Kevin P. Becker

PKC-dependent Activation of Sphingosine Kinase 1 and Translocation to the Plasma Membrane EXTRACELLULAR RELEASE OF SPHINGOSINE-1-PHOSPHATE INDUCED BY PHORBOL 12-MYRISTATE 13-ACETATE (PMA)

Sphingosine-1-phosphate (S1P) is a highly bioactive sphingolipid involved in diverse biological processes leading to changes in cell growth, differentiation, motility, and survival. S1P generation is regulated via sphingosine kinase (SK), and many of its effects are mediated through extracelluar action on G-protein-coupled receptors. In this study, we have investigated the mechanisms regulating SK, where this occurs in the cell, and whether this leads to release of S1P extracellularly. The protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), induced early activation of SK in HEK 293 cells, and this activation was more specific to the membrane-associated SK. Therefore, we next investigated whether PMA induced translocation of SK to the plasma membrane. PMA induced translocation of both endogenous and green fluorescent protein (GFP)-tagged human SK1 (hSK1) to the plasma membrane. PMA also induced phosphorylation of GFP-hSK1. The PMA-induced translocation was abrogated by preincubation with known PKC inhibitors (bisindoylmaleimide and calphostin-c) as well as by the indirect inhibitor of PKC, C6-ceramide, supporting a role for PKC in mediating translocation of SK to the plasma membrane. SK activity was not necessary for translocation, because a dominant negative G82D mutation also translocated in response to PMA. Importantly, PKC regulation of SK was accompanied by a 4-fold increase in S1P in the media. These results demonstrate a novel mechanism by which PKC regulates SK and increases secretion of S1P, allowing for autocrine/paracrine signaling.

Role of human sphingosine-1-phosphate phosphatase 1 in the regulation of intra- and extracellular sphingosine-1-phosphate levels and cell viability

Sphingosine-1-phosphate (S1P) is a highly bioactive lipid that exerts numerous biological effects both intracellularly as a second messenger and extracellularly by binding to its G-protein-coupled receptors of the endothelial differentiation gene family (S1P receptors-(1–5)). Intracellularly, at least two enzymes, sphingosine kinase and S1P phosphatase, regulate the activity of S1P by governing the phosphorylation status of S1P. To study the regulation of S1P levels, we cloned the human isoform of S1P phosphatase 1 (hSPPase1). The hSPPase1 has 78% homology to the mouse SPPase at the amino acid level with 6–8 possible transmembrane domains. Confocal microscopy revealed green fluorescent protein-tagged hSPPase1, expressed in either MCF7 or HEK293 cells, co-localized to endoplasmic reticulum with calreticulin. According to Northern blot analysis, hSPPase1 is expressed in most tissues, with the strongest levels found in the highly vascular tissues of placenta and kidney. Transient overexpression of hSPPase1 exhibited a 2-fold increase in phosphatase activity against S1P and dihydro-S1P, indicating that the expressed protein was functional. Small interfering RNA (siRNA) knockdown of endogenous hSPPase1 drastically reduced hSPPase1 mRNA levels, as confirmed by reverse transcription PCR, and resulted in an overall 25% reduction of in vitro phosphatase activity in the membrane fractions. Sphingolipid mass measurements in hSPPase1 siRNA knockdown cells revealed a 2-fold increase of S1P levels and concomitant decrease in sphingosine. In vivo labeling of hSPPase1 siRNA-treated cells showed accumulation of S1P within cells, as well as significantly increased secretion of S1P into the media, indicating that hSPPase1 regulates secreted S1P. In addition, siRNA-induced knockdown of hSPPase1 endowed resistance to tumor necrosis factor-α and the chemotherapeutic agent daunorubicin. Collectively, these data suggest that regulation of hSPPase1 with the resultant changes in cellular and secreted S1P could have important implications to cell proliferation, angiogenesis, and apoptosis.

cPKC-dependent sequestration of membrane-recycling components in a subset of recycling endosomes.

In addition to the classical role of protein kinase C (PKC) as a mediator of transmembrane signals initiated at the plasma membrane, there is also significant evidence to suggest that a more sustained PKC activity is necessary for a variety of long term cellular responses. To date, the subcellular localization of PKC during sustained activation has not been extensively studied. We report here that long term activation of PKC (1 h) leads to the selective translocation of classical PKC isoenzymes, α and βII, to a juxtanuclear compartment. Jux-tanuclear translocation of PKC required an intact C1 and C2 domain, and occurred in a microtubule-dependent manner. This juxtanuclear compartment was localized close to the Golgi complex but displayed no overlap with Golgi markers, and was resistant to dispersal with Golgi disrupting agents, brefeldin A and nocodazole. Further characterization revealed that PKCα and βII translocated to a compartment that colocalized with the small GTPase, rab11, which is a marker for the subset of recycling endosomes concentrated around the microtubule-organizing center/centrosome. Analysis of the functional consequence of cPKC translocation on membrane recycling demonstrated a cPKC-dependent sequestration of transferrin, a marker of membrane recycling, in the cPKC compartment. These results identify a novel site for cPKC translocation and define a novel function for the sustained activation of PKCα and βII in regulation of recycling components.

