scholarly journals Activation of MAP kinase-activated protein kinase 2 in human neutrophils after phorbol ester or fMLP peptide stimulation

Blood ◽  
1996 ◽  
Vol 87 (12) ◽  
pp. 5287-5296 ◽  
Author(s):  
YL Zu ◽  
Y Ai ◽  
A Gilchrist ◽  
ME Labadia ◽  
RI Sha'afi ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Enzymatic Activity ◽  
Map Kinase ◽  
Phorbol Ester ◽  
Map Kinases ◽  
Phosphorylation Site ◽  
Human Neutrophils ◽  
Inhibitory Peptide

In response to extracellular stimulation, one of the earliest events in human neutrophils is protein phosphorylation, which mediates signal transduction and leads to the regulation of cellular functions. Mitogen- activated protein (MAP) kinases are rapidly activated by a variety of mitogens, cytokines, and stresses. The activated MAP kinases in turn regulate their substrate molecules by phosphorylation. MAP kinase- activated protein (MAPKAP) kinase 2, a Ser/Thr kinase, has been shown to be phosphorylated by p38 MAP kinase both in vivo and in vitro. Phosphorylation of the Thr-334 site of MAPKAP kinase 2 results in a conformational change with subsequent activation of the enzyme. To better define the role of MAPKAP kinase 2 in the activation of human neutrophils, its enzymatic activity was measured after stimulation by either a phorbol ester (phorbol myristate acetate [PMA]), a potent protein kinase C activator, or the tripeptide fMLP, which is a chemotactic factor. The in vitro kinase assays indicate that both PMA and fMLP stimulated a transient increase in the enzymatic activity of cellular MAPKAP kinase 2. The induced kinase activation was concentration-dependent and reached a maximum at 5 minutes for PMA and 1 minute for fMLP. To identify potential substrate molecules for MAPKAP kinase 2, a highly active kinase mutant was generated by mutating the MAP kinase phosphorylation site in the C-terminal region. The replacement of threonine 334 with alanine resulted in a marked augmentation of catalytic activity. Analysis of in vitro protein phosphorylation in the presence of the active kinase indicates that a 60-kD cytosolic protein (p60) was markedly phosphorylated and served as the major substrate for MAPKAP kinase 2 in human neutrophils. Based on the MAPKAP kinase 2 phosphorylation site of Hsp27, a competitive inhibitory peptide was synthesized. This competitive inhibitory peptide specifically inhibited MAPKAP kinase 2 enzymatic activity, as well as the in vitro and in vivo kinase-induced p60 phosphorylation. To assess the contribution of MAPKAP kinase 2 in neutrophil function, the oxidative burst response after manipulation of endogenous kinase activity was measured. Intracellular delivery of the competitive inhibitory peptide into human neutrophils reduced both PMA- and fMLP- stimulated superoxide anion production. Thus, the results strongly suggest that MAPKAP kinase 2 is involved in the activation of human neutrophils.

Phosphorylation of caldesmon by ERK MAP kinases in smooth muscle

2000 ◽  
Vol 278 (4) ◽  
pp. C718-C726 ◽  
Author(s):  
Jason C. Hedges ◽  
Brian C. Oxhorn ◽  
Michael Carty ◽  
Leonard P. Adam ◽  
Ilia A. Yamboliev ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Map Kinase ◽  
Map Kinases ◽  
Isometric Force ◽  
Phosphorylation Site ◽  
Smooth Muscle Contraction ◽  
Tracheal Smooth Muscle ◽  
Muscarinic Agonists

Phosphorylation of h-caldesmon has been proposed to regulate airway smooth muscle contraction. Both extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinases phosphorylate h-caldesmon in vitro. To determine whether both enzymes phosphorylate caldesmon in vivo, phosphorylation-site-selective antibodies were used to assay phosphorylation of MAP kinase consensus sites. Stimulation of cultured tracheal smooth muscle cells with ACh or platelet-derived growth factor increased caldesmon phosphorylation at Ser789 by about twofold. Inhibiting ERK MAP kinase activation with 50 μM PD-98059 blocked agonist-induced caldesmon phosphorylation completely. Inhibiting p38 MAP kinases with 25 μM SB-203580 had no effect on ACh-induced caldesmon phosphorylation. Carbachol stimulation increased caldesmon phosphorylation at Ser789 in intact tracheal smooth muscle, which was blocked by the M2 antagonist AF-DX 116 (1 μM). AF-DX 116 inhibited carbachol-induced isometric contraction by 15 ± 1.4%, thus dissociating caldesmon phosphorylation from contraction. Activation of M2 receptors leads to activation of ERK MAP kinases and phosphorylation of caldesmon with little or no functional effect on isometric force. P38 MAP kinases are also activated by muscarinic agonists, but they do not phosphorylate caldesmon in vivo.

