scholarly journals Role of Cryptococcus neoformans Rho1 GTPases in the PKC1 Signaling Pathway in Response to Thermal Stress

2012 ◽  
Vol 12 (1) ◽  
pp. 118-131 ◽  
Author(s):  
Woei C. Lam ◽  
Kimberly J. Gerik ◽  
Jennifer K. Lodge
Keyword(s):  
Thermal Stress ◽  
Heat Shock ◽  
Protein Kinase ◽  
Cryptococcus Neoformans ◽  
Signaling Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Cell Integrity ◽  
Wild Type ◽  
Rho Proteins ◽  
Content Type

ABSTRACTTo initiate and establish infection in mammals, the opportunistic fungal pathogenCryptococcus neoformansmust survive and thrive upon subjection to host temperature. Primary maintenance of cell integrity is controlled through the protein kinase C1 (PKC1) signaling pathway, which is regulated by a Rho1 GTPase inSaccharomyces cerevisiae. We identified threeC. neoformansRho GTPases, Rho1, Rho10, and Rho11, and have begun to elucidate their role in growth and activation of the PKC1 pathway in response to thermal stress. Western blot analysis revealed that heat shock of wild-type cells resulted in phosphorylation of Mpk1 mitogen-activated protein kinase (MAPK). Constitutive activation of Rho1 caused phosphorylation of Mpk1 independent of temperature, indicating its role in pathway regulation. A strain with a deletion ofRHO10also displayed this constitutive Mpk1 phosphorylation phenotype, while one with a deletion ofRHO11yielded phosphorylation similar to that of wild type. Surprisingly, like arho10Δ strain, a strain with a deletion of bothRHO10andRHO11displayed temperature sensitivity but mimicked wild-type phosphorylation, which suggests that Rho10 and Rho11 have coordinately regulated functions. Heat shock-induced Mpk1 phosphorylation also required the PKC1 pathway kinases Bck1 and Mkk2. However, Pkc1, thought to be the major regulatory kinase of the cell integrity pathway, was dispensable for this response. Together, our results argue that Rho proteins likely interact via downstream components of the PKC1 pathway or by alternative pathways to activate the cell integrity pathway inC. neoformans.

scholarly journals Cross Talk between the Cell Wall Integrity and Cyclic AMP/Protein Kinase A Pathways in Cryptococcus neoformans

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Maureen J. Donlin ◽  
Rajendra Upadhya ◽  
Kimberly J. Gerik ◽  
Woei Lam ◽  
Laura G. VanArendonk ◽  
...  
Keyword(s):  
Cell Wall ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinase A ◽  
Cryptococcus Neoformans ◽  
Signaling Pathway ◽  
Cell Wall Integrity ◽  
Wild Type ◽  
Content Type ◽  
Kinase A

ABSTRACTCryptococcus neoformans is a fungal pathogen of immunocompromised people that causes fatal meningitis. The fungal cell wall is essential to viability and pathogenesis ofC. neoformans, and biosynthesis and repair of the wall is primarily controlled by the cell wall integrity (CWI) signaling pathway. Previous work has shown that deletion of genes encoding the four major kinases in the CWI signaling pathway, namely,PKC1,BCK1,MKK2, andMPK1results in severe cell wall phenotypes, sensitivity to a variety of cell wall stressors, and for Mpk1, reduced virulence in a mouse model. Here, we examined the global transcriptional responses to gene deletions ofBCK1,MKK2, andMPK1compared to wild-type cells. We found that over 1,000 genes were differentially expressed in one or more of the deletion strains, with 115 genes differentially expressed in all three strains, many of which have been identified as genes regulated by the cyclic AMP (cAMP)/protein kinase A (PKA) pathway. Biochemical measurements of cAMP levels in the kinase deletion strains revealed significantly less cAMP in all of the deletion strains compared to the wild-type strain. The deletion strains also produced significantly smaller capsules than the wild-type KN99 strain did under capsule-inducing conditions, although the levels of capsule they shed were similar to those shed by the wild type. Finally, addition of exogenous cAMP led to reduced sensitivity to cell wall stress and restored surface capsule to levels near those of wild type. Thus, we have direct evidence of cross talk between the CWI and cAMP/PKA pathways that may have important implications for regulation of cell wall and capsule homeostasis.IMPORTANCECryptococcus neoformans is a fungal pathogen of immunocompromised people that causes fatal meningitis. The fungal cell wall is essential to viability and pathogenesis ofC. neoformans, and biosynthesis and repair of the wall are primarily controlled by the cell wall integrity (CWI) signaling pathway. In this study, we demonstrate that deletion of any of three core kinases in the CWI pathway impacts not only the cell wall but also the amount of surface capsule. Deletion of any of the kinases results in significantly reduced cellular cyclic AMP (cAMP) levels, and addition of exogenous cAMP rescues the capsule defect and some cell wall defects, supporting a direct role for the CWI pathway in regulation of capsule in conjunction with the cAMP/protein kinase A pathway.

