Related Experiment Videos
Novel human ZAKI-4 isoforms: hormonal and tissue-specific regulation and function as calcineurin inhibitors
Xia Cao1, Fukushi Kambe, Takashi Miyazaki
1Department of Endocrinology and Metabolism, Division of Molecular and Cellular Adaptation, Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601, Japan.
Abstract:
We identified a thyroid hormone [3,5,3'-tri-iodothyronine (T(3))]-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, alpha, beta1 and beta2, in human brain by 5'- and 3'-RACE (rapid amplification of cDNA ends). The alpha 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 beta1 and beta2 encode an identical protein product, beta, which differs from alpha in its N-terminus. Since alpha and beta 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 alpha transcript is expressed exclusively in the brain, whereas beta transcripts are expressed ubiquitously, most abundantly in brain, heart, skeletal muscle and kidney. It was also demonstrated that human skin fibroblasts express both alpha and beta transcripts, raising the question of which transcript is up-regulated by T(3). It was revealed that T(3) markedly induced the expression of alpha isoform but not of beta. This T(3)-mediated increase in the alpha 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.
Insights
Thyroid hormone (T3) up-regulates the ZAKI-4 alpha transcript in human skin fibroblasts, decreasing calcineurin activity. This highlights distinct hormonal and tissue-specific regulation of ZAKI-4 isoforms, forming a complex signaling network.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- ZAKI-4 is a thyroid hormone (T3)-responsive gene identified in human fibroblasts.
- It encodes proteins with a conserved motif that inhibits calcineurin phosphatase activity.
- Calcineurin is a key enzyme in cellular signaling pathways.
Purpose of the Study:
- To investigate the different transcripts of the ZAKI-4 gene in the human brain.
- To characterize the expression patterns and regulation of ZAKI-4 isoforms.
- To elucidate the role of ZAKI-4 in thyroid hormone signaling and calcineurin inhibition.
Main Methods:
- 5'- and 3'-RACE (rapid amplification of cDNA ends) were used to identify ZAKI-4 transcripts in human brain.
- Protein isoforms were predicted based on transcript sequences.
- Expression profiles were analyzed across 12 human tissues using quantitative methods.
- The effect of T3 on ZAKI-4 expression and calcineurin activity was assessed in human fibroblasts.
Main Results:
- Three ZAKI-4 transcripts (alpha, beta1, beta2) were identified in the human brain, generated from a single gene on chromosome 6.
- Alpha and beta isoforms differ in their N-termini but share a C-terminal calcineurin-inhibitory motif.
- Alpha transcript is brain-specific, while beta transcripts are ubiquitously expressed, with high levels in brain, heart, muscle, and kidney.
- T3 markedly induced alpha transcript expression in fibroblasts, leading to decreased calcineurin activity, while beta expression remained unaffected.
- Both isoforms associate with and inhibit calcineurin activity.
Conclusions:
- ZAKI-4 gene expression is regulated by alternative initiation and splicing, producing distinct isoforms.
- ZAKI-4 isoforms exhibit tissue-specific and hormonal regulation, particularly the T3-mediated induction of the alpha isoform.
- The differential regulation of ZAKI-4 isoforms contributes to a complex signaling network through calcineurin inhibition.