Following in vitro activation of mitogen-activated protein kinases by mass spectrometry and tryptic peptide analysis: purifying fully activated p38 mitogen-activated protein kinase α

Szafranska, Anna E.; Luo, Xuemei; Dalby, Kevin N.

doi:10.1016/j.ab.2004.09.039

Cited by 10 publications

(22 citation statements)

References 24 publications

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“…5B of Ref. 10) because it is generally difficult to obtain high phosphorylation stoichiometry with the wild type MAPK by in vitro activation (30). A second intriguing possibility could be due to different origins of p38␣ proteins because the Xenopus p38␣ homologue was used by Groom et al (10), and ours is of a mammalian origin.…”

Section: Resultsmentioning

confidence: 99%

Mitogen-activated Protein Kinase (MAPK) Phosphatase 3-mediated Cross-talk between MAPKs ERK2 and p38α

Zhang

Wang

2011

Journal of Biological Chemistry

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MAPK phosphatase 3 (MKP3) is highly specific for ERK1/2 inactivation via dephosphorylation of both phosphotyrosine and phosphothreonine critical for enzymatic activation. Here, we show that MKP3 is able to effectively dephosphorylate the phosphotyrosine, but not phosphothreonine, in the activation loop of p38␣ in vitro and in intact cells. The catalytic constant of the MKP3 reaction for p38␣ is comparable with that for ERK2. Remarkably, MKP3, ERK2, and phosphorylated p38␣ can form a stable ternary complex in solution, and the phosphatase activity of MKP3 toward p38␣ substrate is allosterically regulated by ERK2-MKP3 interaction. This suggests that MKP3 not only controls the activities of ERK2 and p38␣ but also mediates cross-talk between these two MAPK pathways. The crystal structure of bisphosphorylated p38␣ has been determined at 2.1 Å resolution. Comparisons between the phosphorylated MAPK structures reveal the molecular basis of MKP3 substrate specificity.MAPK pathways convert different extracellular stimuli into specific cellular responses and mediate various physiological processes, including cellular proliferation, apoptosis, differentiation, and stress responses. There are three major mammalian MAPK subfamilies: ERK, JNK and p38 (1-6). The ERKs are typically activated by growth factors and phorbol ester, whereas JNK and p38 MAPKs are primarily activated by cytokines and environmental stress. MAPK activity is tightly controlled by phosphorylation and dephosphorylation. Full activation of the MAPKs requires phosphorylation on both threonine and tyrosine residues in the TXY motif by their specific upstream dual specificity kinases (MAPK kinases). After activation, each MAPK phosphorylates a distinct spectrum of substrates, which include key regulatory enzymes, cytoskeletal proteins, nuclear receptors, regulators of apoptosis, and many transcription factors.The regulated dephosphorylation of MAPKs plays a key role in determining the magnitude and duration of kinase activation and hence the physiological outcome of signaling. MAPK phosphatases (MKPs) 2 are important negative regulators of MAPK signaling. MKPs belong to a family of dual specificity phosphatases and specifically dephosphorylate both threonine and tyrosine residues in the activation loop of MAPKs. In mammalian cells, there are 10 distinct catalytically active MKPs. Several of these MKPs display distinct in vivo substrate preferences for the various MAPKs. For example, it has been shown that MKP3 selectively targets ERK, whereas MKP5 shows a preference for JNK and p38 (7-9). Therefore, there must be a fine regulatory mechanism for MAPK-MKP recognition.MKP3 is localized predominantly in the cytoplasm and is highly specific for ERK1/2 inactivation (10 -13). MKP3 contains a conserved catalytic domain in its C terminus and a less conserved MAPK binding domain at its N terminus (supplemental Fig. S1). The inactive ERK1/2, but not p38 and JNK MAPKs, can form a tight complex with MKP3 via the interaction between the N-terminal domain of MKP3 and the C-l...

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Section: Resultsmentioning

confidence: 99%

Mitogen-activated Protein Kinase (MAPK) Phosphatase 3-mediated Cross-talk between MAPKs ERK2 and p38α

Zhang

Wang

2011

Journal of Biological Chemistry

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“…Compounds targeting p38α must be highly selective in order to avoid adverse side effects triggered by off-target kinase inhibition. Although in vitro p38 inhibition assays exist, they are hampered by the need to activate the MAPK either in vivo, e.g., following lipopolysaccharide treatment, or through in vitro MKK6 trans -phosphorylation (Szafranska et al., 2005) and the short lived nature of the activated enzyme. By promoting a long-lasting activation of p38α, GRA24 challenges the natural-negative-feedback mechanisms that prevent the MAPK activation ad infinitum and offers a powerful in vitro tool to screen for small-molecule p38α inhibitors.…”

