scholarly journals Protein Kinase C Signaling Mediates a Program of Cell Cycle Withdrawal in the Intestinal Epithelium

2000 ◽  
Vol 151 (4) ◽  
pp. 763-778 ◽  
Author(s):  
Mark R. Frey ◽  
Jennifer A. Clark ◽  
Olga Leontieva ◽  
Joshua M. Uronis ◽  
Adrian R. Black ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Intestinal Epithelium ◽  
Molecular Mechanisms ◽  
Model Systems ◽  
Intestinal Epithelial ◽  
Kinase C

Members of the protein kinase C (PKC) family of signal transduction molecules have been widely implicated in regulation of cell growth and differentiation, although the underlying molecular mechanisms involved remain poorly defined. Using combined in vitro and in vivo intestinal epithelial model systems, we demonstrate that PKC signaling can trigger a coordinated program of molecular events leading to cell cycle withdrawal into G0. PKC activation in the IEC-18 intestinal crypt cell line resulted in rapid downregulation of D-type cyclins and differential induction of p21waf1/cip1 and p27kip1, thus targeting all of the major G1/S cyclin-dependent kinase complexes. These events were associated with coordinated alterations in expression and phosphorylation of the pocket proteins p107, pRb, and p130 that drive cells to exit the cell cycle into G0 as indicated by concomitant downregulation of the DNA licensing factor cdc6. Manipulation of PKC isozyme levels in IEC-18 cells demonstrated that PKCα alone can trigger hallmark events of cell cycle withdrawal in intestinal epithelial cells. Notably, analysis of the developmental control of cell cycle regulatory molecules along the crypt–villus axis revealed that PKCα activation is appropriately positioned within intestinal crypts to trigger this program of cell cycle exit–specific events in situ. Together, these data point to PKCα as a key regulator of cell cycle withdrawal in the intestinal epithelium.

Mitogen stimulation of Na+-H+ exchange: differential involvement of protein kinase C

1987 ◽  
Vol 253 (2) ◽  
pp. C219-C229 ◽  
Author(s):  
L. L. Muldoon ◽  
G. A. Jamieson ◽  
A. C. Kao ◽  
H. C. Palfrey ◽  
M. L. Villereal
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Types ◽  
Intact Cells ◽  
Kinase C ◽  
Protein Kinase C Activity ◽  
Human Fibroblast Strains ◽  
Stimulation Of

The mitogen-induced activation of Na+-H+ exchange was investigated in two cultured human fibroblast strains (HSWP and WI-38 cells) that, based on previous studies, differed in their response to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (L. M. Vincentini and M. L. Villereal, Proc. Natl. Acad. Sci. USA 82: 8053-8056, 1985). The role of protein kinase C in the activation of Na+-H+ exchange was investigated by comparing the effects of TPA on Na+ influx, in vitro phosphorylation, and in vivo phosphorylation in both cell types. Although both cell types have significant quantities of protein kinase C activity that can be activated by TPA in intact cells, the addition of TPA to intact cells stimulates Na+ influx in WI-38 cells but not in HSWP cells, indicating that in HSWP cells the stimulation of protein kinase C is not sufficient to activate the Na+-H+ exchanger. Cells were then depleted of protein kinase C activity by chronic treatment with high doses of TPA. Both HSWP and WI-38 cells were rendered protein kinase C deficient by this treatment as determined by in vitro and in vivo phosphorylation studies. Protein kinase C-deficient HSWP cells lose the ability for TPA to inhibit the serum-induced activation of Na+-H+ exchange, but there is no reduction in the stimulation of Na+ influx by serum, bradykinin, vasopressin, melittin, or vanadate, indicating that protein kinase C activity is not necessary for the mitogen-induced activation of Na+-H+ exchange in HSWP cells by agents known to stimulate phosphatidylinositol turnover (G. A. Jamieson and M. Villereal. Arch. Biochem. Biophys. 252: 478-486, 1987). In contrast, depletion of protein kinase C activity in WI-38 cells significantly reduces both the TPA- and the serum-induced activation of the Na+-H+ exchange system, suggesting that protein kinase C activity is necessary for at least a portion of the mitogen-induced activation of the Na+-H+ exchanger in WI-38 cells. These results indicate that the mechanisms for regulating Na+-H+ exchange can differ dramatically between different types of fibroblasts.

