The present studies were designed to clarify important questions concerning major splice variants of the PI4KIIIα enzyme. The orthologue of this enzyme, Stt4p is essential for yeast and increasing evidence suggests that it is also critical in maintaining the functional integrity of mammalian cells. There are several interesting features of this protein: first it is primarily located in the endoplasmic reticulum where it was shown to regulate ER exit sites [32
]. Curiously, in spite of its ER localization, PI4KIIIα seems to be responsible for the maintenance of the plasma membrane phosphoinositide pools [34
], similarly to the yeast Stt4p protein [18
]. This might be related to the enrichment of the enzyme in signaling domains at ER-PM contact zones as shown in the yeast [35
], a finding yet to be demonstrated in mammalian cells. Second, recent studies have demonstrated that PI4KIIIα is essential for HCV viral replication in liver [9
] and possibly also for the replication of other small RNA viruses [36
], thus gaining significant interest as a druggable target.
There are two splice variants of PI4KIIIα listed in GenBank (GI:4505807= Isoform 1, GI:155030226= Isoform 2). The shorter isoform was the first PI4KIIIα clone isolated from humans [20
]. Since the smaller splice variant has never been shown to exist endogenously at the protein level, it was important to know whether it indeed is present and if so, whether it contributes to the PI4K activity of mammalian cells. Our analysis did not confirm the existence of the short splice variant of PI4KIIIα, and also proved that even if it existed, this protein would be catalytically inactive. Consistent with this finding, unlike the full-length protein, this short splice variant was unable to restore HCV replication in OR6 cells with downregulated PI4KIIIα. These data together make it quite unlikely that the short splice variant of the PI4KIIIα has any major functional significance.
Quantitative analysis of the PI4KA mRNA copy numbers in several cell lines revealed a wide range, showing the most abundant message in K562 cells and HEK293 cells substantially higher than in COS-7 cells. In fact, earlier studies concluded that the enzyme was not expressed in COS-7 cells, which is consistent with the low level of the protein in these cells [37
]. In the present study we were able to detect the low endogenous level of PI4KIIIα in COS-7 cells, although both K562 cells and HEK293 cells showed significantly higher levels of the protein. However, these differences in protein levels did not reflect the proportions found in the number of transcripts. This discrepancy can be caused by the non-linearity of the Western analysis, but most likely is due to differences in translational efficiency and protein stability between the cell lines. This aspect of regulation was beyond the scope of the current studies. The other important question that arises is the nature of the shorter transcripts detected in the K562 cells. These could represent shorter forms generated from the PI4KA gene or from the two pseudogenes. Since our Northern analysis cannot discriminate between these possibilities, we could not determine with certainty the contribution of these different sources to these smaller transcripts. However, our qPCR analysis suggested a contribution to these transcripts by the PI4KAP2 pseudogene, in agreement with the existence of a number of ESTs in the database corresponding to transcripts from the pseudogenes. Therefore, we concluded that the pseudogenes contribute to the transcript heterogeneity in K562 cells where chromosomal amplification likely yields abundant transcripts generated from this chromosomal region.
Our analysis of the genomic organization of the PI4KA gene in human Chr22 revealed important details with potential implications in human disease. First, the gene is located in the 22q11.2 locus that is known for its genetic instability. This is due to the presence of low copy sequence repeats (LCRs) that can result in recombination defects causing “22q11.2 deletion syndromes” [38
]. Intriguingly, these repeats contain two partial copies of the PI4KA gene containing most (but not all) exons (exon 24 and from exon 33 upward) comprising part of what encodes the PI4KIIIα catalytic domain. These observations are even more revealing in light of the high expression of PI4KA transcript (together with smaller transcript variants) in the K562 erythroleukemia cell line along with a number of other genes, all of which are located in the 22q11.2 locus. It is also notable that several genes that are found highly expressed in correlation with PI4KA are located in 22q11.2 and are involved in FGF signaling (see ). CRKL is a member of the Crk family of adaptor proteins that is a downstream target of Fgf receptors 1 and 2 and is a target of tyrosine phosphorylation by the BCR-Abl tyrosine kinase in CML cells [39
]. THAP7 is a histone tail-binding protein that represses transcription by recruiting the HDAC3 histone deacetylase to transcriptional complexes [41
] and a close relationship between Fgf signaling and histone deacetylation has been documented [42
]. In this context it is notable that downregulation of PI4KIIIα in zebrafish embryos results in defective Fgf signaling [44
]. However, it will require further studies to determine whether PI4KA haploinsufficiency contributes to some of the symptoms associated with 22q11.2 deletion syndromes.
In summary, the present study shows that the functionally relevant PI4KIIIα protein is the larger isoform 2 and that isoform 1, even if exists, is present at undetectable levels and encodes a catalytically inactive protein. These studies help to clarify the significance of the distinct isoforms listed in the GenBank and to draw attention to this enzyme as a potentially important candidate in several forms of human diseases associated with the human Chr22q11 region.