In the present study, we first report two forms of human phosphoserine aminotransferase (PSAT) cDNA (HsPSATα and HsPSATβ). HsPSATα has a predicted open reading frame comprising 324 amino acids, encoding a 35.2 kDa protein (PSATα), whereas HsPSATβ consists of an open reading frame comprising 370 amino acids that encodes a 40 kDa protein (PSATβ). PSATα is identical with PSATβ, except that it lacks 46 amino acids between Val290 and Ser337 of PSATβ, which is encoded by the entire exon 8 (138 bp). Both PSATα and PSATβ can functionally rescue the deletion mutation of the Saccharomyces cerevisiae counterpart. Reverse transcriptase–PCR analysis revealed that the expression of PSATβ mRNA was more dominant when compared with PSATα mRNA in all human cell lines tested. PSATβ was easily detected in proportion to the level of mRNA; however, PSATα was detected only in K562 and HepG2 cells as a very faint band. The relative enzyme activity of glutathione S-transferase (GST)–PSATβ expressed in Escherichia coli appeared to be 6.8 times higher than that of GST–PSATα. PSAT mRNA was expressed at high levels (approx. 2.2 kb) in the brain, liver, kidney and pancreas, and very weakly expressed in the thymus, prostate, testis and colon. In U937 cells, the levels of PSAT mRNA and protein appeared to be up-regulated to support proliferation. Accumulation of PSAT mRNA reached a maximum in the S-phase of Jurkat T-cells. These results demonstrate that although two isoforms of human PSAT can be produced by alternative splicing, PSATβ rather than PSATα is the physiologically functional enzyme required for the phosphorylated pathway, and indicate that the human PSAT gene is regulated depending on tissue specificity as well as cellular proliferation status with a maximum level expression in the S-phase.
- alternative splicing
- cell-cycle progression
- human phosphoserine aminotransferase gene
- l-serine synthesis
- phosphoserine aminotransferase α and β
- tissue specificity
The nucleotide sequences for the human phosphoserine aminotransferase α and β cDNAs have been deposited in the GenBank® Nucleotide Sequence Database under the accession numbers AF113132 and AY131232 respectively.
Abbreviations used: CNS, central nervous system; FBS, foetal bovine serum; GST, glutathione S-transferase; HBSS, Hanks balanced salt solution; ORF, open reading frame; PHGDH, 3-phosphoglycerate dehydrogenase; PSAT, phosphoserine aminotransferase; RT, reverse transcriptase; TdR, thymidine deoxyribose.
- The Biochemical Society, London ©2003