Involvement of PKC-alpha in PDGF-mediated mitogenic signaling in human mesangial cells

1993 ◽  
Vol 265 (5) ◽  
pp. F634-F642 ◽  
Author(s):  
G. G. Choudhury ◽  
P. Biswas ◽  
G. Grandaliano ◽  
H. E. Abboud
Keyword(s):  
Dna Synthesis ◽  
Mesangial Cells ◽  
Signal Transduction Pathway ◽  
Human Mesangial Cells ◽  
Cell Extracts ◽  
Pkc Isoforms ◽  
Dependent Protein ◽  
Pkc Alpha

Platelet-derived growth factor (PDGF) is a potent mitogen for a variety of cells. The calcium/phospholipid-dependent protein kinase C (PKC) represents a major signal transduction pathway for many growth stimuli including PDGF. Various isoforms of PKC are differentially expressed in the same or in different cells and tissues, and diverse stimuli may selectively activate one or more PKC isoforms. We studied the effect of PDGF on DNA synthesis and on the activity of PKC in human mesangial cells and vascular pericytes in the glomerular microvascular bed. PKC activity was measured as the amount of phosphorylated myelin basic protein-derived peptide substrate in the absence and presence of an inhibitor, a peptide spanning the pseudosubstrate region of PKC. PDGF (15 ng/ml) stimulated PKC activity within 5 min, and the effect was sustained for 60 min. Pretreatment of mesangial cells with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), an inhibitor of PKC, abolished the stimulation of PKC and DNA synthesis in response to PDGF. This effect of H-7 was specific, because H-7 did not inhibit the tyrosine phosphorylation of the PDGF receptor in vivo when added to the cells or the in vitro kinase activity in the PDGF beta-receptor immunoprecipitates. Utilizing isotype-specific antibodies against PKC-alpha, -beta, or -gamma for immunoprecipitation of PDGF-treated mesangial cell extracts, followed by assay of PKC activity, we demonstrated the activation of PKC-alpha only. Northern blot analysis of mRNA prepared from mesangial cells also revealed two transcripts, 3.7 kb and 1.8 kb, that hybridized with cDNA specific for PKC-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of cadmium on the actin cytoskeleton in renal mesangial cells

2013 ◽  
Vol 91 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Douglas M. Templeton ◽  
Ying Liu
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
Mesangial Cells ◽  
Focal Adhesions ◽  
Filamentous Actin ◽  
Human Mesangial Cells ◽  
Dependent Protein

We provide an overview of our studies on cadmium and the actin cytoskeleton in mesangial cells, from earlier work on the effects of Cd2+ on actin polymerization in vivo and in vitro, to a role of disruption or stabilization of the cytoskeleton in apoptosis and apoptosis-like death. More recent studies implicate cadmium-dependent association of gelsolin and the Ca2+/calmodulin-dependent protein kinase II (CaMK-II) with actin filaments in cytoskeletal effects. We also present previously unpublished data concerning cadmium and the disruption of focal adhesions. The work encompasses studies on rat, mouse, and human mesangial cells. The major conclusions are that Cd2+ acts independently of direct effects on cellular Ca2+ levels to nevertheless activate Ca2+-dependent proteins that shift the actin polymerization–depolymerization in favour of depolymerization. Cadmium-dependent translocation of CaMK-IIδ, gelsolin, and a 50 kDa gelsolin cleavage fragment to the filamentous (F-)actin cytoskeleton appear to be involved. An intact filamentous actin cytoskeleton is required to initiate apoptotic and apoptotic-like death, but F-actin depolymerization is an eventual result.

scholarly journals Cyclic AMP-dependent protein kinase inhibits the activity of myogenic helix-loop-helix proteins.

