Ste20-related Protein Kinase LOSK (SLK) Controls Microtubule Radial Array in Interphase

Interphase microtubules are organized into a radial array with centrosome in the center. This organization is a subject of cellular regulation that can be driven by protein phosphorylation. Only few protein kinases that regulate microtubule array in interphase cells have been described. Ste20-like protein kinase LOSK (SLK) was identified as a microtubule and centrosome-associated protein. In this study we have shown that the inhibition of LOSK activity by dominant-negative mutant K63R-ΔT or by LOSK depletion with RNAi leads to unfocused microtubule arrangement. Microtubule disorganization is prominent in Vero, CV-1, and CHO-K1 cells but less distinct in HeLa cells. The effect is a result neither of microtubule stabilization nor of centrosome disruption. In cells with suppressed LOSK activity centrosomes are unable to anchor or to cap microtubules, though they keep nucleating microtubules. These centrosomes are depleted of dynactin. Vero cells overexpressing K63R-ΔT have normal dynactin “comets” at microtubule ends and unaltered morphology of Golgi complex but are unable to polarize it at the wound edge. We conclude that protein kinase LOSK is required for radial microtubule organization and for the proper localization of Golgi complex in various cell types.

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Signal transduction pathways of IL-1β-mediated iNOS in pulmonary vascular smooth muscle cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.2001.281.4.l816 ◽

2001 ◽

Vol 281 (4) ◽

pp. L816-L823 ◽

Cited By ~ 14

Author(s):

Jonathan D. Finder ◽

Jennifer L. Petrus ◽

Andrew Hamilton ◽

Raphael T. Villavicencio ◽

Bruce R. Pitt ◽

...

Keyword(s):

Smooth Muscle ◽

Protein Kinase ◽

Pulmonary Artery ◽

Smooth Muscle Cells ◽

Intracellular Signaling ◽

Muscle Cells ◽

Dominant Negative ◽

Janus Kinase ◽

Dominant Negative Mutant ◽

Pulmonary Artery Smooth Muscle

Interleukin (IL)-1β is an important early mediator of inflammation in pulmonary artery smooth muscle cells. We previously reported that a geranylgeranyltransferase inhibitor elevated basal levels of inducible nitric oxide synthase (iNOS) and enhanced IL-1β-mediated induction, suggesting that Rac or Rho small G proteins are candidates for antagonism of such induction. In this study, overexpression of constitutively active Rac1 or its dominant negative mutant did not affect IL-1β induction of iNOS. Alternatively, treatment with Clostridium botulinum C3 exoenzyme, which ADP-ribosylates Rho, was associated with superinduction of iNOS, suggesting an inhibitory role for Rho. IL-1β activated the three mitogen-activated protein kinase (extracellular signal-regulated kinases 1 and 2, c-Jun NH2-terminal kinase/stress-activated protein kinase, and p38) and the Janus kinase (JAK)-signal transducer and activator of transcription pathways. The former two pathways were not associated with IL-1β-mediated iNOS induction, whereas the latter two appeared to have inhibitory roles in iNOS expression. These data suggest that a broad intracellular signaling response to IL-1β in rat pulmonary artery smooth muscle cells results in elevated levels of iNOS that is opposed by the geranylgeranylated small G protein Rho as well as the p38 and JAK2 pathways.

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Molecular mechanisms involved in the regulation of cytokine production by muramyl dipeptide

Biochemical Journal ◽

10.1042/bj20061704 ◽

2007 ◽

Vol 404 (2) ◽

pp. 179-190 ◽

Cited By ~ 130

Author(s):

Mark Windheim ◽

Christine Lang ◽

Mark Peggie ◽

Lorna A. Plater ◽

Philip Cohen

Keyword(s):