scholarly journals Rac-PAK Signaling Stimulates Extracellular Signal-Regulated Kinase (ERK) Activation by Regulating Formation of MEK1-ERK Complexes

2002 ◽  
Vol 22 (17) ◽  
pp. 6023-6033 ◽  
Author(s):  
Scott T. Eblen ◽  
Jill K. Slack ◽  
Michael J. Weber ◽  
Andrew D. Catling
Keyword(s):  
Protein Kinase ◽  
Signaling Pathway ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Wild Type ◽  
Erk Activation ◽  
Extracellular Signal Regulated Kinase ◽  
Signaling Complex ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

ABSTRACT Utilizing mutants of extracellular signal-regulated kinase 2 (ERK2) that are defective for intrinsic mitogen-activated protein kinase or ERK kinase (MEK) binding, we have identified a convergent signaling pathway that facilitates regulated MEK-ERK association and ERK activation. ERK2-Δ19-25 mutants defective in MEK binding could be phosphorylated in response to mitogens; however, signaling from the Raf-MEK pathway alone was insufficient to stimulate their phosphorylation in COS-1 cells. Phosphorylation of ERK2-Δ19-25 but not of wild-type ERK2 in response to Ras V12 was greatly inhibited by dominant-negative Rac. Activated forms of Rac and Cdc42 could enhance the association of wild-type ERK2 with MEK1 but not with MEK2 in serum-starved adherent cells. This effect was p21-activated kinase (PAK) dependent and required the putative PAK phosphorylation sites T292 and S298 of MEK1. In detached cells placed in suspension, ERK2 was complexed with MEK2 but not with MEK1. However, upon replating of cells onto a fibronectin matrix, there was a substantial induction of MEK1-ERK2 association and ERK activation, both of which could be inhibited by dominant-negative PAK1. These data show that Rac facilitates the assembly of a mitogen-activated protein kinase signaling complex required for ERK activation and that this facilitative signaling pathway is active during adhesion to the extracellular matrix. These findings reveal a novel mechanism by which adhesion and growth factor signals are integrated during ERK activation.

scholarly journals Schizosaccharomyces pombeRho2p GTPase Regulates Cell Wall α-Glucan Biosynthesis through the Protein Kinase Pck2p

2000 ◽  
Vol 11 (12) ◽  
pp. 4393-4401 ◽  
Author(s):  
Teresa M. Calonge ◽  
Kentaro Nakano ◽  
Manuel Arellano ◽  
Ritsuko Arai ◽  
Satoshi Katayama ◽  
...  
Keyword(s):  
Cell Wall ◽  
Protein Kinase ◽  
Signaling Pathway ◽  
Cell Integrity ◽  
Wild Type ◽  
Morphological Alterations ◽  
Kinase C ◽  
Glucan Synthesis ◽  
Synthetically Lethal ◽  
Structural Polymer

