The family of human choline/ethanolamine kinases comprises two genes, CHKA
that codify for three enzymes, ChoKα1 (52 kDa), ChoKα2 (50 kDa) and ChoKβ1 (45 kDa). ChoKα1 and ChoKα2 are almost identical, except for a stretch of 18 extra amino acids in ChoKα1, as they result from differential splicing from the same gene, CHKA
. While the implication of ChoKα1 in the regulation of cell growth and cancer has been extensively demonstrated 
, preliminary evidence suggest that ChoKβ may not be involved in carcinogenesis since it is not overexpressed in breast cancer cell lines 
nor in the TRAMP mouse prostate cancer model 
A distinct human gene family has been described that codifies for EtnK activity 
, suggesting that EtnK1 is the main enzyme involved in PE homeostasis. The ChoK/EtnK domain confers to ChoKα and ChoKβ the ability to function as both ChoK and EtnK activity under cell-free conditions 
, but it is still unknown if these previously characterized enzymes showed any selectivity to each branch of the Kennedy pathway in the de novo
synthesis of PC or PE. It has been recently described the different specificity towards Cho and Etn of the two isoforms of Cho/EtnK of Tripanosoma brucei
. Whereas Tb
Cho/Etn1 displays only EtnK activity, Tb
Cho/Etn2 displays both ChoK and EtnK activities in vitro 
. These results are in keeping with those described for murine EtnK1 that is Etn specific and EtnK2 that displays a dual ChoK/EtnK function 
. On the other hand, whereas murine Pcyt1α and Pcyt1β are involved in PC biosynthesis, Pcyt2 is focused to PE only 
However, it is not yet fully understood which ChoK isoform, if any, contributes in vivo
in each pathway to maintain the normal homeostasis of both PC and PE in biological membranes. The results shown here confirm that both enzymes have the ability to phosphorylate choline and ethanolamine under cell-free conditions, either as recombinant proteins produced in E. coli
, or in cell extracts from mammalian cells. However, we found that under whole cell conditions ChoKα1 has the ability to function as both ChoK and EtnK, but ChoKβ only affects the production of PEtn. These findings of different roles for α and β isoforms are in keeping with the information from the recently generated Knock Out (KO) mice for ChoKα and ChoKβ genes 
. Thus, ChoKβ KO mice (rmd
mice) are viable, but develop a rostrocaudal muscular dystrophy, while normal PC lipid levels are found in most tissues analyzed except in hindlimb skeletal muscle 
. Therefore, ChoKα is sufficient to maintain normal PC levels in most tissues. By contrast, the lack of ChoKα results in embryonic lethality, and ChoKα+/−
heterozygous mice display an accumulation of Cho and a reduction in PCho in liver and testis, suggesting that there is no ChoKβ compensation for PC biosynthesis in vivo
. These results suggest different roles in vivo
for both ChoKα and ChoKβ isoforms. Furthermore, the attenuated levels in PE found in ChoKα+/−
heterozygous mice suggest the involvement of ChoKα not only in the biosynthesis of PC but also in the PE pathway. This is also consistent with the fact that in ChoKβ KO mice, PE levels are unaffected, indicating that PE homeostasis is fully maintained with the EtnK1 and ChoKα proteins intact.
The group of Ishidate has provided valuable information about the in vitro
activity of ChoK from different mouse tissues, and they have postulated that the most active form for choline kinase activity is the α/α homodimer followed by α/β heterodimers, being the β/β homodimer the least active form 
. The in vivo
results shown here are partially in keeping with this hypothesis. The in vivo
activity of ChoKβ is focused in PE biosynthesis and displays higher Km for choline than ChoKα, this could be the reason why β/β dimers show low ChoK activity. When ChoKβ was overexpressed we observed an increase in the intracellular levels of PEtn but not of PCho. However no differences were found between both isoforms for the generation of PEtn, since both display similar EtnK activity.
PCho has been proposed to promote mitogenesis in mammalian cells 
. In keeping with this, magnetic resonance spectroscopy techniques have revealed higher levels of phosphomonoesthers in tumoral samples when compared to their normal counterparts 
. Moreover, overexpression of ChoKα1 is oncogenic 
, and enhanced ChoKα activity is a frequent feature in tumoral samples compared to normal tissues 
. Taken together all these results strongly indicate that ChoKα1 activity and PCho levels have a strong implication in cancer. Furthermore, overexpression of ChoKα1 results in an increase in EtnK activity and PEtn levels. However the latter effect by itself is not sufficient to induce cell transformation, since overexpression of ChoKβ does not induce enhanced colony formation in soft-agar or tumor growth in nude mice. These results are consistent with the hypothesis that it is the production of PCho what is linked to cell proliferation and transformation mediated by ChoK, and that the production of PEtn may not be sufficient or relevant in this process.
