Mystique is a new insulin-like growth factor-I-regulated PDZ-LIM domain protein that promotes cell attachment and migration and suppresses Anchorage-independent growth

Abstract

By comparing differential gene expression in the insulin-like growth factor (IGF)-IR null cell fibroblast cell line (R- cells) with cells overexpressing the IGF-IR (R+ cells), we identified the Mystique gene expressed as alternatively spliced variants. The human homologue of Mystique is located on chromosome 8p21.2 and encodes a PDZ LIM domain protein (PDLIM2). GFP-Mystique was colocalized at cytoskeleton focal contacts with alpha-actinin and beta1-integrin. Only one isoform of endogenous human Mystique protein, Mystique 2, was detected in cell lines. Mystique 2 was more abundant in nontransformed MCF10A breast epithelial cells than in MCF-7 breast carcinoma cells and was induced by IGF-I and cell adhesion. Overexpression of Mystique 2 in MCF-7 cells suppressed colony formation in soft agarose and enhanced cell adhesion to collagen and fibronectin. Point mutation of either the PDZ or LIM domain was sufficient to reverse suppression of colony formation, but mutation of the PDZ domain alone was sufficient to abolish enhanced adhesion. Knockdown of Mystique 2 with small interfering RNA abrogated both adhesion and migration in MCF10A and MCF-7 cells. The data indicate that Mystique is an IGF-IR-regulated adapter protein located at the actin cytoskeleton that is necessary for the migratory capacity of epithelial cells.

Figure 1.

Mystique is expressed as at least three variants and encodes a protein with homology to an emerging family of PDZ-LIM domain proteins. (A) A Northern blot of R+ and R– cell RNA was probed with mouse Mystique cDNA followed by a probe to detect 18S RNA as a loading control. (B) A murine multiple tissue Northern blot was probed with mouse Mystique and β-actin cDNA probes. (C) The PDZ and LIM domains in human Mystique isoforms are illustrated. (D) Mystique PDZ and LIM domains were aligned with those of RIL, ALP, and CLP-36. Identical amino acids are shaded dark gray and similar amino acids are shaded in light gray.

Figure 2.

Mystique is a cytoskeleton-associated protein. (A) HeLa cells were transiently transfected with GFP-tagged Mystique 1, 2, or 3a and examined by fluorescence microscopy. (B) COS cells were transiently transfected with HA-tagged Mystique isoforms and TX-100–soluble proteins were extracted from cells (left) and TX-100–insoluble proteins (right) for Western blotting with anti-HA antibody. (C) Western blots were prepared with TX-100–soluble and –insoluble protein fractions extracted from R+ and R– cells and probed with anti-Mystique antiserum or anti-β-actin antibody. (D) Western blots prepared with TX-100–soluble and –insoluble protein extracts from MRC-5, MCF-7, HeLa, DU145, and Jurkat cells were probed with anti-Mystique antiserum and with anti-β-actin antibody.

Figure 3.

Localization of HA-Mystique 2 relative to α-actinin, paxillin, β1-integrin, and phosphotyrosine in MCF-7 cells. MCF-7 cells transiently expressing HA-tagged Mystique 2 were grown on collagen-coated coverslips. Cells were fixed and stained with either anti-HA antibody or anti-Mystique antiserum as indicated, which was detected by Cy2-labeled secondary antibody (green). Cells also were stained with either α-actinin, paxillin, β1-integrin, or phosphotyrosine antibodies as indicated, which were detected with Cy3-labeled secondary antibody (red). Amplified areas from the merged images are shown in the far-right panels.

Figure 4.

Stable overexpression of Mystique 2 inhibits anchorage-independent cell growth. (A) Point mutations of HA-Mystique 2 were generated as outlined in Materials and Methods. The amino acid positions of the mutations in the PDZ (L80K) and LIM (CC313–316SS) domains are illustrated. (B) Three MCF-7 cell clones that stably expressed either WT or the different Mystique 2 mutants were isolated and tested for expression of HA-Mystique by Western blotting with the anti-HA antibody. (C) MCF-7 clones were assessed for proliferation rates in monolayer culture >7 d. Wild-type Mystique 2-overexpressing cells (broken lines) were compared with Neo control cells (solid lines). Data are presented as the mean and SD of counts from quadruplicate wells and represent one of at least three independent experiments with similar results. (D) Mystique 2 WT and mutant-overexpressing MCF-7 clones were compared with Neo control clones for clonogenic growth in soft agarose. Each clone was plated in triplicate wells, and after 10 d the number of colonies from five fields per well was counted. The mean number of colonies per field for each well of each clone was averaged for each transfected construct (i.e., the means of WT clones 1–3 were averaged) and compared with the means of Neo clones 1–3 by using the Students t-test (*p < 0.01).

Figure 5.

Mystique is highly expressed in MCF10A cells and is induced by IGF-I or ECM adhesion. (A) Cell lysates were prepared from MCF10A, MCF-7, and R+ and R– cells cultured in complete medium and assessed for Mystique 2 expression by using the anti-Mystique antiserum. Blots were reprobed with anti-RACK1 anti body as a loading control. (B) MCF10A or MCF-7 cells were starved from serum (24 h) before stimulation with IGF-I for the indicated times. Cell lysates were prepared for Western blotting with anti-Mystique antiserum or anti-β-actin as a loading control. (C) R+, MCF-7, or MCF10A cells were detached from tissues culture plates for 24 h before replating. At the indicated times, cell lysates were prepared for Western blotting with anti-Mystique antiserum and anti β-actin as a loading control.

Figure 6.

Overexpression of Mystique 2 enhances MCF-7 cell attachment, which requires an intact PDZ domain. (A) MCF-7 cell clones expressing Neo or WT Mystique 2 (WT) were allowed to adhere to either collagen (10 μg/ml) or fibronectin (5 μg/ml) for 15 or 30 min as indicated. Attached cells were stained with crystal violet, which was measured by absorbance at 595 nm. The mean absorbance from four wells was averaged for three clones of Neo and WT each, and then the means of each set of clones were compared with the means of Neo clones by using the Student's t-test. (B) Adhesion assays were carried out as in A with three clones each of MCF-7 cells expressing either Neo, WT, or Mystique-2 mutants. The means of each clone were compared with Neo by using Student's t-test (*p < 0.01, **p < 0.001). (C) GFP-fused Mystique 2 mutants were transiently transfected into MCF-7 cells, which also were incubated with TRITC-phalloidin to visualize the actin cytoskeleton. Cells were examined and photographed using fluorescence microscopy. (D) Cell lysates from MCF-7 cells transfected with either HA-tagged Mystique 2, or Mystique 2 mutants, or Neo control as indicated were immunoprecipitated with anti-α-actinin antibody and then analyzed by Western blotting with anti-HA or anti-α-actinin antibodies as indicated.

Figure 7.

Silencing of Mystique disrupts cell attachment and suppresses motility. (A) MCF10A or MCF-7 cells were transfected with Mystique or control siRNA oligonucleotides and analyzed 3 d after transfection by Western blotting for expression of Mystique. After 60 h transfection with siRNA, MCF10A and MCF-7 cells were assayed for their ability to attach to either collagen or fibronectin (B) and to migrate toward complete media (C). For adhesion and motility assays, the means and standard deviations were calculated from quadruplicate (adhesion) or triplicate (motility) wells and compared using Student's t-test (*p < 0.01, **p < 0.001).