scholarly journals Phosphorylation of TPL-2 on Serine 400 Is Essential for Lipopolysaccharide Activation of Extracellular Signal-Regulated Kinase in Macrophages

2007 ◽  
Vol 27 (21) ◽  
pp. 7355-7364 ◽  
Author(s):  
M. J. Robinson ◽  
S. Beinke ◽  
A. Kouroumalis ◽  
P. N. Tsichlis ◽  
S. C. Ley
Keyword(s):  
Kinase Activity ◽  
Mitogen Activated Protein Kinase ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Control Step ◽  
Cytokine Tumor Necrosis Factor ◽  
Mek Kinase ◽  
Lps Activation ◽  
Stimulation Of

ABSTRACT Tumor progression locus 2 (TPL-2) kinase is essential for Toll-like receptor 4 activation of the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) and for upregulation of the inflammatory cytokine tumor necrosis factor (TNF) in lipopolysaccharide (LPS)-stimulated macrophages. LPS activation of ERK requires TPL-2 release from associated NF-κB1 p105, which blocks TPL-2 access to its substrate, the ERK kinase MEK. Here we demonstrate that TPL-2 activity is also regulated independently of p105, since LPS stimulation was still needed for TPL-2-dependent activation of ERK in Nfkb1 −/− macrophages. In wild-type macrophages, LPS induced the rapid phosphorylation of serine (S) 400 in the TPL-2 C-terminal tail. Mutation of this conserved residue to alanine (A) blocked the ability of retrovirally expressed TPL-2 to induce the activation of ERK in LPS-stimulated Nfkb1 −/− macrophages. TPL-2S400A expression also failed to reconstitute LPS activation of ERK and induction of TNF in Map3k8 −/− macrophages, which lack endogenous TPL-2. Consistently, the S400A mutation was found to block LPS stimulation of TPL-2 MEK kinase activity. Thus, induction of TPL-2 MEK kinase activity by LPS stimulation of macrophages requires TPL-2 phosphorylation on S400, in addition to its release from NF-κB1 p105. Oncogenic C-terminal truncations of TPL-2 that remove S400 could promote its transforming potential by eliminating this critical control step.

scholarly journals Lipopolysaccharide Activation of the TPL-2/MEK/Extracellular Signal-Regulated Kinase Mitogen-Activated Protein Kinase Cascade Is Regulated by IκB Kinase-Induced Proteolysis of NF-κB1 p105

2004 ◽  
Vol 24 (21) ◽  
pp. 9658-9667 ◽  
Author(s):  
S. Beinke ◽  
M. J. Robinson ◽  
M. Hugunin ◽  
S. C. Ley
Keyword(s):  
Kinase Activity ◽  
Extracellular Signal Regulated Kinase ◽  
Iκb Kinase ◽  
Map Kinase Cascade ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Kinase Cascade ◽  
Mek Kinase ◽  
Lps Activation ◽  
Stimulation Of

ABSTRACT The MEK kinase TPL-2 (also known as Cot) is required for lipopolysaccharide (LPS) activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase cascade in macrophages and consequent upregulation of genes involved in innate immune responses. In resting cells, TPL-2 forms a stoichiometric complex with NF-κB1 p105, which negatively regulates its MEK kinase activity. Here, it is shown that lipopolysaccharide (LPS) stimulation of primary macrophages causes the release of both long and short forms of TPL-2 from p105 and that TPL-2 MEK kinase activity is restricted to this p105-free pool. Activation of TPL-2, MEK, and ERK by LPS is also demonstrated to require proteasome-mediated proteolysis. p105 is known to be proteolysed by the proteasome following stimulus-induced phosphorylation of two serines in its PEST region by the IκB kinase (IKK) complex. Expression of a p105 point mutant, which is not susceptible to signal-induced proteolysis, in RAW264.7 macrophages impairs LPS-induced release of TPL-2 from p105 and its subsequent activation of MEK. Furthermore, expression of wild-type but not mutant p105 reconstitutes LPS stimulation of MEK and ERK phosphorylation in primary NF-κB1-deficient macrophages. Consistently, pharmacological blockade of IKK inhibits LPS-induced release of TPL-2 from p105 and TPL-2 activation. These data show that IKK-induced p105 proteolysis is essential for LPS activation of TPL-2, thus revealing a novel function of IKK in the regulation of the ERK MAP kinase cascade.

scholarly journals Insulin-independent, MAPK-dependent stimulation of NKCC activity in skeletal muscle

2001 ◽  
Vol 281 (2) ◽  
pp. R561-R571 ◽  
Author(s):  
Jennifer A. Wong ◽  
Aidar R. Gosmanov ◽  
Edward G. Schneider ◽  
Donald B. Thomason
Keyword(s):  
Skeletal Muscle ◽  
Atpase Activity ◽  
Insulin Treatment ◽  
Ex Vivo ◽  
Mitogen Activated Protein Kinase ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Significant Pathway ◽  
Stimulation Of

