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Mitogen-inducible gene 6 is an endogenous inhibitor of HGF/Met-induced cell migration and neurite growth.

Pante G, Thompson J, Lamballe F, Iwata T, Ferby I, Barr FA, Davies AM, Maina F, Klein R - J. Cell Biol. (2005)

Bottom Line: Here we report a mechanism by which mitogen-inducible gene 6 (Mig6; also called Gene 33 and receptor-associated late transducer) negatively regulates HGF/Met-induced cell migration.The effect is observed by Mig6 overexpression and is reversed by Mig6 small interfering RNA knock-down experiments; this indicates that endogenous Mig6 is part of a mechanism that inhibits Met signaling.Because Mig6 also is induced by HGF stimulation, our results suggest that Mig6 is part of a negative feedback loop that attenuates Met functions in different contexts and cell types.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Neurobiology, Max Planck Institute of Neurobiology, 82152 Munich-Martinsried, Germany.

ABSTRACT
Hepatocyte growth factor (HGF)/Met signaling controls cell migration, growth and differentiation in several embryonic organs and is implicated in human cancer. The physiologic mechanisms that attenuate Met signaling are not well understood. Here we report a mechanism by which mitogen-inducible gene 6 (Mig6; also called Gene 33 and receptor-associated late transducer) negatively regulates HGF/Met-induced cell migration. The effect is observed by Mig6 overexpression and is reversed by Mig6 small interfering RNA knock-down experiments; this indicates that endogenous Mig6 is part of a mechanism that inhibits Met signaling. Mig6 functions in cells of hepatic origin and in neurons, which suggests a role for Mig6 in different cell lineages. Mechanistically, Mig6 requires an intact Cdc42/Rac interactive binding site to exert its inhibitory action, which suggests that Mig6 acts, at least in part, distally from Met, possibly by inhibiting Rho-like GTPases. Because Mig6 also is induced by HGF stimulation, our results suggest that Mig6 is part of a negative feedback loop that attenuates Met functions in different contexts and cell types.