Dynamic Sequestration of the Recycling Compartment by Classical Protein Kinase C

It has been previously shown that upon sustained stimulation (30-60 min) with phorbol esters, protein kinase C (PKC)α andβII become sequestered in a juxtanuclear region, the pericentrion. The activation of PKC also results in sequestration of transferrin, suggesting a role for PKC in regulating endocytosis and sequestration of recycling components. In this work we characterize the pericentrion as a PKC-dependent subset of the recycling compartment. We demonstrate that upon sustained stimulation of PKC, both protein (CD59, caveolin) and possibly also lipid (Bodipy-GM1) cargo become sequestered in a PKC-dependent manner. This sequestration displayed a strict temperature requirement and was inhibited below 32 °C. Treatment of cells with phorbol myristate acetate for 60 min led to the formation of a distinct membrane structure. PKC sequestration and pericentrion formation were blocked by hypertonic sucrose as well as by potassium depletion (inhibitors of clathrin-dependent endocytosis) but not by nystatin or filipin, which inhibit clathrin-independent pathways. Interestingly, it was also observed that some molecules that internalize through clathrin-independent pathways (CD59, Bodipy-GM1, caveolin) also sequestered to the pericentrion upon sustained PKC activation, suggesting that PKC acted distal to the site of internalization of endocytic cargo. Together these results suggest that PKC regulates sequestration of recycling molecules into this compartment, the pericentrion.

Status Quo—standard-of-care Medical and Radiation Therapy for Glioblastoma

AbstractThere will be approximately 10,000 new cases of glioblastoma diagnosed in the United States this year alone. Although a relatively rare cancer, these aggressive tumors lead to a disproportionate amount of cancer morbidity and mortality. The current standard treatment for a glioblastoma consists of surgery for cytoreduction and/or biopsy followed by chemoradiation and adjuvant temozolomide. Without treatment, most patients will die of their disease within 3 months of diagnosis. Surgical intervention can extend survival to 9 to 10 months, and this can be lengthened to 12 months with the addition of adjuvant radiation. In a 2005 landmark clinical trial, Stupp et al demonstrated that temozolomide, an oral DNA-alkylating chemotherapeutic agent, when added to radiation, can improve survival to 14.6 months. Although the effect on survival is modest, this treatment course represents a significant improvement over chemotherapy agents widely used for the 3 previous decades. This review will focus on the development of temozolomide and its use along with radiation therapy as the current standard treatment for glioblastoma.

Isoenzyme-specific Translocation of Protein Kinase C (PKC)βII and not PKCβI to a Juxtanuclear Subset of Recycling Endosomes INVOLVEMENT OF PHOSPHOLIPASE D

Elucidation of isoenzyme-specific functions of individual protein kinase C (PKC) isoenzymes has emerged as an important goal in the study of this family of kinases, but this task has been complicated by modest substrate specificity and high homology among the individual members of each PKC subfamily. The classical PKCβI and PKCβII isoenzymes provide a unique opportunity because they are the alternatively spliced products of the β gene and are 100% identical except for the last 50 of 52 amino acids. In this study, it is shown that green fluorescent protein-tagged PKCβII and not PKCβI translocates to a recently described juxtanuclear site of localization for PKCα and PKCβII isoenzymes that arises with sustained stimulation of PKC. Mechanistically, translocation of PKCβII to the juxtanuclear region required kinase activity. PKCβII, but not PKCβI, was found to activate phospholipase D within this time frame. Inhibitors of phospholipase D (1-butanol and a dominant negative construct) prevented the translocation of PKCβII to the juxtanuclear region but not to the plasma membrane, thus demonstrating a role for phospholipase D in the juxtanuclear translocation of PKCβII. Taken together, these results define specific biochemical and cellular actions of PKCβII when compared with PKCβI.

