Apoptosis plays an important role during neuronal development, and defects in apoptosis may underlie various neurodegenerative disorders. To characterize molecular mechanisms that regulate neuronal apoptosis, the contributions to cell death of mitogen-activated protein (MAP) kinase family members, including ERK (extracellular signal-regulated kinase), JNK (c-JUN NH2-terminal protein kinase), and p38, were examined after withdrawal of nerve growth factor (NGF) from rat PC-12 pheochromocytoma cells. NGF withdrawal led to sustained activation of the JNK and p38 enzymes and inhibition of ERKs. The effects of dominant-interfering or constitutively activated forms of various components of the JNK-p38 and ERK signaling pathways demonstrated that activation of JNK and p38 and concurrent inhibition of ERK are critical for induction of apoptosis in these cells. Therefore, the dynamic balance between growth factor-activated ERK and stress-activated JNK-p38 pathways may be important in determining whether a cell survives or undergoes apoptosis.

https://science.sciencemag.org/content/sci/270/5240/1326.full.pdf

Opposing Effects of ERK and JNK-p38 MAP Kinases on Apoptosis

Biologic basis for interleukin-1 in disease

Mitogen-activated protein (MAP) kinases comprise a family of ubiquitous proline-directed, protein-serine/threonine kinases, which participate in signal transduction pathways that control intracellular events including acute responses to hormones and major developmental changes in organisms. MAP kinases lie in protein kinase cascades. This review discusses the regulation and functions of mammalian MAP kinases. Nonenzymatic mechanisms that impact MAP kinase functions and findings from gene disruption studies are highlighted. Particular emphasis is on ERK1/2.

/pdf/mitogen-activated-protein-map-kinase-pathways-regulation-and-3nsauvepr2.pdf

Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions.

Multicellular organisms have three well-characterized subfamilies of mitogen-activated protein kinases (MAPKs) that control a vast array of physiological processes. These enzymes are regulated by a characteristic phosphorelay system in which a series of three protein kinases phosphorylate and activate one another. The extracellular signal-regulated kinases (ERKs) function in the control of cell division, and inhibitors of these enzymes are being explored as anticancer agents. The c-Jun amino-terminal kinases (JNKs) are critical regulators of transcription, and JNK inhibitors may be effective in control of rheumatoid arthritis. The p38 MAPKs are activated by inflammatory cytokines and environmental stresses and may contribute to diseases like asthma and autoimmunity.

https://science.sciencemag.org/content/sci/298/5600/1911.full.pdf

Mitogen-Activated Protein Kinase Pathways Mediated by ERK, JNK, and p38 Protein Kinases

PD 098059 Is a Specific Inhibitor of the Activation of Mitogen-activated Protein Kinase Kinase in Vitro and in Vivo

Production of interleukin-1 and tumour necrosis factor from stimulated human monocytes is inhibited by a new series of pyridinyl-imidazole compounds. Using radiolabelled and radio-photoaffinity-labelled chemical probes, the target of these compounds was identified as a pair of closely related mitogen-activated protein kinase homologues, termed CSBPs. Binding of the pyridinyl-imidazole compounds inhibited CSBP kinase activity and could be directly correlated with their ability to inhibit cytokine production, suggesting that the CSBPs are critical for cytokine production.

A protein kinase involved in the regulation of inflammatory cytokine biosynthesis.

To identify functional domains of the 886-amino acid human recombinant cAMP-specific phosphodiesterase (PDE) subtype A (rhPDE4A), we engineered the expression of seven mutant proteins containing both NH2- and COOH-terminal truncations. The level of rhPDE4A protein expression in yeast was monitored by immunoblotting using enzyme-specific antisera. Biochemical profiles of the mutant proteins were compared with those of the full-length protein or a fully active truncated form of the enzyme (rhPDE4A Met265-886), lacking the first 264 amino acids. The smallest catalytically active fragment generated was Met332-722, which at 45 kDa is less than half the mass of the full-length enzyme (approximately 110 kDa) but spans the most highly conserved region of the PDE superfamily. Two prototypical PDE4 inhibitors, rolipram and RP 73401, inhibited cAMP hydrolyzing activity of all truncated forms of the enzyme, with IC50 values of 70-2000 nM and 0.2-0.6 nM, respectively. [3H](R)-Rolipram bound to two sites on Met265-886, a high affinity site (Kd1 = 0.7 +/- 0.3 nM) and a low affinity site (Kd2 = 34 +/- 10 nM). Interestingly, [3H](R)-rolipram failed to bind to Met332-886 with high affinity, indicating that high affinity binding is not required for inhibition of enzyme activity. Low affinity rolipram binding was still present in Met332-886 (Kd = 101 +/- 7 nM). In contrast to [3H](R)-rolipram, [3H]RP 73401 bound to a single class of high affinity sites on Met265-886 (Kd = 0.4 +/- 0.1 nM). Further truncation of the enzyme to Met332-886 had no effect on [3H]RP 73401 binding (Kd = 0.2 +/- 0.03 nM). We conclude that the catalytic center of rhPDE4A lies between amino acids 332 and 722. Furthermore, amino acids 265-332 may form a high affinity binding site for rolipram that is outside of the catalytic domain. As a more likely alternative, these amino acids may not form a distinct binding site but instead may be required for the recombinant enzyme to assume a conformation that binds rolipram at the catalytic domain with a high affinity.