1992 ◽  
Vol 12 (10) ◽  
pp. 4478-4485 ◽  
Author(s):  
L Li ◽  
R Heller-Harrison ◽  
M Czech ◽  
E N Olson
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Transcriptional Activation ◽  
Consensus Sequence ◽  
Signal Transduction Pathway ◽  
Specific Gene ◽  
Dependent Protein Kinase ◽  
Dependent Protein

Differentiation of skeletal muscle cells is inhibited by the cyclic AMP (cAMP) signal transduction pathway. Here we report that the catalytic subunit of cAMP-dependent protein kinase (PKA) can substitute for cAMP and suppress muscle-specific transcription by silencing the activity of the MyoD family of regulatory factors, which includes MyoD, myogenin, myf5, and MRF4. Repression by the PKA catalytic (C) subunit is directed at the consensus sequence CANNTG, the target for DNA binding and transcriptional activation by these myogenic regulators. Phosphopeptide mapping of myogenin in vitro and in vivo revealed two PKA phosphorylation sites, both within the basic region. However, repression of myogenin function by PKA does not require direct phosphorylation of these sites but instead involves an indirect mechanism with one or more intermediate steps. Regulation of the transcriptional activity of the MyoD family by modulation of the cAMP signaling pathway may account for the inhibitory effects of certain peptide growth factors on muscle-specific gene expression and may also determine the responsiveness of different cell types to myogenic conversion by these myogenic regulators.

Cyclic AMP-dependent protein kinase inhibits the activity of myogenic helix-loop-helix proteins

1992 ◽  
Vol 12 (10) ◽  
pp. 4478-4485
Author(s):  
L Li ◽  
R Heller-Harrison ◽  
M Czech ◽  
E N Olson
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Transcriptional Activation ◽  
Consensus Sequence ◽  
Signal Transduction Pathway ◽  
Specific Gene ◽  
Dependent Protein Kinase ◽  
Dependent Protein

Differentiation of skeletal muscle cells is inhibited by the cyclic AMP (cAMP) signal transduction pathway. Here we report that the catalytic subunit of cAMP-dependent protein kinase (PKA) can substitute for cAMP and suppress muscle-specific transcription by silencing the activity of the MyoD family of regulatory factors, which includes MyoD, myogenin, myf5, and MRF4. Repression by the PKA catalytic (C) subunit is directed at the consensus sequence CANNTG, the target for DNA binding and transcriptional activation by these myogenic regulators. Phosphopeptide mapping of myogenin in vitro and in vivo revealed two PKA phosphorylation sites, both within the basic region. However, repression of myogenin function by PKA does not require direct phosphorylation of these sites but instead involves an indirect mechanism with one or more intermediate steps. Regulation of the transcriptional activity of the MyoD family by modulation of the cAMP signaling pathway may account for the inhibitory effects of certain peptide growth factors on muscle-specific gene expression and may also determine the responsiveness of different cell types to myogenic conversion by these myogenic regulators.

PDGF and IL-1 induce and activate specific protein kinase C isoforms in mesangial cells

1996 ◽  
Vol 271 (1) ◽  
pp. F108-F113 ◽  
Author(s):  
M. B. Ganz ◽  
B. Saksa ◽  
R. Saxena ◽  
K. Hawkins ◽  
J. R. Sedor
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
De Novo ◽  
Mesangial Cells ◽  
De Novo Synthesis ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Alpha Beta ◽  
Pkc Alpha

In vitro and in vivo data suggest a remarkable plasticity in the differentiated phenotype of intrinsic glomerular cells, which after injury express new structures and functions. We have shown that a protein kinase C (PKC) isoform, beta II, is expressed in diseased but not normal glomeruli. Since intrarenal cytokine synthesis has been implicated in the pathogenesis of progressive glomerular injury, we have hypothesized that these mediators induce a change in isoform profile. To test this hypothesis in vitro, we have determined whether platelet-derived growth factor (PDGF) and interleukin-1 (IL-1) alter the expression or activation of PKC isoforms in cultured mesangial cells (MCs). By immunoblot and ribonuclease (RNase) protection assays, both PDGF and IL-1 induce as early as 2 h de novo synthesis of PKC-beta II. Since MCs constitutively express PKC-alpha, -beta I, and -zeta, we also determined whether IL-1 or PDGF alter the activity of these isoforms. PDGF maximally induced translocation of PKC-alpha (10 min), -beta I (90 min), -epsilon (120 min), and -zeta (120 min) from the cytosolic to the membrane fraction. IL-1, in contrast, did not alter the distribution of alpha, beta I, or epsilon at any time measured but did induce PKC-zeta translocation. These data suggest inflammatory mediators regulate PKC isoform activity in diseased glomeruli both by de novo synthesis of unexpressed isoforms and by activation of constitutively expressed PKC isoforms.

Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells

1985 ◽  
Vol 5 (8) ◽  
pp. 2051-2060
Author(s):  
B W Stillman ◽  
Y Gluzman
Keyword(s):  
Dna Synthesis ◽  
Simian Virus 40 ◽  
Nuclear Extract ◽  
Simian Virus ◽  
T Antigen ◽  
Cell Extracts ◽  
293 Cells ◽  
Dna Strands

Soluble extracts prepared from the nucleus and cytoplasm of human 293 cells are capable of efficient replication and supercoiling of added DNA templates that contain the origin of simian virus 40 replication. Extracts prepared from human HeLa cells are less active than similarly prepared extracts from 293 cells for initiation and elongation of nascent DNA strands. DNA synthesis is dependent on addition of purified simian virus 40 tumor (T) antigen, which is isolated by immunoaffinity chromatography of extracts from cells infected with an adenovirus modified to produce large quantities of this protein. In the presence of T antigen and the cytoplasmic extract, replication initiates at the origin and continues bidirectionally. Initiation is completely dependent on functional origin sequences; a plasmid DNA containing an origin mutation known to affect DNA replication in vivo fails to replicate in vitro. Multiple rounds of DNA synthesis occur, as shown by the appearance of heavy-heavy, bromodeoxyuridine-labeled DNA products. The products of this reaction are resolved, but are relaxed, covalently closed DNA circles. Addition of a nuclear extract during DNA synthesis promotes the negative supercoiling of the replicated DNA molecules.

Differential regulation of angiotensin II and losartan binding sites in glomeruli and mesangial cells

1994 ◽  
Vol 266 (3) ◽  
pp. F384-F393 ◽  
Author(s):  
D. Chansel ◽  
T. Bizet ◽  
S. Vandermeersch ◽  
P. Pham ◽  
B. Levy ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Binding Sites ◽  
Mesangial Cells ◽  
Present Report ◽  
Mrna Levels ◽  
Ang Ii ◽  
Experimental Conditions ◽  
Human Mesangial Cells

The aim of the present report was to examine the effect of several agents on angiotensin II (ANG II) and losartan receptors using 125I-[Sar1,Ala8]ANG II and [3H]losartan as radiolabeled ligand, respectively. ANG II receptors were downregulated in glomeruli from rats infused with ANG II during 3 wk or rats receiving losartan orally during 1 wk. The number of sites (Bmax) was reduced, but the dissociation constant (Kd) value was unchanged. Losartan receptors were downregulated in glomeruli from rats receiving losartan, but remained unchanged in glomeruli from rats infused with ANG II. Since in vivo administration of losartan results in increase of plasma ANG II and formation of metabolites, in vitro studies using human mesangial cells were performed to better analyze the present findings. Treatment of mesangial cells during 4 days by ANG II, losartan, or its metabolite, EXP-3174, also produced downregulation of 125I-[Sar1,Ala8]ANG II binding sites with a decreased Bmax and unchanged Kd value. Only treatment of mesangial cells by ANG II or EXP-3174 produced downregulation of [3H]losartan binding sites. In contrast, exposure of these cells to losartan resulted in upregulation of [3H]losartan binding sites. Under all conditions, only Bmax was modified. Whereas internalization of [3H]losartan in mesangial cells was negligible under all experimental conditions, there was an increase of the percentage of internalized 125I-[Sar1,Ala8]ANG II after exposure of the cells to ANG II or AT1 antagonists. No change was observed in mesangial cell AT1 receptor mRNA levels. This study demonstrates that 1) AT1 mRNA is expressed in human mesangial cells; 2) the characteristics of 125I-[Sar1,Ala8]ANG II and [3H]losartan binding sites in rat glomeruli and human mesangial cells are different, with Kd and Bmax values greater in both preparations when [3H]losartan was utilized; 3) both types of binding sites obey different regulations, and the effects of losartan in vivo are due in part to the associated increase in plasma ANG II levels and the transformation of the drug into its metabolite, EXP-3174; 4) downregulation of AT1 receptors does not depend on changes in mRNA expression but is associated with increased relative internalization.

scholarly journals Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells.

