Steven L. Pelech

Mitogen-activated protein kinases: versatile transducers for cell signaling

Mitogen-activated protein (MAP) kinases are proline-directed serine/threonine protein kinases that are activated via phosphorylation of their own tyrosine residues. Highly conserved during eukaryotic evolution, they serve as common signaling components in distinct transduction pathways initiated by many stimuli. They have been implicated in the control of a broad spectrum of cellular events but are particularly known for their possible roles in cell cycle progression and the control of meiosis.

Signal transduction via phosphatidylcholine cycles

Abstract Phosphatidylcholine turnover cycles can generate second messengers (including diacylglycerol and arachidonic acid). Guanine nucleotide-binding proteins are implicated in the coupling of agonist receptors to the activation of phospholipases A 2 , C and D. In many instances, these agonists also stimulate the resynthesis of phosphatidylcholine via activation of CTP:phosphocholine cytidylyltransferase.

Identification of Kinase-Phosphatase Signaling Modules Composed of p70 S6 Kinase-Protein Phosphatase 2A (PP2A) and p21-activated Kinase-PP2A

A growing body of evidence indicates that regulation of protein-serine/threonine phosphatase 2A (PP2A) involves its association with other cellular and viral proteins in multiprotein complexes. PP2A-containing protein complexes may exist that contribute to PP2A’s important regulatory role in many cellular processes. To identify such protein complexes, PP2A was partially purified from rat brain soluble extracts following treatment with a reversible cross-linker to stabilize large molecular size forms of PP2A. Compared with native (uncross-linked) PP2A, cross-linked PP2A revealed an enrichment of p70 S6 kinase and two p21-activated kinases (PAK1 and PAK3) in the PP2A complex, indicating these kinases may associate with PP2A. The existence of protein kinase-PP2A complexes in rat brain soluble extracts was further substantiated by the following results: 1) independent immunoprecipitation of the kinases revealed that PP2A co-precipitated with p70 S6 kinase and the two PAK isoforms; 2) glutathione S-transferase fusion proteins of p70 S6 kinase and PAK3 each isolated PP2A; and 3) PAK3 and p70 S6 kinase bound to microcystin-Sepharose (an affinity resin for PP2A-PP1). Cumulatively, these findings provide evidence for association of PP2A with p70 S6 kinase, PAK1, and PAK3 in the context of the cellular environment. Moreover, together with the recent reports describing associations of PP2A with Ca2+/calmodulin-dependent protein kinase IV (Westphal, R. S., Anderson, K. A., Means, A. R., and Wadzinski, B. E. (1998) Science 280, 1258–1261) and casein kinase IIα (Heriche, J. K., Lebrin, F., Rabilloud, T., Leroy, D., Chambaz, E. M., and Goldberg, Y. (1997)Science 276, 952–955), the present data provide compelling evidence for the existence of protein kinase-PP2A signaling modules as a new paradigm for the control of various intracellular signaling cascades.

Ischemic Preconditioning Activates MAPKAPK2 in the Isolated Rabbit Heart: Evidence for Involvement of p38 MAPK

