The programmed death-ligand 1 (PD-L1), by binding to PD-1 on the surface of immune cells, activates a major immune checkpoint pathway. Elevated expression of PD-L1 in tumor cells mediates tumor-induced T-cell exhaustion and immune suppression; therefore protect the survival of tumor cells. Although blockade of the PD-1/PD-L1 axis exhibits great potential in cancer treatment, mechanisms driving the up-regulation of PD-L1 in tumor cells remain not fully understood. Here we found that type Iγ phosphatidylinositol 4-phosphate (PtdIns(4)P) 5-kinase (PIPKIγ) is required for PD-L1 expression in triple negative breast cancer cells. Depletion of PIPKIγ inhibits both intrinsic and induced PD-L1 expression. Results from further analyses suggest that PIPKIγ promotes the transcription of the PD-L1 gene by activating the NF-κB pathway in these cells. These results demonstrate that PIPKIγ-dependent expression of PD-L1 is likely important for the progression of triple negative breast cancer.

Phosphatidylinositol 4,5 bisphosphate (PI4,5P(2)) is a critical second messenger that regulates a myriad of diverse cellular activities including modulation of the actin cytoskeleton, vesicle trafficking, focal adhesion formation, and nuclear events. In order to effectively regulate these disparate cellular events, synthesis of PI4,5P(2) by phosphatidylinositol phosphate kinases (PIP kinases) must be both spatially and temporally regulated. Two subfamilies of PIP kinases, types I and II, allow the generation of PI4,5P(2) from independent pools of substrate, PI(4)P and PI(5)P respectively. In turn, type I and II PIP kinases show different subcellular localization and thus are involved in distinct signaling pathways. Additionally, several type I isoforms, and their splice variants, have now been shown to be differentially localized throughout the cell and to be involved in the synthesis of PI4,5P(2) at distinct sites. These findings implicate PIP kinases as the major regulators of PI4,5P(2)-mediated events, making them key signaling enzymes in a variety of processes. Understanding the mechanisms regulating spatial and temporal synthesis of PI4,5P(2) by PIP kinases is vital for understanding these processes as a whole. This review examines both structural and regulatory features that modulate activity, localization, and substrate usage of PIPKs.

Engagement of integrin receptors with the extracellular matrix induces the formation of focal adhesions (FAs). Dynamic regulation of FAs is necessary for cells to polarize and migrate. Key interactions between FA scaffolding and signaling proteins are dependent on tyrosine phosphorylation. However, the precise role of tyrosine phosphorylation in FA development and maturation is poorly defined. Here, we show that phosphorylation of type Iγ phosphatidylinositol phosphate kinase (PIPKIγ661) on tyrosine 644 (Y644) is critical for its interaction with talin, and consequently, localization to FAs. PIPKIγ661 is specifically phosphorylated on Y644 by Src. Phosphorylation is regulated by focal adhesion kinase, which enhances the association between PIPKIγ661 and Src. The phosphorylation of Y644 results in an ∼15-fold increase in binding affinity to the talin head domain and blocks β-integrin binding to talin. This defines a novel phosphotyrosine-binding site on the talin F3 domain and a “molecular switch” for talin binding between PIPKIγ661 and β-integrin that may regulate dynamic FA turnover.

Membrane ruffle formation requires remodeling of cortical actin filaments, a process dependent upon the small G-protein Rac. Growth factors stimulate actin remodeling and membrane ruffling by integration of signaling pathways that regulate actin-binding proteins. Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) regulates the activity of many actin-binding proteins and is produced by the type I phosphatidylinositol phosphate kinases (PIPKIs). Here we show in MG-63 cells that only the PIPKI␣ isoform is localized to platelet-derived growth factor (PDGF)-induced membrane ruffles. Further, expression of kinase dead PIPKI␣, which acts as a dominant negative mutant, blocked membrane ruffling, suggesting that PIPKI␣ and PIP 2 participate in ruffling. To explore this, PIPKI␣ was overexpressed in serumstarved cells and stimulated with PDGF. In serumstarved cells, PIPKI␣ expression did not stimulate actin remodeling, but when these cells were stimulated with PDGF, actin rapidly reorganized into foci but not membrane ruffles. PIPKI␣-mediated formation of actin foci was independent of both Rac1 and phosphatidylinositol 3-kinase activities. Significantly, coexpression of dominant active Rac1 with PIPKI␣ in PDGF-stimulated cells resulted in membrane ruffling. The PDGF-and Rac1-stimulated ruffling was inhibited by expression of kinase-dead PIPKI␣. Combined, these data support a model where the localized production of PIP 2 by PIPKI␣ is necessary for actin remodeling, whereas formation of membrane ruffles required Rac signaling.

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