1985 ◽  
Vol 5 (8) ◽  
pp. 2051-2060 ◽  
Author(s):  
B W Stillman ◽  
Y Gluzman
Keyword(s):  
Dna Synthesis ◽  
Simian Virus 40 ◽  
Nuclear Extract ◽  
Simian Virus ◽  
T Antigen ◽  
Cell Extracts ◽  
293 Cells ◽  
Dna Strands

Soluble extracts prepared from the nucleus and cytoplasm of human 293 cells are capable of efficient replication and supercoiling of added DNA templates that contain the origin of simian virus 40 replication. Extracts prepared from human HeLa cells are less active than similarly prepared extracts from 293 cells for initiation and elongation of nascent DNA strands. DNA synthesis is dependent on addition of purified simian virus 40 tumor (T) antigen, which is isolated by immunoaffinity chromatography of extracts from cells infected with an adenovirus modified to produce large quantities of this protein. In the presence of T antigen and the cytoplasmic extract, replication initiates at the origin and continues bidirectionally. Initiation is completely dependent on functional origin sequences; a plasmid DNA containing an origin mutation known to affect DNA replication in vivo fails to replicate in vitro. Multiple rounds of DNA synthesis occur, as shown by the appearance of heavy-heavy, bromodeoxyuridine-labeled DNA products. The products of this reaction are resolved, but are relaxed, covalently closed DNA circles. Addition of a nuclear extract during DNA synthesis promotes the negative supercoiling of the replicated DNA molecules.

scholarly journals The kinase DYRK1A phosphorylates the transcription factor FKHR at Ser329 in vitro, a novel in vivo phosphorylation site

2001 ◽  
Vol 355 (3) ◽  
pp. 597-607 ◽  
Author(s):  
Yvonne L. WOODS ◽  
Graham RENA ◽  
Nick MORRICE ◽  
Andreas BARTHEL ◽  
Walter BECKER ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Protein Kinase ◽  
Phosphorylation Site ◽  
Control Of Gene Expression ◽  
Cell Extracts ◽  
Dual Specificity ◽  
Dependent Protein ◽  
Phosphoinositide 3 Kinase

Forkhead in rhabdomyosarcoma (FKHR) is a transcription factor that has been implicated in the control of gene expression by insulin, as well as the regulation of apoptosis by survival factors. These signals trigger the protein kinase B (PKB)-catalysed phosphorylation of FKHR at three residues (Thr24, Ser256 and Ser319) by a phosphoinositide 3-kinase-dependent pathway that results in the nuclear exit and inactivation of this transcription factor. Here, we have identified a conserved residue (Ser329) as a novel in vivo phosphorylation site on FKHR. Ser329 phosphorylation also decreases the ability of FKHR to stimulate gene transactivation and reduces the proportion of FKHR present in the nucleus. However, unlike the residues targetted by PKB, Ser329 is phosphorylated in unstimulated HEK-293cells, and phosphorylation is not increased by stimulation with insulin-like growth factor-1 or by transfection with 3-phosphoinositide-dependent protein kinase-1. We have also purified a protein kinase to near homogeneity from rabbit skeletal muscle that phosphorylates FKHR at Ser329 specifically and identified it as DYRK1A (dual-specificity tyrosine-phosphorylated and regulated kinase 1A). We find that FKHR and DYRK1A co-localize in discrete regions of the nucleus and can be co-immunoprecipitated from cell extracts. These experiments suggest that DYRK1A may phosphorylate FKHR at Ser329in vivo.

