scholarly journals Ser-3 is important for regulating Mos interaction with and stimulation of mitogen-activated protein kinase kinase.

1995 ◽  
Vol 15 (9) ◽  
pp. 4727-4734 ◽  
Author(s):  
M Chen ◽  
J A Cooper
Keyword(s):  
Protein Kinase ◽  
Catalytic Domain ◽  
Mitogen Activated Protein Kinase ◽  
Early Embryos ◽  
Mitogen Activated Protein ◽  
Hybrid Test ◽  
Two Hybrid ◽  
Autophosphorylation Site ◽  
Protein Kinase Kinase

Mos is a germ cell-specific serine/threonine protein kinase that activates mitogen-activated protein kinase (MAPK) through MAPK kinase (MKK). In Xenopus oocytes, Mos synthesis is required for progesterone-induced activation of MAPK and maturation promoting factor. Injection of Mos or active MAPK causes mitotic arrest in early embryos, suggesting that Mos also acts via MKK and MAPK to induce the arrest of unfertilized eggs in metaphase of meiosis II. We have investigated whether Mos activity is regulated by phosphorylation. Previous studies have identified Ser-3 as the principal autophosphorylation site. We show that Mos interacts with the catalytic domain of MKK in a Saccharomyces cerevisiae two-hybrid test. Acidic substitutions of the sites phosphorylated by Mos in MKK reduce the interaction, implying that the complex may dissociate after phosphorylation of MKK by Mos. Furthermore, the Mos-MKK interaction requires Mos kinase activity, suggesting that Mos autophosphorylation may be involved in the interaction. Substitution of Ser-3 of Mos with Ala reduces the interaction with MKK and also reduces both the activation of MKK by Mos in vitro and cleavage arrest induced by Mos fusion protein in Xenopus embryos. By contrast, substitution of Ser-3 by Glu, an acidic amino acid that mimics phosphoserine, fosters the Mos interaction with MKK and permits activation of MKK in vitro and Mos-induced cleavage arrest. Moreover, the Glu-3 substitution increases the interaction of a kinase-inactive Mos mutant with MKK. Taken together, these results suggest that an important step in Mos activation involves the phosphorylation at Ser-3, which promotes Mos interaction with and activation of MKK.

scholarly journals An analysis of the phosphorylation and activation of extracellular-signal-regulated protein kinase 5 (ERK5) by mitogen-activated protein kinase kinase 5 (MKK5) in vitro

2003 ◽  
Vol 372 (2) ◽  
pp. 567-575 ◽  
Author(s):  
Nimesh MODY ◽  
David G. CAMPBELL ◽  
Nick MORRICE ◽  
Mark PEGGIE ◽  
Philip COHEN
Keyword(s):  
Protein Kinase ◽  
Catalytic Domain ◽  
Similar Rate ◽  
Mitogen Activated Protein Kinase ◽  
Glutathione S Transferase ◽  
293 Cells ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Protein Kinase Kinase

MKK5 expressed as a glutathione S-transferase fusion protein in human embryonic kidney 293 cells activated full-length extracellular-signal-regulated protein kinase (ERK)5 (ERK5wt) as well as the isolated catalytic domain (ERK5cat) in vitro. Activation was accompanied by the phosphorylation of Thr219 and Tyr221, the former residue being phosphorylated preferentially. ERK5cat phosphorylated at Thr219, but not Tyr221, possessed 10% of the activity of the doubly phosphorylated protein towards myelin basic protein, whereas ERK5cat phosphorylated at Tyr221 alone was much less active. Activated ERK5 phosphorylated itself at a number of residues, including Thr28, Ser421, Ser433, Ser496, Ser731 and Thr733. ERK5 phosphorylated at Thr219, but not Tyr221, phosphorylated itself at a similar rate to ERK5 phosphorylated at both Thr219 and Tyr221. Activated ERK5 also phosphorylated mitogen-activated protein kinase kinase 5 (MKK5) extensively at Ser129, Ser137, Ser142 and Ser149, which are located within the region in MKK5 that is thought to interact with ERK5.

