Kevin P Becker

Medical University of South Carolina, Charleston, SC, United States

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Publications (9)41.86 Total impact

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    ABSTRACT: It has been previously shown that upon sustained stimulation (30-60 min) with phorbol esters, protein kinase C (PKC) alpha and betaII 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 degrees 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.
    Journal of Biological Chemistry 09/2006; 281(31):22321-31. · 4.65 Impact Factor
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    ABSTRACT: In a previous study, we showed that protein kinase C betaII (PKC betaII) translocated to a novel juxtanuclear compartment as observed in several cell types (Becker, K. P., and Hannun, Y. A. (2003) J. Biol. Chem. 278, 52747-52754). In this study, we noted the absence of this translocation in MCF-7 breast cancer cells, and we examined the mechanisms underlying this selectivity of response. We show that sustained stimulation of PKC betaII with 4beta-phorbol 12-myristate 13-acetate (PMA) resulted in accumulation of ceramide in MCF-7 cells but not in those cells that showed juxtanuclear translocation of PKC betaII. Addition of exogenous ceramides or formation of endogenous ceramide by the action of bacterial sphingomyelinase prevented PMA-induced translocation of PKC betaII in HEK 293 cells. On the other hand, inhibition of ceramide accumulation with fumonisin B1 restored the ability of PMA to induce translocation of PKC betaII in MCF-7 cells. Taken together, the results showed that endogenous ceramide is both necessary and sufficient for preventing juxtanuclear translocation of PKC betaII in response to PMA. Investigation of the mechanisms of ceramide generation in response to PMA revealed that PMA activated the salvage pathway of ceramide formation and not the de novo pathway. This conclusion was based on the following: 1) the ability of fumonisin B1 but not myriocin to inhibit ceramide formation, 2) the ability of PMA to induce increases in palmitate-labeled ceramide only under chase labeling but not acute pulse labeling, 3) the induction of the levels of sphingosine but not dihydrosphingosine in response to PMA, and 4) induction of sphingomyelin hydrolysis in response to PMA. Together, these results define a novel pathway of regulated formation of ceramide, the salvage pathway, and they define a role for this pathway in regulating juxtanuclear translocation of PKC betaII.
    Journal of Biological Chemistry 02/2005; 280(4):2606-12. · 4.65 Impact Factor
  • Kevin P Becker, Yusuf A Hannun
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    ABSTRACT: 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 PKCbetaI and PKCbetaII isoenzymes provide a unique opportunity because they are the alternatively spliced products of the beta 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 PKCbetaII and not PKCbetaI translocates to a recently described juxtanuclear site of localization for PKCalpha and PKCbetaII isoenzymes that arises with sustained stimulation of PKC. Mechanistically, translocation of PKCbetaII to the juxtanuclear region required kinase activity. PKCbetaII, but not PKCbetaI, 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 PKCbetaII to the juxtanuclear region but not to the plasma membrane, thus demonstrating a role for phospholipase D in the juxtanuclear translocation of PKCbetaII. Taken together, these results define specific biochemical and cellular actions of PKCbetaII when compared with PKCbetaI.
    Journal of Biological Chemistry 08/2004; 279(27):28251-6. · 4.65 Impact Factor
  • Kevin P. Becker, Yusuf A. Hannun
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    ABSTRACT: 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.
    Journal of Biological Chemistry 07/2004; 279(27):28251-28256. · 4.65 Impact Factor
  • Kevin P Becker, Yusuf A Hannun
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    ABSTRACT: 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, alpha and betaII, to a juxtanuclear compartment. Juxtanuclear 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 PKCalpha and betaII 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 PKCalpha and betaII in regulation of recycling components.
    Journal of Biological Chemistry 01/2004; 278(52):52747-54. · 4.65 Impact Factor
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    ABSTRACT: 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-alpha 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.
    Journal of Biological Chemistry 10/2003; 278(36):34541-7. · 4.65 Impact Factor
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    ABSTRACT: Ceramidases deacylate ceramides, important intermediates in the metabolic pathway of sphingolipids. In this study, we report the cloning and characterization of a novel mouse alkaline ceramidase (maCER1) with a highly restricted substrate specificity. maCER1 consists of 287 amino acids, and it has a 28 and 32% identity to the Saccharomyces alkaline ceramidases (YPC1p and YDC1p) and the human alkaline phytoceramidase, respectively. Reverse transcriptase-PCR analysis demonstrated that maCER1 was predominantly expressed in skin. maCER1 was localized to the endoplasmic reticulum as revealed by immunocytochemistry. In vitro biochemical characterization determined that maCER1 hydrolyzed D-erythro-ceramide exclusively but not D-erythro-dihydroceramide or D-ribo-phytoceramide. Similar to other alkaline ceramidases, maCER1 had an alkaline pH optimum of 8.0, and it was activated by Ca2+ but inhibited by Zn2+,Cu2+, and Mn2+. maCER1 was also inhibited by sphingosine, one of its products. Metabolic labeling studies showed that overexpression of maCER1 caused a decrease in the incorporation of radiolabeled dihydrosphingosine into ceramide and complex sphingolipids but led to a concomitant increase in sphingosine-1-P (S1P) in HeLa cells. Mass measurement showed that overexpression of maCER1 selectively lowered the cellular levels of D-erythro-C24:1-ceramide, but not other ceramide species and caused an increase in the levels of S1P. Taken together, these data suggest that maCER1 is a novel alkaline ceramidase with a stringent substrate specificity and that maCER1 is selectively expressed in skin and may have a role in regulating the levels of bioactive lipids ceramide and S1P, as well as complex sphingolipids.
    Journal of Biological Chemistry 09/2003; 278(33):31184-91. · 4.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Ceramidases deacylate ceramides, important intermediates in the metabolic pathway of sphingolipids. In this study, we report the cloning and characterization of a novel mouse alkaline ceramidase (maCER1) with a highly restricted substrate specificity. maCER1 consists of 287 amino acids, and it has a 28 and 32% identity to the Saccharomyces alkaline ceramidases (YPC1p and YDC1p) and the human alkaline phytoceramidase, respectively. Reverse transcriptase-PCR analysis demonstrated that maCER1 was predominantly expressed in skin. maCER1 was localized to the endoplasmic reticulum as revealed by immunocytochemistry. In vitro biochemical characterization determined that maCER1 hydrolyzed d-erythro-ceramide exclusively but not d-erythro-dihydroceramide or d-ribo-phytoceramide. Similar to other alkaline ceramidases, maCER1 had an alkaline pH optimum of 8.0, and it was activated by Ca2+ but inhibited by Zn2+,Cu2+, and Mn2+. maCER1 was also inhibited by sphingosine, one of its products. Metabolic labeling studies showed that overexpression of maCER1 caused a decrease in the incorporation of radiolabeled dihydrosphingosine into ceramide and complex sphingolipids but led to a concomitant increase in sphingosine-1-P (S1P) in HeLa cells. Mass measurement showed that overexpression of maCER1 selectively lowered the cellular levels of d-erythro-C24:1-ceramide, but not other ceramide species and caused an increase in the levels of S1P. Taken together, these data suggest that maCER1 is a novel alkaline ceramidase with a stringent substrate specificity and that maCER1 is selectively expressed in skin and may have a role in regulating the levels of bioactive lipids ceramide and S1P, as well as complex sphingolipids.
    Journal of Biological Chemistry 08/2003; 278(33):31184-31191. · 4.65 Impact Factor
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    ABSTRACT: 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, C(6)-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.
    Journal of Biological Chemistry 10/2002; 277(38):35257-62. · 4.65 Impact Factor