PMC

A, computer molecular modeling of the LIMD2–ILK–aParvin protein complex. B, a proposed working model for LIMD2 functional roles in assembly of and normal function of the ILK–PINCH–Parvin complex in the integrin-linked signaling pathway.

A, LIMD2 expression in a spectrum of melanoma cancer cells by Western blot analysis. B, knockdown of LIMD2. C, migration potentials of melanoma cells. The statistics were performed and P value is indicated. D, morphologic changes in melanoma cancer cells. Bars,100 μm.

A, breast cancer cell lines express endogenous LIMD2. B, ectopic expression of LIMD2 in MCF-7 cells. C, siRNA-mediated LIMD2 knockdown in MDA-MB-231 cells (the Si2 and Si5 are 2 independent cell clones). Migration potentials of each cell line. Data are presented as mean ± SD. T test was performed and P value is indicated. D, morphology of the cells. Bar, 100 μm. E, soft agar colony formation assays of each cell line.

A, LIMD2 and the LIM-only protein family. B, LIMD2 is most closely related to CRP1. C, the PINCH1-LIM1 and LIMD2 LIM domains are homologous. The zinc-chelating residues are highlighted in red; the conserved amino acids are highlighted in gray. D, antibodies robustly detect LIMD2 protein. *, nonspecific binding. E, TPC1 cells were transfected with myc-LIMD2 then fixed and stained with both anti-myc tag antibody (red) or anti-LIMD2 mAb (green). The cells were counterstained with DAPI to highlight the nucleus (blue). The red arrows indicate concentrations of Myc- and LIMD2 costaining signal, which is at the leading edge of the cells and also is present in streaks reminiscent of vinculin-containing adhesion plaques.

A, ¹H-¹⁵N HSQC spectrum of LIMD2 with backbone assignment: BMRB accession number 18778. Purple represents aliased signals. B, superimposition of the 10 best structures calculated by ARIA (PDB accession code 2LZU) with an RMSD of 0.32 A for LIMD2 residues 39-96aa over backbone atoms. See for structure statistics. Zinc is colored in gold and side chains of the zinc-coordinated residues are shown in green. C, ribbon representation of LIMD2. D, alignment of LIMD2 (red) with PINCH1-LIM1 (cyan PDB code 3F6Q), PINCH2-LIM1 (green PDB code 3IXE), and PINCH1-LIM4 (orange PDB code 1NYP). E, side chains of F60, F65, and F86 from a LIMD2 hydrophobic core. F, electrostatic surface of LIMD2. The asymmetric charge distribution in LIMD2 is shown.

A, LIMD2 is highly expressed in thyroid cancer cells. B, migration potentials of the thyroid cancer cells. Data are presented as mean ± SD. C, images of thyroid carcinoma cell line XTC-1 and thyroid papillary carcinoma cell line TPC-1 taken from the migration assay. Bar, 100 mm. D, LIMD2 mRNA is higher in thyroid cancer samples with extrathyroidal invasion (n = 49). Total RNA was extracted from 252 papillary thyroid cancer samples using the TRIzol reagent (Invitrogen). TaqMan gene expression assay (Applied Biosystems) was used to quantitate LIMD2 mRNA expression levels. E, representative immunostaining results in papillary thyroid cancer samples using the aLIMD2 (mAb).

LIMD2 and PINCH1 directly associate with ILK, LIMD2 affects cell migration through ILK. A, domain architecture of ILK. The N-terminus of ILK contains ankyrin repeat domain (ARD) and the C-terminus of ILK contains a kinase domain. B, ILK protein binds to GST-LIMD2 and GST-PINCH1-LIM1 but not GST alone. C, LIMD2 protein binds to GST-ILK (182-452aa). D, LIMD2 stimulates cell migration in ILK^þ/þ fibroblast but not in ILK ^−/− fibroblast. Lentivirus pLU vector (GFP) and pLU-LIMD2 were infected on ILK^þ/þ and ILK ^−/− fibroblast cells. Migration assay was performed on pLU and LIMD2 virus infected ILK^þ/þ and ILK ^−/− fibroblast cells. Images were taken from the migrated cells. Bar, 100 μm. E, endogenous ILK protein were detected in ILK^þ/þ fibroblast cells but not in ILK ^−/− cells by Western blot analysis using aILK (rAb). Exogenous LIMD2 proteins were detected in LIMD2 virus– infected fibroblast cells by Western blot analysis using aLIMD2 (mAb). F, ectopic LIMD2 expression promotes ILK^þ/þ fibroblast cells migration. Three independent experiments were performed. Data, mean ± SD. P value is indicated.

LIMD2 increases ILK kinase activity in a dose-dependent manner. A, coomassie blue-stained polyacrylamide gel showing highly purified ILK–GST fusion protein used in the ILK kinase assay. B, dose-dependent phosphorylation of the ILK substrate myosin phosphatase target subunit 1 (MYPT1) on Thr696 in the presence of increasing concentrations of purified LIMD2. ILK and LIMD2 were mixed and preincubated for 30 minutes at room temperature before addition of the substrate. The amounts of ILK and MYPT1 were kept constant at 50 and 500 ng/reaction, respectively, and increasing amounts of LIMD2 were added as indicated. C, concentration curve similar to that shown in B, with LIMD2 concentrations increased stepwise from 5 to 50 ng. Reactions were carried out as described for B. D, concentration curve similar to that shown in C, with LIMD2 concentrations increased stepwise from 1 to 10 and 20 ng (molar ratio of 0.1–1.9). Reactions were carried out as described for B. Data in the bar graph are normalized to the signal observed in the presence of ILK alone. E, effect of the specific small molecule inhibitor of ILK, QLT0267, on the phosphorylation of MYPT by ILK-LIMD2. ILK-LIMD2 complexes were preincubated at the indicated concentrations as described above, and QLT0267 was added at a final concentration of 1.0 μmol/L just before initiation of the kinase reaction. Data in the bar graph are normalized to the signal observed in the presence of ILK alone.