Na+-K+-Cl−cotransporter (NKCC) activity in quiescent skeletal muscle is modest. However, ex vivo stimulation of muscle for as little as 18 contractions (1 min, 0.3 Hz) dramatically increased the activity of the cotransporter, measured as the bumetanide-sensitive 86Rb influx, in both soleus and plantaris muscles. This activation of cotransporter activity remained relatively constant for up to 10-Hz stimulation for 1 min, falling off at higher frequencies (30-Hz stimulation for 1 min). Similarly, stimulation of skeletal muscle with adrenergic receptor agonists phenylephrine, isoproterenol, or epinephrine produced a dramatic stimulation of NKCC activity. It did not appear that stimulation of NKCC activity was a reflection of increased Na+-K+-ATPase activity because insulin treatment did not stimulate NKCC activity, despite insulin's well-known stimulation of Na+-K+-ATPase activity. Stimulation of NKCC activity could be blocked by pretreatment with inhibitors of mitogen-activated protein kinase (MAPK) kinase 1/2 (MEK1/2) activity, indicating that activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) MAPKs may be required. These data indicate a regulated NKCC activity in skeletal muscle that may provide a significant pathway for potassium transport into skeletal muscle fibers.

scholarly journals MEK-1 phosphorylation by MEK kinase, Raf, and mitogen-activated protein kinase: analysis of phosphopeptides and regulation of activity.

1994 ◽  
Vol 5 (2) ◽  
pp. 193-201 ◽  
Author(s):  
A M Gardner ◽  
R R Vaillancourt ◽  
C A Lange-Carter ◽  
G L Johnson
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Peptide Mapping ◽  
Mitogen Activated Protein Kinase ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Regulation Of Activity ◽  
Mek Kinase ◽  
Mutant Kinase

MEK-1 is a dual threonine and tyrosine recognition kinase that phosphorylates and activates mitogen-activated protein kinase (MAPK). MEK-1 is in turn activated by phosphorylation. Raf and MAPK/extracellular signal-regulated kinase kinase (MEKK) independently phosphorylate and activate MEK-1. Recombinant MEK-1 is also capable of autoactivation. Purified recombinant wild type MEK-1 and a mutant kinase inactive MEK-1 were used as substrates for MEKK, Raf, and autophosphorylation. MEK-1 phosphorylation catalyzed by Raf, MEKK, or autophosphorylation resulted in activation of MEK-1 kinase activity measured by phosphorylation of a mutant kinase inactive MAPK. Phosphoamino acid analysis and peptide mapping identified similar MEK-1 tryptic phosphopeptides after phosphorylation by MEK kinase, Raf, or MEK-1 autophosphorylation. MEK-1 is phosphorylated by MAPK at sites different from that for Raf and MEKK. Phosphorylation of MEK-1 by MAPK does not affect MEK-1 kinase activity. Several phosphorylation sites present in MEK-1 immunoprecipitated from 32P-labeled cells after stimulation with epidermal growth factor were common to the in vitro phosphorylated enzyme. The major site of MAPK phosphorylation in MEK-1 is threonine 292. Mutation of threonine 292 to alanine eliminates 90% of MAPK catalyzed phosphorylation of MEK-1 but does not influence MEK-1 activity. The results demonstrate that MEKK and Raf regulate MEK-1 activity by phosphorylation of common residues and thus, two independent protein kinases converge at MEK-1 to regulate the activity of MAPK.

scholarly journals Activation of the Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Pathway by Conventional, Novel, and Atypical Protein Kinase C Isotypes

1998 ◽  
Vol 18 (2) ◽  
pp. 790-798 ◽  
Author(s):  
Dorothee C. Schönwasser ◽  
Richard M. Marais ◽  
Christopher J. Marshall ◽  
Peter J. Parker
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Mapk Cascade ◽  
Mitogen Activated Protein Kinase ◽  
Extracellular Signal Regulated Kinase ◽  
Erk Mapk ◽  
Kinase C ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Stimulation Of

ABSTRACT Phorbol ester treatment of quiescent Swiss 3T3 cells leads to cell proliferation, a response thought to be mediated by protein kinase C (PKC), the major cellular receptor for this class of agents. We demonstrate here that this proliferation is dependent on the activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) cascade. It is shown that dominant-negative PKC-α inhibits stimulation of the ERK/MAPK pathway by phorbol esters in Cos-7 cells, demonstrating a role for PKC in this activation. To assess the potential specificity of PKC isotypes mediating this process, constitutively active mutants of six PKC isotypes (α, β1, δ, ɛ, η, and ζ) were employed. Transient transfection of these PKC mutants into Cos-7 cells showed that members of all three groups of PKC (conventional, novel, and atypical) are able to activate p42 MAPK as well as its immediate upstream activator, the MAPK/ERK kinase MEK-1. At the level of Raf, the kinase that phosphorylates MEK-1, the activation cascade diverges; while conventional and novel PKCs (isotypes α and η) are potent activators of c-Raf1, atypical PKC-ζ cannot increase c-Raf1 activity, stimulating MEK by an independent mechanism. Stimulation of c-Raf1 by PKC-α and PKC-η was abrogated for RafCAAX, which is a membrane-localized, partially active form of c-Raf1. We further established that activation of Raf is independent of phosphorylation at serine residues 259 and 499. In addition to activation, we describe a novel Raf desensitization induced by PKC-α, which acts to prevent further Raf stimulation by growth factors. The results thus demonstrate a necessary role for PKC and p42 MAPK activation in 12-O-tetradecanoylphorbol-13-acetate induced mitogenesis and provide evidence for multiple PKC controls acting on this MAPK cascade.

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