Christian F. Meyer

Persistent Activation of c-Jun N-terminal Kinase 1 (JNK1) in γ Radiation-induced Apoptosis

The c-Jun N-terminal kinases (JNK) are activated by various stimuli, including UV light, interleukin-1, tumor necrosis factor-α (TNF-α), and CD28 costimulation. Induction of JNK by TNF-α, a strong apoptosis inducer, implies a possible role of JNK in the regulation of programmed cell death. Present studies show that lethal doses of γ radiation (GR) induced JNK activities at the early phase of apoptosis in Jurkat T-cells. We demonstrate that JNK1 was activated by either the T-cell activation signals, anti-CD28 monoclonal antibody plus phorbol 12-myristate 13-acetate (PMA), or the apoptosis-inducing treatment, GR; however, the induction patterns were different. In contrast to the rapid and transient JNK1 activation caused by CD28 signaling plus PMA, GR induced a delayed and persistent JNK1 activation. This implies a distinct regulatory mechanism and specific function of JNK1 in irradiated cells. The nuclear and cytosolic JNK1 activities were simultaneously increased in the irradiated cells without an evident change in the protein levels. The abilities of GR to induce JNK1 activation and DNA fragmentation were correlated. Peripheral blood lymphocytes were more sensitive to GR than Jurkat cells in JNK1 induction. The responsiveness of JNK1 to GR suggests the involvement of JNK1 in the initiation of the apoptosis process.

Interaction between c-Rel and the Mitogen-activated Protein Kinase Kinase Kinase 1 Signaling Cascade in Mediating B Enhancer Activation

The Rel family of transcription factors are important mediators of various cytokine stimuli such as interleukin (IL)-1, tumor necrosis factor (TNF)-α, and CD28 costimulation in T cell effector responses. These stimuli induce Rel family DNA-binding activity to the κB enhancer and CD28 response elements of many cytokine gene promoters leading to cytokine production. Consistent with the importance of Rel family induction during immune responses, c-Rel knockout mice exhibit profound defects in T cell functions including IL-2 secretion and T cell proliferative responses to CD28 plus T cell receptor costimulation. The novel protein kinases, c-Jun NH-terminal kinases (JNKs)/stress-activated protein kinases, are also activated by TNF-α, IL-1, and CD28 costimulation. Because of the common regulation of c-Rel and JNK1 by these agents in T cells, we investigated the role of JNK1 in c-Rel activation. We found that MAP kinase kinase kinase (MEKK) 1, a JNK1 activator, induced transcription from the human immunodeficiency virus-1 long terminal repeat and IL-2Rα promoters in a κB-dependent manner. Coexpression of IκBα, a c-Rel inhibitor, inhibited the MEKK1-induced transcriptional activity. JNK1 synergized with MEKK1 in activating transcription from a κB-driven heterologous promoter. Furthermore, JNK1 associated with c-Rel in vivo in Jurkat T cells by coimmunoprecipitation assays and bound directly to c-Rel in a yeast two-hybrid assay. c-Rel also competed with c-Jun in in vitro kinase assays. However, JNK1 did not phosphorylate c-Rel, NF-κB, and IκBα in vitro, indicating that c-Rel may serve as a docking molecule to allow JNK1 phosphorylation of certain Rel-associated proteins. Transactivation of the IL-2Rα and HIV-κB-driven promoters by c-Rel was augmented by coexpression of MEKK1. These results demonstrate the first significant role for the MEKK1 kinase cascade module in c-Rel-mediated transcription.

Interaction of Hematopoietic Progenitor Kinase 1 with Adapter Proteins Crk and CrkL Leads to Synergistic Activation of c-Jun N-Terminal Kinase

ABSTRACT Hematopoietic progenitor kinase 1 (HPK1), a mammalian Ste20-related protein kinase, is an upstream activator of c-Jun N-terminal kinase (JNK). In order to further characterize the HPK1-mediated JNK signaling cascade, we searched for HPK1-interacting proteins that could regulate HPK1. We found that HPK1 interacted with Crk and CrkL adaptor proteins in vitro and in vivo and that the proline-rich motifs within HPK1 were involved in the differential interaction of HPK1 with the Crk proteins and Grb2. Crk and CrkL not only activated HPK1 but also synergized with HPK1 in the activation of JNK. The HPK1 mutant (HPK1-PR), which encodes the proline-rich region alone, blocked JNK activation by Crk and CrkL. Dominant-negative mutants of HPK1 downstream effectors, including MEKK1, TAK1, and SEK1, also inhibited Crk-induced JNK activation. These results suggest that the Crk proteins serve as upstream regulators of HPK1. We further observed that the HPK1 mutant HPK1-KD(M46), which encodes the kinase domain with a point mutation at lysine-46, and HPK1-PR blocked interleukin-2 (IL-2) induction in Jurkat T cells, suggesting that HPK1 signaling plays a critical role in IL-2 induction. Interestingly, HPK1 phosphorylated Crk and CrkL, mainly on serine and threonine residues in vitro. Taken together, our findings demonstrate the functional interaction of HPK1 with Crk and CrkL, reveal the downstream pathways of Crk- and CrkL-induced JNK activation, and highlight a potential role of HPK1 in T-cell activation.

