Abstract
The Cpi-17 (ppp1r14) gene family is an evolutionarily conserved, vertebrate specific group of protein phosphatase 1 (PP1) inhibitors. When phosphorylated, Cpi-17 is a potent inhibitor of myosin phosphatase (MP), a holoenzyme complex of the regulatory subunit Mypt1 and the catalytic subunit PP1. Myosin phosphatase dephosphorylates the regulatory myosin light chain (Mlc2) and promotes actomyosin relaxation, which in turn, regulates numerous cellular processes including smooth muscle contraction, cytokinesis, cell motility, and tumor cell invasion. We analyzed zebrafish homologs of the Cpi-17 family, to better understand the mechanisms of myosin phosphatase regulation. We found single homologs of both Kepi (ppp1r14c) and Gbpi (ppp1r14d) in silico, but we detected no expression of these genes during early embryonic development. Cpi-17 (ppp1r14a) and Phi-1 (ppp1r14b) each had two duplicate paralogs, (ppp1r14aa and ppp1r14ab) and (ppp1r14ba and ppp1r14bb), which were each expressed during early development. The spatial expression pattern of these genes has diverged, with ppp1r14aa and ppp1r14bb expressed primarily in smooth muscle and skeletal muscle, respectively, while ppp1r14ab and ppp1r14ba are primarily expressed in neural tissue. We observed that, in in vitro and heterologous cellular systems, the Cpi-17 paralogs both acted as potent myosin phosphatase inhibitors, and were indistinguishable from one another. In contrast, the two Phi-1 paralogs displayed weak myosin phosphatase inhibitory activity in vitro, and did not alter myosin phosphorylation in cells. Through deletion and chimeric analysis, we identified that the difference in specificity for myosin phosphatase between Cpi-17 and Phi-1 was encoded by the highly conserved PHIN (phosphatase holoenzyme inhibitory) domain, and not the more divergent N- and C- termini. We also showed that either Cpi-17 paralog can rescue the knockdown phenotype, but neither Phi-1 paralog could do so. Thus, we provide new evidence about the biochemical and developmental distinctions of the zebrafish Cpi-17 protein family.
Highlights
Teleosts are a large infraclass of ray-finned fish, with 30,000 species, that comprise approximately 95% of fish and 50% of vertebrate species [1]
A simple phylogenetic comparison of the PHIN domain of the zebrafish genes and the human genes is consistent with a model of whole genome duplication, leading to duplicated paralogs, some of which are retained in the genome, while others are lost (Figure 1)
Both paralogs of Cpi-17 and both paralogs of Phi-1 were expressed at various developmental stages, while no expression was detected for ppp1r14c or ppp1r14d during early development
Summary
Teleosts are a large infraclass of ray-finned fish (class Actinopterygii), with 30,000 species, that comprise approximately 95% of fish and 50% of vertebrate species [1] Certain teleosts, such as medaka (Oryzias latipes) and zebrafish (Danio rerio), have become important genetic model organisms of vertebrate development and human disease, which allows researchers to link fish and human gene functions. Duplicated genes can remain redundant, acquire new functions not seen in the ancestral gene, or subfunctionalize by splitting the ancestral gene’s function [11] This subfunctionalization can occur due to changes in the activity of the protein product, or by changes in gene expression of the paralogs [11]
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