Protein Kinase ◽

Molecular Mechanisms ◽

Transforming Growth Factor ◽

Kinase Inhibitor ◽

Muramyl Dipeptide ◽

Dominant Negative ◽

Signalling Pathways ◽

Dominant Negative Mutant ◽

Protein Kinase Activity ◽

P38α Mapk

MDP (muramyl dipeptide), a component of peptidoglycan, interacts with NOD2 (nucleotide-binding oligomerization domain 2) stimulating the NOD2–RIP2 (receptor-interacting protein 2) complex to activate signalling pathways important for antibacterial defence. Here we demonstrate that the protein kinase activity of RIP2 has two functions, namely to limit the strength of downstream signalling and to stabilize the active enzyme. Thus pharmacological inhibition of RIP2 kinase with either SB 203580 [a p38 MAPK (mitogen-activated protein kinase) inhibitor] or the Src family kinase inhibitor PP2 induces a rapid and drastic decrease in the level of the RIP2 protein, which may explain why these RIP2 inhibitors block MDP-stimulated downstream signalling and the production of IL-1β (interleukin-1β) and TNFα (tumour necrosis factor-α). We also show that RIP2 induces the activation of the protein kinase TAK1 (transforming-growth-factor-β-activated kinase-1), that a dominant-negative mutant of TAK1 inhibits RIP2-induced activation of JNK (c-Jun N-terminal kinase) and p38α MAPK, and that signalling downstream of NOD2 or RIP2 is reduced by the TAK1 inhibitor (5Z)-7-oxozeaenol or in TAK1-deficient cells. We also show that MDP activates ERK1 (extracellular-signal-regulated kinase 1)/ERK2 and p38α MAPK in human peripheral-blood mononuclear cells and that the activity of both MAPKs and TAK1 are required for MDP-induced signalling and production of IL-1β and TNFα in these cells. Taken together, our results indicate that the MDP–NOD2/RIP2 and LPS (lipopolysaccharide)–TLR4 (Toll-like receptor 4) signalling pathways converge at the level of TAK1 and that many subsequent events that lead to the production of pro-inflammatory cytokines are common to both pathways.

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Epithelial injury induces Egr-1 and Fos expression by a pathway involving protein kinase C and ERK

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1999.276.2.g322 ◽

1999 ◽

Vol 276 (2) ◽

pp. G322-G330 ◽

Cited By ~ 7

Author(s):

Brian K. Dieckgraefe ◽

Danielle M. Weems

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Dominant Negative ◽

Mitogen Activated Protein Kinase ◽

Dominant Negative Mutant ◽

Mrna Levels ◽

Mobility Shift ◽

Erk Activation ◽

Kinase C

The signaling pathways activated in response to gastrointestinal injury remain poorly understood. Previous work has implicated the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase as a mediator of wound-signal transduction and a possible regulator of epithelial restitution. Monolayer injury resulted in rapid activation of p42 and p44 ERK. Injury-induced ERK activation was blocked by protein kinase C inhibition or by disruption of the cell cytoskeleton. Significant increases in Fos and early growth response (Egr)-1 mRNA levels were stimulated by injury, peaking by 20 min. ERK activation and the induction of Egr-1 mRNA were inhibited in a dose-dependent fashion with PD-98059. Fos mRNA expression was partially blocked by PD-98059. Western blot analysis demonstrated strong expression and nuclear localization of Fos and Egr after wounding. Electrophoretic mobility shift assays demonstrated that nuclear extracts contained a protein that specifically bound double-stranded oligonucleotides containing the Egr consensus binding element. Gel supershift assays demonstrated that the protein-DNA complexes were recognized by anti-Egr antibody. Inhibition of injury-induced ERK activation by PD-98059 or direct interference with Egr by expression of a dominant negative mutant led to significantly reduced in vitro monolayer restitution.

