Nuclear annexin II negatively regulates growth of LNCaP cells and substitution of ser 11 and 25 to glu prevents nucleo-cytoplasmic shuttling of annexin II

Abstract

Background: Annexin II heavy chain (also called p36, calpactin I) is lost in prostate cancers and in a majority of prostate intraepithelial neoplasia (PIN). Loss of annexin II heavy chain appears to be specific for prostate cancer since overexpression of annexin II is observed in a majority of human cancers, including pancreatic cancer, breast cancer and brain tumors. Annexin II exists as a heterotetramer in complex with a protein ligand p11 (S100A10), and as a monomer. Diverse cellular functions are proposed for the two forms of annexin II. The monomer is involved in DNA synthesis. A leucine-rich nuclear export signal (NES) in the N-terminus of annexin II regulates its nuclear export by the CRM1-mediated nuclear export pathway. Mutation of the NES sequence results in nuclear retention of annexin II.

Results: Annexin II localized in the nucleus is phosphorylated, and the appearance of nuclear phosphorylated annexin II is cell cycle dependent, indicating that phosphorylation may play a role in nuclear entry, retention or export of annexin II. By exogenous expression of annexin II in the annexin II-null LNCaP cells, we show that wild-type annexin II is excluded from the nucleus, whereas the NES mutant annexin II localizes in both the nucleus and cytoplasm. Nuclear retention of annexin II results in reduced cell proliferation and increased doubling time of cells. Expression of annexin II, both wild type and NES mutant, causes morphological changes of the cells. By site-specific substitution of glutamic acid in the place of serines 11 and 25 in the N-terminus, we show that simultaneous phosphorylation of both serines 11 and 25, but not either one alone, prevents nuclear localization of annexin II.

Conclusion: Our data show that nuclear annexin II is phosphorylated in a cell cycle-dependent manner and that substitution of serines 11 and 25 inhibit nuclear entry of annexin II. Aberrant accumulation of nuclear annexin II retards proliferation of LNCaP cells.

Figure 1

Annexin II is phosphorylated in nuclear extract of K562 cells. Human K562 cells were fractionated into nuclear extract (NE) and cytosolic extract (CE). One aliquot of NE and CE was treated with CIAP prior to SDS-PAGE. Untreated CE and NE were subjected to incubation in the phosphatase buffer without CIAP. 20 μg protein from each extract were subjected to SDS-PAGE and immunoblotting with anti-annexin II antisera. Positions of the phosphorylated (upper two) and dephosphorylated annexin II are indicated by arrows.

Figure 2

Phosphorylation of nuclear annexin II and cell cycle distribution. Human K562 cells were subjected to centrifugal elutriation followed by flow cytometry to determine the cell cycle distribution of phosphorylated annexin II. Cells enriched in each indicated phase were fractionated to cytosolic extract (CE) and nuclear extract (NE). Twenty μg protein from each sample were subjected to SDS-PAGE followed by immunoblotting with anti-annexin II antibody. Immunoblot of PGK was used as an internal control. The flow cytometric profile of each fraction is presented in the upper panels and the percentage of cells in each cell cycle phase is indicated below the immunoblot under each fraction.

Figure 3

Immunoblot analysis of annexin II in prostate cancer cells. Cell lysates of NIH3T3 (lane 1), SkBr-3 (lane 2), DU-145 (lane 3), PC-3 (lane 4) and LNCaP (passage 181, lane 5; passage 142, lane 6; passage 92, lane 7; passage 34, lane 8) were subjected to western blot analysis with mouse anti-human monoclonal anti-annexin II antibody as described in materials and methods. Lane 9 is the protein molecular markers used for SDS-PAGE. The position of annexin II band is indicated on the right side of the panel.

Figure 4

Northern blot analysis of annexin II in prostate cancer cells. Prior to RNA extraction, LNCaP cells of both low level and high level of confluence were treated with 10 nM dihydrotestosterone (DHT) for 2 days. 20 μg RNA were extracted from PC-3 cells (lane 1), DU-145 cells (lane 2) and LNCaP cells treated with (lanes 3, 6) or without (lanes 4, 5) DHT. [³²P]-labeled annexin II cDNA was used as probe (Panel A). The location of full length annexin II cDNA is indicated by an arrow on the left side of the panel. The membrane from panel A was probed with [³²P]-labeled GAPDH cDNA for normalization of the annexin II level (Panel B).

Figure 5

Localization of wild type and NES mutant annexin II in transiently transfected LNCaP cells. Transiently transfected LNCaP cells expressing GFP alone, GFP-wild type (GFP-WT) annexin II or GFP-NES mutant (GFP-NES) annexin II were fixed and subjected to immunocytochemistry with anti-annexin II antibody as described in materials and methods. The images were scanned using laser scanning confocal microscope (LSCM). The green color represents auto-fluorescence of GFP, the red color represents the rhodamine staining of annexin II antigen-antibody complex, and the yellow color is the overlay of GFP and annexin II images. Bar: 25 μm.

Figure 6

Distribution of GFP and GFP-fused wild type or NES mutant annexin II in LNCaP cells. LNCaP cells expressing GFP, GFP-wild type annexin II (GFP-WT), and GFP-NES mutant annexin II (GFP-NES) were fractionated into cytosolic extract (CE) and nuclear extract (NE). Each extract was subjected to SDS-PAGE and immunoblot with anti-GFP (Panel A) and anti-PGK antibody (panel B). Cytosolic and nuclear extracts from the GFP-NES cells were subjected to potato acid phosphatase (PACP) treatment as described in materials and methods, and an immunoblot analysis was performed for annexin II (Panel C).

Figure 7

Morphology of LNCaP cells transfected with wild type annexin II and NES-annexin II. Untransfected LNCaP and stable clones of vector-alone, GFP-WT and GFP-NES cells were cultured as described in materials and methods. Light microscopy images of each of the cultures are shown.

Figure 8

Accumulation of annexin II in the nucleus retards cell proliferation. Parental LNCaP cells and stable clones of vector-alone (GFP), GFP-WT or GFP-NES mutant annexin II were cultured as described in materials and methods for up to 8 days. Two different GFP-NES clones (NES-1 and NES-4) were included. The cell numbers on the indicated days were obtained from a standard curve (data not shown) according to the OD_570–690 values. The experiment was done in triplicate and the data were analyzed using GraphPad Prism 3.0. Cell doubling time was determined for each culture, and the values were: LNCaP (30.32 ± 2.1 hours), GFP (33.8 ± 4.6 hours), GFP-WT (34.5 ± 5.1 hours) and GFP-NES (48.1 ± 8.3 hours).

Figure 9

Site-directed change of ser11 and ser25 to glutamic acid prevents nuclear entry of annexin II. GFP-fused annexin II containing Ser to Ala or Ser to Glu either singly or in combination were generated as described in materials and methods. Transient transfection of LNCaP cells was performed followed by treatment with or without LMB. Confocal microscopic observations were made and representative images are shown. Bar: 10 μm.