Evidence That SHIP-1 Contributes to Phosphatidylinositol 3,4,5-Trisphosphate Metabolism in T Lymphocytes and Can Regulate Novel Phosphoinositide 3-Kinase Effectors

Abstract

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)P3; the lipid substrate for both SHIP and PTEN) as well as saturated protein kinase B (PKB) phosphorylation. Lower levels of PI(3,4,5)P3 and higher levels of phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2) 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)P3 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)P3 and the activity of downstream phosphoinositide 3-kinase effectors in T lymphocytes.

Restoration of SHIP-1 activity in human leukemic cells modifies NF-κB activation pathway and cellular survival upon oxidative stress

“…CEM cells contained protein for SHIP-1, but barely detectable levels of SHIP-2, as previously described (Freeburn et al, 2002) (Figure 4). Although SHIP-1 has been described as a negative regulator of immune receptor and cytokine and growth factor receptor signaling (Krystal, 2000), a recent work proposed that SHIP-1 has a positive role in TLR4 signaling, notably by positively regulating NF-kB-dependent gene transcription in response to LPS stimulation (Fang et al, 2004).…”

“…Differential expression of the lipid phosphatase SHIP-1 in CEM and Jurkat cells Previous works have shown that the Jurkat leukemic cell line is deficient in SHIP-1 at the protein level, but that CEM cells express the protein normally (Bruyns et al, 1999;Freeburn et al, 2002;Horn et al, 2004). To confirm that difference in our cell lines, we carried out Western blotting analysis with specific antibodies raised against SHIP-1 and another inositol lipid phosphatase, namely SHIP-2.…”

“…Cells lacking SHIP-1 (i.e. Jurkat cells) present a very high basal level of PtdIns(3,4,5)P 3 and a subsequent constitutive Akt activation, which is not the case for SHIP-1-positive cells (Freeburn et al, 2002;Horn et al, 2004). This difference might in turn influence a very large number of cellular events, including IKK activation (Cantley, 2002).…”

Phosphoinositide Lipid Phosphatases: Natural Regulators of Phosphoinositide 3-Kinase Signaling in T Lymphocytes

“…For example, SHIP is tyrosine-phosphorylated in response to TCR and CD28 ligation (29). Moreover, expression of a constitutively active SHIP mutant in the leukemic T cell line Jurkat regulates constitutive PtdIns(3,4,5)P 3 levels and CD28-activated PI3K effectors (30). In addition, SHIP interacts with Tec kinase and inhibits its function in T cells (31) as well as participates in a negative signaling complex by associating with the adaptor protein LAT (32).…”

Localization of Fas/CD95 into the Lipid Rafts on Down-Modulation of the Phosphatidylinositol 3-Kinase Signaling Pathway

“…S1). It is noteworthy that 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 the phosphatase and tensin homologue deleted on chromosome 10 (PTEN), which normally counterbalance the PI3K-mediated de novo generation of PIP3 and hence the level of Akt phosphorylation (29). Thus, in the Jurkat cell line, the significant decrease of the Akt phosphorylation observed following edelfosine addition to cell culture could be associated to a direct down-modulation of the PI3K activity or to the abrogation of the PIP3 docking action.…”