Molecular cloning, expression, and characterization of PTPA, a protein that activates the tyrosyl phosphatase activity of protein phosphatase 2A.
PTPA, or phosphotyrosyl phosphatase activator, is a protein that stimulates the tyrosyl phosphatase activity of protein phosphatase 2A in an ATP, Mg(2+)-requiring reaction (Cayla, X., Goris, J., Hermann, J., Hendrix, P., Ozon, R., and Merevede, W. (1990) Biochemistry 29, 658-667). We constructed oligonucleotide probes based on the amino acid sequences of peptides isolated from purified PTPA and used them to probe rabbit muscle and human heart cDNA libraries. A putative full-length clone was isolated from the rabbit skeletal muscle as well as from the human heart library. The nucleotide sequence of both clones contains an open reading frame of 969 nucleotides starting from an assigned initial ATG codon and encodes for a protein of 323 amino acids. The predicted rabbit and human PTPA protein sequences show an identity of 96.6%. The predicted protein matched all the peptide sequences obtained from the rabbit skeletal muscle protein. Bacterially expressed protein, as well as the in vitro reticulocyte lysate translation product, comigrated with the purified 37-kDa protein on sodium dodecyl sulfate-polyacrylamide gels. Both proteins reacted with immunopurified, anti-PTPA polyclonal antiserum. The recombinant protein was a soluble and active protein. Northern blot analysis revealed two transcripts of 2.8 and 4 kilobases, respectively, in human placenta but only one 2.8-kilobase transcript in rabbit and rat tissues. High levels of PTPA mRNA were detected in testis, which contrasted with the low levels present in skeletal muscle.
Cayla
X
X
Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit te Leuven, Belgium.
Van Hoof
C
C
Bosch
M
M
Waelkens
E
E
Vandekerckhove
J
J
Peeters
B
B
Merlevede
W
W
Goris
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004789:Enzyme Activation; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D014176:Protein Biosynthesis; D054648:Protein Phosphatase 2; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
D018076:DNA, Complementary; D009838:Oligodeoxyribonucleotides; D011506:Proteins; D011994:Recombinant Proteins; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D017027:Protein Tyrosine Phosphatases; C087440:PTPA protein, human