Background
The ICK gene encodes an evolutionarily conserved Ser/Thr kinase in the CMGC group of the kinome, clustering in a subgroup with closely related MAK (male germ cell-associated protein kinase) and more distantly related MOK (MAPK/MAK/MRK overlapping kinase) [
1]. ICK was first identified and named MRK (MAK-related protein kinase) after cloning of its cDNA from heart [
2]). ICK expression was higher in the embryonic myocardium during organogenesis than in the adult tissue [
2]. Decreasing expression of ICK in Colo205 cells stops proliferation and causes cell cycle arrest in G1 due to an increase in p21
Cip [
3]. Colo205 cells greatly overexpress ICK mRNA in comparison to other lines in the NCI60, suggesting an acquired addiction to ICK for proliferation in this line. ICK mRNA is detectable in normal intestinal epithelium only in the region for lineage specification and proliferation [
4].
ICK has to be phosphorylated in a TDY motif (residues 157-159) within the activation loop to be fully active. Phosphorylation of Y159 can occur by autophosphorylation, but at least phosphorylation of T157 requires transphosphorylation by another kinase [
1]. ICK is a substrate for a T157-kinase related to CDK-activating kinase with gene name CCRK (cell cycle regulated kinase, [GenBank: NM_001039803]). CCRK (NM_001039803) unequivocally has T157 kinase activity because wild type but not a kinase-defective mutant phosphorylates T157 in cells [
1]. Decreasing CCRK expression ~80% markedly inhibited proliferation of HCT116 and U2OS cells without a significant, specific change in G1, M, or G2/M populations but modestly increased the population with sub-G1 DNA content, suggesting increased apoptosis [
5]. Other reports support a role for CCRK in molecular carcinogenesis of ovarian cancer [
6]. CCRK-specific gene silencing causes ovarian cancer cells to arrest in G1 [
6]. Recently, CCRK was identified as an interactor of Broadminded in Sonic hedgehog pathways [
7].
Discussion
The full intergenic segment (constructs ICK-1 and FBX9-1, respectively) was active in both orientations in all six of the lines, suggesting that ICK and FBX9 share a bidirectional promoter. Analyses in the different lines show elements in the common SspIb to PstIb fragment are important for bidirectional activity, and may account for the correlated expression of FBX9 and ICK in microarray data that motivated this study. Our analyses show that the intergenic segment is not a constitutive, bidirectional promoter because the FBX9 activity relative to ICK is variable. This report extends our knowledge of ICK regulation: (i) ICK shares a bi-directional promoter with an uncharacterized F-box protein, (ii) the putative ICK 5' start is in a GC-rich region containing a CpG island that is active as a promoter, (iii) a minimal promoter can be regulated by expression of FOXA and β-catenin.
ICK is conserved and almost all metazoans and some unicellular species (including S. pombe) have homologs of both MAK and ICK. Human ICK/MRK and human MAK are nearly identical in the kinase domain. Danio rerio has one gene that encodes a protein more similar to ICK than MAK. This genome is an anomaly, as other teleost fishes have both ICK and MAK genes. ICK message is highly expressed in developing retina in zebra fish (ZDB-GENE-030131-7279). Interestingly, ICK or MAK expression is greatly increased in retinal cancer compared to normal retina (SAGE/cDNA Virtual Northern) according to data at the Cancer Genome Anatomy Project).
Our prior work established ICK as the prototype for a group of CDK and MAPK like protein kinases regulated by phosphorylation in a TDY motif [
1,
29]. No canonical MAP kinase cascades have yet emerged for activation of ICK, in its limited study. An alternative mechanism is transcriptional regulation followed by activation by active protein kinases. The ICK homolog in
S. cerevisiae is regulated by transcription, and is subsequently phosphorylated in the TXY motifs dependent upon yeast CAK [
29].
In an insightful commentary, Adachi and Lieber [
30] noted that of twenty, functional bidirectional promoters reported in the literature at the time, several directed transcription of genes implicated in DNA repair: including BRCA1/NBR2, DNA-PKcs/MCM4, ATM/NPAT, DHFR/MSH3, and Ku86/TERP. While not unique to this class, they concluded placement of genes into bidirectional promoters is a common scenario for DNA repair genes. Clearly, this correlation does not imply anything about function of FBX9 or ICK. Nevertheless, this is of interest since ICK has interactors that may have some role in DNA repair [
29].
FBX9 is predicted to encode an F-box protein [
31]. F-box proteins contain a conserved domain that interacts directly with Skp1 as one of the components of a SCF (Skip, Cullin, F-box) ubiquitin ligase. The F-box protein provides a specific interaction that specifically recruits a substrate, possibly in a specific form (phosphorylated or un-phosphorylated) for degradation by linkage to ubiquitin. The substrate specificity of FBX9 is unknown.
FBX9 could produce three forms (403-447 residues) based on predicted transcripts. FBX9 has a possible homolog in
S. cerevisiae named Hrt3p (E = 2.9e-14 over 334 residues), discovered in a single genome search of
S. cerevisiae using SSEARCH
http://fasta.bioch.virginia.edu. Reciprocally, a search of NCBI human reference proteins with Hrt3p using SSEARCH finds FBX9 as the very first hit. Hrt3p is a putative nuclear ubiquitin ligase component based on large-scale studies (see Saccharomyces Genome Database (Stanford University, Leland, CA). Hrt3p interacts with Cdc53p and Skp1p by affinity capture mass spectrometry [
32], and shows dosage lethality with
cdc34.