14-3-3 Isotypes facilitate coupling of protein kinase C-ζ to Raf-1: negative regulation by 14-3-3 phosphorylation

2000 ◽  
Vol 345 (2) ◽  
pp. 297-306 ◽  
Author(s):  
Paulus C. J. VAN DER HOEVEN ◽  
José C. M. VAN DER WAL ◽  
Paula RUURS ◽  
Marc C. M. VAN DIJK ◽  
Wim J. VAN BLITTERSWIJK
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Complex Formation ◽  
Dependent Manner ◽  
Cos Cells ◽  
Kinase C ◽  
Pkc Ζ ◽  
Co Precipitation

14-3-3 Proteins may function as adapters or scaffold in signal-transduction pathways. We found previously that protein kinase C-ζ (PKC-ζ) can phosphorylate and activate Raf-1 in a signalling complex [van Dijk, Hilkmann and van Blitterswijk (1997) Biochem. J. 325, 303-307]. We report now that PKC-ζ-Raf-1 interaction is mediated by 14-3-3 proteins in vitro and in vivo. Co-immunoprecipitation experiments in COS cells revealed that complex formation between PKC-ζ and Raf-1 is mediated strongly by the 14-3-3β and -θ isotypes, but not by 14-3-3ζ. Far-Western blotting revealed that 14-3-3 binds PKC-ζ directly at its regulatory domain, where a S186A mutation in a putative 14-3-3-binding domain strongly reduced the binding and the complex formation with 14-3-3β and Raf-1. Treatment of PKC-ζ with lambda protein phosphatase also reduced its binding to 14-3-3β in vitro. Preincubation of an immobilized Raf-1 construct with 14-3-3β facilitated PKC-ζ binding. Together, the results suggest that 14-3-3 binds both PKC-ζ (at phospho-Ser-186) and Raf-1 in a ternary complex. Complex formation was much stronger with a kinase-inactive PKC-ζ mutant than with wild-type PKC-ζ, supporting the idea that kinase activity leads to complex dissociation. 14-3-3β and -θ were substrates for PKC-ζ, whereas 14-3-3ζ was not. Phosphorylation of 14-3-3β by PKC-ζ negatively regulated their physical association. 14-3-3β with its putative PKC-ζ phosphorylation sites mutated enhanced co-precipitation between PKC-ζ and Raf-1, suggesting that phosphorylation of 14-3-3 by PKC-ζ weakens the complex in vivo. We conclude that 14-3-3 facilitates coupling of PKC-ζ to Raf-1 in an isotype-specific and phosphorylation-dependent manner. We suggest that 14-3-3 is a transient mediator of Raf-1 phosphorylation and activation by PKC-ζ.

scholarly journals Phosphorylation of the Aspergillus oryzae Woronin body protein, AoHex1, by protein kinase C: evidence for its role in the multimerization and proper localization of the Woronin body protein

2007 ◽  
Vol 405 (3) ◽  
pp. 533-540 ◽  
Author(s):  
Praveen Rao Juvvadi ◽  
Jun-ichi Maruyama ◽  
Katsuhiko Kitamoto
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Aspergillus Oryzae ◽  
Body Protein ◽  
Kinase C ◽  
Woronin Body ◽  
Pkc Inhibitors ◽  
Oligomeric Forms

Woronin body, a specialized peroxisome, is a unique organelle involved in septal pore sealing and protecting filamentous fungus from excessive cytoplasmic bleeding. We recently characterized the Aohex1 gene encoding the major protein of the Woronin body in the fungus Aspergillus oryzae. Although three-dimensional microscopy revealed plugging of the septal pore by Woronin body, the mechanism of its formation remains unknown. We report here a reduction in the oligomeric forms (dimeric and tetrameric) of AoHex1 upon λ-phosphatase treatment, which indicated that AoHex1 phosphorylation in vivo facilitates its oligomerization. Concomitant with the presence of a highly conserved predicted PKC (protein kinase C)-phosphorylatable site (Ser151), the recombinant AoHex1 was phosphorylated by PKC in vitro and the administration of the PKC inhibitors, bisindolylmaleimide I and chelerythrine, resulted in the reduction of the oligomeric forms of AoHex1 in vivo. While spherical dot-like Woronin bodies were visualized by expressing the dsred2–Aohex1 and egfp (enhanced green fluorescent protein)–Aohex1 constructs in A. oryzae, treatment with the PKC inhibitors caused an abnormal localization to ring-like structures. In addition to the reduced phosphorylation of the mutagenized recombinant AoHex1[S151A] (Ser151 to alanine substitution) by PKC in vitro, the overexpression of Aohex1[S151A] as dsred2 fusion against the wild-type background also showed reduction of the oligomeric forms of the endogenous AoHex1 and its perturbed localization to ring-like structures in vivo. In conclusion, the present study implicates the relevance of PKC-dependent phosphorylation of the Woronin body protein, AoHex1, for its multimerization and proper localization.