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Activation of IκB Kinase β by Protein Kinase C Isoforms

Molecular and Cellular Biology ◽

10.1128/mcb.19.3.2180 ◽

1999 ◽

Vol 19 (3) ◽

pp. 2180-2188 ◽

Cited By ~ 281

Author(s):

Maria-José Lallena ◽

María T. Diaz-Meco ◽

Gary Bren ◽

Carlos V. Payá ◽

Jorge Moscat

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Critical Role ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Cell Functions ◽

Kinase C ◽

Iκb Kinase ◽

Tnf Α

ABSTRACT The atypical protein kinase C (PKC) isotypes (λ/ιPKC and ζPKC) have been shown to be critically involved in important cell functions such as proliferation and survival. Previous studies have demonstrated that the atypical PKCs are stimulated by tumor necrosis factor alpha (TNF-α) and are required for the activation of NF-κB by this cytokine through a mechanism that most probably involves the phosphorylation of IκB. The inability of these PKC isotypes to directly phosphorylate IκB led to the hypothesis that ζPKC may use a putative IκB kinase to functionally inactivate IκB. Recently several groups have molecularly characterized and cloned two IκB kinases (IKKα and IKKβ) which phosphorylate the residues in the IκB molecule that serve to target it for ubiquitination and degradation. In this study we have addressed the possibility that different PKCs may control NF-κB through the activation of the IKKs. We report here that αPKC as well as the atypical PKCs bind to the IKKs in vitro and in vivo. In addition, overexpression of ζPKC positively modulates IKKβ activity but not that of IKKα, whereas the transfection of a ζPKC dominant negative mutant severely impairs the activation of IKKβ but not IKKα in TNF-α-stimulated cells. We also show that cell stimulation with phorbol 12-myristate 13-acetate activates IKKβ, which is entirely dependent on the activity of αPKC but not that of the atypical isoforms. In contrast, the inhibition of αPKC does not affect the activation of IKKβ by TNF-α. Interestingly, recombinant active ζPKC and αPKC are able to stimulate in vitro the activity of IKKβ but not that of IKKα. In addition, evidence is presented here that recombinant ζPKC directly phosphorylates IKKβ in vitro, involving Ser177 and Ser181. Collectively, these results demonstrate a critical role for the PKC isoforms in the NF-κB pathway at the level of IKKβ activation and IκB degradation.

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Role of MKK3 and p38 MAPK in cytokine-induced death of insulin-producing cells

Biochemical Journal ◽

10.1042/bj20050814 ◽

2005 ◽

Vol 393 (1) ◽

pp. 129-139 ◽

Cited By ~ 15

Author(s):

Natalia Makeeva ◽

Jason W. Myers ◽

Nils Welsh

Keyword(s):

Nitric Oxide ◽

Cell Death ◽

Protein Kinase ◽

P38 Mapk ◽

Dominant Negative ◽

Mitogen Activated Protein Kinase ◽

Nitric Oxide Donor ◽

Cell Leukaemia ◽

Dominant Negative Mutant

The aim of the present investigation was to elucidate further the importance of p38 MAPK (mitogen-activated protein kinase) in nitric oxide- and cytokine-induced β-cell death. For this purpose, isolated human islets were treated with d-siRNA (diced small interfering RNA) and then exposed to the nitric oxide donor DETA/NONOate [2,2′-(hydroxynitrosohydrazono)bis-ethanamine]. We observed that cells treated with p38α-specific d-siRNA, but not with d-siRNA targeting GL3 (a firefly luciferase siRNA plasmid) or PKCδ (protein kinase Cδ), were protected against nitric oxide-induced death. This was paralleled by an increased level of Bcl-XL (B-cell leukaemia/lymphoma-X long). For an in-depth study of the mechanisms of p38 activation, MKK3 (MAPK kinase 3), MKK6 and their dominant-negative mutants were overexpressed in insulin-producing RIN-5AH cells. In transient transfections, MKK3 overexpression resulted in increased p38 phosphorylation, whereas in stable MKK3-overexpressing RIN-5AH clones, the protein levels of p38 and JNK (c-Jun N-terminal kinase) were decreased, resulting in unaffected phospho-p38 levels. In addition, a long-term MKK3 overexpression did not affect cell death rates in response to the cytokines interleukin-1β and interferon-γ, whereas a short-term MKK3 expression resulted in increased cytokine-induced RIN-5AH cell death. The MKK3-potentiating effect on cytokine-induced cell death was abolished by a nitric oxide synthase inhibitor, and MKK3-stimulated p38 phosphorylation was enhanced by inhibitors of phosphatases. Finally, as the dominant-negative mutant of MKK3 did not affect cytokine-induced p38 phosphorylation, and as wild-type MKK3 did not influence p38 autophosphorylation, it may be that p38 is activated by MKK3/6-independent pathways in response to cytokines and nitric oxide. In addition, it is likely that a long-term increase in p38 activity is counteracted by both a decreased expression of the p38, JNK and p42 genes as well as an increased dephosphorylation of p38.

