CDNA Cloning And MRNA Expression Analysis Of The Genes Coding For The Regulatory Subunits Of PP2A-B″ In Carassius Auratus

Phosphorylation and dephosphorylation of proteins are major intracellular control mechanisms in eukaryotes.Phosphatases play a key role in various process of life by the dephosphorylation.As a major intracellular phosphatase,the protein serine/threonine phosphatase 2A (PP2A) controls the phosphorylation of numerous proteins involved in cell signaling and is an important regulator of cell growth through its lots of regulatory subunits.The cDNAs for the three regulatory subunit isoforms:B" alpha(PR130),beta(PR67) and gamma(G5PR) of PP-2A were cloned from the ovary of goldfish(Carassius auratus) using RT-PCR and rapid-amplification of cDNA ends(RACE).The partial cDNA for PP2A-B"α(1781bp) contains an open reading frame of 426 amino acids, and the full length cDNA clones for PP2A-B"βandγconsist of 2561bp, 1885bp and encode a protein of 581aa,457aa respectively.Both cDNAs and the predicted amino acid sequences show a high homology to the correspondingα,βandγsubunits from other species and the presence of such homology suggests that the three regulatory subunits are highly conserved in evolution.Amino acid sequence alignment of the three subunits shows that bothαandβsubunits have a higher homology when compared withγsubunit.Such results implies thatαandβsubunits may have similar action mode but theγsubunit may adopt a different one.Like other members of B" family,the predicted proteins of PP2A-B"α,βandγin goldfish also bear two EF-hand calcium-binding motifs at the C-terminal.Sequence comparison of the goldfish PP2A- B"α,β,γ subunits with murine PP2A-B'γ1 reveal that the two EF-hand calcium-binding motifs may play an important role in the assembly of goldfish PP2A holoenzyme.In addition,as Ca²⁺ regulatory elements, the EF-HAND motifs may also regulate PP2A activity.Analysis of the expression levels of the three mRNAs with RT-PCR reveals tissue and developmental specificities.For PP2A-B"α,the highest mRNA level was detected in ovary and muscle,the intermediate level in brain,testis,and heart,further reduced in liver and kidney,and the lowest in the gill.The tissue specificity of PP2A-B"βis similar to that of PP2A- B"αexcept that higher level of PP2A-B"βmRNA was found in testis.In contrast to PP2A-B"βandβ,PP2A-B"γmRNA displayed much less difference among the 8 tissues examined.These results imply that the PP2A-B" subunits may be important in both gonad development and germ cell maturation in goldfish.During goldfish development, PP2A-B"αandβmRNAs displayed distinct developmental expression patterns.In contrast,PP2A-B"γwas relatively even-distributed at different stages.These results suggest that the regulatory B"α,βandγsubunits of PP2A may have different functions.While PP2A-B" may only act as the regulatory subunit of PP-2A,PP2A-B"αandβmay also have functions independent of PP-2A besides acting as regulatory subunits.