scholarly journals PfPK6, a novel cyclin-dependent kinase/mitogen-activated protein kinase-related protein kinase from Plasmodium falciparum

2000 ◽  
Vol 347 (1) ◽  
pp. 255-263 ◽  
Author(s):  
Valerie BRACCHI-RICARD ◽  
Sailen BARIK ◽  
Cherie DELVECCHIO ◽  
Christian DOERIG ◽  
Ratna CHAKRABARTI ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Protein Kinase ◽  
Catalytic Domain ◽  
Sequence Similarity ◽  
Small Subunit ◽  
Mitogen Activated Protein Kinase ◽  
Cdna Clones ◽  
Cyclin Dependent Kinase ◽  
Mitogen Activated Protein

We have isolated a novel protein kinase cDNA, PfPK6, by differential display RT-PCR (DDRT-PCR) of mRNA obtained from different asexual erythrocytic stages of Plasmodium falciparum, which shows sequence similarity to both cyclin-dependent kinase (CDK) and mitogen-activated protein kinase (MAPK) family members. The 915 bp open reading frame (ORF) is interrupted by seven introns and encodes a 305-residue polypeptide with a predicted molecular mass of 35848 Da. Several cDNA clones with some of the intron sequences were isolated, indicating alternate or defective splicing of PfPK6 transcripts because the gene seems to be a single copy located on chromosome 13. The similarity of the catalytic domain of PfPK6 to those of CDK2 and MAPK is 57.3% and 49.6%, respectively. The signature PSTAIRE (single-letter amino acid codes) CDK motif is changed to SKCILRE in PfPK6. The TXY residues that are phosphorylated in MAPKs for their activation are T173PT in PfPK6. Three size classes of PfPK6 transcripts of 6.5, 2.0 and 1.1 kb are up-regulated during the transition of P. falciparum from ring to trophozoite. Western blot analysis suggested the expression of a 35 kDa polypeptide in trophozoites and schizonts. Immunofluorescence studies indicated both nuclear and cytoplasmic localization of PfPK6 in trophozoite, schizont and segmenter stages. In vitro, recombinant PfPK6 phosphorylated itself and also exogenous substrates, histone and the small subunit of the malarial ribonucleotide reductase (R2). The kinase activity of PfPK6 is sensitive to CDK inhibitors such as olomoucine and roscovitine. PfPK6 showed a preference for Mn2+ over Mg2+ ions as a cofactor. The Lys38 → Arg mutant is severely defective in its interaction with ATP and bivalent cations and somewhat defective in catalytic rate for R2 phosphorylation.

Activation of mitogen-activated protein kinase kinase by v-Raf in NIH 3T3 cells and in vitro

Science ◽  
1992 ◽  
Vol 257 (5075) ◽  
pp. 1404-1407 ◽  
Author(s):  
P Dent ◽  
W Haser ◽  
T. Haystead ◽  
L. Vincent ◽  
T. Roberts ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mitogen Activated Protein Kinase ◽  
3T3 Cells ◽  
Mitogen Activated Protein ◽  
Nih 3T3 ◽  
Protein Kinase Kinase ◽  
Nih 3T3 Cells

scholarly journals Activation of a Nuclear Cdc2-Related Kinase within a Mitogen-Activated Protein Kinase-Like TDY Motif by Autophosphorylation and Cyclin-Dependent Protein Kinase-Activating Kinase

2005 ◽  
Vol 25 (14) ◽  
pp. 6047-6064 ◽  
Author(s):  
Zheng Fu ◽  
Melanie J. Schroeder ◽  
Jeffrey Shabanowitz ◽  
Philipp Kaldis ◽  
Kasumi Togawa ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nuclear Localization ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Mitogen Activated Protein Kinase ◽  
Intestinal Cell ◽  
Dependent Protein Kinase ◽  
Mitogen Activated Protein ◽  
Dependent Protein