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NF-κB Activation by Double-Stranded-RNA-Activated Protein Kinase (PKR) Is Mediated through NF-κB-Inducing Kinase and IκB Kinase

Molecular and Cellular Biology ◽

10.1128/mcb.20.4.1278-1290.2000 ◽

2000 ◽

Vol 20 (4) ◽

pp. 1278-1290 ◽

Cited By ~ 250

Author(s):

Maryam Zamanian-Daryoush ◽

Trine H. Mogensen ◽

Joseph A. DiDonato ◽

Bryan R. G. Williams

Keyword(s):

Protein Kinase ◽

Kinetic Studies ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Null Cell ◽

Necrosis Factor Alpha ◽

Double Stranded Rna ◽

Iκb Kinase ◽

Tnf Α ◽

Factor Alpha

ABSTRACT The interferon (IFN)-inducible double-stranded-RNA (dsRNA)-activated serine-threonine protein kinase (PKR) is a major mediator of the antiviral and antiproliferative activities of IFNs. PKR has been implicated in different stress-induced signaling pathways including dsRNA signaling to nuclear factor kappa B (NF-κB). The mechanism by which PKR mediates activation of NF-κB is unknown. Here we show that in response to poly(rI) · poly(rC) (pIC), PKR activates IκB kinase (IKK), leading to the degradation of the inhibitors IκBα and IκBβ and the concomitant release of NF-κB. The results of kinetic studies revealed that pIC induced a slow and prolonged activation of IKK, which was preceded by PKR activation. In PKR null cell lines, pIC failed to stimulate IKK activity compared to cells from an isogenic background wild type for PKR in accord with the inability of PKR null cells to induce NF-κB in response to pIC. Moreover, PKR was required to establish a sustained response to tumor necrosis factor alpha (TNF-α) and to potentiate activation of NF-κB by cotreatment with TNF-α and IFN-γ. By coimmunoprecipitation, PKR was shown to be physically associated with the IKK complex. Transient expression of a dominant negative mutant of IKKβ or the NF-κB-inducing kinase (NIK) inhibited pIC-induced gene expression from an NF-κB-dependent reporter construct. Taken together, these results demonstrate that PKR-dependent dsRNA induction of NF-κB is mediated by NIK and IKK activation.

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Coordinate Regulation of IκB Kinases by Mitogen-Activated Protein Kinase Kinase Kinase 1 and NF-κB-Inducing Kinase

Molecular and Cellular Biology ◽

10.1128/mcb.18.12.7336 ◽

1998 ◽

Vol 18 (12) ◽

pp. 7336-7343 ◽

Cited By ~ 181

Author(s):

Shino Nemoto ◽

Joseph A. DiDonato ◽

Anning Lin

Keyword(s):