Recent studies suggest that p38 mitogen-activated protein kinase (MAPK) may be involved in ischemic preconditioning (PC). To further test this possibility, the regulation of MAPK-activated protein kinase 2 (MAPKAPK2), a kinase immediately downstream from p38 MAPK, and the activity of c-Jun NH(2)-terminal kinase (JNK), a second MAPK, were examined in preconditioned hearts. Isolated, perfused rabbit hearts were subjected to 20 to 30 minutes of global ischemia. Ventricular biopsies before treatment and after 20 minutes of ischemia were homogenized, and the activities of MAPKAPK2 and JNK were evaluated. For the MAPKAPK2 experiments, 7 groups were studied, as follows: control hearts; preconditioned hearts; hearts treated with 500 nmol/L R(-) N(6)-(2-phenylisopropyl) adenosine (PIA), an A(1)-adenosine receptor agonist; preconditioned hearts pretreated with 100 micromol/L 8-(p-sulfophenyl) theophylline (SPT), an adenosine receptor antagonist; preconditioned hearts also treated with SB 203580, a potent inhibitor of p38 MAPK activation; hearts treated with 50 ng/mL anisomycin (a p38 MAPK/JNK activator); and hearts treated with both anisomycin (50 ng/mL) and the tyrosine kinase inhibitor genistein (50 micromol/L). MAPKAPK2 activity was not altered in control hearts after 20 minutes of global ischemia. By contrast, there was a 3.8-fold increase in activity during ischemia in preconditioned hearts. Activation of MAPKAPK2 in preconditioned hearts was blocked by both SPT and SB 203580. MAPKAPK2 activity during ischemia increased 3.5-fold and 3.3-fold in hearts pretreated with PIA or anisomycin, respectively. MAPKAPK2 activation during ischemia in hearts pretreated with anisomycin was blocked by genistein. In separate hearts, anisomycin mimicked the anti-infarct effect of PC, and that protection was abolished by genistein. JNK activity was measured in control and preconditioned hearts. There was a comparable, modest decline in activity during 30 minutes of global ischemia in both groups. As a positive control, a third group of hearts was treated with anisomycin before global ischemia, and in these, JNK activity increased by 290% above baseline. These results confirm that the p38 MAPK/MAPKAPK2 pathway is activated during ischemia only if the heart is in a preconditioned state. These data further support p38 MAPK as an important signaling component in ischemic PC.

Chemotactic Peptide N-formyl-Met-Leu-Phe Activation of p38 Mitogen-activated Protein Kinase (MAPK) and MAPK-activated Protein Kinase-2 in Human Neutrophils

Activation of polymorphonuclear leukocytes (PMN) by chemotactic peptides initiates a series of functional responses that serve to eliminate pathogens. The intermediate steps that link engagement of the chemoattractant receptor to the microbicidal responses involve protein kinases that have yet to be identified. In this study we detected in human PMN the presence of p38 mitogen-activated protein kinase (MAPK), which became rapidly tyrosine phosphorylated and activated in response to the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). Pretreatment of PMN with wortmannin, a phosphatidylinositol 3-kinase inhibitor, or bis-indolylmaleimide, a protein kinase C antagonist, resulted in partial inhibition of p38 phosphorylation upon fMLP stimulation. Similarly, phosphorylation of p38 was only partially inhibited when the fMLP-induced cytosolic calcium transient was prevented. Stimulation of PMN by the chemoattractant also resulted in the rapid phosphorylation and activation of MAPK-activated protein kinase-2 (MAPKAPK-2), which was completely inhibited by the specific p38 inhibitor, SB203580. The physical interaction of p38 with MAPKAPK-2 was studied by coimmunoprecipitation. These two kinases were found to be associated in unstimulated PMN but dissociated upon activation of the cells by fMLP. Together these findings demonstrate the activation of p38 by chemotactic peptides in human PMN by a process involving phosphatidylinositol 3-kinase, protein kinase C, and calcium. p38, in turn, is an upstream activator of MAPKAPK-2.

Stress-induced Inhibition of ERK1 and ERK2 by Direct Interaction with p38 MAP Kinase

We have identified a direct physical interaction between the stress signaling p38α MAP kinase and the mitogen-activated protein kinases ERK1 and ERK2 by affinity chromatography and coimmunoprecipitation studies. Phosphorylation and activation of p38α enhanced its interaction with ERK1/2, and this correlated with inhibition of ERK1/2 phosphotransferase activity. The loss of epidermal growth factor-induced activation and phosphorylation of ERK1/2 but not of their direct activator MEK1 in HeLa cells transfected with the p38α activator MKK6(E) indicated that activated p38α may sequester ERK1/2 and sterically block their phosphorylation by MEK1.

