PD-1 Decreases PTEN Protein Stability While Increasing PTEN Phosphatase Activity by Inhibiting CK2.

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.