Section: Discussionmentioning

confidence: 99%

Structural Basis for the Subversion of MAP Kinase Signaling by an Intrinsically Disordered Parasite Secreted Agonist

et al. 2017

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SummaryThe causative agent of toxoplasmosis, the intracellular parasite Toxoplasma gondii, delivers a protein, GRA24, into the cells it infects that interacts with the mitogen-activated protein (MAP) kinase p38α (MAPK14), leading to activation and nuclear translocation of the host kinase and a subsequent inflammatory response that controls the progress of the parasite. The purification of a recombinant complex of GRA24 and human p38α has allowed the molecular basis of this activation to be determined. GRA24 is shown to be intrinsically disordered, binding two kinases that act independently, and is the only factor required to bypass the canonical mitogen-activated protein kinase activation pathway. An adapted kinase interaction motif (KIM) forms a highly stable complex that competes with cytoplasmic regulatory partners. In addition, the recombinant complex forms a powerful in vitro tool to evaluate the specificity and effectiveness of p38α inhibitors that have advanced to clinical trials, as it provides a hitherto unavailable stable and highly active form of p38α.

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“…DNA sequences encoding Mus musculus mitogen-activated protein kinase 14 - p38MAPKα (GenBank accession number NM_011951) was cloned into the pET14B vector to express an N -terminal, His tagged p38MAPKα in E. coli BL21 (DE3) pLysS cells. The enzyme was expressed and purified as described previously (34). The activated, MAPKs were all stored in buffer S [25 mM HEPES (pH 7.5), 50 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 2 mM DTT and 10% (v/v) glycerol] at -80 °C.…”

Section: Methodsmentioning

confidence: 99%

Development of JNK2-Selective Peptide Inhibitors That Inhibit Breast Cancer Cell Migration

et al. 2011

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Despite their lack of selectivity towards c-Jun N-terminal kinase (JNK) isoforms, peptides derived from the JIP (JNK Interacting Protein) scaffolds linked to the cell-penetrating peptide TAT are widely used to investigate JNK-mediated signaling events. To engineer an isoform-selective peptide inhibitor, several JIP-based peptide sequences were designed and tested. A JIP sequence connected through a flexible linker to either the N-terminus of an inverted TAT sequence (JIP10-Δ-TATi), or to a poly-arginine sequence (JIP10-Δ-R9) enabled the potent inhibition of JNK2 (IC50~90 nM) and exhibited 10-fold selectivity for JNK2 over JNK1 and JNK3. Examination of both peptides in HEK293 cells revealed a potent ability to inhibit the induction of both JNK activation and c-Jun phosphorylation in cells treated with anisomycin. Notably, Western blot analysis indicates that only a fraction of total JNK must be activated to elicit robust c-Jun phosphorylation. To examine the potential of each peptide to selectively modulate JNK2 signaling in vivo, their ability to inhibit the migration of Polyoma Middle-T Antigen Mammary Tumor (PyVMT) cells was assessed. PyVMTjnk2-/- cells exhibit a lower migration potential compared to PyVMTjnk2+/+ cells, and this migration potential is restored through the over-expression of GFP-JNK2α. Both JIP10-Δ-TATi and JIP10-Δ-R9 inhibit the migration of PyVMTjnk2+/+ cells and PyVMTjnk2-/- cells expressing GFP-JNK2α. However, neither peptide inhibits the migration of PyVMTjnk2-/- cells. A control form of JIP10-Δ-TATi containing a single leucine to arginine mutation lacks ability to inhibit JNK2 in vitro cell-free and cell-based assays and does not inhibit the migration of PyVMTjnk2+/+ cells. Together, these data suggest that JIP10-Δ-TATi and JIP10-Δ-R9 inhibit the migration of PyVMT cells through the selective inhibition of JNK2. Finally, the mechanism of inhibition of a D-retro-inverso JIP peptide, previously reported to inhibit JNK, was examined and found to inhibit p38MAPKα in an in vitro cell-free assay with little propensity to inhibit JNK isoforms.

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Following in vitro activation of mitogen-activated protein kinases by mass spectrometry and tryptic peptide analysis: purifying fully activated p38 mitogen-activated protein kinase α

Cited by 10 publications

References 24 publications

Mitogen-activated Protein Kinase (MAPK) Phosphatase 3-mediated Cross-talk between MAPKs ERK2 and p38α

Mitogen-activated Protein Kinase (MAPK) Phosphatase 3-mediated Cross-talk between MAPKs ERK2 and p38α

Structural Basis for the Subversion of MAP Kinase Signaling by an Intrinsically Disordered Parasite Secreted Agonist

Development of JNK2-Selective Peptide Inhibitors That Inhibit Breast Cancer Cell Migration

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