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 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 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.

Identification of the autophosphorylation sites and characterization of their effects in the protein kinase DYRK1A

2001 ◽  
Vol 359 (3) ◽  
pp. 497-505 ◽  
Author(s):  
Sunke HIMPEL ◽  
Pascal PANZER ◽  
Klaus EIRMBTER ◽  
Hanna CZAJKOWSKA ◽  
Muhammed SAYED ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Mammalian Cells ◽  
Catalytic Domain ◽  
Molecular Model ◽  
Enzymic Activity ◽  
Mitogen Activated Protein Kinase ◽  
Kinase Family ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

Protein kinases of the DYRK (‘dual-specificity tyrosine-regulated kinase’) family are characterized by a conserved Tyr-Xaa-Tyr motif (Tyr-319–Tyr-321) in a position exactly corresponding to the activation motif of the mitogen-activated protein kinase (MAP kinase) family (Thr-Xaa-Tyr). In a molecular model of the catalytic domain of DYRK1A, the orientation of phosphorylated Tyr-321 is strikingly similar to that of Tyr-185 in the known structure of the activated MAP kinase, extracellular-signal-regulated kinase 2. Consistent with our model, substitution of Tyr-321 but not of Tyr-319 by phenylalanine markedly reduced the enzymic activity of recombinant DYRK1A expressed in either Escherichia coli or mammalian cells. Direct identification of phosphorylated residues by tandem MS confirmed that Tyr-321, but not Tyr-319, was phosphorylated. When expressed in COS-7 cells, DYRK1A was found to be fully phosphorylated on Tyr-321. A catalytically inactive mutant of DYRK1A contained no detectable phosphotyrosine, indicating that Tyr-321 is autophosphorylated by DYRK1A. MS identified Tyr-111 and Ser-97 as additional autophosphorylation sites in the non-catalytic N-terminal domain of bacterially expressed DYRK1A. Enzymic activity was not affected in the DYRK1A-Y111F mutant. The present experimental data and the molecular model indicate that the activity of DYRK1A is dependent on the autophosphorylation of a conserved tyrosine residue in the activation loop.

scholarly journals Phosphorylation of the ARE-binding protein DAZAP1 by ERK2 induces its dissociation from DAZ

2006 ◽  
Vol 399 (2) ◽  
pp. 265-273 ◽  
Author(s):  
Simon Morton ◽  
Huei-Ting Yang ◽  
Ntsane Moleleki ◽  
David G. Campbell ◽  
Philip Cohen ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Rna Binding ◽  
Binding Protein ◽  
Mitogen Activated Protein Kinase ◽  
Raw 264.7 ◽  
Raw 264.7 Macrophages ◽  
Hek 293 ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

A protein in RAW 264.7 macrophages, which became phosphorylated in response to LPS (lipopolysaccharide), was identified as the RNA-binding protein called DAZAP1 [DAZ (deleted in azoospermia)-associated protein 1]. The phosphorylation of this protein was prevented by specific inhibition of MKK1 [MAPK (mitogen-activated protein kinase) kinase 1], indicating that it was phosphorylated via the classical MAPK cascade. Further experiments showed that DAZAP1 was phosphorylated stoichiometrically in vitro by ERK2 (extracellular-signal-regulated protein kinase 2) at two Thr-Pro sequences (Thr269 and Thr315), and that both sites became phosphorylated in HEK-293 (human embryonic kidney 293) cells in response to PMA or EGF (epidermal growth factor), or RAW 264.7 macrophages in response to LPS. Phosphorylation induced by each stimulus was prevented by two structurally distinct inhibitors of MKK1 (PD184352 and U0126), demonstrating that DAZAP1 is a physiological substrate for ERK1/ERK2. The mutation of Thr269 and Thr315 to aspartate or the phosphorylation of these residues caused DAZAP1 to dissociate from its binding partner DAZ. DAZ interacts with PABP [poly(A)-binding protein] and thereby stimulates the translation of mRNAs containing short poly(A) tails [Collier, Gorgoni, Loveridge, Cooke and Gray (2005) EMBO J. 24, 2656–2666]. In the present study we have shown that DAZ cannot bind simultaneously to DAZAP1 and PABP, and suggest that the phosphorylation-induced dissociation of DAZ and DAZAP1 may allow the former to stimulate translation by interacting with PABP.

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