Mapping the functional domains of human recombinant phosphodiesterase 4A: structural requirements for catalytic activity and rolipram binding.

Human platelet cGI-PDE: expression in yeast and localization of the catalytic domain by deletion mutagenesis

CSBP/p38 is a MAP kinase that is activated in response to stress, endotoxin, interleukin 1 and tumor necrosis factor. Using a catalytically inactive mutant (D168A) of human CSBP2 as the bait in a yeast two-hybrid screen, a kinase has been cloned which shares ∩ 70 % amino acid identity to MAPKAP kinase-2, and thus was designated MAPKAP kinase-3. The binding of CSBP to MAPKAP kinase-3 was confirmed in vitro by the precipitation of epitope-tagged CSBP1, CSBP2 and CSBP2(D168A) and endogenous CSBP from mammalian cells by a bacterially-expressed GST-MAPKAP kinase-3 fusion protein and in vivo by co-precipitation of the epitope-tagged proteins co-expressed in HeLa cells. MAPKAP kinase-3 was phosphorylated by both CSBP1 and CSBP2, and was then able to phosphorylate HSP27 in vitro. Treatment of HeLa cells with sorbitol or TNF resulted in activation of CSBP and MAPKAP kinase-3 and activation of MAPKAP kinase-3 could be blocked by pre-incubation of cells with 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole, a specific inhibitor of CSBP kinase activity. These data suggest that MAPKAP kinase-3 is activated by stress and cytokines and is a novel substrate of CSBP both in vitro and in vivo. The use of MAPKAP kinase-3 in screens for the identification of pharmaceutically active compounds is disclosed.

Methods of the identification of pharmaceutically active compounds

Isolated cDNA clones from human brain (frontal cortex) cDNA libraries that encode an unique subtype of the low Km, cAMP-specific phosphodiesterases (PDE IVs) are disclosed. Analysis of the distribution of hPDE IVB mRNA expression in various human tissues using a nonconserved fragment of the cDNA as a probe revealed a restricted pattern of expression, with an ∩4-kb mRNA detected in brain, heart, lung and skeletal muscle and not in placenta, liver, kidney or pancreas. Furthermore, an additional ∩5-kb hPDE IVB- related mRNA species was detected in brain tissue. Expression of hPDE IV?B? in a genetically-engineered PDE-deficient strain of the yeast Saccharomyces cerevisiae resulted in the overproduction of cAMP PDE activity which displayed the expected kinetic characteristics for a PDE IV: (1) low Km (4.3νM) for cAMP,2) high Km (>3 mM) for cGMP, and 3) sensitivity to rolipram (Ki = 0.085 νM), a selective inhibitor of PDE IV. Recombinant hPDE IVB also bound [?3?H]R-rolipram saturably and with a high affinity. Analysis of [3H]R-rolipram binding data revealed curvilinear Scatchard plots, suggesting the presence of two non-interacting high affinity rolipram binding sites (K?d? = 0.4 and 6nM) or a negatively cooperative interaction among multiple binding sites. This novel enzyme is particularly useful for screening candidate compounds for their ability to serve as potential anti-depressant, antiasthmatic or anti-inflammatory agents.

Megan M. McLaughlin

Papers

A protein kinase involved in the regulation of inflammatory cytokine biosynthesis.

Mapping the functional domains of human recombinant phosphodiesterase 4A: structural requirements for catalytic activity and rolipram binding.

Human platelet cGI-PDE: expression in yeast and localization of the catalytic domain by deletion mutagenesis

Methods of the identification of pharmaceutically active compounds

Human brain phosphodiesterase