The intestinal epithelium has advantages for studies of differentiation, one being the segregation of the epithelium into defined zones containing stem cells, zones for proliferating transit cells, and a zone of non-proliferating differentiated enterocytes [
33]. Other differentiated progeny, enteroendocrine cells, goblet cells and Paneth cells, derive from the same stem cells and assume characteristic positions in the epithelium. The epithelium is also constantly turned over during adult life. Since transcription factors regulate differentiation and are relatively easy to study, a large fund of knowledge existed for transcription factors in the gut that could suggest functions for ICK. This was a major motivation for our study. We found that FOXA1 and FOXA2, β-catenin activate an ICK reporter. These factors are known to regulate proliferation and differentiation in the intestinal epithelium [
24,
34,
35].
Recently, mutation of ICK was linked to neonatal deaths in humans. A study of a cohort of malformed newborns in Old Order Amish families revealed R272Q mutation of ICK as the probable cause of a severe recessive, endocrine/cerebro/osteodysplasia (ECO) syndrome [
36]. R272Q mutation causes loss of nuclear localization and kinase activity of ICK [
1,
36]. Abnormalities occurred in multiple systems, including bone, brain, and endocrine tissues [
36].
If the R272Q mutation in ICK can be confirmed as causally related to the ECO syndrome, ICK is unequivocally required for normal development. The finding warrants testing a similar knock-in mutation in mouse. MAK has been knocked out in mice with no phenotype noted except for reduced fertility and reduced sperm motility [
37]. Lack of a clear phenotype for a MAK knockout may be due to presence of ICK. However, the mild motility phenotype mentioned for sperm may be significant.
A single ICK/MAK homolog (LmxMPK9) in
Leishmania mexicana regulates morphogenesis of the flagella [
38]. Loss of LmxMP9 causes elongated flagella whereas overexpression of LmxMPK9 causes shortened or no flagella [
38]. Genetic studies of flagella morphogenesis in
Chlamydomonas reinhardtii identified a CCRK homolog as well as a homolog of MOK [
39] as having function in flagellar morphogenesis.
These links to flagella phenotypes seem abstruse for human disease except for the fact that there is a major developmental pathway in cells that respond to Sonic hedgehog that depends on primary cilia [
7]. CCRK interacts with Broadminded (Bromi) in the Sonic hedgehog pathway. We believe the cluster of genes ICK, MAK, and MOK may be regulated by CCRK and play a role in Sonic hedgehog signaling that was preceded in evolution by roles in flagellar morphogenesis in unicellular eukaryotes.
Another possible function for ICK is cell cycle regulation. The related kinase in budding yeast Ime2p (inducer of meiosis 2) controls a checkpoint that times meiotic S-phase and controls meiotic progression [
40,
41]. ICK can affect the cell cycle since reducing its expression in Colo205 cells causes arrest in G1 [
3].
The interactors suggest leads for ICK function to the degree that the functions of the interactors are understood [
29]. One interactor is multifunctional PP5, a protein phosphatase that recognizes substrates by a docking domain. The best established roles of PP5 are in control of apoptosis by inhibition of ASK1 [
42]; in the cell cycle by suppressing a pathway regulating the expression of p21(waf1) [
43]; in DNA repair by dephosphorylation of substrate DNA-PK [
44]; and in ATR-mediated checkpoint activation via an unknown substrate [
45].
The second ICK interactor we identified is the protein in literature BAT3 (NCBI designation) or Scythe or BAG6, whose functional roles are becoming clearer even if its names are not. All three names are common. ICK phosphorylates BAT3/Scythe at T1080 in vitro and in situ [
29]. BAT3 functions demands more study. The name Scythe came from ability of the protein to bind reaper in
in vitro capture experiments [
46], leading to several reports supporting the idea that BAT3 functions in apoptosis [
47]. BAT3, for example, can interact with an inter-membrane mitochondrial protein apoptosis-inducing factor, which seemed to fit the apoptosis-function hypothesis [
47]. A Deletion of BAT3 (-, -) does cause lethality and major abnormalities in development, and not surprisingly increased apoptosis in tissues. This is also consistent, but increased apoptosis may result indirectly, not because of a proposed model that BAT3 is a direct apoptotic regulator. BAT3 (-, -) fibroblasts are not very different from wild type fibroblasts in propensity to apoptose except to a very few stimuli. BAT3 is not directly functioning in any known apoptosis cascades. A second literature supports function of BAT3 as a co-chaperone with Hsp70 and regulation of protein stability and ubiquitin-dependent degradation [
48]. The kinases ICK, MAK, and MOK bind a chaperone Cdc37/p50, a nonexclusive partner of Hsp90 [
49].
Finding many interactions for BAT3 suggests a scaffolding domain. We believe a unifying hypothesis for the defects in development in the BAT3 (-, -) mouse may come in the future from vigorous study of its nuclear functions. BAT3 contains a nuclear localization sequence [
50]. Recent work establishes that nuclear retention of BAT3 can be dependent upon cellular transformation [
51]. In the nucleus, BAT3 and SET1A form a complex with Boris to modulate H3K4 histone dimethylation marks and gene expression [
52]. The latter discovery fits nicely with nuclear localization of BAT3 and transformation, abnormalities in development, and the high expression of BAT3 and MAK that occurs during spermatogenesis [
53,
54]. H3K and H3K4 methylation interplay to regulate gene activation [
55]. Nuclear function of BAT3 is also indicated by its requirement for p53 acetylation in response to DNA damage [
56]. Certain BAT3 genetic variations are strongly linked to susceptibility to lung cancer [
57].
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
TWS conceived the project, obtained the Sanger clone, directed the project, performed all of the bioinformatics analyses, interpreted the data, and wrote the manuscript. PBS and MWW performed the co-expression experiments in HEK293T cells. SMC provided the motivation to embark on studies of ICK, provided expertise on roles of β-catenin in intestinal epithelium, and helped supervise the project. All authors read and approved the final manuscript.