Abstract
CEP-1347 (KT7515) promotes neuronal survival at dosages that inhibit activation of the c-Jun amino-terminal kinases (JNKs) in primary embryonic cultures and differentiated PC12 cells after trophic withdrawal and in mice treated with 1-methyl-4-phenyl tetrahydropyridine. In an effort to identify molecular target(s) of CEP-1347 in the JNK cascade, JNK1 and known upstream regulators of JNK1 were co-expressed in Cos-7 cells to determine whether CEP-1347 could modulate JNK1 activation. CEP-1347 blocked JNK1 activation induced by members of the mixed lineage kinase (MLK) family (MLK3, MLK2, MLK1, dual leucine zipper kinase, and leucine zipper kinase). The response was selective because CEP-1347 did not inhibit JNK1 activation in cells induced by kinases independent of the MLK cascade. CEP-1347 inhibition of recombinant MLK members in vitro was competitive with ATP, resulting in IC(50) values ranging from 23 to 51 nm, comparable to inhibitory potencies observed in intact cells. In addition, overexpression of MLK3 led to death in Chinese hamster ovary cells, and CEP-1347 blocked this death at doses comparable to those that inhibited MLK3 kinase activity. These results identify MLKs as targets of CEP-1347 in the JNK signaling cascade and demonstrate that CEP-1347 can block MLK-induced cell death.
Highlights
CEP-1347 (KT7515) promotes neuronal survival at dosages that inhibit activation of the c-Jun amino-terminal kinases (JNKs) in primary embryonic cultures and differentiated PC12 cells after trophic withdrawal and in mice treated with 1-methyl-4-phenyl tetrahydropyridine
We previously reported that differentiated PC12 cells are protected from UVinduced death in the presence of CEP-1347 [28], SH-SY5Y cells were chosen for these studies because the MLK3 antibody readily recognizes human but not rodent MLK3
CEP-1347 is a potent inhibitor of neuronal cell death induced by a variety of stress stimuli both in vitro and in vivo
Summary
Reagents—Dulbecco’s modified Eagle’s medium (catalog no. 11995040) was purchased from Life Technologies, Inc. Cos-7 Cell Transfection—Cos-7 cells were plated to 80% confluency in 60-mm dishes and transfected with 2 g of test cDNA and 2 g of hemagglutinin (HA)-tagged JNK1 constructs using LipofectAMINE as recommended by the provider (Life Technologies, Inc.). Further washing as described above was followed by incubation with the phospho-MKK4 antibody at a 1:500 dilution in Superblock for 1 h at 37 °C. After repeated washing (five times), wells were treated with goat anti-rabbit alkaline phosphatase at 1:2000 in 2% bovine serum albumin/TBS for 1 h at 37 °C This incubation was followed by three washes with TTBS and three washes with TBS. Lysate from Cos-7 cells overexpressing MLK3 or from SH-SY5Y cells treated with UV irradiation were run on a Nupage 3– 8% Tris acetate gel, transferred to Millipore polyvinylidene difluoride membrane and probed with an MLK3 antibody The enhanced chemiflorescense blots were quantified on a Molecular Dynamics PhosphorImager
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