The above results suggest that the two ChoK isoforms investigated, besides their similarity in their primary sequences, are implicated in different metabolic pathways. Thus while ChoKα1 impinges into both PC and PE synthesis, ChoKβ affects only PE synthesis. Furthermore, the transformation capacity seems to be exclusive to the ChoKα isoform. However, since in the human HEK293T, Sk-Br-3 and H1299 cell lines, ChoKα1 overexpression produces elevated levels of both PCho and PEtn, while similar ChoKβ overexpression results only in higher levels of PEtn but normal levels of PCho, we can not rule out the possibility that cell transformation requires both ChoK and EtnK activities.
Consistent with the idea that links oncogenic activity to the function of ChoKα, but not ChoKβ, the antiproliferative and antitumoral activity of MN58b was only associated to the activity of ChoKα. Furthermore, as previously reported, the in vivo
treatment with MN58b results in a specific decrease of PCho levels in the tumours but no significant effect on the levels of PEtn 
. Previous results from our group have demonstrated that MN58b also inhibits choline transport 
. However this effect has a little influence in the antiproliferative and antitumoral activity of the drug since HC-3, a much more potent inhibitor of choline transporters, is far less potent as an antiproliferative agent than MN58b 
. Furthermore, MN58b has a differential effect on either normal or tumor cells, a strong demonstration of a differential activity due to ChoK inhibition 
. These results are also in keeping with the observation that ChoKα but not ChoKβ is a downstream target of oncogenic molecules such as Ras and RhoA. Thus, while Ras activates ChoKα through Ral-GDS and PI3K 
, and RhoA activates ChoKα through ROCK 
, none of these oncogenic GTPases affect ChoKβ activity under similar conditions. Again, these results indicate that although both ChoK enzymes are able to phosphorylate both choline and ethanolamine under cell-free systems conditions, they display different affinities for these substrates, and in whole-cell assays conditions they are governed by distinct regulatory pathways.
Finally, the involvement of ChoKβ in breast and lung cancer has been studied, ChoKα and β mRNA levels were determined by Q-PCR. All tumour-derived cell lines assayed significantly overexpress ChoKα mRNA, while no changes were found in the expression of ChoKβ. Similar results have been recently reported, using semi-quantitative PCR in breast cancer cell lines 
. In addition, it has been recently described that elevated mRNA levels of ChoKα is a poor prognostic factor in lung cancer 
. These results suggest that ChoKα but not ChoKβ is playing a crucial role in human carcinogenesis.
The results shown here suggest that ChoKβ and its produced metabolites are not implicated in human cell transformation. Therefore, all the efforts aimed at dilucidating the involvement of ChoK activity in the diagnosis, prognosis and treatment of cancer have to be focused in the ChoKα isoform. In keeping with this, recently the use of specific monoclonal anti-ChoKα antibodies has been proposed as a diagnostic tool in human cancer 
. Furthermore, the newly designed antitumoral agents are expected to be more specific and hence less toxic than the actual drugs used in conventional chemotherapy. Due to the high structural homology displayed by both choline kinase proteins, the search for new anticancer agents based on their ability to interfere with ChoK activity, must exhibit stronger antiproliferative activity based on their specificity towards the ChoKα isoform.
The lack of specific inhibition of ChoKβ by these newly designed compounds represents a new feature to take into account for the chemical improvement of ChoKα inhibitors with potential antitumoral activity. Furthermore, non-specific drugs affecting ChoKβ may result in a muscular disease produced by the lack of cell membrane lipid reparation in muscle tissue.
In addition, it has been recently demonstrated using a genetic approach that the specific inhibition of ChoKα by shRNA displays antiproliferative and antitumoral activity The high specificity of this technology provides definitive evidence of an antitumoral strategy based on ChoKα inhibition, supporting previous results with the pharmacological inhibitors. 
Thus, despite the high homology and similar activity displayed under cell-free conditions, ChoKα1 and ChoKβ isoforms show a different substrate ability and behave very differently under in vivo conditions, suggesting that in human cells, ChoKβ behaves as an EtnK and its overexpression is not able to induce higher intracellular levels of PCho. As a consequence, ChoKβ has no effect on cell proliferation and does not contribute to oncogenic transformation. Finally, ChoKα but not ChoKβ, should be used as the molecular target for the design of anticancer drugs aiming at interfering with choline kinase acitivity.