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.

The conversion of chemical energy into mechanical force by AAA+ (ATPases associated with diverse cellular activities) ATPases is integral to cellular processes, including DNA replication, protein unfolding, cargo transport, and membrane fusion1. The AAA+ ATPase motor cytoplasmic dynein regulates ciliary trafficking2, mitotic spindle formation3, and organelle transport4, and dissecting its precise functions has been challenging due to its rapid timescale of action and the lack of cell-permeable, chemical modulators. Here we describe the discovery of ciliobrevins, the first specific small-molecule antagonists of cytoplasmic dynein. Ciliobrevins perturb protein trafficking within the primary cilium, leading to their malformation and Hedgehog signaling blockade. Ciliobrevins also prevent spindle pole focusing, kinetochore-microtubule attachment, melanosome aggregation, and peroxisome motility in cultured cells. We further demonstrate the ability of ciliobrevins to block dynein-dependent microtubule gliding and ATPase activity in vitro. Ciliobrevins therefore will be useful reagents for studying cellular processes that require this microtubule motor and may guide the development of additional AAA+ ATPase superfamily inhibitors.

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.

antagonist ͉ cancer ͉ Gli ͉ medulloblastoma

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.

Summary Investigating therapeutic “outliers” that show exceptional responses to anti-cancer treatment can uncover biomarkers of drug sensitivity. We performed preclinical trials investigating primary murine acute myeloid leukemias (AMLs) generated by retroviral insertional mutagenesis in KrasG12D “knock-in” mice with the MEK inhibitor PD0325901 (PD901). One outlier AML responded and exhibited intrinsic drug resistance at relapse. Loss of wild-type (WT) Kras enhanced the fitness of the dominant clone and rendered it sensitive to MEK inhibition. Similarly, human colorectal cancer cell lines with increased KRAS mutant allele frequency are more sensitive to MAP kinase inhibition, and CRISPR-Cas9-mediated replacement of WT KRAS with a mutant allele sensitized heterozygous mutant HCT116 cells to treatment. In a prospectively characterized cohort of patients with advanced cancer, 642 of 1168 (55%) with KRAS mutations exhibited allelic imbalance. These studies demonstrate that serial genetic changes at the Kras/KRAS locus are frequent in cancer, and modulate competitive fitness and MEK dependency.

Clathrin-coated vesicles mediate sorting and intracellular transport of membrane-bound proteins. The formation of these coats is initiated by the assembly of adaptor proteins (AP), which specifically bind to membrane cargo proteins via recognition of endocytic sorting motifs. The lipid signaling molecule phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) is critical for this process, as it serves as both a targeting and regulatory factor. PI(4,5)P 2 is synthesized by type I phosphatidylinositol phosphate kinases (PIPKI). We have discovered a direct interaction between the 2-subunit of the AP2 complex and PIPKI␥661 via a yeast two-hybrid screen. This interaction was confirmed using both the 2-subunit in glutathione S-transferase pulldowns and via coimmunoprecipitation of endogenous PIPKI␥661 with the AP2 complex from HEK293 cells. The interaction is mediated, in vivo, by a tyrosine-based motif in the 26-amino acid tail of PIPKI␥661. Because AP2 regulates endocytosis of transferrin receptor from the plasma membrane, we also examined a role for PIPKI␥661 using a flow cytometry endocytosis assay. We observed that stable expression of wild type PIPKI␥661 in Madin-Darby canine kidney cells enhanced transferrin uptake, whereas stable expression of kinase-dead PIPKI␥661 had an inhibitory effect. Neither condition affected the overall cellular level of PI(4,5)P 2 . RNA interference-based knockdown of PIPKI␥661 in HeLa cells also had an inhibitory effect on transferrin endocytosis using the same assay system. Collectively, this evidence implies an important role for PIPKI␥661 in the AP2-mediated endocytosis of transferrin.

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.