scholarly journals Pertussis Toxin Shows Distinct Early Signalling Events in Platelet-Activating Factor–, Leukotriene B4–, and C5a-Induced Eosinophil Homotypic Aggregation In Vitro and Recruitment In Vivo

Blood ◽  
1997 ◽  
Vol 89 (12) ◽  
pp. 4566-4573 ◽  
Author(s):  
Mauro M. Teixeira ◽  
Mark A. Giembycz ◽  
Mark A. Lindsay ◽  
Paul G. Hellewell
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Pertussis Toxin ◽  
Platelet Activating Factor ◽  
Leukotriene B4 ◽  
Protein Kinase Inhibitors ◽  
Eosinophil Recruitment ◽  
Kinase C

Abstract The present study was performed to investigate the early signalling events responsible for eosinophil activation in response to platelet-activating factor (PAF ), C5a, and leukotriene B4 (LTB4 ). We evaluated the effect of pertussis toxin (PTX) on eosinophil aggregation in vitro and cutaneous eosinophil recruitment in vivo. Further studies using the protein kinase inhibitors Ro 31-8220 and staurosporine were performed in vitro to assess in more detail the early signalling events induced by these three mediators. Our results show that C5a and LTB4 signal predominantly or exclusively through a PTX-sensitive G protein that is negatively modulated by protein kinase C, possibly at the level of phospholipase C-β. In contrast, PAF activates eosinophils independent of Gi by a mechanism that is abolished by Ro 31-8220, a selective protein kinase C inhibitor. In addition, these results show for the first time that a receptor-operated event on the eosinophil is essential for chemoattractant-induced eosinophil recruitment in vivo.

Phospholipase D: enzymology, mechanisms of regulation, and function

1997 ◽  
Vol 77 (2) ◽  
pp. 303-320 ◽  
Author(s):  
J. H. Exton
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phosphatidic Acid ◽  
G Protein ◽  
Phospholipase D ◽  
Tyrosine Kinases ◽  
Rho Proteins ◽  
Kinase C

Phospholipase D exists in various forms that differ in their regulation but predominantly hydrolyze phosphatidylcholine. The Ca(2+)-dependent isozymes of protein kinase C regulate phospholipase D in vitro and play a major role in its control by growth factors and G protein-linked agonists in vivo. Recent studies have demonstrated that small G proteins of the ADP-ribosylation factor (ARF) and Rho families activate the enzyme in vitro, and evidence is accumulating that they also are involved in its control in vivo. Both types of G protein play important roles in cellular function, and the possible mechanisms by which they are activated by agonists are discussed. There is also emerging evidence of the control of phospholipase D and Rho proteins by soluble tyrosine kinases and novel serine/threonine kinases. The possible role of these kinases in agonist regulation of phospholipase D is discussed. The function of phospholipase D in cells is still poorly defined. Postulated roles of phosphatidic acid produced by phospholipase D action include the activation of Ca(2+)-independent isoforms of protein kinase C, the regulation of growth and the cytoskeleton in fibroblasts, and control of the respiratory burst in neutrophils. Another important function of phosphatidic acid is to act as a substrate for a specific phospholipase A2 to generate lysophosphatidic acid, which is becoming increasingly recognized as a major intercellular messenger. Finally, it is possible that the phospholipid changes induced in various cellular membranes by phospholipase D may per se play an important role in vesicle trafficking and other membrane-associated events.

scholarly journals Regulation of Impaired Protein Kinase C Signaling by Chemokines in Murine Macrophages during Visceral Leishmaniasis

2005 ◽  
Vol 73 (12) ◽  
pp. 8334-8344 ◽  
Author(s):  
Ranadhir Dey ◽  
Arup Sarkar ◽  
Nivedita Majumder ◽  
Suchandra Bhattacharyya (Majumdar) ◽  
Kaushik Roychoudhury ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Leishmania Donovani ◽  
Disease Process ◽  
Kinase C ◽  
Parasitic Burden ◽  
Infected Cells

ABSTRACT The protein kinase C (PKC) family regulates macrophage function involved in host defense against infection. In the case of Leishmania donovani infection, the impairment of PKC-mediated signaling is one of the crucial events for the establishment of parasite into the macrophages. Earlier reports established that C-C chemokines mediated protection against leishmaniasis via the generation of nitric oxide after 48 h. In this study, we investigated the role of MIP-1α and MCP-1 in the regulation of impaired PKC activity in the early hours (6 h) of infection. These chemokines restored Ca2+-dependent PKC activity and inhibited Ca2+-independent atypical PKC activity in L. donovani-infected macrophages under both in vivo and in vitro conditions. Pretreatment of macrophages with chemokines induced superoxide anion generation by activating NADPH oxidase components in infected cells. Chemokine administration in vitro induced the migration of infected macrophages and triggered the production of reactive oxygen species. In vivo treatment with chemokines significantly restricted the parasitic burden in livers as well as in spleens. Collectively, these results indicate a novel regulatory role of C-C chemokines in controlling the intracellular growth and multiplication of L. donovani, thereby demonstrating the antileishmanial properties of C-C chemokines in the disease process.

Heme and Iron Chelators Inhibit HIV-1 Through The Induction Of Heme Oxygenase 1, Ferroportin and IKBα

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2198-2198
Author(s):  
Namita Kumari ◽  
Sergei A Nekhai
Keyword(s):  
Cell Cycle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Heme Oxygenase ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Iron Chelators ◽  
Heme Oxygenase 1 ◽  
Kinase C ◽  
Hiv 1

Abstract Background Recently, HIV-1 infection was shown to be efficiently inhibited in macrophages and T-cells treated with hemin that was added extracellularly 1,2. Hemin administration to humanized transgenic mice significantly reduced HIV-1 viral load 1. Suppression of HIV-1 by hemin was mediated through the induction of (HO-1)1, via a protein kinase C-dependent pathway2. The inhibitory effect of hemin could be reversed by protoporphyrin, an HO-1 inhibitor 2. Induction of heme oxygenase-1 (HO-1) by hemin was shown to inhibit HIV-1. We recently analyzed the role of HO-1 in protecting LPS-treated human macrophages against HIV-1 infection3. LPS-treated macrophages were negative for mature virions, expressed HO-1 and produced MIP1α, MIP1β and LD78β chemokines which led to a decreased CCR5 expression. Treatment with HO-1 inhibitor SnPP IX (tin protoporphyrin IX) increased HIV-1 replication and decreased secretion of MIP1α, MIP1β, and LD78β chemokines. HO-1 also affects several proteins involved in cell cycle progression, and cell cycle is critical for HIV-1 progression. Hypoxia leads to induction and stabilization of HIF-1α and is inhibitory to HIV-1 replication. NF-kB is important for basal and Tat-activated HIV-1 transcription. Here we analyzed factors involved in HIV-1 transcription affected by HO-1 expression. Results HIV-1 replication was reduced in THP1 cells treated with hemin. Subsequent treatment with hepcidin restored HIV-1 replication, suggesting that ferroportin plays a key role in the HIV-1 inhibition. Stable ferroportin knock down in THP1 cells led to the inability of hemin to inhibit HIV-1, again suggesting that ferroportin plays a key role in this process. In hemin-treated THP-1 cells, expression of p21, HIF-1α and IKBα mRNA was induced. The expression of IKBα, an inhibitor of NF-kB, reduced the level of p65 subunit of NF-kB. We obtained similar results in THP-1 cell treated with iron chelators, which also induced the expression of IKBα, HIF-1 and p21. THP-1 cells treated with hemin or iron chelators were arrested in G1 phase of cell cycle. Stable HIF-1a knockdown in promonocytic THP-1 cells increased HIV replication suggesting that HIF-1 might be a restriction factor for HIV-1. In contrast to iron chelators that inhibited enzymatic activity of CDK2 without affecting its protein level, hemin treatment reduced CDK2 expression at mRNA and protein levels. Conclusions Induction of HIF-1 regulatory pathway and iron export by ferroportin might protect hemin-treated THP-1 cells from HIV-1 infection. Additional molecular mechanisms of heme-mediated HIV-1 inhibition might also include NF-kB inhibition by IKBα and CDK2 inhibition leading to the inhibition of HIV-1 transcription. Our results point to novel therapeutics, such as the use of hemin and iron chelators, both of which are FDA approved for treatment for acute porphyries and iron overload. Acknowledgments This project was supported by NIH Research Grants 1SC1GM082325, 2G12RR003048, and P30HL107253. Literature 1. Devadas K, Dhawan S. Hemin activation ameliorates HIV-1 infection via heme oxygenase-1 induction. J Immunol. 2006;176(7):4252-4257. 2. Devadas K, Hewlett IK, Dhawan S. Lipopolysaccharide suppresses HIV-1 replication in human monocytes by protein kinase C-dependent heme oxygenase-1 induction. J Leukoc Biol. 2010;87(5):915-924. 3. Zhou ZH, Kumari N, Nekhai S, et al. Heme oxygenase-1 induction alters chemokine regulation and ameliorates human immunodeficiency virus-type-1 infection in lipopolysaccharide-stimulated macrophages. Biochem Biophys Res Commun. 2013;435(3):373-377. Disclosures: No relevant conflicts of interest to declare.

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