Schizosaccharomyces pombe rho1+andrho2+genes are involved in the control of cell morphogenesis, cell integrity, and polarization of the actin cytoskeleton. Although both GTPases interact with each of the twoS. pombe protein kinase C homologues, Pck1p and Pck2p, their functions are distinct from each other. It is known that Rho1p regulates (1,3)β-d-glucan synthesis both directly and through Pck2p. In this paper, we have investigated Rho2p signaling and show that pck2Δ andrho2Δ strains display similar defects with regard to cell wall integrity, indicating that they might be in the same signaling pathway. We also show that Rho2 GTPase regulates the synthesis of α-d-glucan, the other main structural polymer of the S. pombe cell wall, primarily through Pck2p. Although overexpression of rho2+in wild-type or pck1Δ cells is lethal and causes morphological alterations, actin depolarization, and an increase in α-d-glucan biosynthesis, all of these effects are suppressed in a pck2Δ strain. In addition, genetic interactions suggest that Rho2p and Pck2p are important for the regulation of Mok1p, the major (1–3)α-d-glucan synthase. Thus, a rho2Δ mutation, like pck2Δ, is synthetically lethal with mok1–664, and the mutant partially fails to localize Mok1p to the growing areas. Moreover, overexpression of mok1+inrho2Δ cells causes a lethal phenotype that is completely different from that of mok1+overexpression in wild-type cells, and the increase in α-glucan is considerably lower. Taken together, all of these results indicate the presence of a signaling pathway regulating α-glucan biosynthesis in which the Rho2p GTPase activates Pck2p, and this kinase in turn controls Mok1p.

scholarly journals Attaching and Effacing Bacterial Effector NleC Suppresses Epithelial Inflammatory Responses by Inhibiting NF-κB and p38 Mitogen-Activated Protein Kinase Activation

2011 ◽  
Vol 79 (9) ◽  
pp. 3552-3562 ◽  
Author(s):  
Ho Pan Sham ◽  
Stephanie R. Shames ◽  
Matthew A. Croxen ◽  
Caixia Ma ◽  
Justin M. Chan ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Epithelial Cells ◽  
Intestinal Inflammation ◽  
Inflammatory Responses ◽  
Mitogen Activated Protein Kinase ◽  
Loop Model ◽  
Wild Type ◽  
Content Type ◽  
Mitogen Activated Protein ◽  
The Impact

ABSTRACTEnteropathogenicEscherichia coli(EPEC) and enterohemorrhagicE. coliare noninvasive attaching and effacing (A/E) bacterial pathogens that cause intestinal inflammation and severe diarrheal disease. These pathogens utilize a type III secretion system to deliver effector proteins into host epithelial cells, modulating diverse cellular functions, including the release of the chemokine interleukin-8 (IL-8). While studies have implicated the effectors NleE (non-locus of enterocyte effacement [LEE]-encoded effector E) and NleH1 in suppressing IL-8 release, by preventing NF-κB nuclear translocation, the impact of these effectors only partially replicates the immunosuppressive actions of wild-type EPEC, suggesting another effector or effectors are involved. Testing an array of EPEC mutants, we identified the non-LEE-encoded effector C (NleC) as also suppressing IL-8 release. Infection by ΔnleCEPEC led to exaggerated IL-8 release from infected Caco-2 and HT-29 epithelial cells. NleC localized to EPEC-induced pedestals, with signaling studies revealing NleC inhibits both NF-κB and p38 mitogen-activated protein kinase (MAPK) activation. UsingCitrobacter rodentium, a mouse-adapted A/E bacterium, we found that ΔnleCand wild-typeC. rodentium-infected mice carried similar pathogen burdens, yet ΔnleCstrain infection led to worsened colitis. Similarly, infection with ΔnleCC. rodentiumin a cecal loop model induced significantly greater chemokine responses than infection with wild-type bacteria. These studies thus advance our understanding of how A/E pathogens subvert host inflammatory responses.

