Analysis Differential Expression Patterns Of The Protein Phosphatase-1 And -2A In The Vertebrates

Protein Phosphorylation and dephosphorylation is a fundamental regulatory mechanism in enkaryotes. Dephosphorylation through protein phosphatases play a critical part in regulating intracellular processes in response to extracellular signals, mediated by hormones, growth factors, cytokines and antigens, and to intracellular signals such as those associated with DNA damage and those influencing the cell cycle. In eukaryotes, dephosphorylation at the serine/threonine residues is largely executed by four major protein phosphatases: Phosphatase (PP)-1, -2A,-2B, -2C, and some minor phosphatases: PP-4, -5, -6, and -7. About 80% of intracellular protein serine/threonine phosphatase activity has been attributed to PP-1 and PP-2A. Using goldfish, allotetraploid fish,common carp/red crucian carp, triploid crucian carp, and white mouse as experimental materials, and using RT-PCR, Real-time PCR, Westem blot and immunofluorescence cytochemistry as experimental strategies,Ⅰhas investigated the differential expression patterns of the catalytic subunits for PP1 and PP2A in the above-mentions vertebrates and obtained following results:(1) Partial eDNA sequences for PPla (823 bp),PP1β(419 bp),and PP2A a (408 bp) of Goldfish were initially determined using deduced oligo primer and PCR amplification, which offered experiment basis for our further study of PP1 and PP2A expression in goldfish.(2) In gold fish, the expression patterns of the catalytic subunits for PP1 and PP2A were analysized at the mRNA and protein levels in liver, muscle, ovary, testis, brain, kidney, heart, gill and tail. It was found that the highest level of PPlc mRNA was observed in testis, and slightly lower PP1c mRNA was detected in ovary, kidney, heart, liver, and brain, and an even lower level of PP1c mRNA was found in muscle, gill and tail. For PP2Ac mRNA, the highest level of expression was detected in gonads. A slightly lower level of PP2Ac mRNA was detected in kidney. An even lower level of PP2Ac mRNA was detected in liver, brain, heart. The lowest level of PP2Ac mRNA was observed in muscle, gill and tail. At the protein levels, both PPlc and PP2Ac displayed differential expression in different tissues examined. The most striking feature is that both muscle and heart had either barely detectable or undetectable PP2Ac.(3) PP1c and PP2Ac have their temporal and special expression patterns during embryonic development of goldfish. Westem blot revealed that the expression of both PP1c and PP2Ac were gradually enhanced from blastula to hatching and peaked at hatching. Immunocytochemistry analysis demonstrated that both PP1c and PP2Ac were strongly expressed in all different development stages of the goldfish embryo. Inhibition of PP1 and PP2A activity with calyculin A lead to massive apoptosis of blastula cells and embryonic lethality.(4) In the allotetraploid fish and their distal parents: common carp and red crucian carp as well triploid crucian carp, differential expression patterns of PP1c and PP2Ac were also observed in brain, heart, muscle, kidney, liver and gonads. The expression patterns were similar to that in the goldfish tissues, so were true with PP1e and PP2Ac in 11 different mouse tissues.(5) PP1c and PP2Ac have expressions in the following 11 tissues— liver, brain, kidney, heart, muscle, spleen, ovary, testis, breast, lung, stomach. For mRNA level, the relatively higher expression was found in gonads and brain, the expression in liver, spleen, kidney, heart and breast took the second place, the lower level was detected in muscle and stomach. For protein level, the higher expression was found in the brain; the lower level was detected in the ovary, testis, kidney, liver, breast, spleen, heart, lung; similarly, the lowest level was observed in muscle and stomach. The localization study showed the expression of PPlc and PP2Ac in the tissues and cellular has its specificity.(6) A careful study of the expression patterns of PP1c and PP2Ac in the eyes of goldfish and mouse demonstrated that higher levels of expression of PP1c and PP2Ac were detected in the retina of both organisms. In the cornea, while high levels of PP1 and PP2A were also observed in mouse cornea, the lowest levels of expression of PP1c and PP2Ac were found in goldfish cornea. In all four compartments of gold fish and mouse eye, PP1 seems to be a more abundant protein serine/threonine phosphatase. From the localization study on the embryo eyes' development of the fertilized 48-hour goldfish and the mouse, it was found that the expression of PP1c and PP2Ac was quite high in epithelium cells and fiber cells of the goldfish embryo eyes, but wasrelatively lower in retina cells and cornea. To the mouse, the expression pattern was similar.In summary, both PP1c and PP2C were highly expressed in most tissues examined from goldfish to mouse. A differential expression pattern was observed in different tissues of all organisms examined. Both PP1c and PP2Ac play an important role in regulating embryonic development. Inhibition of PP1 and PP2A activity lead to goldfish embryonic lethality at the blastula stage.