Regulation of OATP1B1 Function by Tyrosine Kinase-mediated Phosphorylation.

Purpose: OATP1B1 (SLCO1B1) is the most abundant and pharmacologically relevant uptake transporter in the liver and a key mediator of xenobiotic clearance. However, the regulatory mechanisms that determine OATP1B1 activity remain uncertain, and as a result, unexpected drug-drug interactions involving OATP1B1 substrates continue to be reported, including several involving tyrosine kinase inhibitors (TKI).

Experimental Design: OATP1B1-mediated activity in overexpressing HEK293 cells and hepatocytes was assessed in the presence of FDA-approved TKIs, while rosuvastatin pharmacokinetics in the presence of an OATP1B1 inhibiting TKI were measured .

A kinome-wide screen identifies a CDKL5-SOX9 regulatory axis in epithelial cell death and kidney injury.

Renal tubular epithelial cells (RTECs) perform the essential function of maintaining the constancy of body fluid composition and volume. Toxic, inflammatory, or hypoxic-insults to RTECs can cause systemic fluid imbalance, electrolyte abnormalities and metabolic waste accumulation- manifesting as acute kidney injury (AKI), a common disorder associated with adverse long-term sequelae and high mortality. Here we report the results of a kinome-wide RNAi screen for cellular pathways involved in AKI-associated RTEC-dysfunction and cell death.

Transcription factor ZNF148 is a negative regulator of human muscle differentiation.

Muscle differentiation is a complex process in which muscle progenitor cells undergo determination and eventually cellular fusion. This process is heavily regulated by such master transcription factors as MYOD and members of the MEF2 family. Here, we show that the transcription factor ZNF148 plays a direct role in human muscle cell differentiation.

Targeting Histone Demethylases in MYC-Driven Neuroblastomas with Ciclopirox.

Histone lysine demethylases facilitate the activity of oncogenic transcription factors, including possibly MYC. Here we show that multiple histone demethylases influence the viability and poor prognosis of neuroblastoma cells, where MYC is often overexpressed. We also identified the approved small-molecule antifungal agent ciclopirox as a novel pan-histone demethylase inhibitor.

Blocking an N-terminal acetylation-dependent protein interaction inhibits an E3 ligase.

N-terminal acetylation is an abundant modification influencing protein functions. Because ∼80% of mammalian cytosolic proteins are N-terminally acetylated, this modification is potentially an untapped target for chemical control of their functions. Structural studies have revealed that, like lysine acetylation, N-terminal acetylation converts a positively charged amine into a hydrophobic handle that mediates protein interactions; hence, this modification may be a druggable target.

A phosphotyrosine switch regulates organic cation transporters.

Membrane transporters are key determinants of therapeutic outcomes. They regulate systemic and cellular drug levels influencing efficacy as well as toxicities. Here we report a unique phosphorylation-dependent interaction between drug transporters and tyrosine kinase inhibitors (TKIs), which has uncovered widespread phosphotyrosine-mediated regulation of drug transporters.

Multikinase Inhibitors Induce Cutaneous Toxicity through OAT6-Mediated Uptake and MAP3K7-Driven Cell Death.

The use of multikinase inhibitors (MKI) in oncology, such as sorafenib, is associated with a cutaneous adverse event called hand-foot skin reaction (HFSR), in which sites of pressure or friction become inflamed and painful, thus significantly impacting quality of life. The pathogenesis of MKI-induced HFSR is unknown, and the only available treatment options involve dose reduction or discontinuation of therapy, which have negative effects on primary disease management. To investigate the underlying mechanisms by which sorafenib promotes keratinocyte cytotoxicity and subsequent HFSR induction, we performed a transporter-directed RNAi screen in human epidermal keratinocytes and identified SLC22A20 (OAT6) as an uptake carrier of sorafenib.

Serine 350 of human pregnane X receptor is crucial for its heterodimerization with retinoid X receptor alpha and transactivation of target genes in vitro and in vivo.

The human pregnane X receptor (hPXR), a member of the nuclear receptor superfamily, senses xenobiotics and controls the transcription of genes encoding drug-metabolizing enzymes and transporters. The regulation of hPXR's transcriptional activation of its target genes is important for xenobiotic detoxification and endobiotic metabolism, and hPXR dysregulation can cause various adverse drug effects. Studies have implicated the putative phosphorylation site serine 350 (Ser(350)) in regulating hPXR transcriptional activity, but the mechanism of regulation remains elusive.

Mitigation of acute kidney injury by cell-cycle inhibitors that suppress both CDK4/6 and OCT2 functions.