scholarly journals Active MAP Kinase in Mitosis: Localization at Kinetochores and Association with the Motor Protein CENP-E

1998 ◽  
Vol 142 (6) ◽  
pp. 1547-1558 ◽  
Author(s):  
Maja Zecevic ◽  
Andrew D. Catling ◽  
Scott T. Eblen ◽  
Luigina Renzi ◽  
James C. Hittle ◽  
...  
Keyword(s):  
Map Kinase ◽  
Map Kinases ◽  
Phosphorylation Site ◽  
Motor Protein ◽  
Nuclear Envelope Breakdown ◽  
Chromosome Congression ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

To investigate possible involvement of the mitogen-activated protein (MAP) kinases ERK1 and ERK2 (extracellular signal-regulated kinases) in somatic cell mitosis, we have used indirect immunofluorescence with a highly specific phospho-MAP kinase antibody and found that a portion of the active MAP kinase is localized at kinetochores, asters, and the midbody during mitosis. Although the aster labeling was constant from the time of nuclear envelope breakdown, the kinetochore labeling first appeared at early prometaphase, started to fade during chromosome congression, and then disappeared at midanaphase. At telophase, active MAP kinase localized at the midbody. Based on colocalization and the presence of a MAP kinase consensus phosphorylation site, we identified the kinetochore motor protein CENP-E as a candidate mitotic substrate for MAP kinase. CENP-E was phosphorylated in vitro by MAP kinase on sites that are known to regulate its interactions with microtubules and was found to associate in vivo preferentially with the active MAP kinase during mitosis. Therefore, the presence of active MAP kinase at specific mitotic structures and its interaction with CENP-E suggest that MAP kinase could play a role in mitosis at least in part by altering the ability of CENP-E to mediate interactions between chromosomes and microtubules.

scholarly journals Protein kinase D inhibits plasma membrane Na+/H+exchanger activity

1999 ◽  
Vol 277 (6) ◽  
pp. C1202-C1209 ◽  
Author(s):  
Robert S. Haworth ◽  
James Sinnett-Smith ◽  
Enrique Rozengurt ◽  
Metin Avkiran
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Protein Kinase D ◽  
Wild Type ◽  
Ph Indicator

The regulation of plasma membrane Na+/H+exchanger (NHE) activity by protein kinase D (PKD), a novel protein kinase C- and phorbol ester-regulated kinase, was investigated. To determine the effect of PKD on NHE activity in vivo, intracellular pH (pHi) measurements were made in COS-7 cells by microepifluorescence using the pH indicator cSNARF-1. Cells were transfected with empty vector (control), wild-type PKD, or its kinase-deficient mutant PKD-K618M, together with green fluorescent protein (GFP). NHE activity, as reflected by the rate of acid efflux ( J H), was determined in single GFP-positive cells following intracellular acidification. Overexpression of wild-type PKD had no significant effect on J H(3.48 ± 0.25 vs. 3.78 ± 0.24 mM/min in control at pHi 7.0). In contrast, overexpression of PKD-K618M increased J H (5.31 ± 0.57 mM/min at pHi 7.0; P < 0.05 vs. control). Transfection with these constructs produced similar effects also in A-10 cells, indicating that native PKD may have an inhibitory effect on NHE in both cell types, which is relieved by a dominant-negative action of PKD-K618M. Exposure of COS-7 cells to phorbol ester significantly increased J H in control cells but failed to do so in cells overexpressing either wild-type PKD (due to inhibition by the overexpressed PKD) or PKD-K618M (because basal J Hwas already near maximal). A fusion protein containing the cytosolic regulatory domain (amino acids 637–815) of NHE1 (the ubiquitous NHE isoform) was phosphorylated in vitro by wild-type PKD, but with low stoichiometry. These data suggest that PKD inhibits NHE activity, probably through an indirect mechanism, and represents a novel pathway in the regulation of the exchanger.