Isoenzyme-specific translocation of PKCbII and not PKCbI to a Juxtanuclear subset of recycling endosomes. Involvement of phospholipase D

Elucidation of isoenzyme-specific functions of individual protein kinase C (PKC) isoenzymes has emerged as an important goal in the study of this family of kinases, but this task has been complicated by modest substrate specificity and high homology among the individual members of each PKC subfamily. The classical PKCβI and PKCβII isoenzymes provide a unique opportunity because they are the alternatively spliced products of the β gene and are 100% identical except for the last 50 of 52 amino acids. In this study, it is shown that green fluorescent protein-tagged PKCβII and not PKCβI translocates to a recently described juxtanuclear site of localization for PKCα and PKCβII isoenzymes that arises with sustained stimulation of PKC. Mechanistically, translocation of PKCβII to the juxtanuclear region required kinase activity. PKCβII, but not PKCβI, was found to activate phospholipase D within this time frame. Inhibitors of phospholipase D (1-butanol and a dominant negative construct) prevented the translocation of PKCβII to the juxtanuclear region but not to the plasma membrane, thus demonstrating a role for phospholipase D in the juxtanuclear translocation of PKCβII. Taken together, these results define specific biochemical and cellular actions of PKCβII when compared with PKCβI.

Coupling of thromboxane A2 receptor isoforms to Gα13: effects on ligand binding and signalling

Previous subtyping of thromboxane A2 (TXA2) receptors in platelets and vascular smooth muscle cells was based on pharmacological criteria. Two distinct carboxy-terminal splice variants for TXA2 receptors exist and they couple to several different G protein alpha subunits including Galpha13, but it has not been established whether either or both isoforms interact with and signal through it. We sought to determine: (1) which TXA2 receptor isoforms exist in vascular smooth muscle, (2) if Galpha13 is present in vascular smooth muscle and (3) if Galpha13 interacts with either or both of the two TXA2 receptor isoforms as determined by changes in ligand binding properties and generation of intracellular signals. Both TXA2 receptor isoforms and Galpha13 were found in vascular smooth muscle cells. Both the alpha and beta isoforms of the TXA2 receptors were transiently transfected with or without Galpha13 into COS-7 (radioligand binding assays) or CHO cells (agonist induced Na+/H+ exchange). Co-expression of each receptor isoform with Galpha13 significantly (P<0.05) increased the affinity of each receptor for the two agonists, I-BOP and ONO11113, and decreased the affinity of the receptor for the antagonists, SQ29,548 and L657,925. I-BOP stimulated Na+/H+ exchange in vascular smooth muscle cells. Co-expression of Galpha13 with each TXA2 receptor isoform in CHO cells resulted in a significant (P<0.04) agonist induced increase in Na+/H+ exchange compared to cells not transfected with Galpha13. The results support the possibility that the previous classification of TXA2 receptor subtypes based on pharmacological criteria reflect unique interactions with specific G protein alpha subunits.

CLONING AND CHARACTERIZATION OF AN ENDOGENOUS COS-7 CELL THROMBOXANE A2 RECEPTOR

A cDNA for a thromboxane A2 (TXA2) receptor was cloned from an SV40 transformed African Green Monkey kidney cell line (COS-7). The sequence is 98% homologous with the isoform of the human TXA2 receptor and has agonist and antagonist ligand binding characteristics that are not significantly different from the human receptor. Stimulation of the COS-7 cells with the TXA2 receptor agonist, ONO 11113 resulted in a significant increase in cAMP formation that was blocked by a receptor antagonist. The results raise the question of the utility of the COS-7 cell line for studies of cloned and expressed TXA2 receptor signalling mechanisms.

Adjuvant chemotherapy and overall survival in adult medulloblastoma

Background Although chemotherapy is used routinely in pediatric medulloblastoma (MB) patients, its benefit for adult MB is unclear. We evaluated the survival impact of adjuvant chemotherapy in adult MB. Methods Using the National Cancer Data Base, we identified patients aged 18 years and older who were diagnosed with MB in 2004-2012 and underwent surgical resection and adjuvant craniospinal irradiation (CSI). Patients were divided into those who received adjuvant CSI and chemotherapy (CRT) or CSI alone (RT). Predictors of CRT compared with RT were evaluated with univariable and multivariable logistic regression. Survival analysis was limited to patients receiving CSI doses between 23 and 36 Gy. Overall survival (OS) was evaluated using the Kaplan-Meier estimator, log-rank test, multivariable Cox proportional hazards modeling, and propensity score matching. Results Of the 751 patients included, 520 (69.2%) received CRT, and 231 (30.8%) received RT. With median follow-up of 5.0 years, estimated 5-year OS was superior in patients receiving CRT versus RT (86.1% vs 71.6%, P < .0001). On multivariable analysis, after controlling for risk factors, CRT was associated with superior OS compared with RT (HR: 0.53; 95%CI: 0.32-0.88, P = .01). On planned subgroup analyses, the 5 year OS of patients receiving CRT versus RT was improved for M0 patients (P < .0001), for patients receiving 36 Gy CSI (P = .0007), and for M0 patients receiving 36 Gy CSI (P = .0008). Conclusions This national database analysis demonstrates that combined postoperative chemotherapy and radiotherapy are associated with superior survival for adult MB compared with radiotherapy alone, even for M0 patients who receive high-dose CSI.