scholarly journals Involvement of the Mannose Receptor and p38 Mitogen-Activated Protein Kinase Signaling Pathway of the Microdomain of the Integral Membrane Protein after Enteropathogenic Escherichia coli Infection

2012 ◽  
Vol 80 (4) ◽  
pp. 1343-1350 ◽  
Author(s):  
Zhihua Liu ◽  
Yanlei Ma ◽  
Mary Pat Moyer ◽  
Peng Zhang ◽  
Chenzhang Shi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Kinase ◽  
Signaling Pathway ◽  
P38 Mapk ◽  
Mannose Receptor ◽  
Integral Membrane Protein ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Content Type ◽  
Mitogen Activated Protein

ABSTRACTThe microdomain of the integral membrane protein (MIMP) has been shown to adhere to mucin and to antagonize the adhesion of enteropathogenicEscherichia coli(EPEC) to epithelial cells; however, the mechanism has not been fully elucidated. In this study, we further identified the receptor of MIMP on NCM460 cells and investigated the mechanism (the p38 mitogen-activated protein kinase [MAPK] pathway) following the interaction of MIMP and its corresponding receptor, mannose receptor. We first identified the target receptor of MIMP on the surfaces of NCM460 cells using immunoprecipitation-mass spectrometry technology. We also verified the mannose receptor and examined the degradation and activation of the p38 MAPK signaling pathway. The results indicated that MIMP adhered to NCM460 cells by binding to the mannose receptor and inhibited the phosphorylation of p38 MAPK stimulated after EPEC infection via inhibition of the Toll-like receptor 5 pathway. These findings indicated that MIMPs relieve the injury of NCM460 cells after enteropathogenicE. coliinfection through the mannose receptor and inhibition of the p38 MAPK signaling pathway, both of which may therefore be potential therapeutic targets for intestinal diseases, such as inflammatory bowel disease.

scholarly journals TRAF6-Dependent Mitogen-Activated Protein Kinase Activation Differentially Regulates the Production of Interleukin-12 by Macrophages in Response to Toxoplasma gondii

2004 ◽  
Vol 72 (10) ◽  
pp. 5662-5667 ◽  
Author(s):  
Nicola J. Mason ◽  
Jim Fiore ◽  
Takashi Kobayashi ◽  
Katherine S. Masek ◽  
Yongwon Choi ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Toxoplasma Gondii ◽  
Signaling Pathways ◽  
Intracellular Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Interleukin 12 ◽  
Wild Type ◽  
Kinase Activation ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

ABSTRACT The production of interleukin-12 (IL-12) is critical to the development of innate and adaptive immune responses required for the control of intracellular pathogens. Many microbial products signal through Toll-like receptors (TLR) and activate NF-κB family members that are required for the production of IL-12. Recent studies suggest that components of the TLR pathway are required for the production of IL-12 in response to the parasite Toxoplasma gondii; however, the production of IL-12 in response to this parasite is independent of NF-κB activation. The adaptor molecule TRAF6 is involved in TLR signaling pathways and associates with serine/threonine kinases involved in the activation of both NF-κB and mitogen-activated protein kinase (MAPK). To elucidate the intracellular signaling pathways involved in the production of IL-12 in response to soluble toxoplasma antigen (STAg), wild-type and TRAF6−/− mice were inoculated with STAg, and the production of IL-12(p40) was determined. TRAF6−/− mice failed to produce IL-12(p40) in response to STAg, and TRAF6−/− macrophages stimulated with STAg also failed to produce IL-12(p40). Studies using Western blot analysis of wild-type and TRAF6−/− macrophages revealed that stimulation with STAg resulted in the rapid TRAF6-dependent phosphorylation of p38 and extracellular signal-related kinase, which differentially regulated the production of IL-12(p40). The studies presented here demonstrate for the first time that the production of IL-12(p40) in response to toxoplasma is dependent upon TRAF6 and p38 MAPK.

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