Abstract
Human prostatic acid phosphatase (PAcP) is a prostate epithelium-specific differentiation antigen. In prostate carcinomas, the cellular PAcP is decreased. We investigated its functional role in these cells. Several lines of evidence support the hypothesis that cellular PAcP functions as a neutral protein-tyrosine phosphatase and is involved in regulating prostate cell growth. In this study, we identify its in vivo substrate. Our results demonstrated that, in different human prostate cancer cell lines, the phosphotyrosine (Tyr(P)) level of a 185-kDa phosphoprotein (pp185) inversely correlates with the cellular activity of PAcP. On SDS-PAGE, this pp185 co-migrates with the c-ErbB-2 oncoprotein. Immunodepletion experiments revealed that c-ErbB-2 protein is the major pp185 in cells. Results from subclones of LNCaP cells indicated the lower the cellular PAcP activity, the higher the Tyr(P) levels of c-ErbB-2. This inverse correlation was further observed in PAcP cDNA-transfected cells. In clone 33 LNCaP cells, L-(+)-tartrate suppresses the cellular PAcP activity and causes an elevated Tyr(P) level of c-ErbB-2 protein. Epidermal growth factor stimulates the proliferation of LNCaP cells, which concurs with a decreased cellular PAcP activity as well as an increased Tyr(P) level of c-ErbB-2. Biochemically, PAcP dephosphorylates c-ErbB-2 at pH 7.0. The results thus suggest that cellular PAcP down-regulates prostate cell growth by dephosphorylating Tyr(P) on c-ErbB-2 oncoprotein in those cells.
Highlights
Protein tyrosine phosphorylation and dephosphorylation play a key role in regulating the proliferation and differentiation of normal and tumor cells [1, 2]
prostatic acid phosphatase (PAcP) Expression and Tyrosine Phosphorylation of pp185 in Different Prostate Cancer Cells—Previous studies demonstrated that cellular PAcP could function as a neutral PTPase in prostate cancer cells [19, 22, 23]
L-(ϩ)-Tartrate Effect on Tyrosine Phosphorylation of c-ErbB2—Since results from PAcP cDNA transfection studies revealed that an additional PAcP expression correlates with a decreased Tyr(P) level on c-ErbB-2 protein in prostate cancer cells, we investigated this relationship further by treating clone 33 LNCaP cells with L-(ϩ)-tartrate, a classic inhibitor of PAcP [23, 30]
Summary
Materials—FBS, RPMI 1640 culture medium, gentamicin, EGF, and horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgGs were purchased from Life Technologies, Inc. Protein molecular weight standard marker, acrylamide, and a protein assay kit were obtained from Bio-Rad. Polyclonal antiErbB-2 Abs (C-18 and K-15) for immunoblotting, monoclonal antiErbB-2 Ab (9G6) for immunoprecipitation, and normal mouse IgG were purchased from Santa Cruz Biotechnology, Inc. Clone 81 LNCaP cells were transfected with the same expression vector containing human PAcP cDNA. Since L-(ϩ)-tartrate is a classic inhibitor of PAcP and since greater than 90% of L-(ϩ)-tartrate-sensitive acid phosphatase in LNCaP cells can be precipitated by anti-PAcP antiserum, the L-(ϩ)-tartrate-sensitive acid phosphatase activity was used to represent the PAcP activity [30]. An aliquot of total lysate protein (50 g) or the supernatant of immunoprecipitated complexes in SDS-PAGE sample buffer was electrophoresed and transferred to a nitrocellulose filter (Micron Separation Inc., MA). After two washes with TBST buffer (i.e. 20 mM Tris-buffered saline, pH 7.5, containing 0.1% Tween 20), the filters were reacted with specific Abs, and the signal was detected by the ECL method
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