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Related in: MedlinePlus

The CRIB domain of Mig6 is necessary and sufficient to inhibit HGF-induced cell migration. (A) Mig6 binds Grb2. GST-Mig6FL and GST-Mig6ΔCRIB purified fusion proteins were used to pull down endogenous Grb2 from MLP29 total cell lysate. The fusion proteins were incubated for 1 h with total (Tot.) cell lysate and pulled down using glutathione-sepharose beads. Pulled-down proteins were eluted from the beads and analyzed by SDS-PAGE. Western blot (W.B.) analysis using an α-Grb2 antibody shows that Grb2 associates with Mig6, and that the association is independent of Mig6's CRIB domain. A GST-PAKBD fusion protein was used as a negative control. (B) Mig6 reduces the levels of Cdc42-GTP. The GST-Ack-CRIB–purified fusion protein was used to pull down (P.D.) the active form (GTP-bound) of transfected GFP-Cdc42 at different time points of HGF stimulation in the presence or the absence of Mig6 expression plasmid. MLP29 cells were transfected with GFP-Cdc42 plus GFP (left half) or GFP-Cdc42 plus Mig6FL-V5 expression plasmids (right half), starved for 48 h in 0.1% FBS, stimulated with 40 ng/ml of HGF for the indicated times, and lysed. The GST-Ack-CRIB protein was incubated with the cell lysates, pulled down using glutathione-sepharose beads, eluted from the beads, and analyzed by SDS-PAGE. Western blot (W.B.) analysis using an anti-GFP–specific antibody showed that exogenous GFP-Cdc42 is activated upon 15 and 30 min (′) of HGF stimulation. The expression of Mig6 significantly reduced exogenous GFP-Cdc42 activation upon HGF stimulation (compare 15- and 30-min time points, left and right part of the blot). Western blot analysis on total cell lysates (TCL) using an anti-GFP–specific antibody was used to control that comparable amounts of total GFP-Cdc42 were used for the pull down. (C) Schematic representation of the different GST-Mig6 deletion mutants. 14-3-3, 14-3-3 binding region; AH, Ack homology domain; PDZ, PDZ target site; SH3BM, SH3 binding site. (D, left blot) GST-Mig6FL, GST-Mig6ΔCRIB, GST-Mig6NT, and GST-Mig6CRIB were transfected in MLP29 cells and pulled down using glutathione-Sepharose beads (Glut.). The proteins were eluted from the beads and analyzed by SDS-PAGE. Western blot (W.B.) analysis using an α-GST antibody shows that the Mig6 fusion proteins were expressed at similar levels. Transfection with YFP expression plasmid was used as a negative control for the α-GST antibody. (D, right blot) GST-Mig6FL plus GFP-Cdc42*, GFP-Cdc42*, or GST-Mig6FL plus GFP expression plasmids were transfected in MLP29 cells. The cells were lysed, and total cell lysates analyzed by SDS-PAGE. Western blot analysis using an anti-GST– (top panel) and an anti-GFP (bottom panel) specific–antibody shows that the exogenous Mig6 and Cdc42* (arrowheads) are expressed at equal levels. Transfection of GFP expression plasmid was used as a negative control for the anti-GST and anti-GFP antibodies. Asterisk in panel D denotes an unspecific cross-reactive protein that is detected with anti-GST antibodies. Quantification of cell migration expressed as fold of induction over unstimulated cells in the presence of 10% FBS (E and G) or HGF (F and H). MLP29 cells were left untransfected or were transfected with the indicated GST-Mig6 expression plasmids (E and F) or cotransfected with a combination of GFP-Cdc42* and GST-Mig6FL expression plasmids as indicated (G and H) and seeded onto the upper compartment of the Boyden chamber. The NH2-terminal half of Mig6 (Mig6NT) and its CRIB domain (Mig6CRIB) were able to inhibit HGF-mediated migration significantly compared with control cells (for both P = 0.001, t test) (panel F). **, P < 0.01. In contrast, Mig6ΔCRIB was not able to reduce HGF-mediated migration significantly compared with untransfected control (P = 0.88, t test). The inhibition of HGF-mediated migration due to GST-Mig6FL expression was rescued significantly by the coexpression of GFP-Cdc42* (P = 0.76), but not of GFP expression plasmids (average of three separate experiments; P < 0.0001). 10% FBS-induced cell migration was not affected by any of the Mig6-expressing constructs (E and G).
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fig7: The CRIB domain of Mig6 is necessary and sufficient to inhibit HGF-induced cell migration. (A) Mig6 binds Grb2. GST-Mig6FL and GST-Mig6ΔCRIB purified fusion proteins were used to pull down endogenous Grb2 from MLP29 total cell lysate. The fusion proteins were incubated for 1 h with total (Tot.) cell lysate and pulled down using glutathione-sepharose beads. Pulled-down proteins were eluted from the beads and analyzed by SDS-PAGE. Western blot (W.B.) analysis using an α-Grb2 antibody shows that Grb2 associates with Mig6, and that the association is independent of Mig6's CRIB domain. A GST-PAKBD fusion protein was used as a negative control. (B) Mig6 reduces the levels of Cdc42-GTP. The GST-Ack-CRIB–purified fusion protein was used to pull down (P.D.) the active form (GTP-bound) of transfected GFP-Cdc42 at different time points of HGF stimulation in the presence or the absence of Mig6 expression plasmid. MLP29 cells were transfected with GFP-Cdc42 plus GFP (left half) or GFP-Cdc42 plus Mig6FL-V5 expression plasmids (right half), starved for 48 h in 0.1% FBS, stimulated with 40 ng/ml of HGF for the indicated times, and lysed. The GST-Ack-CRIB protein was incubated with the cell lysates, pulled down using glutathione-sepharose beads, eluted from the beads, and analyzed by SDS-PAGE. Western blot (W.B.) analysis using an anti-GFP–specific antibody showed that exogenous GFP-Cdc42 is activated upon 15 and 30 min (′) of HGF stimulation. The expression of Mig6 significantly reduced exogenous GFP-Cdc42 activation upon HGF stimulation (compare 15- and 30-min time points, left and right part of the blot). Western blot analysis on total cell lysates (TCL) using an anti-GFP–specific antibody was used to control that comparable amounts of total GFP-Cdc42 were used for the pull down. (C) Schematic representation of the different GST-Mig6 deletion mutants. 14-3-3, 14-3-3 binding region; AH, Ack homology domain; PDZ, PDZ target site; SH3BM, SH3 binding site. (D, left blot) GST-Mig6FL, GST-Mig6ΔCRIB, GST-Mig6NT, and GST-Mig6CRIB were transfected in MLP29 cells and pulled down using glutathione-Sepharose beads (Glut.). The proteins were eluted from the beads and analyzed by SDS-PAGE. Western blot (W.B.) analysis using an α-GST antibody shows that the Mig6 fusion proteins were expressed at similar levels. Transfection with YFP expression plasmid was used as a negative control for the α-GST antibody. (D, right blot) GST-Mig6FL plus GFP-Cdc42*, GFP-Cdc42*, or GST-Mig6FL plus GFP expression plasmids were transfected in MLP29 cells. The cells were lysed, and total cell lysates analyzed by SDS-PAGE. Western blot analysis using an anti-GST– (top panel) and an anti-GFP (bottom panel) specific–antibody shows that the exogenous Mig6 and Cdc42* (arrowheads) are expressed at equal levels. Transfection of GFP expression plasmid was used as a negative control for the anti-GST and anti-GFP antibodies. Asterisk in panel D denotes an unspecific cross-reactive protein that is detected with anti-GST antibodies. Quantification of cell migration expressed as fold of induction over unstimulated cells in the presence of 10% FBS (E and G) or HGF (F and H). MLP29 cells were left untransfected or were transfected with the indicated GST-Mig6 expression plasmids (E and F) or cotransfected with a combination of GFP-Cdc42* and GST-Mig6FL expression plasmids as indicated (G and H) and seeded onto the upper compartment of the Boyden chamber. The NH2-terminal half of Mig6 (Mig6NT) and its CRIB domain (Mig6CRIB) were able to inhibit HGF-mediated migration significantly compared with control cells (for both P = 0.001, t test) (panel F). **, P < 0.01. In contrast, Mig6ΔCRIB was not able to reduce HGF-mediated migration significantly compared with untransfected control (P = 0.88, t test). The inhibition of HGF-mediated migration due to GST-Mig6FL expression was rescued significantly by the coexpression of GFP-Cdc42* (P = 0.76), but not of GFP expression plasmids (average of three separate experiments; P < 0.0001). 10% FBS-induced cell migration was not affected by any of the Mig6-expressing constructs (E and G).

