Novel human ZAKI-4 isoforms: hormonal and tissue-specific regulation and function as calcineurin inhibitors

Abstract

We identified a thyroid hormone [3,5,3′-tri-iodothyronine (T3)]-responsive gene, ZAKI-4, in cultured human skin fibroblasts. It belongs to a family of genes that encode proteins containing a conserved motif. The motif binds to calcineurin and inhibits its phosphatase activity. In the present study, we have demonstrated three different ZAKI-4 transcripts, α, β1 and β2, in human brain by 5′- and 3′-RACE (rapid amplification of cDNA ends). The α transcript was identical with the one that we originally cloned from human fibroblasts and the other two are novel. The three transcripts are generated by alternative initiation and splicing from a single gene on the short arm of chromosome 6. It is predicted that β1 and β2 encode an identical protein product, β, which differs from α in its N-terminus. Since α and β contain an identical C-terminal region harbouring the conserved motif, both isoforms are suggested to inhibit calcineurin activity. Indeed, each isoform associates with calcineurin A and inhibits its activity in a similar manner, suggesting that the difference in N-terminus of each isoform does not affect the inhibitory function on calcineurin. An examination of the expression profile of the three transcripts in 12 human tissues revealed that the α transcript is expressed exclusively in the brain, whereas β transcripts are expressed ubiquitously, most abundantly in brain, heart, skeletal muscle and kidney. It was also demonstrated that human skin fibroblasts express both α and β transcripts, raising the question of which transcript is up-regulated by T3. It was revealed that T3 markedly induced the expression of α isoform but not of β. This T3-mediated increase in the α isoform was associated with a significant decrease in endogenous calcineurin activity. These results suggest that the expression of ZAKI-4 isoforms is subjected to distinct hormonal as well as tissue-specific regulation, constituting a complex signalling network through inhibition of calcineurin.