Protein Kinase ◽

Interleukin 1 ◽

Dominant Negative ◽

Mitogen Activated Protein Kinase ◽

Dominant Negative Mutant ◽

Necrosis Factor Alpha ◽

Tnf Α ◽

Ikk Complex ◽

Mitogen Activated Protein ◽

Protein Kinase Kinase

ABSTRACT IκB kinases (IKKα and IKKβ) are key components of the IKK complex that mediates activation of the transcription factor NF-κB in response to extracellular stimuli such as inflammatory cytokines, viral and bacterial infection, and UV irradiation. Although NF-κB-inducing kinase (NIK) interacts with and activates the IKKs, the upstream kinases for the IKKs still remain obscure. We identified mitogen-activated protein kinase kinase kinase 1 (MEKK1) as an immediate upstream kinase of the IKK complex. MEKK1 is activated by tumor necrosis factor alpha (TNF-α) and interleukin-1 and can potentiate the stimulatory effect of TNF-α on IKK and NF-κB activation. The dominant negative mutant of MEKK1, on the other hand, partially blocks activation of IKK by TNF-α. MEKK1 interacts with and stimulates the activities of both IKKα and IKKβ in transfected HeLa and COS-1 cells and directly phosphorylates the IKKs in vitro. Furthermore, MEKK1 appears to act in parallel to NIK, leading to synergistic activation of the IKK complex. The formation of the MEKK1-IKK complex versus the NIK-IKK complex may provide a molecular basis for regulation of the IKK complex by various extracellular signals.

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BMPs signal alternately through a SMAD or FRAP–STAT pathway to regulate fate choice in CNS stem cells

The Journal of Cell Biology ◽

10.1083/jcb.200211021 ◽

2003 ◽

Vol 161 (5) ◽

pp. 911-921 ◽

Cited By ~ 142

Author(s):

Prithi Rajan ◽

David M. Panchision ◽

Laura F. Newell ◽

Ronald D.G. McKay

Keyword(s):

Stem Cells ◽

Smooth Muscle ◽

Cell Fate ◽

Mammalian Target Of Rapamycin ◽

Neural Crest Cell ◽

Cell Types ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Glial Differentiation ◽

Cytoplasmic Signals

The ability of stem cells to generate distinct fates is critical for the generation of cellular diversity during development. Central nervous system (CNS) stem cells respond to bone morphogenetic protein (BMP) 4 by differentiating into a wide variety of dorsal CNS and neural crest cell types. We show that distinct mechanisms are responsible for the generation of two of these cell types, smooth muscle and glia. Smooth muscle differentiation requires BMP-mediated Smad1/5/8 activation and predominates where local cell density is low. In contrast, glial differentiation predominates at high local densities in response to BMP4 and is specifically blocked by a dominant-negative mutant Stat3. Upon BMP4 treatment, the serine-threonine kinase FKBP12/rapamycin-associated protein (FRAP), mammalian target of rapamycin (mTOR), associates with Stat3 and facilitates STAT activation. Inhibition of FRAP prevents STAT activation and glial differentiation. Thus, glial differentiation by BMP4 occurs by a novel pathway mediated by FRAP and STAT proteins. These results suggest that a single ligand can regulate cell fate by activating distinct cytoplasmic signals.

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Role of the atypical protein kinase Cζ in regulation of 5′-AMP-activated protein kinase in cardiac and skeletal muscle

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00009.2009 ◽

2009 ◽

Vol 297 (2) ◽

pp. E349-E357 ◽

Cited By ~ 19

Author(s):

John R. Ussher ◽

Jagdip S. Jaswal ◽

Cory S. Wagg ◽

Heather E. Armstrong ◽

David G. Lopaschuk ◽

...

Keyword(s):

