Simultaneous detection of 19 drugs of abuse on dried urine spot by liquid chromatography-tandem mass spectrometry.

BACKGROUND: The lack of specificity of immunoassays for drugs of abuse testing (DAT), and concerns over its cost in conjunction with reflex confirmatory tests prompted us to investigate the combinatorial use of dried urine spot (DUS) and LC-MS/MS as an alternative. METHODS: The method development and validation were performed in accordance with the guidelines published by FDA and CLSI. RESULTS: In this study we established and validated the precision, accuracy, and linearity of our DUS-LC-MS/MS method, and assessed the recovery, interference, and carryover as well. The linearity check for all 19 analytes demonstrated slopes between 0.94 and 1.04, and R(2) always greater than 0.99. Between-batch CV for QC at 4 difference levels ranged from 1.1% to 10%, where CV of LLOQ ranged from 1.2% to 12.8% and CV of ULOQ ranged from 0.8% to 5.1%. A concordance study with patient specimens between our method and GC-MS demonstrated 80.8% to 100% agreement. Stability of DUS specimens was assessed up to 30 days and the measured concentrations ranged from 94% to 114% of the 100 ng/mL urine calibrator used for this assessment. CONCLUSIONS: We established and validated a DUS-LC-MS/MS method for DAT that conforms to the guidelines dictated by FDA, CLSI, and SAMHSA. While our method with high sensitivity and specificity provides an alternative diagnostic utility to EMIT immunoassays, it also offers superior solutions in specimen transportation, preservation, and storage. The benefits of our method are apparent in reducing turnaround time and test costs that result in better patient care.

Regulation of I(kappa)B kinase complex by phosphorylation of (gamma)-binding domain of I(kappa)B kinase (beta) by Polo-like kinase 1.

IkappaB kinase (IKK) complex is a key regulator of NF-kappaB pathways. Signal-induced interaction of the IKKgamma (NEMO) subunit with the C-terminal IKKgamma/NEMO-binding domain (gammaBD) of IKKbeta is an essential interaction for IKK regulation. Underlying regulatory mechanism(s) of this interaction are not known. Phosphorylation of gammaBD has been suggested to play a regulatory role for IKK activation. However, a kinase that phosphorylates gammaBD has not been identified. In this study, we used a C-terminal fragment of IKKbeta as substrate and purified Polo-like kinase 1 (Plk1) from HeLa cell extracts by standard chromatography as a gammaBD kinase. Plk1 phosphorylates serines 733, 740, and 750 in the gammaBD of IKKbeta in vitro. Phosphorylating gammaBD with Plk1 decreased its affinity for IKKgamma in pulldown assay. We generated phosphoantibodies against serine 740 and showed that gammaBD is phosphorylated in vivo. Expressing a constitutively active Plk1 in mammalian cells reduced tumor necrosis factor (TNF)-induced IKK activation, resulting in decreased phosphorylation of endogenous IkappaBalpha and reduced NF-kappaB activation. To activate endogenous Plk1, cells were treated with nocodazole, which reduced TNF-induced IKK activation, and increased the phosphorylation of gammaBD. Knocking down Plk1 in mammalian cells restored TNF-induced IKK activation in nocodazole-treated cells. Activation of Plk1 inhibited TNF-induced expression of cyclin D1. In cells in which Plk1 was knocked down, TNFalpha increased expression of cyclin D1 and the proportion of cells in the S phase of the cell cycle. Taken together, this study shows that phosphorylation regulates the interaction of gammaBD of IKKbeta with IKKgamma and therefore plays a critical role for IKK activation. Moreover, we identify Plk1 as a gammaBD kinase, which negatively regulates TNF-induced IKK activation and cyclin D1 expression, thereby affecting cell cycle regulation. Untimely activation of cyclin D1 by TNFalpha can provide a potential mechanism for an involvement of TNFalpha in inflammation-induced cancer.

Role for KAP1 serine 824 phosphorylation and sumoylation/desumoylation switch in regulating KAP1-mediated transcriptional repression.