Phosphorylation of Argonaute 2 at serine-387 facilitates its localization to processing bodies

2008 ◽  
Vol 413 (3) ◽  
pp. 429-436 ◽  
Author(s):  
Yan Zeng ◽  
Heidi Sankala ◽  
Xiaoxiao Zhang ◽  
Paul R. Graves
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Kinase Inhibitor ◽  
Mapk Pathway ◽  
Phosphorylation Site ◽  
Mitogen Activated Protein Kinase ◽  
Processing Bodies ◽  
Mitogen Activated Protein

Ago (Argonaute) proteins are essential effectors of RNA-mediated gene silencing. To explore potential regulatory mechanisms for Ago proteins, we examined the phosphorylation of human Ago2. We identified serine-387 as the major Ago2 phosphorylation site in vivo. Phosphorylation of Ago2 at serine-387 was significantly induced by treatment with sodium arsenite or anisomycin, and arsenite-induced phosphorylation was inhibited by a p38 MAPK (mitogen-activated protein kinase) inhibitor, but not by inhibitors of JNK (c-Jun N-terminal kinase) or MEK [MAPK/ERK (extracellular-signal-regulated kinase) kinase]. MAPKAPK2 (MAPK-activated protein kinase-2) phosphorylated bacterially expressed full-length human Ago2 at serine-387 in vitro, but not the S387A mutant. Finally, mutation of serine-387 to an alanine residue or treatment of cells with a p38 MAPK inhibitor reduced the localization of Ago2 to processing bodies. These results suggest a potential regulatory mechanism for RNA silencing acting through Ago2 serine-387 phosphorylation mediated by the p38 MAPK pathway.

scholarly journals c-Jun N-terminal phosphorylation correlates with activation of the JNK subgroup but not the ERK subgroup of mitogen-activated protein kinases.

1994 ◽  
Vol 14 (10) ◽  
pp. 6683-6688 ◽  
Author(s):  
A Minden ◽  
A Lin ◽  
T Smeal ◽  
B Dérijard ◽  
M Cobb ◽  
...  
Keyword(s):  
Map Kinase ◽  
Map Kinases ◽  
New Members ◽  
Protein Map ◽  
Mitogen Activated Protein ◽  
Extracellular Stimuli ◽  
Terminal Site ◽  
Stimulation Of

c-Jun transcriptional activity is stimulated by phosphorylation at two N-terminal sites: Ser-63 and -73. Phosphorylation of these sites is enhanced in response to a variety of extracellular stimuli, including growth factors, cytokines, and UV irradiation. New members of the mitogen-activated protein (MAP) kinase group of signal-transducing enzymes, termed JNKs, bind to the activation domain of c-Jun and specifically phosphorylate these sites. However, the N-terminal sites of c-Jun were also suggested to be phosphorylated by two other MAP kinases, ERK1 and ERK2. Despite these reports, we find that unlike the JNKs, ERK1 and ERK2 do not phosphorylate the N-terminal sites of c-Jun in vitro; instead they phosphorylate an inhibitory C-terminal site. Furthermore, the phosphorylation of c-Jun in vivo at the N-terminal sites correlates with activation of the JNKs but not the ERKs. The ERKs are probably involved in the induction of c-fos expression and thereby contribute to the stimulation of AP-1 activity. Our study suggests that two different branches of the MAP kinase group are involved in the stimulation of AP-1 activity through two different mechanisms.

scholarly journals BI-D1870 is a specific inhibitor of the p90 RSK (ribosomal S6 kinase) isoforms in vitro and in vivo

2006 ◽  
Vol 401 (1) ◽  
pp. 29-38 ◽  
Author(s):  
Gopal P. Sapkota ◽  
Lorna Cummings ◽  
Felicity S. Newell ◽  
Christopher Armstrong ◽  
Jennifer Bain ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Phorbol Ester ◽  
Glycogen Synthase ◽  
Specific Inhibitor ◽  
Camp Response Element Binding ◽  
S6 Kinase ◽  
Ribosomal S6 Kinase

