GRIM-19 inhibits v-Src-induced cell motility by interfering with cytoskeletal restructuring

Abstract

GRIM-19 (Gene associated with Retinoid-Interferon-induced Mortality 19) is a novel tumor suppressor regulated by interferon/retinoid combination. We have recently shown that GRIM-19 inhibits v-Src-induced oncogenic transformation and metastatic behavior of cells. Oncogenic v-Src induces cell motility by cytoskeletal remodeling, especially the formation of podosomes and. Here, we show that GRIM-19 inhibited the v-Src-induced cell motility by inhibiting cytoskeletal remodeling, that is, podosome formation. We also show that the N terminus of GRIM-19 played a major role in this process and identified critical residues in this region. More importantly, we show that tumor-associated GRIM-19 mutations disrupted its ability to inhibit v-Src-induced cell motility. These actions appear to occur independently of STAT3, a known target of GRIM-19-mediated inhibition. Lastly, tumor-associated GRIM-19 mutants significantly lost their ability to control v-Src-induced metastases in vivo, indicating the biological and pathological significance of these observations.

Fig. 1. GRIM-19 suppressed v-Src-induced morphologic changes and cytoskeleton remodeling

(A) Phase contrast photomicrographs of 3Y1 cells expressing GRIM-19, v-Src, v-Src/GRIM-19 and control vector. The graph below show the percentage of cells with rounded morphology as quantified from various fields. Magnification: 10×. Mean percentages and SE were plotted. (B) Immunofluorescent images of cells expressing, control vector GRIM-19, v-Src, and v-Src/GRIM-19 showing actin network (Red), exogenous GRIM-19 (green) and nucleus (Blue). Magnification: 100×. Scale bar = 50µm (C) Immunofluorescent images of cells expressing control vector, GRIM-19, v-Src, and v-Src/GRIM-19 showing dynamic microtubules (green), stable microtubules (red) and nucleus (blue). Magnification: 100×. Scale bar = 50µm.

Fig. 2. GRIM-19 suppressed phosphorylation of cortactin by v-Src

Representative Western blot profiles of total cellular lysates probed with the indicated antibodies and signals generated using ECL. Typically, duplicate gels were run in parallel and after transfer, the membrane was cut horizontally aided by protein molecular weight marker front (pre-stained) and incubated with the following antibodies separately; a) cortactin (1:2000) or phosphorylated cortactin (1:1000), b) Myc-tag (1:1000), c) Actin (1:2000) and d) Src (1:1000) or phosphorylated Src (1:1000). In some cases, a membrane was first probed with an antibody that detects the phosphorylated form of the protein (pY⁴²¹ Cortactin or pY⁴¹⁶ v-Src), then striped and reprobed with an antibody to detect the total protein (Cortactin or Src).

Fig. 3. Podosome formation in v-Src-transformed cells expressing GRIM-19 proteins

(A&C) Immunofluorescent images of actin network (Red) and nucleus (blue) in 3Y1 cells expressing v-Src and v-Src/GRIM-19 combinations. Magnification: 100×. Scale bar = 50µm. (B&D) A semi-quantitative representation of cells showing podosome-like feature in the indicated gene combinations. The percentage of cells with a rounded appearance, based on actin network, was counted manually from atleast 5 independent fields, each field containing ~70 cells. Rounded appearance of cells was counted as positive for podosome.

Fig. 4. Effect of GRIM-19 on v-Src-induced phosphorylation of cortactin

(A&B) Representative Western blots of total and phosphorylated cortactin in v-Src-transformed 3Y1 cells co-expressing wildtype or mutant GRIM-19 proteins. A semi-quantitative representation of phosphorylated cortactin levels in the respective cell lines. The band intensity of phosphorylated cortactin was normalized using band intensity of total cortactin. Bar represents mean band intensity ±SE from 4 independent samples is shown in each case.

Fig. 5. Effect of GRIM-19 on v-Src-dependent cell motility

(A) Injury-induced migration of cells into the wounded area was monitored. Magnification: 100×. White line indicates the edge of injured site. Note the rapid migration of v-Src-expressing cells, but not the controls, into the wounded area. The wildtype and mutant GRIM-19 proteins were expressed using lentiviral vectors and their impact on cell motility was measured. Note suppression of v-Src-induced cell motility in the presence of wildtype GRIM-19. The mutant proteins have lost such suppressive activity to varying degrees. (B) A quantified view of the inhibitory effects of GRIM-19 on cell motility. Distance migrated from the edge of the monolayer to the center of the denuded area 4h after the induction of injury was calculated from a number of independent samples (n=8) and plotted.