Functional activity of RLIM/Rnf12 is regulated by phosphorylation-dependent nucleocytoplasmic shuttling

Abstract

The X-linked gene Rnf12 encodes the ubiquitin ligase really interesting new gene (RING) finger LIM domain-interacting protein (RLIM)/RING finger protein 12 (Rnf12), which serves as a major sex-specific epigenetic regulator of female mouse nurturing tissues. Early during embryogenesis, RLIM/Rnf12 expressed from the maternal allele is crucial for the development of extraembryonic trophoblast cells. In contrast, in mammary glands of pregnant and lactating adult females RLIM/Rnf12 expressed from the paternal allele functions as a critical survival factor for milk-producing alveolar cells. Although RLIM/Rnf12 is detected mostly in the nucleus, little is known about how and in which cellular compartment(s) RLIM/Rnf12 mediates its biological functions. Here we demonstrate that RLIM/Rnf12 protein shuttles between nucleus and cytoplasm and this is regulated by phosphorylation of serine S214 located within its nuclear localization sequence. We show that shuttling is important for RLIM to exert its biological functions, as alveolar cell survival activity is inhibited in cells expressing shuttling-deficient nuclear or cytoplasmic RLIM/Rnf12. Thus regulated nucleocytoplasmic shuttling of RLIM/Rnf12 coordinates cellular compartments during mammary alveolar cell survival.

FIGURE 1:

RLIM localizes in the nuclear and cytoplasmic compartments in cells. (A) RLIM is found predominantly in the nuclei of cells in culture. αT3, HeLa, MCF7, and HEK293 cells stained with antibodies directed against RLIM. (B) Western blot on total, nuclear, and cytoplasmic fractions prepared from αT3 cells using antibodies directed against RLIM, Lhx3, and tubulin. (C) Expression of RLIM in human breast epidermis. Note that RLIM is expressed in the nucleus and cytoplasm in the basal layer in undifferentiated human breast epithelial cells (red arrows), whereas differentiating cells (black arrows) express RLIM mainly in the nucleus. Scale bar, 20 μm. (D) Immunocytochemical staining of undifferentiated and differentiated primary HFK cells with RLIM antibodies. Specific staining along the plasma membrane in undifferentiated cells is indicated by arrows.

FIGURE 2:

RLIM is a nucleocytoplasmic shuttling protein. (A) Structure of RLIM and Rnf6 proteins showing a leucine-zipper-like domain (L-Zip-L), putative NLS (green), NES (red), BD (gray), and RING finger (RING, black). The percentage of amino acid identity between each region is indicated. (B) The NLS and NES in RLIM and Rnf6 are functional. Plasmids expressing GFP-NLS and GFP-NES sequences of RLIM (NLS_RLIM and NES_RLIM) and Rnf6 (NLS_Rnf6 and NES_Rnf6) were overexpressed in HeLa cells, and the fusion proteins were visualized using anti-GFP antibodies (green). (C) The putative NLS of RLIM is required for nuclear localization. Staining of Myc-tagged full-length or mutant protein deleted of the putative NLS domain (Myc-RLIMΔNLS) was overexpressed in HeLa cells and stained with anti-Myc antibodies (green). Nuclei were stained with Toto 3 dye (blue), and the cytoplasm was stained for actin with rhodamine–phalloidin (red). (D) RLIM shuttles between the nucleus and cytoplasm in heterokaryon assays. Human HeLa cells were transfected with Myc-RLIM or Myc-RLIMΔNES lacking the NES (white arrows) and fused with untransfected mouse NIH3T3 cells (red arrows). Arrows in PEG-treated cells point at nuclei in heterokaryons (circled). Note Myc-RLIM in the nuclei of NIH3T3 cells in a heterokaryon, indicating protein shuttling, whereas Myc-RLIMΔNES is retained in the nuclei of transfected HeLa cells. (E) The unique NLS of RLIM confers nuclear localization of RLIM. Cells transfected with a Myc-Rnf6 construct in which the NLS was replaced with that of RLIM (Myc-Rnf6ΔNLS_NLSRLIM) and a Myc-RLIM construct in which the NLS was replaced with that of Rnf6 (Myc-RLIMΔNLS_NLSRnf6) or SV40 (Myc-RLIMΔNLS_NLSSV40). Cells were stained with anti-Myc antibody (green), Toto 3 dye (blue), and rhodamine-phalloidin (red).

