Deficiency of PTEN in Jurkat T cells causes constitutive localization of Itk to the plasma membrane and hyperresponsiveness to CD3 stimulation

Pleckstrin homology (PH) domain binding to D3-phosphorylated phosphatidylin ositides (PI) provides a reversible means of recruiting proteins to the pla sma membrane, with the resultant change in subcellular localization playing a key role in the activation of multiple intracellular signaling pathways. Previously we found that the T-cell-specific PH domain-containing kinase I tk is constitutively membrane associated in Jurkat T cells. This distributi on was unexpected given that the closely related B-cell kinase, Btk, is alm ost exclusively cytosolic. In addition to constitutive membrane association of Itk, unstimulated JTAg T cells also exhibited constitutive phosphorylat ion of Akt on Ser-473, an indication of elevated basal levels of the phosph atidylinositol 3-kinase (PI3K) products PI-3,4-P-2 and PI-3,4,5-P-3 in the plasma membrane. Here we describe a defect in expression of the D3 phosphoi nositide phosphatase, PTEN, in Jurkat and JTAg T cells that leads to unregu lated PH domain interactions with the plasma membrane. Inhibition of D3 pho sphorylation by PI3K inhibitors, or by expression of PTEN, blocked constitu tive phosphorylation of Akt on Ser-473 and caused Itk to redistribute to th e cytosol. The PTEN-deficient cells were also hyperresponsive to T-cell rec eptor (TCR) stimulation, as measured by Itk kinase activity, tyrosine phosp horylation of phospholipase C-gamma 1, and activation of Erk compared to th ose in PTEN-replete cells. These data support the idea that PH domain-media ted association with the plasma membrane is required for Itk activation, pr ovide evidence for a negative regulatory role of PTEN in TCR stimulation, a nd suggest that signaling models based on results from Jurkat T-cell lines may underestimate the role of PI3K in TCR signaling.