Skeletal Muscle ◽

Protein Kinase ◽

Muscle Cells ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Minor Role ◽

Atypical Protein Kinase ◽

Downstream Target ◽

Ampk Phosphorylation ◽

Amp Activated Protein Kinase

During metabolic stress, phosphorylation and activation of 5′-AMP-activated protein kinase (AMPK) becomes a major regulator of cellular energy metabolism in heart and skeletal muscle. Despite this, the upstream regulation of AMPK in both heart and muscle is poorly understood. Recent work has implicated the atypical protein kinase Cζ (PKCζ) as a regulator of AMPK in endothelial cells via phosphorylation of LKB1, an upstream AMPK kinase (AMPKK). Our goal was to determine the potential role PKCζ plays in regulating AMPK in cardiac and skeletal muscle. Cultures of H9c2 myocytes (cardiac) and C2C12 myotubes (skeletal muscle) were pretreated with a selective PKCζ pseudosubstrate peptide inhibitor and treated with various AMPK activating agents to determine whether PKCζ regulates AMPK. PKCζ activity was also examined in isolated working rat hearts subjected to ischemia. We show that PKCζ is not involved in regulating threonine 172 AMPK phosphorylation induced by metformin or phenformin in either cardiac or skeletal muscle cells but is involved in 5-aminoimidazole-4-carboxamine-1-β-d-ribofuranoside (AICAR)-induced AMPK phosphorylation in cardiac muscle cells. Activation of PKCζ with high palmitate concentrations is also insufficient to increase AMPK phosphorylation. Furthermore, we show that the ischemia-induced activation of AMPK is not accompanied by increased PKCζ activity. Finally, we show that PKCζ may actually be a downstream target of AMPK in skeletal muscle, since adenoviral expression of a dominant-negative mutant of AMPK prevented metformin- and AICAR-induced phosphorylation of PKCζ. We conclude that PKCζ plays a very minor role in the regulation of AMPK in cardiac and skeletal muscle and may actually be a downstream target of AMPK in skeletal muscle.

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Requirement of Atypical Protein Kinase Cλ for Insulin Stimulation of Glucose Uptake but Not for Akt Activation in 3T3-L1 Adipocytes

Molecular and Cellular Biology ◽

10.1128/mcb.18.12.6971 ◽

1998 ◽

Vol 18 (12) ◽

pp. 6971-6982 ◽

Cited By ~ 269

Author(s):

Ko Kotani ◽

Wataru Ogawa ◽

Michihiro Matsumoto ◽

Tadahiro Kitamura ◽

Hiroshi Sakaue ◽

...

Keyword(s):

Protein Kinase ◽

Glucose Uptake ◽

Glucose Transporter ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Pleckstrin Homology Domain ◽

Dependent Manner ◽

Insulin Stimulation ◽

Negative Effects ◽

Stimulation Of

ABSTRACT Phosphoinositide (PI) 3-kinase contributes to a wide variety of biological actions, including insulin stimulation of glucose transport in adipocytes. Both Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, and atypical isoforms of protein kinase C (PKCζ and PKCλ) have been implicated as downstream effectors of PI 3-kinase. Endogenous or transfected PKCλ in 3T3-L1 adipocytes or CHO cells has now been shown to be activated by insulin in a manner sensitive to inhibitors of PI 3-kinase (wortmannin and a dominant negative mutant of PI 3-kinase). Overexpression of kinase-deficient mutants of PKCλ (λKD or λΔNKD), achieved with the use of adenovirus-mediated gene transfer, resulted in inhibition of insulin activation of PKCλ, indicating that these mutants exert dominant negative effects. Insulin-stimulated glucose uptake and translocation of the glucose transporter GLUT4 to the plasma membrane, but not growth hormone- or hyperosmolarity-induced glucose uptake, were inhibited by λKD or λΔNKD in a dose-dependent manner. The maximal inhibition of insulin-induced glucose uptake achieved by the dominant negative mutants of PKCλ was ∼50 to 60%. These mutants did not inhibit insulin-induced activation of Akt. A PKCλ mutant that lacks the pseudosubstrate domain (λΔPD) exhibited markedly increased kinase activity relative to that of the wild-type enzyme, and expression of λΔPD in quiescent 3T3-L1 adipocytes resulted in the stimulation of glucose uptake and translocation of GLUT4 but not in the activation of Akt. Furthermore, overexpression of an Akt mutant in which the phosphorylation sites targeted by growth factors are replaced by alanine resulted in inhibition of insulin-induced activation of Akt but not of PKCλ. These results suggest that insulin-elicited signals that pass through PI 3-kinase subsequently diverge into at least two independent pathways, an Akt pathway and a PKCλ pathway, and that the latter pathway contributes, at least in part, to insulin stimulation of glucose uptake in 3T3-L1 adipocytes.

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