Activation of the c-Jun N-terminal kinase pathway by a novel protein kinase related to human germinal center kinase

Abstract

The c-Jun N-terminal kinase (JNK), or stress-activated protein kinase plays a crucial role in cellular responses stimulated by environmental stress and proinflammatory cytokines. However, the mechanisms that lead to the activation of the JNK pathway have not been elucidated. We have isolated a cDNA encoding a novel protein kinase that has significant sequence similarities to human germinal center kinase (GCK) and human hematopoietic progenitor kinase 1. The novel GCK-like kinase (GLK) has a nucleotide sequence that encodes an ORF of 885 amino acids with 11 kinase subdomains. Endogenous GLK could be activated by UV radiation and proinflammatory cytokine tumor necrosis factor alpha. When transiently expressed in 293 cells, GLK specifically activated the JNK, but not the p42/44(MAPK)/extracellular signal-regulated kinase or p38 kinase signaling pathways. Interestingly, deletion of amino acids 353-835 in the putative C-terminal regulatory region, or mutation of Lys-35 in the putative ATP-binding domain, markedly reduced the ability of GLK to activate JNK. This result indicates that both kinase activity and the C-terminal region of GLK are required for maximal activation of JNK. Furthermore, GLK-induced JNK activation could be inhibited by a dominant-negative mutant of mitogen-activated protein kinase kinase kinase 1 (MEKK1) or mitogen-activated protein kinase kinase 4/SAPK/ERK kinase 1 (SEK1), suggesting that GLK may function upstream of MEKK1 in the JNK signaling pathway.

Figure 1

(A) Deduced amino acid sequences of GLK. The deduced amino acid sequence of GLK is shown. The putative catalytic domain is underlined (..). Three proline-rich regions are doubly underlined (=). Two putative PEST (proline, glutamic acid, serine, and threonine) sequences are singly underlined (—). (B) Amino acid sequence alignment and comparison of GLK with the related protein kinases. The putative catalytic domain of GLK was aligned and compared with the catalytic domains of human GCK, HPK1, PAK1, and S. cerevisiae Ste20 by the dnastar program.

Figure 2

Expression pattern of human GLK mRNA. A filter containing poly(A)⁺ RNA from the indicated tissues was hybridized with a radioactive GLK probe as described. The hybridization with a glyceraldehyde-3-phosphate dehydrogenase (G3PDH) probe was also shown.

Figure 3

Characterization of human GLK. (A) 293 cells were transfected with vector alone (lane 1), an expression vector expressing GLK tagged with an N-terminal flag epitope (lane 2), or a kinase inactive mutant (K35E) (lane 3). Cell lysates were harvested and immunoblotted with mAb M2. (B) Lysates from cells transfected with vector alone (lane 1), wild-type GLK (lane 2), or K35E mutant (lane 3) were prepared. GLK was immunoprecipitated with mAb M2, then used in a kinase assay with MBP as substrate.

Figure 4

Activation of JNK but not ERK or p38 kinase by GLK in transfected 293 cells. (A) 293 cells were cotransfected with either vector alone (lane 1) or GLK (lane 2) and HA epitope-tagged JNK1 plasmid. Alternatively, cells were transfected with JNK1 plasmid and treated with 10 ng/ml anisomycin (lane 3). JNK was immunoprecipitated and kinase assays were performed using GST c-Jun as substrate. (B) 293 cells were cotransfected with either vector alone (lane 1) or GLK (lanes 2 and 3) and HA epitope-tagged JNK1 plasmid. GLK (lane 2) or JNK (lane 3) was immunoprecipitated and used in an in vitro kinase assay using GST c-Jun as substrate. (C) 293 cells were cotransfected with either vector alone (lane 1) or GLK (lane 2) and HA epitope-tagged ERK2 plasmid. Alternatively, cells were transfected with ERK2 and stimulated with epidermal growth factor (EGF) (30 ng/ml) for 10 min (lane 3). Kinase assays were performed using PHAS-I as substrate. (D) 293 cells were cotransfected with either vector alone (lane 1) or GLK (lane 2) and HA epitope-tagged p38 kinase plasmid. Alternatively, cells were transfected with p38 plasmid and treated with UV light (lane 3) or 0.5 M NaCl for 30 min (lane 4). Kinase assays were performed using ATF2 peptide (1–109) as substrate.

Figure 5

Effect of kinase activity and amino acids 353–835 in the C-terminal region of GLK on JNK activation. (A) 293 cells were cotransfected with vector alone (lane 1), wild-type GLK (lane 2), K35E mutant (lane 3), or a GLK C-terminal deletion mutant (ΔGLK) (lane 4) plus JNK. JNK activity was assayed using GST c-Jun as substrate. (B) Lysates containing equal amounts of total protein were run on 10% SDS/PAGE and transferred to a nitrocellulose membrane. The blot was probed with a mAb against the flag epitope followed by rabbit anti-mouse IgG. The blot was developed using the ECL system. (C) Kinase activity of GLK C-terminal deletion mutant. Lysates from cells transfected with vector alone (lane 1) or with GLK C-terminal deletion mutant (ΔGLK) (lane 2) were prepared and recombinant protein was immunoprecipitated with mAb M2 followed by kinase assay with MBP as substrate.

Figure 6

Effect of dominant-negative mutants of MKK4/SEK1 and MEKK1 on GLK-induced JNK activation. (A) 293 cells were cotransfected with vector alone (lane 1), GLK and vector (lane 2), GLK and either wild-type MKK4 (lane 4), or a dominant-negative mutant of MKK4 (lane 3) plus HA epitope-tagged JNK. JNK activities were determined as described. (B) Effect of a dominant-negative mutant of MEKK1 on GLK induced JNK activation. 293 cells were transfected with vector alone (lane 1), GLK and vector (lane 2), GLK and either wild-type MEKK1 (lane 3), or a dominant-negative mutant of MEKK1 (MEKK-DN) (lane 4) plus HA epitope-tagged JNK. JNK activities were determined as described. The data are representative of three experiments performed. (C) Lysates from 293 cells transfected with vector alone (lane 1), wild-type GLK (lane 2), or K35E mutant (lane 3) were prepared. GLK was immunoprecipitated with mAb M2, then used in a kinase assay with a polyhistidine MEKK1 fusion protein as substrate.

Figure 7

Activation of endogenous GLK in HeLa cells. (A) Lysates from 293 cells transfected with either vector alone (lane 1) or GLK plasmid (lane 2) or from different cell lines (lanes 3–7) were immunoblotted with a polyclonal antibody to GLK. (B) HeLa cells were stimulated with UV radiation or TNF-α for the time indicated. Lysates containing equal amounts of total protein were immunoprecipitated with the polyclonal GLK antibody followed by a kinase assay with MBP as substrate.