The regulation of mammalian adenylyl cyclases by protein kinase C.
Item
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Title
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The regulation of mammalian adenylyl cyclases by protein kinase C.
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Identifier
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AAI9510673
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identifier
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9510673
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Creator
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Jacobowitz, Ofer.
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Contributor
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Adviser: Ravi Iyengar
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Date
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1994
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Language
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English
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Publisher
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City University of New York.
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Subject
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Health Sciences, Pharmacology | Biology, Molecular | Chemistry, Biochemistry
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Abstract
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Many hormones and neurotransmitters regulate target organs through the adenylyl cyclase pathway. Hormone receptors activate the heterotrimeric G protein, G{dollar}\sb{lcub}\rm s{rcub}{dollar}, which in turn stimulates the enzyme adenylyl cyclase to produce cAMP. Activation of protein kinase A by cAMP leads to phosphorylation and regulation of metabolic enzymes, ion channels and transcription factors. Many hormones, however, stimulate adenylyl cyclase independently of G{dollar}\sb{lcub}\rm s{rcub}{dollar}. For example, angiotensin II induces steroidogenesis in adrenal glands by raising cAMP levels without activating G{dollar}\sb{lcub}\rm s{rcub}{dollar}. Nerve growth factor, which does not activate G{dollar}\sb{lcub}\rm s{rcub}{dollar} can also raise cAMP levels. I have investigated the molecular basis for these signal transmission pathways.;Since both angiotensin II and nerve growth factor stimulate protein kinase C (PKC) and stimulation of PKC raises cAMP levels in some cells, I decided to test whether PKC activation would stimulate any of the six cloned adenylyl cyclases. Using transient transfection of human embryonal kidney cells (HEK-293), I identified adenylyl cyclase 2 (AC2), an isoform abundant in brain and lung, as the isoform most extensively stimulated by PKC. Phorbol 12-myristate, 13-acetate (PMA), a PKC activator, stimulated basal adenylyl cyclase activity in AC2-expressing cells. This suggested that AC2 may be phosphorylated through PKC activation in absence of G{dollar}\sb{lcub}\rm s{rcub}{dollar} stimulation. To demonstrate phosphorylation, AC2 cDNA was transduced into Sf9 cells by recombinant baculovirus. PMA treatment of AC2-expressing Sf9 cells increased basal activity. This stimulation was blocked by staurosporine. Stimulation was reflected by an increased V{dollar}\sb{lcub}\rm max{rcub}{dollar} and unaltered K{dollar}\sb{lcub}\rm m{rcub}{dollar} of AC2. PMA treatment also potentiated stimulation of AC2 by G{dollar}\sb{lcub}\rm s{rcub}{dollar}-{dollar}\alpha{dollar} and {dollar}\beta\gamma{dollar} subunits. AC2 was epitope-tagged at the N-terminus to permit purification. Tagged-AC2 was also stimulated by PMA treatment of cells. Tagged-AC2 was purified to apparent homogeneity with an anti-epitope antibody affinity column. PMA treatment of {dollar}\sp{lcub}32{rcub}{dollar}P-labeled Sf9 cells expressing tagged-AC2 resulted in enhanced {dollar}\sp{lcub}32{rcub}{dollar}P incorporation into purified, tagged-AC2. Thus, PKC activation results in G{dollar}\sb{lcub}\rm s{rcub}{dollar}-independent stimulation and enhanced phosphorylation of AC2. AC2 may therefore serve as a signal recognition and integration element allowing many external signals to impart some of their cellular effects through the cAMP pathway.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