Caspase-mediated cleavage and functional changes of hematopoietic progenitor kinase 1 (HPK1)

Activation of c-Jun N-terminal kinase (JNK) by Fas ligation is caspase-dependent, suggesting that caspases may regulate activators of the JNK pathway. Here, we report that an upstream activator of JNK, hematopoietic progenitor kinase 1 (HPK1), was cleaved during apoptosis. Cleavage of HPK1 was blocked by peptide inhibitors for caspases. HPK1 was efficiently processed by recombinant caspase 3 in vitro. A conserved caspase recognition site, DDVD (amino acids 382 – 385), was found in the HPK1 protein sequence. By testing HPK1 proteins with in vivo and in vitro cleavage assays, we showed that aspartic acid residue 385 is the target for caspases. HPK1 cleavage separated the amino N-terminal kinase domain from the carboxyl C-terminal regulatory domain, and enhanced HPK1 kinase activity. Unlike the full-length HPK1, the N-terminal cleaved product failed to bind adaptor molecules Grb2 (growth factor receptor-bound protein 2) and Crk (CT10 regulator of kinase). The C-terminal fragment, although having three proline-rich domains, bound to Grb2 and Crk less efficiently than the full-length HPK1 protein. Taken together, the cleavage of HPK1 by caspase profoundly changed its biochemical properties.

Hematopoietic progenitor kinase-1 (HPK1) stress response signaling pathway activates IκB kinases (IKK-α/β) and IKK-β is a developmentally regulated protein kinase

Nuclear factor kappa-B (NF-κB) is a pleiotropic transcription factor that plays a central role in the immune and inflammatory responses, and is also involved in controlling viral transcription and apoptosis. A critical control in the activation of NF-κB is the phosphorylation of its inhibitory factor IκBs by IκB kinases (IKK-α and -β). Here, we present experiments addressing the regulation and global expression of murine IKK-β, and localize the IKK-β gene to mouse chromosome 8A3-A4. IKK-β was expressed primarily in the liver, kidney and spleen, and at lower levels in the other adult tissues. While IKK-β was expressed ubiquitously throughout the mouse embryo at 9.5 days, its expression began to be localized to the brain, neural ganglia, neural tube, and liver in the 12.5-days embryo. At 15.5 days, the expression of IKK-β was further restricted to specific tissues of the embryo, suggesting that IKK-β is a developmentally regulated protein kinase. Interestingly, IKK-β phosphorylated IκB constitutively, whereas IKK-α was not active in the absence of cell stimulation. Moreover, both IKK-α and -β were activated by hematopoietic progenitor kinase-1 (HPK1) and MAPK/ERK kinase kinase-1 (MEKK1) specifically, suggesting that IκB/NF-κB is regulated through the HPK1-MEKK1 stress response signaling pathway.

Allelotyping analyses of synchronous primary and metastasis CIN colon cancers identified different subtypes

In colorectal cancer, the molecular alterations that lead to metastasis are not clearly established, probably because of their high genetic complexity. To identify combinations of genetic changes involved in tumor progression and metastasis, we focused on chromosome instable (CIN) colon cancers. We compared by allelotyping of 33 microsatellites, the genomic alterations of 38 primary colon tumors with the synchronously resected matched liver metastases (CLM). We observed that (i) the number of patients with alterations at certain loci did not differ significantly between the whole primary tumor and the paired CLM, (ii) a group of patients had fewer alterations in the metastasis when compared with the matched primary tumor. A 2‐way hierarchical unsupervised clustering of the allelotyping data revealed 2 tumor subtypes that have different levels of CIN (CIN‐High, CIN‐Low). Both subtypes have a minimal common set of alterations at chromosomes 8p, 17p and 18q, but does not include alteration at 5q or mutation at K‐Ras. These 2 subtypes were also observed using a collection of 104 independent primary CIN colon tumors. In addition, we found a third subtype, consisting of tumors with a very low number of alterations not associated with specific loci (CIN‐Very Low). We found that colon carcinogenesis may require a minimal set of alterations and that, in contrast to the current hypothesis, the level of CIN does not correlate with tumor progression. Therefore, our results suggest that metastasis potential could be present at very early stages of tumor development.

Cdx1 homeobox gene during human colon cancer progression.

The Cdx1 homeobox gene encodes an intestine-specific transcription factor with a pro-oncogenic function in vitro. Here we have analysed the pattern of Cdx1 in human colon cancer progression. Cdx1 expression remains at a high level in the majority of the polyps and it is even overexpressed in more than one-third of the specimens, consistent with the fact that the gene is an intestine-specific target of oncogenic pathways. However, Cdx1 decreases in one-fifth of the polyps, which is reminiscent of the loss of expression previously reported in the majority of carcinomas. Allelic imbalance analysis demonstrates that the Cdx1 locus located on chromosome 5q is a major site of genomic rearrangement in colorectal cancers, and that the frequency of the rearrangements increases during polyps to carcinoma progression. Allelic imbalance at the Cdx1 locus occurs in relation to, although not invariably in association with, the rearrangements at the APC locus on the same chromosomal arm. Xenografts of primary human colon carcinomas indicate that the level of Cdx1 mRNA correlates with the intensity of allelic imbalance. Together, these data show that Cdx1 exhibits a complex pattern during colorectal cancer progression. Given that Cdx1 has a pro-oncogenic function in vitro, the maintenance of a high level of expression in polyps, and even its overexpression in one-third of the specimens, suggest that this homeobox gene may be an important factor in the process toward malignant transformation during the first steps of tumorigenesis.