Selective in Vivo Inhibition of Mitogen-activated Protein Kinase Activation Using Cell-permeable Peptides

Abstract

The extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinases (MAPKs), is essential for cellular proliferation and differentiation, and thus there exists great interest to develop specific and selective inhibitors of this enzyme. Whereas small molecule inhibitors PD098095 and U0126 have been used to study MAPK/ERK kinase (MEK), their target selectivity has been questioned recently. The cross-reactivity of ATP-directed inhibitors with other protein kinases prompted us to develop structure-based selective peptide inhibitors of ERK activation. Based on a MEK1-derived peptide, we developed inhibitors of ERK activation in vitro and in vivo. The inclusion of either an alkyl moiety or a membrane-translocating peptide sequence facilitated the cellular uptake of the peptide inhibitor and prevented ERK activation in 4-phorbol 12-myristate 13-acetate-stimulated NIH 3T3 cells or nervegrowthfactor-treatedPC12cellsinaconcentration-dependent manner. In addition, cell-permeable peptides inhibited ERK-mediated activation of the transcriptional activity of ELK1. The peptides did not have an inhibitory effect on the activity of two other closely related classes of MAPKs, c-Jun amino-terminal kinase or p38 protein kinase. Thus, these peptides may serve as valuable tools for investigating ERK activation and for selective investigation of ERK-mediated responses. With the knowledge of other kinase interacting domains, it would be possible to design cell-permeable inhibitors for investigating diverse cellular signaling mechanisms and for possible therapeutic applications.Protein phosphorylation plays a critical role in cellular signaling in response to a variety of hormones, growth factors, neurotransmitters, and a wide range of stimuli. Mitogen-activated protein kinases (MAPKs) 1 play a pivotal role in these processes, particularly in stimulus-mediated cellular responses (1-3). The activation of these enzymes requires a cascade-like mechanism in which each MAPK is phosphorylated on two amino acid residues (Thr/Tyr) by an upstream protein kinase, MAPKK (MEK), and the latter in turn is phosphorylated on two amino acid residues (Ser/Thr) by a third protein kinase, MAPKK kinase (MEKK). There are at least three such protein kinase modules in mammalian cells as follows: extracellular signal-regulated kinases (ERKs), c-Jun amino-terminal kinases (JNKs), and the p38 MAP kinases (p38). The dual phosphorylation of MAPKs by MEKs is necessary for their activation (4) and is considered an essential step in the signaling pathways in response to growth factors and mitogenic stimuli, stress-causing agents, and cytokines. For phosphorylation-dependent activation of MAPKs to occur, MAPK must first associate with its cognate upstream kinase, MEK. Thus, disrupting this interaction using a peptide derived from an association domain of either enzyme (5, 6) would be predicted to block the activation of the downstream protein kinase. To test this hypothesis, we evaluated the ability of a peptide corresponding to the ami...

Src SH3/2 Domain-mediated Peripheral Accumulation of Src and Phospho-myosin Is Linked to Deregulation of E-cadherin and the Epithelial-Mesenchymal Transition

“…However, expression of active c-Src in KM12C/Src527F cells impaired E-cadherin localization between cells, although occasionally E-cadherin appeared to localize to fragmented structures between two cells ( Figure 6A, b). In contrast, when KM12C/Src527F cells were treated with the MEK inhibitor (UO126), an ERK activation inhibitor peptide II (Kelemen et al, 2002) or the MLCK inhibitor (ML7), E-cadherin localization was restored between most cells ( Figure 6A, c, d, and f). Treatment with the ROCK inhibitor (Y27632) also caused distribution of E-cadherin to cell-cell contacts when the cells were switched to high calcium ( Figure 6A, e).…”

A novel cellular survival factor – the B2 subunit of vacuolar H+-ATPase inhibits apoptosis

“…Ste-MEK1-13 was reported to have an IC 50 of 1373-3075 mM for inhibiting ERK both in vitro and in vivo. 21 This specific ERK inhibitor also increased both cytochrome c and AIF release (Figure 6b) and reduced the degree of Bad phosphorylation (Figure 6c) during serum starvation despite B2 overexpression. As shown, Ste-MEK1-13-induced apoptotic morphological changes in 293B2 cells after serum deprivation for 24 h at a concentration as low as 7 mM and showed a dose-dependent response in the range of 7-56 mM.…”

“…* , ***Po0.05 and 0.01 when compared to GFP cells; D, DDD Po0.05 and 0.01 when compared to nonstarved corresponding cells (n ¼ 4) interaction with MEK. 21 After serum deprivation for 24 h, control 293GFP cells displayed significant morphologic evidence of apoptosis as shown by annexin V staining (Figure 6a). In contrast, serum-deprived 293B2 cells only became apoptotic after treatment with Ste-MEK1 13 in a range of concentration from 7 to 56 mM.…”

Inhibition of IL-6-dependent growth of myeloma cells by an acidic peptide repressing the gp130-mediated activation of Src family kinases

“…The wide use of acylation in general for peptide delivery (Adachi et al, 1999b;Boehning et al, 2005) and of myristoylation in particular (Eichholtz et al, 1993;Kelemen et al, 2002;Niv et al, 2004) made it likely that also the present peptides entered into the cytoplasm. To confirm this assumption, 100 mM of fluorescence-labelled derivatives of peptide 18AD and its non-myristoylated equivalent were added to suspensions of 7TD1 cells.…”

“…For myristoylated peptides derived from the catalytic domains of different protein kinases and used at a concentration of 10 mM, the threshold for a proof of efficacy against cancer cell lines was set at 40% growth inhibition (Niv et al, 2004). The inhibition of Erk phosphorylation by membranepenetrating MEK1-derived peptides exhibited IC 50 -values of 10-30 mM (Kelemen et al, 2002). In contrast, inhibition by the somatostatin analogs octreotide or RC-160 of the proliferation of myeloma cell lines (Georgii-Hemming et al, 1999), or a human oral carcinoma cell line (Dasgupta et al, 2000), respectively, occurred in the nM concentration range, and the latter peptide was more potent when myristoylated.…”