FIGURE 3:

The NLS of RLIM is phosphorylated at conserved Ser residues. (A) Metabolic labeling of transfected Myc-tagged RLIM protein mutants RLIM-M (amino acids [aa] 206–423), RLIM-C (aa 403–600), and RLIM-MA (aa 206–305) in cells. Note strong phosphorylation of RLIM-M and RLIM-MA deletion mutants, both containing the NLS. (B) Comparison of NLS of RLIM in vertebrates (205–230). Note the high conservation of the NLS. Serines at positions 212, 214, 227, and 229 that may serve as potential phosphorylation sites are indicated by arrows. RSRSP motifs are indicated in red. Predicted α-helices (GOR IV, PSIPRED, and Jnet) are indicated in blue. (C) In vitro phosphorylation of the mouse RLIM-NLS (205–230) fused to GST and RLIM-NLS Ser-to-Ala mutant proteins in which all serine residues were replaced by alanine residues (AAAA, in red) or containing only one serine residue at the indicated position. Note the strong serine phosphorylation at position 214. As loading control the same membrane was hybridized with an antibody directed against GST. (D) Metabolic labeling of Myc-tagged mouse RLIM-NLS (205–230) Ser-to-Ala mutations after transfection of corresponding expression plasmids. Note strong serine phosphorylation at positions 214 and 229. As a loading control the same membrane was hybridized with an antibody directed against Myc.

FIGURE 4:

Nuclear RLIM is phosphorylated at S214. (A) Immunocytochemical staining of undifferentiated and differentiated primary HFK cells with phospho-specific peptide antibodies specifically recognizing the NLS of RLIM phosphorylated on S214 (RLIM-pS214). Note exclusively nuclear staining in cells. (B, C) Immunohistochemical sections of adult human epithelia were stained with non–phospho-specific RLIM antibodies or phospho-specific RLIM-pS214 antibodies. Human breast epidermis is shown in B. Right, magnification of boxed area. Human oral cavity epithelium is shown in C. Note weak staining of basal layer (arrows) in sections stained with the phospho-specific RLIM-pS214 antibodies. Scale bars, 25 μm.

FIGURE 5:

The RLIM-NLS regulates localization of RLIM in a phosphorylation-dependent manner. Expression of Myc-tagged RLIM containing various Ser-to-Ala mutations at positions 212, 214, 227, and 229 in their NLS in the context of the full-length protein. Left, representative images of transfected cells; right, statistical analyses of at least 100 transfected cells. Note that replacement of Ser-214 with alanine (SASS) strongly inhibits nuclear localization and RLIM containing S214 and S229 (ASAS) is mainly localized in the nucleus, similar to wild-type RLIM (SSSS).

FIGURE 6:

Shuttling of RLIM is important for its function of promoting cell motility. (A) Knockdown of RLIM inhibits cell motility. MCF7 cells were infected with lentivirus containing control or short hairpin RNA against RLIM. Top, motility of infected MCF7 cells as measured in Transwell migration assays. Averages ± SEM for five independent measurements. Bottom, representative Western blot of infected cell extracts. The same blot was hybridized with antibodies against RLIM and GAPDH. (B) Function of RLIM to promote cell motility is dependent on nucleocytoplasmic shuttling. MCF7 cells were infected with lentivirus containing RLIM, various RLIM-NLS mutants, and, as control, GFP. Top, motility of infected MCF7 cells as measured in Transwell migration assays. Averages ± SEM for four independent measurements. Note that overexpression of Myc-RLIM wild type but not shuttling-deficient Myc-RLIM mutant proteins promotes cell migration. Bottom, representative Western blot of infected cell extracts. To compare cellular levels of endogenous with lentivirus-mediated expression of RLIM, cells were treated with various concentrations of the proteasome inhibitor lactacystin for 6 h. The same blot was hybridized with antibodies against RLIM recognizing exogenous and endogenous protein and GAPDH.

FIGURE 7:

Nucleocytoplasmic shuttling regulates RLIM's ability to act as alveolar survival factor. (A) RLIM is phosphorylated in nuclei of mammary epithelial cells. Immunohistochemical sections of adult human mammary gland were stained with non–phospho-specific antibodies recognizing RLIM (left) or phospho-specific peptide RLIM-pS214 antibodies (right). Scale bars, 25 μm. (B) Dual localization of RLIM in the nucleus and cytoplasm in basal and luminal primary mammary cells. Primary mammary epithelial cells were isolated from an adult wild-type virgin female mouse and costained with antibodies directed against RLIM and basal marker CK14 or luminal marker CK18. (C) RLIM is associated with the actin cytoskeleton in primary mammary cells. Primary mammary epithelial cells were isolated from an adult wild-type virgin female mouse and costained with antibodies directed against RLIM and rhodamine–phalloidin. Boxed area is shown in higher magnification. (D) Primary mammary epithelial cells that lack RLIM were isolated from adult Rnf12^fl/fl x MMTV-Cre virgin female mice. Cells were infected with lentivirus expressing Myc-tagged wild-type (SSSS) and shuttling-deficient RLIM mutant proteins (SASS) and ΔNES proteins. Cells infected with lentivirus expressing GFP and Myc-tag and uninfected cells were used as control. Averages ± SEM for four independent measurements.