Cloning and expression of PCPTP1 encoding protein tyrosine phosphatase.
A novel cDNA encoding PTP (protein tyrosine phosphatase) was cloned from PC12h cells and designated as PCPTP1 (gene encoding PC12 protein Tyr phosphatase). The longest open reading frame (ORF) of this clone encodes a 656-amino-acid (aa) protein with a single PTP catalytic domain. Western blot analysis using a polyclonal Ab (antibody) raised against the cytoplasmic region of PCPTP1 detected two products, a major 65-kDa and minor 42-kDa protein, designated PCPTP1-MFI and PCPTP1-MVQ, respectively, in PC12h cells. These two proteins correspond to the products translated from the second and fifth methionine of PCPTP1, respectively. The bacterially expressed GST::PCPTP1-MVQ fusion protein had phosphatase activity with pNPP (p-nitrophenyl phosphate) as a substrate. Alignment of the aa sequence of PCPTP1-MVQ with those of other PTP showed the highest similarity to STEP and LC-PTP/HePTP, with 54 and 51% identity, respectively. Northern blot analysis showed only one 3.9-kb transcript in PC12h cells, indicating that PCPTP1 corresponds to this 3.9-kb transcript. The 3.9-kb PCPTP1 mRNA was detected in the brain and adrenal gland, but not in other non-neuronal tissues in adult rats. Two other transcripts of 3.3 and 1.7 kb were also detected in brain. NGF (nerve growth factor) and glucocorticoid are known to bimodally regulate the cell fate decision of sympathoadrenal precursors like PC12 cells, with NGF promoting the neuronal phenotype and glucocorticoid promoting the chromaffin phenotype. Still, both agents decreased the level of PCPTP1 mRNA in PC12h cells. Therefore, it is likely that the decrease in the level of PCPTP1 mRNA might be associated or correlated with cell differentiation in PC12h cells.
Shiozuka
K
K
Molecular Medicine Research Laboratories, Yamanouchi Institute for Drug Discovery Research, Ibaraki, Japan.
Watanabe
Y
Y
Ikeda
T
T
Hashimoto
S
S
Kawashima
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology; D002454:Cell Differentiation; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D005796:Genes; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D016716:PC12 Cells / Q000201:enzymology; D017027:Protein Tyrosine Phosphatases / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D054634:Receptor-Like Protein Tyrosine Phosphatases, Class 7; D014018:Tissue Distribution
D017931:DNA Primers; D018076:DNA, Complementary; D047908:Intracellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D017027:Protein Tyrosine Phosphatases; C513471:Ptprr protein, rat; D054634:Receptor-Like Protein Tyrosine Phosphatases, Class 7