Fig. 2. FRNK inhibits v-Src-stimulated three-dimensional cell invasion. (A) Matrigel (30 µg) invasion assays were performed with the indicated cells for 24 h using a serum stimulus in the lower chamber. Values are means ± SD from four experiments. No cell invasion was detected in the presence of BSA. (B) Representative images (60×) of the lower porous membrane surface from Matrigel invasion assays. Crystal violet-stained cells can be distinguished from the 8 µm membrane pores. (C) Cell invasion through polymerized collagen type I was assayed with the indicated cells for 24 h using a serum stimulus in the lower chamber. Values are means ± SD from two independent experiments. Random invasion activity was assayed in the presence of BSA.

Fig. 4. FRNK inhibits v-Src-stimulated ERK2 and JNK1 but not Akt activation. FRNK does not affect v-Src-stimulated cell growth in vitro. (A–C) Lysates were made from the indicated serum-starved cells. Blotting analyses were performed on whole-cell lysates with phospho-specific antibodies to either activated ERK (pERK), JNK (pJNK) or Akt (pAkt). The corresponding blots were stripped and reprobed with polyclonal antibodies to ERK2, JNK1 and Akt, respectively. (A) ERK2 IPs from the indicated cells were analyzed for in vitro kinase activity by the phosphorylation of myelin basic protein (MBP). (B) IPs using polyclonal antibodies to JNK1 and JNK2 from the indicated cell lysates were analyzed for in vitro kinase activity using GST–ATF-2 as a substrate. (C) Akt IPs from the indicated cells were analyzed for in vitro kinase activity by the phosphorylation of GST–GSK-3. Values in (A–C) are means ± SD from two experiments. (D) Growth of the indicated cells on gelatin-coated dishes in 0.5% serum. Cell counts were performed every 24 h in triplicate. Values are means ± SD from two independent experiments. NIH-3T3 (diamonds), v-Src3T3 (open squares), v-Src FRNK (open triangles) and v-Src FRNK S-1034 (filled circles). (E) Anchorage-independent cell growth in soft agar was evaluated by plating the indicated cells in 10% serum within 0.3% agar on a solidified base. Images show typical fields at 60×.

Fig. 6. FRNK inhibits v-Src-stimulated MMP-2 secretion. (A) Gelatin zymography of conditioned media from equal numbers of NIH-3T3 or v-Src3T3 fibroblasts. The positions of proMMP9, proMMP-2 and active MMP-2 are indicated. (B) Conditioned medium from the indicated cells was analyzed by gelatin zymography and MMP-2 blotting. Whole-cell lysates from the indicated cells were analyzed by MT1-MMP blotting. (C) Conditioned medium from the indicated cells was analyzed for soluble gelatinase activity. Values are expressed as a percentage of an activated MMP-2 standard. (D) Whole-cell lysates from the indicated cells were analyzed for soluble gelatinase activity. (E) Matrigel invasion assays were performed with v-Src3T3s in the presence of recombinant human TIMP-2 at the concentration indicated in the top chamber. Values are means ± SD from two independent experiments.

Fig. 8. JNK activity is important for v-Src-stimulated cell invasion and MMP-2 secretion. (A) Matrigel invasion assays were performed with v-Src3T3s in the presence of DMSO or the indicated concentrations of MEK1 (PD98059) or JNK (SP600125) inhibitors added to both chambers. Values are means ± SD from two experiments. (B and C) v-Src3T3s were mock treated with DMSO or incubated in the presence of 25 µM of either PD98059 or SP600125 for 18 h in starved conditions. Protein lysates were analyzed for (B) ERK2 in vitro kinase activity or (C) JNK in vitro kinase activity. Values are means ± SD from two experiments. Whole-cell lysates were blotted for either activated ERK (pERK) or activated JNK (pJNK1) and re-probed for either total ERK2 or JNK1 protein expression, respectively. (D) Conditioned media from either serum-starved, mock-treated (DMSO) or 25 µM SP600125-treated (18 h) v-Src3T3s were analyzed by gelatin zymography. A conditioned medium sample from the same number of serum-starved NIH-3T3 fibroblasts is shown for comparison.

Fig. 1. Stable FRNK expression in v-Src3T3s does not inhibit cell motility. (A) Equal protein lysates of either NIH-3T3 or v-Src3T3 fibroblasts were analyzed by blotting with a v-Src-specific mAb (top panel) and re-probed with an HA tag mAb to visualize FRNK (clones R2, A2 and O4) or FRNK S-1034 expression (lower panel). (B) c-Src and v-Src were immunoprecipitated (mAb 2-17) from lysates of serum-starved cells and were analyzed for associated in vitro kinase (IVK) activity using GST–FAK-CT as a substrate. (C) Haptotaxis motility was assayed with immobilized FN and the indicated cells. Random cell motility was measured in the presence of BSA. Values are means ± SD from four experiments. (D) Chemotaxis motility was assayed on collagen-coated membranes with the indicated cells using a serum stimulus added to the lower chamber. Random cell motility was measured in the presence of BSA. Values are means ± SD from three experiments. (E) Wound-healing scratch motility assays were performed with the indicated cells plated onto Matrigel-coated glass slides in the presence of serum. Cell migration was assessed by 1 mm grid comparisons of four image sets at 0 and 24 h; representative images are shown.