Hormones and growth factors induce the activation of a number of protein kinases that belong to the AGC subfamily, including isoforms of PKA, protein kinase B (also known as Akt), PKC, S6K p70 (ribosomal S6 kinase), RSK (p90 ribosomal S6 kinase) and MSK (mitogen- and stress-activated protein kinase), which then mediate many of the physiological processes that are regulated by these extracellular agonists. It can be difficult to assess the individual functions of each AGC kinase because their substrate specificities are similar. Here we describe the small molecule BI-D1870, which inhibits RSK1, RSK2, RSK3 and RSK4 in vitro with an IC50 of 10–30 nM, but does not signi-ficantly inhibit ten other AGC kinase members and over 40 other protein kinases tested at 100-fold higher concentrations. BI-D1870 is cell permeant and prevents the RSK-mediated phorbol ester- and EGF (epidermal growth factor)-induced phosphoryl-ation of glycogen synthase kinase-3β and LKB1 in human embry-onic kidney 293 cells and Rat-2 cells. In contrast, BI-D1870 does not affect the agonist-triggered phosphorylation of substrates for six other AGC kinases. Moreover, BI-D1870 does not suppress the phorbol ester- or EGF-induced phosphorylation of CREB (cAMP-response-element-binding protein), consistent with the genetic evidence indicating that MSK, and not RSK, isoforms mediate the mitogen-induced phosphorylation of this transcription factor.

scholarly journals Full-length cardiac Na+/Ca2+ exchanger 1 protein is not phosphorylated by protein kinase A

2011 ◽  
Vol 300 (5) ◽  
pp. C989-C997 ◽  
Author(s):  
Pimthanya Wanichawan ◽  
William E. Louch ◽  
Kristin H. Hortemo ◽  
Bjørg Austbø ◽  
Per Kristian Lunde ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Caspase 3 ◽  
Fluorescent Protein ◽  
Mutational Analysis ◽  
Phosphorylation Site ◽  
Full Length ◽  
Kinase A

The cardiac Na+/Ca2+ exchanger 1 (NCX1) is an important regulator of intracellular Ca2+ homeostasis and cardiac function. Several studies have indicated that NCX1 is phosphorylated by the cAMP-dependent protein kinase A (PKA) in vitro, which increases its activity. However, this finding is controversial and no phosphorylation site has so far been identified. Using bioinformatic analysis and peptide arrays, we screened NCX1 for putative PKA phosphorylation sites. Although several NCX1 synthetic peptides were phosphorylated by PKA in vitro, only one PKA site (threonine 731) was identified after mutational analysis. To further examine whether NCX1 protein could be PKA phosphorylated, wild-type and alanine-substituted NCX1-green fluorescent protein (GFP)-fusion proteins expressed in human embryonic kidney (HEK)293 cells were generated. No phosphorylation of full-length or calpain- or caspase-3 digested NCX1-GFP was observed with purified PKA-C and [γ-32P]ATP. Immunoblotting experiments with anti-PKA substrate and phosphothreonine-specific antibodies were further performed to investigate phosphorylation of endogenous NCX1. Phospho-NCX1 levels were also not increased after forskolin or isoproterenol treatment in vivo, in isolated neonatal cardiomyocytes, or in total heart homogenate. These data indicate that the novel in vitro PKA phosphorylation site is inaccessible in full-length as well as in calpain- or caspase-3 digested NCX1 protein, suggesting that NCX1 is not a direct target for PKA phosphorylation.

scholarly journals Phosphorylation of the c-Fos transrepression domain by mitogen-activated protein kinase and 90-kDa ribosomal S6 kinase

1993 ◽  
Vol 90 (23) ◽  
pp. 10952-10956 ◽  
Author(s):  
R H Chen ◽  
C Abate ◽  
J Blenis
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Mitogen Activated Protein Kinase ◽  
S6 Kinase ◽  
Downstream Signaling ◽  
C Terminus ◽  
Mitogen Activated Protein ◽  
Ribosomal S6 Kinase