Activation of p42 Mitogen-Activated Protein Kinase by Glutamate Receptor Stimulation in Rat Primary Cortical Cultures

Abstract— Recent studies have identified at least two homologous mitogen‐activated protein (MAP) kinases that are activated by phosphorylation of both tyrosine and threonine residues by an activator kinase. To help define the role of these MAP kinases in neuronal signalling, we have used primary cultures derived from fetal rat cortex to assess the regulation of their activity by agonist stimulation of glutamate receptors and by synaptic activity. Regulation was assayed by monitoring changes in both tyrosine phosphorylation on western blots and in vitro kinase activity toward a selective MAP kinase substrate peptide. In initial studies, we found that phorbol ester treatment increased tyrosine phosphorylation of p42 MAP kinase and stimulated MAP kinase activity. A similar response was elicited by three agonists of metabotropic glutamate receptors, i.e., trans‐(±)‐1‐amino‐1,3‐cyclopentane dicarboxylic acid, quisqualate, and (2S,3S,4S)‐α‐(carboxycyclopropyl)glycine. MAP kinase activity and p42 MAP kinase tyrosine phosphorylation were also stimulated by the ionotropic glutamate receptor agonist, kainate, but not by N‐methyl‐d‐aspartate. To examine regulation of MAP kinase by synaptic activity, cultures were treated with picrotoxin, an inhibitor of GABAA receptor‐mediated inhibition that enhances spontaneous excitatory synaptic activity. Treatment of cultures with picrotoxin elicited activation of MAP kinase. This response was blocked by tetrodotoxin, which suppresses synaptic activity. These results demonstrate that p42 MAP kinase is activated by glutamate receptor agonist stimulation and by endogenous synaptic activity.

α-Synuclein activates stress signaling protein kinases in THP-1 cells and microglia

Here we show that alpha-synuclein, a major constituent of Lewy bodies, induces inflammation in human microglial and human THP-1 cells. Secretions from such stimulated THP-1 cells contain increased levels of IL-1beta and TNF-alpha. When stimulated by alpha-synuclein in combination with IFN-gamma, secretions from the cells also become toxic towards SH-SY5Y neuroblastoma cells. The A30P, E46K and A53T alpha-synuclein mutations, which induce Parkinsons disease, are more potent than normal alpha-synuclein in the induction of such cytotoxicity. To investigate the signaling mechanisms evoked, protein phosphorylation profiling was applied. At least 81 target phospho-sites were identified. Large increases were induced in the three major mitogen-activated protein (MAP) kinase pathways: p38 MAP kinase, extracellular regulated protein-serine kinase (ERK)1/2 and c-Jun-N-terminal kinase (JNK). Upregulation occurred within minutes following exposure to alpha-synuclein, which is consistent with a receptor-mediated effect. These findings demonstrate that alpha-synuclein acts as a potent inflammatory stimulator of microglial cells, and that inhibitors of such stimulation might be beneficial in the treatment of Parkinsons disease and other synucleinopathies.

Regulation of Amyloid Precursor Protein Catabolism Involves the Mitogen-Activated Protein Kinase Signal Transduction Pathway

Catabolic processing of the amyloid precursor protein (APP) is subject to regulatory control by protein kinases. We hypothesized that this regulation involves sequential activation of the enzymes mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated protein kinase (ERK). In the present investigation, we provide evidence that MEK is critically involved in regulating APP processing by both nerve growth factor and phorbol esters. Western blot analysis of the soluble N-terminal APP derivative APPsdemonstrated that the synthetic MEK inhibitor PD 98059 antagonized nerve growth factor stimulation of both APPs production and ERK activation in PC12 cells. Moreover, PD 98059 inhibited phorbol ester stimulation of APPs production and activation of ERK in both human embryonic kidney cells and cortical neurons. Furthermore, overexpression of a kinase-inactive MEK mutant inhibited phorbol ester stimulation of APP secretion and activation of ERK in human embryonic kidney cell lines. Most important, PD 98059 antagonized phorbol ester-mediated inhibition of Aβ secretion from cells overexpressing human APP695 carrying the “Swedish mutation.” Taken together, these data indicate that MEK and ERK may be critically involved in protein kinase C and nerve growth factor regulation of APP processing. The mitogen-activated protein kinase cascade may provide a novel target for altering catabolic processing of APP.

Enzyme translocation in the regulation of phosphatidylcholine biosynthesis

Abstract Phosphatidylcholine biosynthesis is regulated by translocation of the rate-limiting enzyme (CTP:phosphocholine cytidylyltransferase) from the cytosol, where it is inactive, to the endoplasmic reticulum (microsomes) where it is activated. The process is regulated by reversible protein phosphorylation and by fatty acids.