The Phosphotyrosine Phosphatase SHP-2 Participates in a Multimeric Signaling Complex and Regulates T Cell Receptor (TCR) coupling to the Ras/Mitogen-activated Protein Kinase (MAPK) Pathway in Jurkat T Cells

Abstract

Src homology 2 (SH2) domain–containing phosphotyrosine phosphatases (SHPs) are increasingly being shown to play critical roles in protein tyrosine kinase–mediated signaling pathways. The role of SHP-1 as a negative regulator of T cell receptor (TCR) signaling has been established. To further explore the function of the other member of this family, SHP-2, in TCR-mediated events, a catalytically inactive mutant SHP-2 was expressed under an inducible promoter in Jurkat T cells. Expression of the mutant phosphatase significantly inhibited TCR-induced activation of the extracellular-regulated kinase (ERK)-2 member of the mitogen-activated protein kinase (MAPK) family, but had no effect on TCR-ζ chain tyrosine phosphorylation or TCR-elicited Ca2+ transients. Inactive SHP-2 was targeted to membranes resulting in the selective increase in tyrosine phosphorylation of three membrane-associated candidate SHP-2 substrates of 110 kD, 55-60 kD, and 36 kD, respectively. Analysis of immunoprecipitates containing inactive SHP-2 also indicated that the 110-kD and 36-kD Grb-2–associated proteins were putative substrates for SHP-2. TCR-stimulation of Jurkat T cells expressing wild-type SHP-2 resulted in the formation of a multimeric cytosolic complex composed of SHP-2, Grb-2, phosphatidylinositol (PI) 3′-kinase, and p110. A significant proportion of this complex was shown to be membrane associated, presumably as a result of translocation from the cytosol. Catalytically inactive SHP-2, rather than the wild-type PTPase, was preferentially localized in complex with Grb-2 and the p85 subunit of PI 3′-kinase, suggesting that the dephosphorylating actions of SHP-2 may regulate the association of these signaling molecules to the p110 complex. Our results show that SHP-2 plays a critical role in linking the TCR to the Ras/MAPK pathway in Jurkat T cells, and also provide some insight into the molecular interactions of SHP-2 that form the basis of this signal transduction process.

The tyrosine phosphatase SHP‐2 regulates differentiation and apoptosis of individual primary T lymphocytes

“…We also present data showing indications that the negative signals transmitted by the adapter-like function of SHP-2 could be mediated via interaction with the ubiquitin ligases of the Cbl family. Earlier work with Jurkat cells transfected with SHP-2 C>S showed that over-expression of catalytic inactive SHP-2 isoforms resulted in reduced TCR-mediated activation of ERK in bulk cultures [18]. Here we extend the finding that SHP-2 C>S leads to impaired activation of ERK also in individual, primary T lymphocytes ( Fig.…”

“…Therefore the catalytic activity is crucial for the TCR-induced activation of ERK1/2. These results achieved in primary T lymphocytes extend the finding of Frearson and Alexander [18], who used Jurkat lymphoma cells. In addition, when analyzing the activation status of T lymphocytes on the basis of the expression of activation-induced receptors CD96, CD25 and CD71 on the cell surface 6, 24 and 48 h after Ag-mediated restimulation, there was no difference in the activation status of cells expressing the different isoforms of SHP-2 ( Fig.…”

“…1A-D). The first dominant-negative isoform is the commonly used C>S mutant ("SHP-2 C>S") [18,30], which inhibits the catalytic activity but retains its function as an adapter-like molecule (Fig. 1B, leftmost).…”

“…However, experiments showing functional consequences of SHP-2 in IL-2 signaling were performed in transfected fibroblasts or in unequally activated T cells, but not in synchronized primary T cells [15,17]. In T lymphocytes SHP-2 becomes phosphorylated after stimulation via the T cell receptor (TCR) and can be found in a multi-protein complex consisting of at least Gab2, Shc, and PI3K [18,19].…”

“…On the one hand, a stimulating effect on the phosphorylation and activation of ERK1/2 has been shown, surprisingly without affecting IL-2 secretion/consumption measured by ELISA [17,18]. On the other hand, SHP-2 has been attributed to relay the inhibitory signals from over-expressed Gab2 [20], although other results show that instead PI3K is mediating this inhibitory effect [21].…”