The proline‐, glutamic acid‐, serine‐ and threonine‐enriched protein tyrosine phosphatase PEP, which is expressed primarily in hematopoietic cells, was recently discovered to be physically associated with the 50‐kDa cytosolic protein tyrosine kinase (PTK) Csk, an important suppressor of Src family PTK, including Lck and Fyn in T cells. We report that this phosphatase has an inhibitory effect on TCR‐induced transcriptional activation of the c‐fos proto‐oncogene and elements from the IL‐2 gene promoter. Catalytically inactive mutants of PEP had no effects in these assays. Expression of PEP also reduced activation of the N‐terminal c‐Jun kinase Jnk2 in response to receptor ligation, but not in response to UV light. In agreement with a more receptor‐proximal site of action, we found that PEP reduced the TCR‐induced increase in tyrosine phosphorylation of an Lck mutant, Lck‐Y505F, which is only phosphorylated on tyrosine 394, the positive regulatory site. Finally, we observed that PEP reduced c‐fos activation in a synergistic manner with Csk, supporting the notion that these two enzymes form a functional team acting on Src family kinases involved in TCR signaling.
A high protein tyrosine phosphatase (PTPase) activity is required to maintain circulating T lymphocytes in a resting phenotype, and to limit the initiation of T cell activation. We report that 15 of the currently known 24 intracellular PTPases are expressed in T cells, namely HePTP, TCPTP, SHP1, SHP2, PEP, PTP‐PEST, PTP‐MEG2, PTEN, PTPH1, PTP‐MEG1, PTP36, PTP‐BAS, LMPTP, PRL‐1 and OV‐1. Most were found in the cytosol and many were enriched at the plasma membrane. Only TCPTP and PTP‐MEG2 had subcellular localizations that essentially excludes them from a direct role in early T cell antigen receptor signaling events. Overexpression of 6 of the PTPases reduced IL‐2 gene activation, 3 of them thereby identified as novel candidates for negative regulators of TCR signaling. Our findings expand the repertoire of PTPases that should be considered for a regulatory role in T cell activation.
Stimulus-induced secretion of bioactive polypeptides is a fundamental aspect of the immune system. Secretory proteins are synthesized in the endoplasmic reticulum and are transported through the Golgi apparatus to the trans-Golgi network, where they are sorted into transport vesicles that bud off and fuse into condensing vacuoles, which subsequently undergo an editing and concentration process to become mature secretory vesicles. In this study, we report that the PTP-MEG2 protein tyrosine phosphatase is located on these vesicles in mast cells. Expression of PTP-MEG2 caused a striking enlargement of these vesicles in both rat basophilic leukemia mast cells and Jurkat T leukemia cells into giant vesicles with diameters of up to several micrometers. The fused vesicles did not acquire markers for other compartments and were adjacent to the trans-Golgi network, contained carboxypeptidase E, chromogranin C, and IL-2, and had an electron-dense core typical of secretory vesicles. Expression of PTP-MEG2 also caused a reduction in the secretion of IL-2 from stimulated Jurkat cells. The effects of PTP-MEG2 on secretory vesicles required the catalytic activity of PTP-MEG2 and was rapidly reversed by pervanadate. We propose that PTP-MEG2 represents a novel connection between tyrosine dephosphorylation and the regulation of secretory vesicles in hematopoietic cells.
The tumor suppressor gene PTEN encodes a 55-kDa enzyme that hydrolyzes both protein phosphotyrosyl and 3-phosphorylated inositol phospholipids in vitro. We have found that the latter activity is physiologically relevant in intact T cells. Expression of active PTEN lead to a 50% loss of transfected cells due to increased apoptosis, which was completely prevented by coexpression of a constitutively active, membrane-bound form of protein kinase B. A mutant of PTEN selectively lacking lipid phosphatase activity, but retaining protein phosphatase activity, had no effects on cell number. Active (but not mutant) PTEN also decreased TCR-induced activation of the mitogen-activated protein kinase ERK2 (extracellular signal-related kinase 2), as seen after inhibition of phosphatidylinositol 3-kinase. Our data indicate that PTEN is a phosphatidylinositol 3-phosphatase in T cells, and we suggest that PTEN may play a role in the regulation of T cell survival and TCR signaling by directly opposing phosphatidylinositol 3-kinase.
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