Cutting edge: TREM-like transcript-1, a platelet immunoreceptor tyrosine-based inhibition motif encoding costimulatory immunoreceptor that enhances, rather than inhibits, calcium signaling via SHP-2.

To date, immunoreceptor tyrosine-based inhibition motifs (ITIMs) have been shown to mediate inhibitory properties. We report a novel triggering receptor expressed on myeloid cells (TREM) family member, TREM-like transcript-1 (TLT1), which differs from the activating members because its cytoplasmic tail contains two ITIMs at Y245 and Y281. A TLT1 splice variant (TLT1sp) encodes a different cytoplasmic tail lacking ITIMs. Both isoforms are expressed in resting platelet alpha-granules, which are up-regulated to the cell surface following activation. TLT1 recruited Src homology 2 domain-containing tyrosine phosphatase (SHP)-2 to the "classical" ITIM (Y281) but not the "nonclassical" ITIM (Y245). In contrast to previously characterized ITIM receptors, TLT1 enhanced, rather than inhibited, FcepsilonRI-mediated calcium signaling in rat basophilic leukemia cells, a property dependent on the SHP-2 recruiting classical Y281 ITIM. Therefore, TLT1 represents a new costimulatory ITIM immunoreceptor and is the second ITIM-bearing receptor to be identified in platelets after platelet endothelial cell adhesion molecule-1.

Identification of pro-interleukin 16 as a novel target of MAP kinases in activated T lymphocytes.

T lymphocyte activation is controlled by a coordinated web of tyrosine and serine kinases. There is a large body of information about tyrosine kinase substrates in T cells but analysis of serine kinase substrates has been more difficult. Recently we described an antiserum that recognizes serine-phosphorylated peptides corresponding to the substrate sequences for AGC serine kinases. This antiserum, termed PAP-1 (phospho antibody for proteomics-1), has proven useful for probing the serine phosphoproteome of antigen receptor-activated T lymphocytes. The present study shows that PAP-1 can also be used to explore serine kinases activated by cytokines and chemokines in T cells. Using PAP-1, together with proteomic analysis, the precursor form of the cytokine IL-16 (ProIL-16) was shown to be phosphorylated on Ser144 in antigen receptor-, SDF1alpha- and IL-2-activated T cells. Genetic and pharmacological-inhibitor experiments showed that the phosphorylation of ProIL-16 is dependent on activation of the kinases Erk1/2. IL-16 is secreted by mitogen-activated T cells, and the biochemical link between ProIL-16 and Erk1/2, revealed by studies with PAP-1, prompted analysis of the role of MAP kinases in this response. We show that TCR-mediated secretion of IL-16 is dependent on MAP kinases. The present study thus reveals how phosphoproteomic analysis opens previously unrecognized avenues for research, and yields novel insights about targets for MAP kinases in T lymphocytes.

Approaches to define antigen receptor-induced serine kinase signal transduction pathways.

In the present report we describe the properties of a novel phospho-specific antiserum that has opened a route to the characterization of antigen receptor-activated serine kinase pathways in lymphocytes. The basis for the present work was that Ser-21 in glycogen synthase kinase 3alpha is robustly phosphorylated following antigen receptor triggering. We predicted accordingly that antigen receptors would also stimulate phosphorylation of other proteins with a similar sequence. To test this idea we raised an antibody against the phospho-peptide RARTSpSFAEP, where pS is a phospho-serine corresponding to the glycogen synthase kinase 3alpha Ser-21 sequence. The resulting antiserum was called phospho antibody for proteomics-1 (PAP-1). The present study describes the properties of PAP-1 and shows that it can reveal quite striking differences in the phospho-proteome of different cell types and is able to pinpoint new targets in important signal transduction pathways. PAP-1 was used to map protein phosphorylations regulated by the antigen receptor in T cells. One of these PAP-1-reactive proteins was purified and revealed to be a previously unrecognized target for antigen receptor signal transduction, namely an "orphan" adapter SLY (Src homology 3 (SH3) domain-containing protein expressed in lymphocytes). The use of sera detecting specific phosphorylation sites is thus proved as a powerful method for the discovery of novel downstream components of antigen receptor signals in T cells.

Evidence that SHIP-1 contributes to phosphatidylinositol 3,4,5-trisphosphate metabolism in T lymphocytes and can regulate novel phosphoinositide 3-kinase effectors.

The leukemic T cell line Jurkat is deficient in protein expression of the lipid phosphatases Src homology 2 domain containing inositol polyphosphate phosphatase (SHIP) and phosphatase and tensin homolog deleted on chromosome ten (PTEN). We examined whether the lack of expression of SHIP-1 and PTEN is shared by other leukemic T cell lines and PBLs. Analysis of a range of cell lines and PBLs revealed that unlike Jurkat cells, two other well-characterized T cell lines, namely CEM and MOLT-4 cells, expressed the 5'-phosphatase SHIP at the protein level. However, the 3-phosphatase PTEN was not expressed by CEM or MOLT-4 cells or Jurkat cells. The HUT78 cell line and PBLs expressed both SHIP and PTEN. Jurkat cells exhibited high basal levels of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3); the lipid substrate for both SHIP and PTEN) as well as saturated protein kinase B (PKB) phosphorylation. Lower levels of PI(3,4,5)P(3) and higher levels of phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)) as well as unsaturated constitutive phosphorylation of PKB were observed in CEM and MOLT-4 cells compared with Jurkat cells. In PBLs and HUT78 cells which express both PTEN and SHIP-1, there was no constitutive PI(3,4,5)P(3) or PKB phosphorylation, and receptor stimuli were able to elicit robust phosphorylation of PKB. Expression of a constitutively active SHIP-1 protein in Jurkat cells was sufficient to reduce both constitutive PKB membrane localization and PKB phosphorylation. Together, these data indicate important differences between T leukemic cells as well as PBLs, regarding expression of key lipid phosphatases. This study provides the first evidence that SHIP-1 can influence the constitutive levels of PI(3,4,5)P(3) and the activity of downstream phosphoinositide 3-kinase effectors in T lymphocytes.