scholarly journals Phosphodiesterase 3A binds to 14-3-3 proteins in response to PMA-induced phosphorylation of Ser428

2005 ◽  
Vol 392 (1) ◽  
pp. 163-172 ◽  
Author(s):  
Mercedes Pozuelo Rubio ◽  
David G. Campbell ◽  
Nicholas A. Morrice ◽  
Carol Mackintosh
Keyword(s):  
Protein Kinase ◽  
Metal Ion ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Cell Extracts ◽  
Mitogen Activated Protein ◽  
Dependent Protein ◽  
Pkc Protein Kinase C

PDE3A (phosphodiesterase 3A) was identified as a phosphoprotein that co-immunoprecipitates with endogenous 14-3-3 proteins from HeLa cell extracts, and binds directly to 14-3-3 proteins in a phosphorylation-dependent manner. Among cellular stimuli tested, PMA promoted maximal binding of PDE3A to 14-3-3 proteins. While p42/p44 MAPK (mitogen-activated protein kinase), SAPK2 (stress-activated protein kinase 2)/p38 and PKC (protein kinase C) were all activated by PMA in HeLa cells, the PMA-induced binding of PDE3A to 14-3-3 proteins was inhibited by the non-specific PKC inhibitors Ro 318220 and H-7, but not by PD 184352, which inhibits MAPK activation, nor by SB 203580 and BIRB0796, which inhibit SAPK2 activation. Binding of PDE3A to 14-3-3 proteins was also blocked by the DNA replication inhibitors aphidicolin and mimosine, but the PDE3A–14-3-3 interaction was not cell-cycle-regulated. PDE3A isolated from cells was able to bind to 14-3-3 proteins after in vitro phosphorylation with PKC isoforms. Using MS/MS of IMAC (immobilized metal ion affinity chromatography)-enriched tryptic phosphopeptides and phosphospecific antibodies, at least five sites on PDE3A were found to be phosphorylated in vivo, of which Ser428 was selectively phosphorylated in response to PMA and dephosphorylated in cells treated with aphidicolin and mimosine. Phosphorylation of Ser428 therefore correlated with 14-3-3 binding to PDE3A. Ser312 of PDE3A was phosphorylated in an H-89-sensitive response to forskolin, indicative of phosphorylation by PKA (cAMP-dependent protein kinase), but phosphorylation at this site did not stimulate 14-3-3 binding. Thus 14-3-3 proteins can discriminate between sites in a region of multisite phosphorylation on PDE3A. An additional observation was that the cytoskeletal cross-linker protein plectin-1 coimmunoprecipitated with PDE3A independently of 14-3-3 binding.

In vitro concatemerization of bacteriophage T7 DNA: Role of DNA synthesis and gene 6 exonuclease

1985 ◽  
Vol 63 (4) ◽  
pp. 237-242 ◽  
Author(s):  
Donald D. Lee ◽  
Paul D. Sadowski
Keyword(s):  
Infected Cell ◽  
Dna Synthesis ◽  
Base Pair ◽  
Bacteriophage T7 ◽  
Cell Extracts ◽  
In Vitro Incubation ◽  
Insight Into

The replication of bacteriophage T7 DNA in vivo proceeds via the synthesis of complex concatemeric intermediates which are joined via the 160 base pair terminal redundancies at either end of the phage chromosome. To gain some insight into the mode of generation of these structures, we have examined the role of DNA synthesis in the formation of concatemeric bacteriophage T7 DNA in vitro. Incubation of mature T7 DNA with T7-infected cell extracts and a deoxynucleoside [32P]triphosphate resulted in the incorporation of significant radioactivity into the DNA. Highest levels of incorporation were at the termini of the DNA and decreased toward the middle of the molecule. Incorporation was dependent upon the presence of the activity of the gene 6 exonuclease and correlated with the generation of concatemeric DNA. A model explaining the role of exonucleolytic degradation and DNA synthesis in the generation of concatemeric DNA is presented.

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