The Protein-tyrosine Phosphatase SHP-2 Associates with Tyrosine-phosphorylated Adhesion Molecule PECAM-1 (CD31)

Kenji Sagawa,Teruaki Kimura,Mark Swieter,Reuben P Siraganian

doi:10.1074/jbc.272.49.31086

Abstract

Aggregation of many cell-surface receptors results in tyrosine phosphorylation of numerous proteins. We previously observed the tyrosine phosphorylation of the platelet/endothelial cell adhesion molecule, PECAM-1 (CD31), after FcepsilonRI stimulation in rat basophilic leukemia RBL-2H3 cells. Here we found that PECAM-1 was also transiently tyrosine-phosphoryated after adherence of these cells to fibronectin. Similarly aggregation of the T cell receptor on Jurkat cells also induced this tyrosine phosphorylation. The protein-tyrosine phosphatase SHP-2 is a widely expressed cytosolic enzyme with two Src homology 2 (SH2) domains. SHP-2, but not the related protein-tyrosine phosphatase SHP-1, associated with PECAM-1. This association of the two proteins correlated with the extent of the tyrosine phosphorylation of PECAM-1. A fusion protein containing the two SH2 domains of SHP-2 precipitated PECAM-1 from cell lysates and also directly bound to phosphorylated PECAM-1. In immune precipitate phosphatase assays, there was tyrosine dephosphorylation of PECAM-1. Therefore, integrin and immune receptor activation results in tyrosine phosphorylation of PECAM-1 and the binding of the protein-tyrosine phosphatase SHP-2, which could regulate receptor-mediated signaling in cells.

Highlights

Aggregation of many cell-surface receptors results in tyrosine phosphorylation of numerous proteins
Association of PECAM-1 with SHP-2—We recently observed that the adhesion molecule PECAM-1 (CD31) became tyrosinephosphorylated after Fc⑀RI aggregation
Experiments studied the association of SHP-1 and SHP-2 when cells were stimulated with more physiological stimulants such as the calcium ionophore A23187 and activation of Fc⑀RI that induce the tyrosine phosphorylation of PECAM-1 (Fig. 1)

Summary

The abbreviations used are

Fc⑀RI, the receptor with high affinity for IgE; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PECAM-1, platelet/endothelial cell adhesion molecule 1 ( called CD31); RBL-2H3, rat basophilic leukemia 2H3 cell line; SH2, Src homology 2 region; SHP-1, SH2 containing protein-tyrosine phosphatase 1; SHP-2, SH2 containing protein-tyrosine phosphatase 2; MOPS, 3-(N-morpholino)propanesulfonic acid; BSA, bovine serum albumin; ITIM, immunoreceptor tyrosine-based inhibiting motif. PECAM-1 is a member of the immunoglobulin superfamily of cell adhesion molecules expressed on platelets, endothelial cells, and cells of the myeloid lineage including leukocytes, monocytes, some T cell subsets, and basophils [8, 9]. SHP-2 associates with tyrosine-phosphorylated epidermal growth factor receptor, the platelet-derived growth factor receptor, insulin receptor substrate-1, Fc⑀RI, and with the T and B cell receptors (18 –20). It becomes tyrosine-phosphorylated upon cell stimulation and may act either as a negative regulator for receptor function or as a positive effector for downstream signaling [21,22,23,24]. The tyrosine phosphorylation of PECAM-1 and the recruitment of protein-tyrosine phosphatases may play an important role in regulating signaling from cell-surface receptors

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION