Reorganization of the Cortical Actin Cytoskeleton during Maturation Division in theTubifexEgg: Possible Involvement of Protein Kinase C

1997 ◽  
Vol 188 (1) ◽  
pp. 110-121 ◽  
Author(s):  
Takashi Shimizu
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Actin Cytoskeleton ◽  
Cortical Actin ◽  
Maturation Division ◽  
Kinase C

scholarly journals Fission Yeast α-Glucan Synthase Mok1 Requires the Actin Cytoskeleton to Localize the Sites of Growth and Plays an Essential Role in Cell Morphogenesis Downstream of Protein Kinase C Function

1999 ◽  
Vol 144 (6) ◽  
pp. 1173-1186 ◽  
Author(s):  
Satoshi Katayama ◽  
Dai Hirata ◽  
Manuel Arellano ◽  
Pilar Pérez ◽  
Takashi Toda
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Actin Cytoskeleton ◽  
Fission Yeast ◽  
Cell Morphogenesis ◽  
Cortical Actin ◽  
Glucan Synthase ◽  
Kinase C ◽  
Downstream Effectors ◽  
Synthetically Lethal

In fission yeast protein kinase C homologues (Pck1 and Pck2) are essential for cell morphogenesis. We have isolated mok1+ in a genetic screen to identify downstream effectors for Pck1/2. mok1+ is essential for viability and encodes a protein that has several membrane-spanning domains and regions homologous to glucan metabolic enzymes. mok1 mutant shows abnormal cell shape, randomization of F-actin and weak cell wall. Biochemical analysis shows that Mok1 appears to have α-glucan synthase activity. Mok1 localization undergoes dramatic alteration during the cell cycle. It localizes to the growing tips in interphase, the medial ring upon mitosis, a double ring before and dense dot during cytokinesis. Double immunofluorescence staining shows that Mok1 exists in close proximity to actin. The subcellular localization of Mok1 is dependent upon the integrity of the F-actin cytoskeleton. Conversely, overexpression of mok1+ blocks the translocation of cortical actin from one end of the cell to the other. pck2 mutant is synthetically lethal with mok1 mutant, delocalizes Mok1 and shows a lower level of α-glucan. These results indicate that Mok1 plays a crucial role in cell morphogenesis interdependently of the actin cytoskeleton and works as one of downstream effectors for Pck1/2.

scholarly journals Host Serine/Threonine Kinases mTOR and Protein Kinase C-α Promote InlB-Mediated Entry of Listeria monocytogenes

2017 ◽  
Vol 85 (7) ◽  
Author(s):  
Manmeet Bhalla ◽  
Daria Law ◽  
Georgina C. Dowd ◽  
Keith Ireton
Keyword(s):  
Protein Kinase C ◽  
Listeria Monocytogenes ◽  
Protein Kinase ◽  
Actin Cytoskeleton ◽  
Bacterial Pathogen ◽  
Human Cell Line ◽  
Content Type ◽  
Kinase C ◽  
Cell Line Hela ◽  
Threonine Kinases

ABSTRACT The bacterial pathogen Listeria monocytogenes causes foodborne illnesses resulting in gastroenteritis, meningitis, or abortion. Listeria induces its internalization into some human cells through interaction of the bacterial surface protein InlB with the host receptor tyrosine kinase Met. InlB-dependent entry requires localized polymerization of the host actin cytoskeleton. The signal transduction pathways that act downstream of Met to regulate actin filament assembly or other processes during Listeria uptake remain incompletely characterized. Here, we demonstrate important roles for the human serine/threonine kinases mTOR and protein kinase C-α (PKC-α) in InlB-dependent entry. Experiments involving RNA interference (RNAi) indicated that two multiprotein complexes containing mTOR, mTORC1 and mTORC2, are each needed for efficient internalization of Listeria into cells of the human cell line HeLa. InlB stimulated Met-dependent phosphorylation of mTORC1 or mTORC2 substrates, demonstrating activation of both mTOR-containing complexes. RNAi studies indicated that the mTORC1 effectors 4E-BP1 and hypoxia-inducible factor 1α (HIF-1α) and the mTORC2 substrate PKC-α each control Listeria uptake. Genetic or pharmacological inhibition of PKC-α reduced the internalization of Listeria and the accumulation of actin filaments that normally accompanies InlB-mediated entry. Collectively, our results identify mTOR and PKC-α to be host factors exploited by Listeria to promote infection. PKC-α controls Listeria entry, at least in part, by regulating the actin cytoskeleton downstream of the Met receptor.

scholarly journals The Small Mr Ras-like GTPase Rap1 and the Phospholipase C Pathway Act to Regulate Phagocytosis inDictyostelium discoideum

1999 ◽  
Vol 10 (2) ◽  
pp. 393-406 ◽  
Author(s):  
David J. Seastone ◽  
Linyi Zhang ◽  
Greg Buczynski ◽  
Patrick Rebstein ◽  
Gerald Weeks ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Protein Tyrosine Kinase ◽  
Dominant Negative ◽  
Wild Type ◽  
Cortical Actin ◽  
Calphostin C ◽  
Kinase C ◽  
Protein Kinase C Inhibitor

The function of the small-Mr Ras-like GTPase Rap1 remains largely unknown, but this protein has been demonstrated to regulate cortical actin-based morphologic changes inDictyostelium and the oxidative burst in mammalian neutrophils. To test whether Rap1 regulates phagocytosis, we biochemically analyzed cell lines that conditionally and modestly overexpressed wild-type [Rap1 WT(+)], constitutively active [Rap1 G12T(+)], and dominant negative [Rap1 S17N(+)] forms of D. discoideum Rap1. The rates of phagocytosis of bacteria and latex beads were significantly higher in Rap1 WT(+) and Rap1 G12T(+) cells and were reduced in Rap1 S17N(+) cells. The addition of inhibitors of protein kinase A, protein kinase G, protein tyrosine kinase, or phosphatidylinositide 3-kinase did not affect phagocytosis rates in wild-type cells. In contrast, the addition of U73122 (a phospholipase C inhibitor), calphostin C (a protein kinase C inhibitor), and BAPTA-AM (an intracellular Ca2+ chelator) reduced phagocytosis rates by 90, 50, and 65%, respectively, suggesting both arms of the phospholipase C signaling pathways played a role in this process. Other protein kinase C–specific inhibitors, such as chelerythrine and bisindolylmaleimide I, did not reduce phagocytosis rates in control cells, suggesting calphostin C was affecting phagocytosis by interfering with a protein containing a diacylglycerol-binding domain. The addition of calphostin C did not reduce phagocytosis rates in Rap1 G12T(+) cells, suggesting that the putative diacylglycerol-binding protein acted upstream in a signaling pathway with Rap1. Surprisingly, macropinocytosis was significantly reduced in Rap1 WT(+) and Rap1 G12T(+) cells compared with control cells. Together our results suggest that Rap1 and Ca2+ may act together to coordinate important early events regulating phagocytosis.

The fission yeast sts5+ gene is required for maintenance of growth polarity and functionally interacts with protein kinase C and an osmosensing MAP-kinase pathway

1996 ◽  
Vol 109 (9) ◽  
pp. 2331-2342 ◽  
Author(s):  
T. Toda ◽  
H. Niwa ◽  
T. Nemoto ◽  
S. Dhut ◽  
M. Eddison ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Map Kinase ◽  
Fission Yeast ◽  
Cell Morphology ◽  
Fluorescent Protein ◽  
Sequence Similarity ◽  
Cell Morphogenesis ◽  
Cortical Actin ◽  
Kinase C

Cell morphogenesis is a fundamental phenomenon that involves understanding a number of biological processes including the developmental program, polarity and cell division. Fission yeast sts5 mutant cells are round rather than cylindrical with cortical actin randomly dispersed. Genetic analyses demonstrate that the sts5+ gene is required for maintenance of cell shape during interphase when the cell normally exhibits polarised growth. The sts5 mutant is not defective in cell wall integrity. Deletion of ppe1+, which encodes a type 2A-like protein phosphatase, shows similar phenotypes to the sts5 mutant and these two mutations are synthetically lethal. Multicopy plasmids containing either the protein kinase C-like gene pck1+ or the protein tyrosine phosphatase pyp1+, an inhibitor of an osmosensing Sty1/Spc1 MAP-kinase, are capable of suppressing the sts5 mutation. Consistent with this, we have found that the wis1 mutation, which is defective in a MAP-kinase kinase of the pathway, suppresses the sts5 mutation. The predicted sts5+ gene product exhibits sequence similarity to two yeast proteins, Dis3 and Ssd1 and a nematode protein, F46E8.6, where the former two yeast proteins have been shown to be involved in cell cycle control and cell morphogenesis. The sts5+ gene is not essential for cell viability, but is absolutely required for polarised growth as the gene disruption showed the same phenotypes as those of the original mutants. Overexpression of the sts5+ gene resulted in altered cell morphology and, cortical actin in these overproducing cells was also abnormal, fainter and often dispersed. Anti-Sts5 antibody specifically detected a 130 kDa protein by western blotting. A green fluorescent protein-Sts5 fusion protein localised in the cytoplasm with a discrete punctate pattern, suggesting that the Sts5 protein is a component of a novel structure. These results have indicated that the Sts5 protein is a crucial determinant of polarised growth and that it functionally interacts with the serine/threonine phosphatase, protein kinase C, and an osmosensing MAP-kinase to maintain cell morphology.

scholarly journals Phorbol ester-induced actin assembly in neutrophils: role of protein kinase C.

1992 ◽  
Vol 116 (3) ◽  
pp. 695-706 ◽  
Author(s):  
G P Downey ◽  
C K Chan ◽  
P Lea ◽  
A Takai ◽  
S Grinstein
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Actin Cytoskeleton ◽  
Protein Phosphorylation ◽  
Neutrophil Activation ◽  
Human Neutrophils ◽  
Actin Assembly ◽  
Membrane Ruffling ◽  
Kinase C

The shape changes and membrane ruffling that accompany neutrophil activation are dependent on the assembly and reorganization of the actin cytoskeleton, the molecular basis of which remains to be clarified. A role of protein kinase C (PKC) has been postulated because neutrophil activation, with the attendant shape and membrane ruffling changes, can be initiated by phorbol esters, known activators of PKC. It has become apparent, however, that multiple isoforms of PKC with differing substrate specificities exist. To reassess the role of PKC in cytoskeletal reorganization, we compared the effects of diacylglycerol analogs and of PKC antagonists on kinase activity and on actin assembly in human neutrophils. Ruffling of the plasma membrane was assessed by scanning EM, and spatial redistribution of filamentous (F)-actin was assessed by scanning confocal microscopy. Staining with NBD-phallacidin and incorporation of actin into the Triton X-100-insoluble ("cytoskeletal") fraction were used to quantify the formation of (F)-actin. [32P]ATP was used to detect protein phosphorylation in electroporated cells. Exposure of neutrophils to 4 beta-PMA (an activator of PKC) induced protein phosphorylation, membrane ruffling, and assembly and reorganization of the actin cytoskeleton, whereas the 4a-isomer, which is inactive towards PKC, failed to produce any of these changes. Moreover, 1,2-dioctanoylglycerol, mezerein, and 3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol, which are nonphorbol activators of PKC, also promoted actin assembly. Although these effects were consistent with a role of PKC, the following observations suggested that stimulation of conventional isoforms of the kinase were not directly responsible for actin assembly: (a) Okadaic acid, an inhibitor of phosphatases 1 and 2A, potentiated PMA-induced protein phosphorylation, but not actin assembly; and (b) PMA-induced actin assembly and membrane ruffling were not prevented by the conventional PKC inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, staurosporine, calphostin C, or sphingosine at concentrations that precluded PMA-induced protein phosphorylation and superoxide production. On the other hand, PMA-induced actin assembly was inhibited by long-chain fatty acid coenzyme A esters, known inhibitors of nuclear PKC (nPKC). We conclude that PMA-induced actin assembly is unlikely to be mediated by the conventional isoforms of PKC, but may be mediated by novel isoforms of the kinase such as nPKC.

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