Identification and characterization of alternative splice products encoded by the human phosphotyrosyl phosphatase activator gene.
The phosphotyrosyl phosphatase activator (PTPA), a protein phosphatase 2A (PP2A) regulatory protein, specifically stimulates the phosphotyrosyl phosphatase activity of PP2A in vitro. Human PTPA is encoded by a single gene, the structure and chromosomal localization of which have been determined in our previous work. In this paper, we report the identification and characterization of six additional splice variants, termed PTPAbeta to PTPAeta, in addition to the originally identified PTPAalpha form. Interestingly, PTPAbeta and PTPAgamma contain a novel exon that had been overlooked in the formerly identified gene structure. As revealed by nested PCR, all these PTPA transcripts are expressed in various human cDNA libraries and cell lines. However, a quantitative approach, using a single PCR reaction followed by detection of the reaction products with a radioactively labeled probe, revealed only PTPAalpha, beta and delta, suggesting that the other transcripts are expressed very poorly. In vitro transcription-translation revealed that only PTPAalpha, beta, delta and epsilon are translated into functional proteins, whereas translation of PTPAgamma, zeta and eta is stopped prematurely due to a frameshift resulting from skipping exon 2, suggesting that the latter isoforms may result from splicing errors. By western analysis of HepG2 and Saos-2 cell extracts, only PTPAalpha and beta were detected. PTPAalpha and beta were expressed as GST fusion proteins in bacteria, and were found to contain the same specific phosphotyrosyl phosphatase stimulatory activity towards PP2A. The identification of this family of PTPA variants adds another level of complexity to the in vivo function(s) of PTPA, opening up the possibility that different isoforms may perform different functions.
Janssens
V
V
Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit Leuven, Belgium.
van Hoof
C
C
Martens
E
E
de Baere
I
I
Merlevede
W
W
Goris
J
J
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005091:Exons; D016368:Frameshift Mutation; D015723:Gene Library; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D014176:Protein Biosynthesis; D020033:Protein Isoforms; D054648:Protein Phosphatase 2; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014018:Tissue Distribution; D014158:Transcription, Genetic
D018076:DNA, Complementary; D020033:Protein Isoforms; D011506:Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; C087440:PTPA protein, human