:10.1074/jbc.274.40.28301

Abstract

The tyrosine phosphatase SHP-1 functions as a negative regulator in hematopoietic cell development, proliferation, and receptor-mediated cellular activation. In Jurkat T cells, a major 68-kDa band and a minor 70-kDa band were immunoprecipitated by a monoclonal antibody against the SHP-1 protein-tyrosine phosphatase domain, while an antibody against the SHP-1 C-terminal 19 amino acids recognized only the 68-kDa SHP-1. The SDS-gel-purified 70-kDa protein was subjected to tryptic mapping and microsequencing, which was followed by molecular cloning. It revealed that the 70-kDa protein, termed SHP-1L, is a C-terminal alternatively spliced form of SHP-1. SHP-1L is 29 amino acids longer than SHP-1, and its 66 C-terminal amino acids are different from SHP-1. The C terminus of SHP-1L contains a proline-rich motif PVPGPPVLSP, a potential Src homology 3 domain-binding site. In contrast to SHP-1, tyrosine phosphorylation of SHP-1L is not detected upon stimulation in Jurkat T cells. This is apparently due to the lack of a single in vivo tyrosine phosphorylation site, which only exists in the C terminus of SHP-1 (Y564). COS cell-expressed glutathione S-transferase-SHP-1L can dephosphorylate tyrosine-phosphorylated ZAP70. At pH 7.4, SHP-1L was shown to be more active than SHP-1 in the dephosphorylation of ZAP70. At pH 5.4, SHP-1L and SHP-1 exhibited similar catalytic activity. It is likely that these two isoforms play different roles in the regulation of hematopoietic cell signal transduction.

Highlights

SHP-1 is a 68-kDa, non-transmembrane protein-tyrosine phosphatase (PTP)1 containing two tandem Src homology (SH2) domains, a catalytic domain, and a C-terminal tail of about 100 amino acid residues [1,2,3,4]
Identification of a 70-kDa Molecule in Jurkat T Cells Immunoprecipitated by Anti-SHP-1—Jurkat T cell lysates were immunoprecipitated with a mAb against the PTP domain of human SHP-1
To determine whether SH2 containing PTPs will bind to a phenylarsine oxide (PAO) column, Nonidet P-40 lysates of Jurkat T cells were passed through the PAO column

Summary

Introduction

SHP-1 ( called HCP, SHPTP1, and PTP1c) is a 68-kDa, non-transmembrane protein-tyrosine phosphatase (PTP)1 containing two tandem Src homology (SH2) domains, a catalytic domain, and a C-terminal tail of about 100 amino acid residues [1,2,3,4]. PTP Activity of GST-SHP-1L and GST-SHP-1—To prepare tyrosinephosphorylated ZAP70, 200 ϫ 106 Jurkat T cells were treated with pervanadate, lysed, and immunoprecipitated with anti-ZAP70 mAb plus protein A beads. When similar immunoprecipitations were carried out by using a polyclonal Ab raised against the C-terminal 19 amino acids of human SHP-1, only the major 68-kDa SHP-1 band was detected.

Results

Conclusion