Characterization of a Prostate-specific Tyrosine Phosphatase by Mutagenesis and Expression in Human Prostate Cancer Cells

Xiu-Qing Zhang,Ming-Shyue Lee,Stanislav Zelivianski,Ming-Fong Lin

doi:10.1074/jbc.m006661200

Xiu-Qing Zhang, Ming-Shyue Lee + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m006661200

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Abstract

The cellular form of human prostatic acid phosphatase (PAcP) is a neutral protein-tyrosine phosphatase (PTP) and may play a key role in regulating the growth and androgen responsiveness of prostate cancer cells. The functional role of the enzyme is at least due in part to its dephosphorylation of c-ErbB-2, an in vivo substrate of the enzyme. In this study, we investigated the molecular mechanism of phosphotyrosine dephosphorylation by cellular PAcP. We mutated several amino acid residues including one cysteine residue that was proposed to be involved in the PTP activity of the enzyme by serving as the phosphate acceptor. The cDNA constructs of mutant enzymes were transiently transfected into C-81 LNCaP and PC-3 human prostate cancer cells that lack the endogenous PAcP expression. The phosphotyrosine level of ErbB-2 in these transfected cells was subsequently analyzed. Our results demonstrated that the phosphotyrosine level of ErbB-2 in cells expressing H12A or D258A mutant PAcP is similar to that in control cells without PAcP expression, suggesting that these mutants are incapable of dephosphorylating ErbB-2. In contrast, cells expressing C183A, C281A, or wild-type PAcP had a decreased phosphotyrosine level of ErbB-2, compared with the control cells. Similar results were obtained from in vitro dephosphorylation of immunoprecipitated ErbB-2 by these mutant enzymes. Furthermore, transient expression of C183A, C281A, or the wild-type enzyme, but not H12A or D258A, decreased the growth rate of C-81 LNCaP cells. The data collectively indicate that His-12 and Asp-258, but not Cys-183 or Cys-281, are required for the PTP activity of PAcP.

Highlights

Protein tyrosine phosphorylation plays a pivotal role in controlling multiple eukaryotic cellular processes including metabolism, proliferation, differentiation, migration, and survival [1,2,3]
Our results demonstrated that the phosphotyrosine level of ErbB-2 in cells expressing H12A or D258A mutant prostatic acid phosphatase (PAcP) is similar to that in control cells without PAcP expression, suggesting that these mutants are incapable of dephosphorylating ErbB-2
The results clearly demonstrated that replacement of His-12 or Asp-258 by Ala abolishes the protein-tyrosine phosphatase (PTP) activity of PAcP toward ErbB-2, whereas mutation of Cys-183 or Cys-281 has no significant effect on the activity

Summary

EXPERIMENTAL PROCEDURES

Reagents and Antibodies—Gentamicin, L-glutamine, fetal bovine serum, RPMI medium 1640, OPTI-MEM I reduced-serum medium, LipofectAMINE PLUS transfection reagent, and BamHI and XbaI restriction enzymes were purchased from Life Technologies, Inc. In addition to changing the desired residue to Ala, the mutations (underlined) create an MvnI restriction site (bold) to facilitate the initial mutant screening. Generation of this restriction site does not result in a change in amino acid. The sequence-confirmed cDNA was subcloned into mammalian expression vectors pcDNA 3.1 and pCEP4 for transfection and expression in prostate cancer cells. About 1 and 6 ␮g of plasmids carrying PAcP cDNAs were used for transfection of the cells in each well and dish, respectively. The supernatant fractions were further incubated with 5– 8 ␮g of monoclonal mouse anti-ErbB-2 antibody preconjugated with protein A-Sepharose for 3 h at 4 °C. In Vitro Dephosphorylation of ErbB-2—ErbB-2 protein was immunoprecipitated from C-81 LNCaP cells as described above. The ErbB-2 immunocomplexes were washed with the lysis buffer and suspended in SDS-sample buffer for further analysis by SDS-PAGE and Western blotting

RESULTS

Mutagenesis and Expression Analysis of PAcP

DISCUSSION