Acute kidney injury (AKI) is a potentially fatal syndrome characterized by a rapid decline in kidney function caused by ischemic or toxic injury to renal tubular cells. The widely used chemotherapy drug cisplatin accumulates preferentially in the renal tubular cells and is a frequent cause of drug-induced AKI. During the development of AKI the quiescent tubular cells reenter the cell cycle.

Stability of the human pregnane X receptor is regulated by E3 ligase UBR5 and serine/threonine kinase DYRK2.

The hPXR (human pregnane X receptor), a major chemical toxin sensor, is a ligand-induced transcription factor activated by various xenobiotics and toxins, resulting in the transcriptional up-regulation of detoxifying enzymes. To date, little is known about the upstream regulation of hPXR. Using MS analysis and a kinome-wide siRNA screen, we report that the E3 ligase UBR5 (ubiquitin protein ligase E3 component n-recognin 5) and DYRK2 (dual-specificity tyrosine-phosphorylation-regulated kinase 2) regulate hPXR stability.

Identification and characterization of phosphorylation sites within the pregnane X receptor protein.

Pregnane X receptor (PXR) is a xenobiotic sensor regulating the expression of genes involved in xenobiotic detoxification and elimination. Phosphorylation plays an important role in modulating PXR activity and several phosphorylation sites have been predicted and characterized in in vitro experiments. Although PXR has been shown to be a phosphoprotein in vivo, the exact residues that are phosphorylated remain elusive.

Piperine activates human pregnane X receptor to induce the expression of cytochrome P450 3A4 and multidrug resistance protein 1.

Activation of the pregnane X receptor (PXR) and subsequently its target genes, including those encoding drug transporters and metabolizing enzymes, while playing substantial roles in xenobiotic detoxification, might cause undesired drug-drug interactions. Recently, an increased awareness has been given to dietary components for potential induction of diet-drug interactions through activation of PXR. Here, we studied, whether piperine (PIP), a major component extracted from the widely-used daily spice black pepper, could induce PXR-mediated expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1).

Cyclin-dependent kinase 4 phosphorylates and positively regulates PAX3-FOXO1 in human alveolar rhabdomyosarcoma cells.

Alveolar rhabdomyosarcoma (ARMS) is an aggressive childhood muscle sarcoma with a 5-year survival rate of less than 30%. More than 80% of ARMSs harbor a PAX3-FOXO1 fusion transcription factor. However, expression of PAX3-FOXO1 in muscle cells alone is not sufficient and requires the loss of function of Ink4a/ARF to promote malignant proliferation of muscle cells in vitro or initiate ARMS tumor formation in vivo.

GUItars: a GUI tool for analysis of high-throughput RNA interference screening data.

Background: High-throughput RNA interference (RNAi) screening has become a widely used approach to elucidating gene functions. However, analysis and annotation of large data sets generated from these screens has been a challenge for researchers without a programming background. Over the years, numerous data analysis methods were produced for plate quality control and hit selection and implemented by a few open-access software packages.

Role of CAR and PXR in xenobiotic sensing and metabolism.

Introduction: The xenobiotic detoxification system, which protects the human body from external chemicals, comprises drug-metabolizing enzymes and transporters whose expressions are regulated by pregnane X receptor (PXR) and the constitutive androstane receptor (CAR). The progress made in a large number of recent studies calls for a timely review to summarize and highlight these key discoveries.

Areas Covered: This review summarizes recent advances in elucidating the roles of PXR and CAR in the xenobiotic detoxification system.

Regulation of Syk by phosphorylation on serine in the linker insert.

The Syk protein-tyrosine kinase is phosphorylated on multiple tyrosines after the aggregation of the B cell antigen receptor. However, metabolic labeling experiments indicate that Syk is inducibly phosphorylated to an even greater extent on serine after receptor ligation. A combination of phosphopeptide mapping and mass spectrometric analyses indicates that serine 291 is a major site of phosphorylation.

The SH3 domain of Lck modulates T-cell receptor-dependent activation of extracellular signal-regulated kinase through activation of Raf-1.

Engagement of the T-cell antigen receptor (TCR) results in the proximal activation of the Src family tyrosine kinase Lck. The activation of Lck leads to the downstream activation of the Ras/Raf/MEK/ERK signaling pathway (where ERK is extracellular signal-related kinase). Under conditions of weak, but not strong, stimulation through the TCR, a version of Lck that contains a single point mutation in the SH3 (Src homology 3) domain (W97ALck) fails to support the activation of ERK, despite initiating signaling through the TCR, as demonstrated by the robust activation of ZAP-70, PLC-gamma, and Ras.