Yoon-Se Kang

Cloning and characterization of a chicken protein tyrosine phosphatase, CPTP1

Protein tyrosine phosphorylation and dephosphorylation are an important regulatory reactions in cell physiology. We have cloned a cDNA that encode a cytosolic protein tyrosine phosphatase (CPTP1) from chicken intestine cDNA library. Amino acid sequence identity between the CPTP1 and low molecular weight form of human placenta enzyme (HPTP1B) was 92%. CPTP1 lacked 13 amino acids in N-terminal region, while it had an additional 48 amino acids in the C-terminal region in comparison with the truncated form of HPTP1B of 321 amino acids. This C-terminal sequence was different from those of all known PTPs. The CPTP1 does not have a membrane targeting or nuclear localization sequences at its C-terminus like other PTPs such as HPTP1B and murine homolog of the human T-cell protein tyrosine phosphatase (MPTP) do. The cloned cDNA has been expressed in E. coli and purified by affinity chromatography. Dephosphorylation kinetics of this enzyme closely resembled those of the known PTPs. The dephosphorylation reaction required a reducing agent such as glutathione and dithiothreitol and was inhibited by sodium vanadate and formaldehyde. Deletion of 72 amino acids from C-terminal side of CPTP1 gene resulted in higher expression in E. coli and more potent phosphatase activity than wild type CPTP1 gene product. This result suggests that the C-terminal region of the CPTP1 protein negatively regulates phosphatase activity. These results also imply that CPTP1 might be a nontransmembrane-type enzyme with a structure and localization specificity distinct from other known cytosolic PTP1B type homolog.

Phosphorylation of chicken protein tyrosine phosphatase 1 by casein kinase II in vitro

The phosphorylation and dephosphorylation of proteins on tyrosyl residues are key regulatory mechanisms of cell growth and signal transduction and are controlled by opposing activities of protein tyrosine kinases and phosphotyrosyl phosphatases (PTPs). We have previously cloned and characterized a nontransmembrane chicken protein tyrosine phosphatase 1 (CPTP1) similar to human placental PTP1B (HPTP1B). CPTP1 contains several phosphorylation sequence motifs (S/T-X-X-D/E) for casein kinase II (CKII), [(I>E>V)-Y-(E>G)-(E>D>P>N)-(I/V>L)] for p56(lck), and (P-E-S-P) for MAP kinase. To examine whether phosphatase activity of CPTP1 could be controlled by phosphorylation, CPTP1 and HPTP1B fusion proteins purified from E. coli were subjected to the in vitro phosphorylation by CKII. Phosphoamino acid analysis revealed that CPTP1 was phosphorylated on both serine and threonine residues by CKII in vitro. In addition, the degree of the phosphorylation of CPTP1 by CKII was shown to be five times higher than that of HPTP1B. Phosphorylation on both serine and threonine residues of CPTP1 in vitro results in an inhibition of its phosphatase activity. This result suggests that phosphorylation of CPTP1 and HPTP1B by CKII might be implicated in the regulation of their catalytic activities in the cell.

Elongation factor-2 in chick embryo is phosphorylated on tyrosine as well as serine and threonine.

An endogenous 95 kDa chick embryo cytosolic protein (p95) was phosphorylated in the presence of [gamma-32P]ATP and the kinase activity for p95 was mostly associated with particulate fraction. Phosphorylation of p95 was prominent in embryos of early developmental stage. Hydrolysis of p95 phosphoprotein yielded phosphotyrosine in addition to phosphothreonine and phosphoserine. Native p95 was also tyrosine-phosphorylated. p95 phosphoprotein was purified by DEAE-Sephacel chromatography and immunoprecipitation with anti-phosphotyrosine antibody and the amino acid sequence was determined. The N-terminal sequence, Val-Asn-Phe-Thr-Val-Asp-Gln-Ile-Arg-Ala-Ile-Met-Asp- Lys-Lys-Ala-Asn-Ile-Arg-Asn-Met-, was found to be identical to those of elongation factor-2 (EF-2) of both rat and hamster. Our results suggest the presence of other EF-2 kinase in chick embryo cell than the previously reported Ca2+/calmodulin-dependent protein kinase III.