ABSTRACT Male germ cell-associated kinase (MAK) and intestinal cell kinase (ICK) are nuclear Cdc2-related kinases with nearly identical N-terminal catalytic domains and more divergent C-terminal noncatalytic domains. The catalytic domain is also related to mitogen-activated protein kinases (MAPKs) and contains a corresponding TDY motif. Nuclear localization of ICK requires subdomain XI and interactions of the conserved Arg-272, but not kinase activity or, surprisingly, any of the noncatalytic domain. Further, nuclear localization of ICK is required for its activation. ICK is activated by dual phosphorylation of the TDY motif. Phosphorylation of Tyr-159 in the TDY motif requires ICK autokinase activity but confers only basal kinase activity. Full activation requires additional phosphorylation of Thr-157 in the TDY motif. Coexpression of ICK with constitutively active MEK1 or MEK5 fails to increase ICK phosphorylation or activity, suggesting that MEKs are not involved. ICK and MAK are related to Ime2p in budding yeast, and cyclin-dependent protein kinase-activating kinase Cak1p has been placed genetically upstream of Ime2p. Recombinant Cak1p phosphorylates Thr-157 in the TDY motif of recombinant ICK and activates its activity in vitro. Coexpression of ICK with wild-type CAK1 but not kinase-inactive CAK1 in cells also increases ICK phosphorylation and activity. Our studies establish ICK as the prototype for a new group of MAPK-like kinases requiring dual phosphorylation at TDY motifs.

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.

Inhibition of Mitogen Activated Protein Kinase Kinase (MEK1) Is Effective Against CLL Cells Cultured in Media Alone or in a Supportive Microenvironment and Is Synergistic with Fludarabine in a Mechanism That Involves Decreased Levels of Reactive Oxygen Species and MCL-1 Protein

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1804-1804
Author(s):  
Giles Best ◽  
Kyle Crassini ◽  
Williams Stevenson ◽  
Stephen P. Mulligan
Keyword(s):  
Reactive Oxygen Species ◽  
Clinical Trials ◽  
Protein Kinase ◽  
Mek Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Oxygen Species ◽  
Mitogen Activated Protein ◽  
Cells Cultured ◽  
Protein Kinase Kinase

Abstract Abstract 1804 Background Despite the high response rates of patients with Chronic Lymphocytic Leukemia (CLL) to the fludarabine (F), cyclophosphamide (C), rituximab (R) regimen, relapsed or refractory disease is common. Novel therapeutic approaches are required that are effective in this setting. Targeting specific signaling molecules is proving an effective strategy for treating patients who are refractory to FCR. Given that the mitogen-activated protein kinase pathway (MAPK) pathway is constitutively active in CLL cells and that inhibitors of mitogen-activated protein kinase kinase (MEK1) in this pathway are in clinical trials for solid tumors, we sought to investigate the potential of MEK1 as a therapeutic target in CLL. Results Inhibition of MEK1/2 using MEK inhibitor I (MEKi; Calbiochem/Merck) induced apoptosis in the MEC1 cell line and in 18 patient samples. Importantly, sensitivity of the patient samples occurred irrespective of ATM/TP53 functional status, of poor prognostic features or of treatment history. MEKi was also effective against 4 CLL patient samples cultured in an in vitro model of the tumor microenvironment, albeit with a significantly higher IC50 than observed against CLL cells cultured in media alone. As fludarabine-based therapies have become the mainstay of CLL treatment, we investigated the effect of combining the MEK inhibitor with this purine analogue. Synergy between MEKi and fludarabine was apparent against the MEC-1 cell line and 10 patient samples. Dose reduction indices (DRI) calculated from the drug combination indicate this synergy was predominantly due to an increase in fludarabine sensitivity. Investigation of the mechanisms of the synergy between MEKi and fludarabine suggests decreased levels of reactive oxygen species (ROS) and expression of the pro-survival protein, MCL-1, may be contributing factors (see figure). Summary These data suggest for the first time that inhibition of MEK1/2 may represent a potential therapeutic option for CLL patients. The efficacy of the MEK inhibitor against CLL cells cultured in the supportive in vitro environment suggest that this approach may also be effective at targeting the proliferative fraction of CLL cells in the tumor microenvironment. As clinical trials of MEK1/2 inhibitors are currently underway in solid tissue malignancies, our data suggest that trials of these agents may also be warranted for high risk CLL. Disclosures: No relevant conflicts of interest to declare.

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