Human prostatic acid phosphatase: structure, function and regulation

Abstract

Human prostatic acid phosphatase (PAcP) is a 100 kDa glycoprotein composed of two subunits. Recent advances demonstrate that cellular PAcP (cPAcP) functions as a protein tyrosine phosphatase by dephosphorylating ErbB-2/Neu/HER-2 at the phosphotyrosine residues in prostate cancer (PCa) cells, which results in reduced tumorigenicity. Further, the interaction of cPAcP and ErbB-2 regulates androgen sensitivity of PCa cells. Knockdown of cPAcP expression allows androgen-sensitive PCa cells to develop the castration-resistant phenotype, where cells proliferate under an androgen-reduced condition. Thus, cPAcP has a significant influence on PCa cell growth. Interestingly, promoter analysis suggests that PAcP expression can be regulated by NF-κB, via a novel binding sequence in an androgen-independent manner. Further understanding of PAcP function and regulation of expression will have a significant impact on understanding PCa progression and therapy.

Figure 1

(a) Sequence alignment of human prostatic acid phosphatases shows that the active sites are evolutionarily conserved in closely related mammals. Only active site residues are shown with the amino acid position. Mature protein starts with lysine (K). Active site histidine (H) and aspartate (D) are highlighted in green, cysteine (C) with pink and glycosylation at asparagine (N) with gray. The number in the parenthesis indicates the percentage of similarity with hPAcP; (b) Sequence alignment of hPAcP with hLAcP, AcPT and rat PAcP shows that the active sites are evolutionarily conserved in closely related mammals. Only active site residues are shown with the amino acid position. Mature protein starts with lysine (K). Active site histidine (H) and aspartate (D) are highlighted in green, arginine (R) with red, cysteine (C) with pink, and glycosylation at asparagine (N) with gray. The hPAcP sequence is given in the first row, subsequent rows display the AcP sequence from other sources. The number in the parenthesis indicates the percentage of similarity with hPAcP.

Figure 2

Association of cPAcP and ErbB-2 phosphorylation levels in androgen-sensitive (AS) LNCaP C-33 and androgen-independent (AI) LNCaP C-81 and PC-3 cells. Knockdown of endogenous PAcP in LNCaP C-33 cells leads to increase ErbB-2 tyrosine phosphorylation and tumorigenicity. Conversely, ectopic expression of cPAcP expression in AR-positive LNCaP C-81 and in PAcP-null PC-3 cells restores their androgen sensitivity, decrease the growth rate and tumorigenicity.

Figure 3

The schematic representation of the PAcP gene promoter. The transcription starts at +1 and the grey box indicates the translational region for PAcP protein (starts at +50 bp). The yellow boxes indicate the Alu repeat. The novel NF-κB binding site is identified in the positive regulatory domain (Green box; at −1245 bp upstream). The computer analysis of the sequence shows at least nine additional putative transcriptional binding sites.

Figure 4

Effects of NaB on cPAcP protein expression and ErbB-2 tyrosyl phosphorylation. LNCaP C-81 cells were plated in different cell densities (0.3, 0.5 and 1 × 10⁶ cells/T25) in regular culture conditions for 2 days and then treated with 1 mM NaB for 48 h. The cells were harvested and the total protein was subjected to western blot analyses. NaB effects on cPAcP protein expression and ErbB-2 phosphorylation at Tyr1221/2 levels were shown. β-actin was detected as a loading control. The data shown is a representative from three sets of independent experiments.

Figure 5

Schematic representation of ErbB-2 signaling and androgen sensitivity regulated by cPAcP in prostate cancer cells. The solid red arrow indicates the classical ligand dependent activation of androgen receptor (AR) pathway. Unbound AR resides in the cytosol in association with heat shock proteins (hsps). Androgen (DHT) enters into the cytoplasm and binds to the AR by displacing associated hsps, which allows the AR to enter into the nucleus, where it dimerizes, recruits various co-regulatory proteins and binds to the androgen response element (ARE), leading to the transcriptional regulation of the target gene. Solid black arrows indicate one of the major cPAcP-regulated pathways in prostate cancer cells with clinical significance. Progression of androgen-sensitive PCa cells towards androgen independence is accompanied by an early decrease/loss of cPAcP expression in PCa cells, results in hyperphosphorylation of HER-2 at tyrosine residues (1221/2 and/or 1248), leading to androgen-independent cell proliferation. Activated HER-2 can transduce its signals via p52Shc (blocked by dominant-negative (DN) HER-2 cDNA transfection or an HER-2 inhibitor, AG879) to activate the downstream ERK/MAPK pathway (blocked by p52Shc Y317F mutant cDNA transfection or an MEK inhibitor, PD 98059 and U0126) through Ras/Raf mediation. These events could also lead to AR phosphorylation and activation, resulting in an increase in androgen-independent cell proliferation. Activated HER-2, via Akt, may also phosphorylate AR. Alternatively, the loss of cPAcP expression results in the accumulation of PI3P, which may lead to activation of the Akt pathway.