Molecular Cloning And Differential Expression Patterns Of The Gene Encoding The PR55/B Family Memembers Of PP2A In Goldfish, Carassius Auratus

The reversible phosphorylation of proteins is an important posttranslational modification in eukaryotes. Phosphatases play a key role in various cellular process by dephosphorylation. Protein phosphatase 2A (PP2A) is one of the major serine/threonine-specific phosphatases and involved in many cellular processes such as cell cycle progression, DNA replication, transcription, and signal transduction. PR55/B family is one of identified regulatory B subunits of PP2A.Using 3'-and 5'-RACE,PR55δ, PR55βand partial PR55γof PR55/B family of PP2A were cloned from goldfish. The full length of PR55δcDNA is 2163 bp, containing an open reading frame (ORF) of 1347 that encodes a deduced protein of 448 amino acids. Full-length PR55βconsists of 1940 bp with an ORF of 1332 nucleotides coding for a deduced protein of 443 amino acids. The partial PR55γcontains 1218 nucleotides, which encodes a deduced part protein of 405 amino acids. Sequence analysis showed that goldfishβ,δandγisoforms of PR55/B family show high identities with their counterparts in other species, thus suggesting the conservation of PR55/B family members through animal kingdom. The PR55/B phylogenetic tree based on the amino acid sequences from various species fits well with the traditional phylogenetic tree. Among the three isoforms of PR55/B family members, the N-termini were significantly divergent between PR55δand PR55β, suggesting that a short, divergent stretch of amino acids may confer specific targeting information. Amino acid sequence analysis through ExPASy and other program revealed that the WD 40 tandem repeats were conserved in PR55δ, PR55βand PR55γisoforms. It is predicted that this domain is important for their binding to the PP2A-A subunits. RT-PCR and Western blot analysis showed that PR55βgene could be only detected in brain and heart, whereas the PR55δwas expressed ubiquitously but non-uniformly in 9 tissues examined. And PR55γgene mRNA specifically expressed in brain and fin. Our demonstration of the PR55βmRNA in heart and the PR55γmRNA in fin provides the first evidence regarding their tissue-specific expression patterns. In addition, goldfish (lower vertebrates) and murine (higher vertebrates) displayed distinct difference in the tissue-specific expression patterns of PR55δ/β/γ. Our results suggest that PR55βmay play an important role in heart and PR55γin fin.The PR55δis highly expressed at both mRNA and protein levels from early to later developmental stages of goldfish. The PR55βmRNA was also detected in goldfish embryo, but we rarely detected any PR55βprotein expression at the 12 different developmental stages examined. Distinct difference exists in the temporal expression patterns of the PR55δ&βbetween lower (goldfish) and higher vertebrates (murine), which suggests that PR55δ/βmay play different roles in regulating the developmental processes of the two types of vertebrates. The PR55γexpression was first detected at neurula stage, and then gradually enhanced. This expression pattern suggests that PR55γmay be important for development of the nervous system.To explore PR55δfunction in regulating development, we expressed the anti-sense RNA from the exogenous PR55δcDNA which can block translation of the PR55δmRNA from the endogenous PR55δgene. When PR55δwas downregulated, the development of goldfish embryos displayed severe abnormality in organogenesis. We observed that during brain differentiation stage, the expression of the antisense PR55δRNA led to absence of a differentiated brain, while the expression of the vector (mock) had little effect on the brain development. In addition, the eye development was also interrupted, displaying microphthalmia in the embryos with reduced PR55δexpression. These results provide the first evidence that the regulatory subunit of PP2A directly controls organogenesis. Our demonstration that downregulation of PR55δby anti-sense RNA led to severe abnormality in the brain and eye development suggests that the specific PP2A activity contributed by PR55δregulatory subunit is crucial for development. In this case, the PP2A containing PR55δregulatory subunit may modulate a set of specific targets important for development that can't be dephosphorylated by PP2A with non-PR55δregulatory subunit.Together, our results suggested that PR55/B family members have versatile functions during goldfish development and tissue homeostasis.