Death-associated protein (DAP)-like kinase (Dlk) (1
) or Zipper interacting protein (ZIP) kinase (2
) is a novel serine/threonine-specific kinase that belongs to the subfamily of DAP kinases. Presently, this kinase subfamily has five members: DAP kinase itself, DAP-related protein kinase, Dlk/ZIP kinase and DAPK-related apoptosis-inducing kinases (DRAK 1 and 2). The first three of these share a high degree of sequence identity (>80%) in their catalytic domains while the latter are more distantly related (~50% sequence identity to the first group). They all differ greatly in their non-catalytic domains (reviewed in 3
). DAP kinase itself plays an important role in interferon γ- and TNFα/fas-induced cell death (5
). Likewise, the DAP-related kinases have been implicated in apoptotic processes. Additionally, DAP kinase might have a tumorsuppressor function as it seems to interfere with tumor progression, particularly metastatic capacity (8
Dlk/ZIP kinase (Dlk, for simplicity) is a nuclear kinase that is ubiquitously expressed and highly conserved (99.7 or 86% identity between rat and mouse or rat and human homologs, respectively) suggesting that it fulfills an important function. Dlk does not induce apoptosis per se
. Rather, induction of cell death by Dlk requires its cytoplasmic retention and association with actin filaments. This can be achieved either by C-terminal truncation (9
) or by coexpression and interaction with pro-apoptotic protein Par-4 (10
). A physiological substrate in this process might be myosin light chain (MLC), since Dlk phosphorylates MLC in vitro
) and phosphorylation of MLC has been implicated in apoptotic membrane blebbing (11
). The function(s) of Dlk in the nucleus remain to be determined. Dlk contains a leucine zipper which mediates homodimerization and interaction with transcription factors ATF-4 (2
) and AATF (12
) and with transcription and splicing factor CDC5 (13
). Additionally, Dlk phosphorylates core histones H3, H4 and, to lesser extent, H2A in vitro
). Together, these data suggest a role in transcription and splicing and, perhaps, in chromatin remodeling.
Histones undergo extensive post-translational modifications, particularly acetylation, methylation, phosphorylation, ubiquitination and ADP-ribosylation (for reviews, see 14
). While the significance of the latter two modifications is less well understood, a growing body of information about the former three allows us to deduce some general principles. Thus, acetylation of the N-terminal tails of core histones H3 and H4 has been implicated in transcriptional activation by facilitating access for the transcriptional machinery to chromatin; methylation has been implicated in long-term transcriptional silencing, and phosphorylation has been connected to chromatin condensation during mitosis. Additionally, a role of H3 phosphorylation in activation of early response genes has been demonstrated. In these latter cases, H3 phosphorylation appears to facilitate recruitment of histone acetyl transferases and subsequent acetylation of H3 at adjacent sites (Lys14) (16
). Perhaps both these modifications cooperate in chromatin remodeling in promoter regions of a subset of genes and thereby contribute to full transcriptional activation. In summary, the various modifications of histones serve different functions in the dynamic changes of chromatin during transcription, replication and recombination, and during mitosis.
Phosphorylation of H3, particularly at Ser10 (and perhaps Ser28) seems to be crucial for progression through mitosis. In mammalian cells, phosphorylation of H3 at Ser10 initiates in late G2, in the pericentromeric region and spreads over the whole chromosome arms thereafter (17
). It assumes its highest level during prometaphase/metaphase when chromosomes are highly condensed, and lasts until anaphase upon which it disappears. The strong correlation of H3 phosphorylation and chromosome condensation suggested a causal relationship. However, in other organisms, particularly plants, H3 phosphorylation occurs on condensed chromosomes. Thus, H3 phosphorylation may serve as a label for mitotic chromosomes as suggested recently by the ‘ready production hypothesis’ (reviewed in 15
Interestingly, H3 or at least Ser10-phosphorylated H3 appeared to be excluded from centromeres, where it is replaced by its relative centromere protein (CENP-A) (18
). CENP-A, a constitutive centromere protein is incorporated into chromatin in late S-phase and seems to provide a specific, epigenetic label for centromeric chromatin serving as platform for assembly of centromere/kinetochore complexes (21
). However, a recent study by Blower et al
) using a novel chromatin-stretching technique showed that centromeres contain both CENP-A and H3, perhaps in alternating arrays. Interestingly, both H3 and its relative CENP-A seem to be phosphorylated by aurora B kinase (24
). However, it is not understood why this kinase phosphorylates H3 in chromosomal arms but not in centromeres where it prefers CENP-A. Obviously, other factors are necessary to mediate the distinction between different substrates in their respective contexts.
In the present study, we have investigated the significance of Dlk as a histone kinase. We found that Dlk phosphorylates H3 at a novel site, Thr11, which was specifically phosphorylated during mitosis. H3 bearing this modification seemed to be enriched in centromeric chromatin while H3 phosphorylated at Ser10 was found in the peripheral regions. Strikingly, Dlk was also associated with centromeres during mitosis and this association coincided precisely with phosphorylation of histone H3 at Thr11 and of CENP-A. We hypothesize that Dlk might be a centromere-specific histone kinase which plays a role in labeling centromeric chromatin for subsequent mitotic processes.