As a multifunctional protein, KRAB domain-associated protein 1 (KAP1) is reportedly subjected to multiple protein posttranslational modifications, including phosphorylation and sumoylation. However, gaps exist in our knowledge of how KAP1 phosphorylation cross-talks with KAP1 sumoylation and what the biological consequence is. Here, we show that doxorubicin (Dox) treatment induces KAP1 phosphorylation at Ser-824 via an ataxia telangiectasia mutated (ATM)-dependent manner, correlating with the transcriptional de-repression of p21WAF1/CIP1 and Gadd45alpha. A S824A substitution of KAP1, which ablates the ATM-induced phosphorylation, results in an increase of KAP1 sumoylation and repression of p21 transcription in Dox-treated cells. By contrast, a S824D mutation of KAP1, which mimics constitutive phosphorylation of KAP1, leads to a decrease of KAP1 sumoylation and stimulation of p21 transcription before the exposure of Dox. We further provide evidence that SENP1 deSUMOylase is involved in activating basal, but not Dox-induced, KAP1 Ser-824 phosphorylation, rendering a stimulation of p21 and Gadd45alpha transcription. Moreover, KAP1 and differential sumoylation of KAP1 were also demonstrated to fine-tune the transcription of three additional KAP1-targeted genes, including Bax, Puma, and Noxa. Taken together, our results suggest a novel role for ATM that selectively stimulates KAP1 Ser-824 phosphorylation to repress its sumoylation, leading to the de-repression of expression of a subset of genes involved in promoting cell cycle control and apoptosis in response to genotoxic stresses.

Doxorubicin down-regulates Kruppel-associated box domain-associated protein 1 sumoylation that relieves its transcription repression on p21WAF1/CIP1 in breast cancer MCF-7 cells.

The role of post-translational modification, such as sumoylation, in modulating the efficacy of doxorubicin (Dox) treatment remains unclear. Transcriptional cofactor KRAB domain-associated protein 1 (KAP1) has been shown to complex with the KRAB zinc finger protein, ZBRK1, to repress the transcription of target genes. Through a combination of proteomic screening and site-directed mutagenesis approaches, we have identified lysines 554, 779, and 804 as the major sumoylation sites in KAP1. We then present evidence that Dox-mediated induction of cell cycle regulator p21 expression is differentially regulated by KAP1 sumoylation status. Moreover, the KAP1 sumoylation level was transiently decreased upon Dox exposure, and transfection with the KAP1 sumoylation mimetic, SUMO-1-KAP1, desensitizes breast cancer MCF-7 cells to Dox-elicited cell death. The sumoylation-dependent stimulation of KAP1 function is achieved by enhancing the methylation of H3-K9 and attenuating the acetylation of H3-K9 and H3-K14 at the p21 core promoter. We also show that occupancy of ZBRK1 response elements located at the p21 promoter by ZBRK1.KAP1 is independent of KAP1 sumoylation. Hence, sumoylation of KAP1 represses p21 transcription via a chromatin-silencing process without affecting interaction between KAP1.ZBRK1 and DNA, thus providing a novel mechanistic basis for the understanding of Dox-induced de-repression of p21 transcription. Taken together, our results suggest that Dox-induced decrease in KAP1 sumoylation is essential for Dox to induce p21 expression and subsequent cell growth inhibition in MCF-7 cells.

Regulation of IkappaB kinase (IKK) complex by IKKgamma-dependent phosphorylation of the T-loop and C terminus of IKKbeta.

The mechanistic relationship of phosphorylation of the C terminus of IKKbeta with phosphorylation of its T-loop kinase domain within the IKK complex remained unclear. We investigated the regulatory role of the serine cluster residing immediately adjacent to the HLH domain and of the serines in the NEMO/IKKgamma-binding domain (NBD/gammaBD) in the C-terminal portion of IKKbeta in MEFs deficient in IKKbeta and IKKalpha and in yeast reconstitution system. We show that phosphorylation events at the C terminus of IKKbeta can be divided into autophosphorylation of the serine cluster adjacent to the HLH domain and phosphorylation of the NBD/gammaBD. Autophosphorylation of the serine cluster occurs immediately after IKK activation and requires IKKgamma. In MEFs, this autophosphorylation does not have the down-regulatory function on the IKK complex that was previously described (1). On the other hand, phosphorylation of the NBD/gammaBD regulates IKKgamma-dependent phosphorylation of the T-loop activation domain in IKKbeta and, hence, IKK complex activation. Our study suggests that, within the IKK complex, modulation of the NBD/gammaBD by IKKgamma is upstream to the T-loop phosphorylation.