Fig. 3. FRNK inhibits the formation of a v-Src–FAK signaling complex, promotes FAK and p130Cas dephosphorylation, but does not affect cell morphology. (A) v-Src3T3 FRNK cells plated onto FN in the presence or absence of serum visualized by phase, actin staining with FITC–phalloidin or indirect staining for focal contact-associated vinculin. The scale bar is 30 µm. (B) FAK was immunoprecipitated (IP) from the indicated serum-starved cells and analyzed for ³²P-associated in vitro kinase (IVK) activity. The FAK IPs were resolved by SDS–PAGE, transferred to a PVDF membrane and exposed to film. Equal levels of FAK and the co-immunoprecipitation of v-Src were visualized by blotting. (C) FAK IPs from the indicated serum-starved cells were analyzed sequentially with anti-phosphotyrosine (pTyr) or with phospho-specific antibodies to FAK Tyr397 (pY397), Tyr861 (pY861) or Tyr925 (pY925). FAK levels were determined by FAK blotting, and these analyses were performed on two sets of IPs. (D) p130Cas IPs from the indicated cells were analyzed sequentially by anti-P.Tyr and p130Cas blotting.

Fig. 5. FRNK inhibits experimental metastasis but not v-Src-stimulated solid tumor growth. (A) Athymic nude mice were injected subcutaneously with the indicated cells and, after 10 days, visible tumor size was measured with calipers. Values are means ± SD from two experiments with eight animals per group. NIH-3T3 (diamonds), v-Src3T3 (open squares), v-Src FRNK (open triangles) and v-Src FRNK S-1034 (filled circles). (B) Protein lysates from the indicated subcutaneously grown tumor tissue were analyzed by FAK (top), v-Src (middle) or HA tag blotting (lower). (C) Representative Bouin’s-fixed lungs from mice 4 weeks after injection with the indicated cells. Multiple tumor nodules on the lung surface can be seen in v-Src3T3- and v-Src FRNK S-1034-injected animals. The scale bar is 1 cm. (D) Representative H&E-stained mouse lung sections from animals injected with the indicated cells. Metastatic tumor cells (arrows) and blood vessels (B) are indicated. (E) Blotting analyses of lung tissue after 4 weeks from animals injected with the indicated cells. FAK blotting was performed on protein lysates (top), v-Src was detected by immunoprecipitation and blotting (middle), and FRNK was detected by HA tag immunoprecipitation and blotting (lower). (F) HA tag blotting analysis was performed on protein lysates of lung tumor nodules from either v-Src FRNK- or v-Src FRNK S-1034-injected mice. The M number is used to identify mice.

Fig. 7. FRNK inhibits MMP-2 gene expresion. (A) Semi-quantitative RT–PCR analysis was performed with RNA isolated from the indicated cells using primer pairs to amplify MMP-2 (449 bp), MMP-9 (433 bp), β-actin (302 bp) or GAPDH (265 bp). The intensity of either the MMP-2 or MMP-9 bands was normalized to both β-actin and GAPDH signals. Values for v-Src3T3s were set to 1. (B) Luciferase activity from a 1686 bp region of the MMP-2 promoter (pT4-Luc-1686) or the empty luciferase vector (pGL2-Basic). Constructs were transiently transfected into human 293T cells along with increasing concentrations of FRNK or FRNK S-1034. Results are expressed as the ratio of luciferase versus Renilla activity. Values are means ± SD from two experiments.

Fig. 9. MMP-2 overexpression rescues the FRNK-mediated blockage of cell invasion. (A) Conditioned media samples from the indicated cells stably overexpressing and secreting either MMP-2 or catalytically inactive MMP-2 (Ala404) were analyzed by His tag blotting (top). v-Src (middle) or HA tag (lower) blotting of whole-cell lysates from the indicated cells. (B) In vitro cleavage and activation of His-tagged MMP-2 by incubation with the catalytic domain of MT1-MMP as visualized by MMP-2 blotting (top) and gelatin zymography (lower). (C) Conditioned media samples from the indicated cells were analyzed by gelatin zymography. The positions of proMMP-9, proMMP-2 and active MMP-2 are indicated. (D) v-Src was immunoprecipitated from the indicated serum-starved cell lysates, and associated in vitro kinase activity (IVK) was measured by GST–FAK-CT phosphorylation. (E) Matrigel (30 µg) invasion assays were performed with the indicated cells for 24 h. Values are means ± SD from two independent experiments. No cell invasion was detected in the presence of BSA.