Abstract
Abstract 2145
Maintenance of peripheral tolerance is essential for homeostasis of the immune system. The newer pathway of the B7:CD28 family consisting of the receptor PD-1 (CD279) and its ligands, PD-L1 (B7-H1; CD274) and PD-L2 (B7-DC; CD273), plays a vital role in the induction and maintenance of T cell anergy and peripheral tolerance. This pathway also regulates the balance between stimulatory and inhibitory signals needed for effective immunity and maintenance of T cell homeostasis but can also compromise anti-tumor and anti-viral immunity. The precise mechanisms via which PD-1 mediates its inhibitory effects in T cells remain elusive. One of the critical signaling pathways targeted by PD-1 is the PI3K/Akt pathway, via which PD-1 mediates inhibitory effects on T cell proliferation, cytokine production and glucose uptake. In contrast to CTLA-4, which affects PI3/Akt signaling by targeting Akt through PP2A activation, PD-1 targets the PI3K/Akt pathway by directly inhibiting activation of PI3K. It has been proposed that PD-1 inhibits PI3K by upregulating PTEN (phosphatase and tensin homologue deleted on chromosome 10), a phosphoinositole 3,4,5-trisphosphatase important for antagonizing PI3K signaling, but the mechanism remains unclear. In the present study we sought to dissect the effects of PD-1 mediated signaling on the regulation of PTEN expression and function. We used highly purified primary human CD4+ T cells and magnetic beads conjugated with monoclonal antibodies (mAbs) against CD3 and CD28 with or without agonist mAb against PD-1. As determined by assessment of PTEN lipid phosphatase activity, stimulation of T cells via TCR/CD3, CD28 and PD-1 resulted in prominent increase of PTEN phosphatase activity compared to T cells stimulated via TCR/CD3 and CD28 without PD-1 signals. Surprisingly, although T cells receiving PD-1 signals displayed increased PTEN phosphatase activity they expressed lower levels of PTEN compared to T cells stimulated via TCR/CD3 and CD28, as determined by both western blot and by flow cytometry. In other cell types, the abundance of PTEN can be regulated transcriptionally and through phosphorylation-dependent modulation of protein stability. Assessment of PTEN mRNA by real time qPCR indicated that PTEN mRNA levels were comparable between T cells stimulated via TCR/CD3/CD28 and T cells stimulated via TCR/CD3/CD28 and PD-1. PTEN possesses a C-terminal non-catalytic regulatory domain, which plays an important role in its biological activity. A cluster of serine/threonine residues within this region is phosphorylated by the serine/threonine kinase casein kinase 2 (CK2). CK2-mediated phosphorylation stabilizes PTEN by preventing its ubiquitin-dependent degradation while decreasing PTEN phosphatase activity. In contrast, impaired phosphorylation in this region results in decreased PTEN protein stability but increased phosphatase activity. Because PD-1 signaling resulted in reduced PTEN protein expression but increased phosphatase activity, we examined whether PD-1 might alter CK2 activity and CK2-mediated phosphorylation of PTEN. In CD4+ T cells stimulated via TCR/CD3 and CD28, CK2 activation was robust and resulted in sustained phosphorylation on PTEN S380/T382/383 cluster. In contrast, T cells receiving PD-1 signals displayed impaired CK2 activity and significantly diminished phosphorylation of PTEN in the S380/T382/383 cluster. To determine whether downregulation of PTEN protein expression via PD-1 was mediated via proteasome-dependent degradation we used the proteasome inhibitor MG132. Stimulation of T cells via TCR/CD3, CD28 and PD-1 in the presence of MG132 prevented PD-1-mediated decrease of PTEN and resulted in reduced PTEN activity. Our results identify CK2 as a new target of PD-1-mediated signaling and reveal an unexpected mechanism via which PD-1 regulates PTEN phosphatase activity and inhibition of the PI3K/Akt pathway.
Disclosures:
No relevant conflicts of interest to declare.
Binding of PTEN to Specific PDZ Domains Contributes to PTEN Protein Stability and Phosphorylation by Microtubule-associated Serine/Threonine Kinases