Phosphorylation of the C terminus of c-Fos has been implicated in serum response element-mediated repression of c-fos transcription after its induction by serum growth factors. The growth-regulated enzymes responsible for this phosphorylation in early G1 phase of the cell cycle and the sites of phosphorylation have not been identified. We now provide evidence that two growth-regulated, nucleus- and cytoplasm-localized protein kinases, 90-kDa ribosomal S6 kinase (RSK) and mitogen-activated protein kinase (MAP kinase), contribute to the serum-induced phosphorylation of c-Fos. The major phosphopeptides derived from biosynthetically labeled c-Fos correspond to phosphopeptides generated after phosphorylation of c-Fos in vitro with both RSK and MAP kinase. The phosphorylation sites identified for RSK (Ser-362) and MAP kinase (Ser-374) are in the transrepression domain. Cooperative phosphorylation at these sites by both enzymes was observed in vitro and reflected in vivo by the predominance of the peptide phosphorylated on both sites, as opposed to singly phosphorylated peptides. This study suggests a role for nuclear RSK and MAP kinase in modulating newly synthesized c-Fos phosphorylation and downstream signaling.

scholarly journals Targeting redox regulatory site of protein kinase B impedes neutrophilic inflammation in lung injury

2018 ◽  
Author(s):  
Po-Jen Chen ◽  
I-Ling Ko ◽  
Chia-Lin Lee ◽  
Hao-Chun Hu ◽  
Fang-Rong Chang ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Lung Injury ◽  
Inflammatory Responses ◽  
Protein Kinase B ◽  
Reduced Form ◽  
Human Neutrophils ◽  
Akt Inhibitor ◽  
Neutrophilic Inflammation

AbstractNeutrophil activation has a pathogenic effect in inflammatory diseases. Protein kinase B (PKB)/AKT regulates diverse cellular responses. However, the significance of AKT in neutrophilic inflammation is still not well understood. Here, we identified CLLV-1 as a novel AKT inhibitor. CLLV-1 inhibited respiratory burst, degranulation, chemotaxis, and AKT phosphorylation in activated human neutrophils and dHL-60 cells. Significantly, CLLV-1 blocked AKT activity and covalently reacted with AKT Cys310 in vitro. The AKT309-313 peptide-CLLV-1 adducts were determined by NMR or mass spectrometry assay. The alkylation agent-conjugated AKT (reduced form) level was also inhibited by CLLV-1. Additionally, CLLV-1 ameliorated lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. CLLV-1 acts as a covalent allosteric AKT inhibitor by targeting AKT Cys310 to restrain inflammatory responses in human neutrophils and LPS-induced ALI in vivo. Our findings provide a mechanistic framework for redox modification of AKT that may serve as a novel pharmacological target to alleviate neutrophilic inflammation.

scholarly journals Mitogen-activated protein kinase kinase 1 (MKK1) is negatively regulated by threonine phosphorylation

1994 ◽  
Vol 14 (3) ◽  
pp. 1594-1602
Author(s):  
A J Rossomando ◽  
P Dent ◽  
T W Sturgill ◽  
D R Marshak
Keyword(s):  
Protein Kinase ◽  
Phosphorylation Site ◽  
Mitogen Activated Protein Kinase ◽  
Dual Specificity ◽  
Mitogen Activated Protein ◽  
Kinase Cascade ◽  
Protein Kinase Cascade ◽  
Protein Kinase Kinase

Mitogen-activated protein kinase kinase 1 (MKK1), a dual-specificity tyrosine/threonine protein kinase, has been shown to be phosphorylated and activated by the raf oncogene product as part of the mitogen-activated protein kinase cascade. Here we report the phosphorylation and inactivation of MKK1 by phosphorylation on threonine 286 and threonine 292. MKK1 contains a consensus phosphorylation site for p34cdc2, a serine/threonine protein kinase that regulates the cell division cycle, at Thr-286 and a related site at Thr-292. p34cdc2 catalyzes the in vitro phosphorylation of MKK1 on both of these threonine residues and inactivates MKK1 enzymatic activity. Both sites are phosphorylated in vivo as well. The data presented in this report provide evidence that MKK1 is negatively regulated by threonine phosphorylation.

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