Mentions: Mig6 was proposed to inhibit EGFR signaling by direct binding to EGFR and ErbB2—by suppressing the EGFR kinase activity—and by a receptor distal mechanism (Anastasi et al., 2003). To begin dissecting the mechanism of Mig6-mediated inhibition of cell migration, we asked whether Mig6 would directly bind Met in MLP29 cells that were stimulated with HGF. In pull-down experiments that used bacterially expressed, GST-tagged, purified, full-length Mig6, we were unable to demonstrate a direct association between Mig6 and Met (unpublished data). Moreover, the minimal region that was mapped in EGFR to be essential for the binding of Mig6 is not found in the amino acid sequence of mouse Met. However, pull-down experiments with GST-tagged Mig6 confirmed the association with growth factor receptor–bound protein 2 (Grb2) (Fig. 7 A), which had been observed previously (Fiorentino et al., 2000). Control pull-downs with the GST-tagged CRIB domain of P21-associated serine/threonine kinase failed to pull down Grb2 (Fig. 7 A). This suggested the possibility that Mig6 may bind Met indirectly by way of Grb2, thereby inhibiting Met in a receptor-proximal fashion.


Mitogen-inducible gene 6 is an endogenous inhibitor of HGF/Met-induced cell migration and neurite growth.

Pante G, Thompson J, Lamballe F, Iwata T, Ferby I, Barr FA, Davies AM, Maina F, Klein R - J. Cell Biol. (2005)

The CRIB domain of Mig6 is necessary and sufficient to inhibit HGF-induced cell migration. (A) Mig6 binds Grb2. GST-Mig6FL and GST-Mig6ΔCRIB purified fusion proteins were used to pull down endogenous Grb2 from MLP29 total cell lysate. The fusion proteins were incubated for 1 h with total (Tot.) cell lysate and pulled down using glutathione-sepharose beads. Pulled-down proteins were eluted from the beads and analyzed by SDS-PAGE. Western blot (W.B.) analysis using an α-Grb2 antibody shows that Grb2 associates with Mig6, and that the association is independent of Mig6's CRIB domain. A GST-PAKBD fusion protein was used as a negative control. (B) Mig6 reduces the levels of Cdc42-GTP. The GST-Ack-CRIB–purified fusion protein was used to pull down (P.D.) the active form (GTP-bound) of transfected GFP-Cdc42 at different time points of HGF stimulation in the presence or the absence of Mig6 expression plasmid. MLP29 cells were transfected with GFP-Cdc42 plus GFP (left half) or GFP-Cdc42 plus Mig6FL-V5 expression plasmids (right half), starved for 48 h in 0.1% FBS, stimulated with 40 ng/ml of HGF for the indicated times, and lysed. The GST-Ack-CRIB protein was incubated with the cell lysates, pulled down using glutathione-sepharose beads, eluted from the beads, and analyzed by SDS-PAGE. Western blot (W.B.) analysis using an anti-GFP–specific antibody showed that exogenous GFP-Cdc42 is activated upon 15 and 30 min (′) of HGF stimulation. The expression of Mig6 significantly reduced exogenous GFP-Cdc42 activation upon HGF stimulation (compare 15- and 30-min time points, left and right part of the blot). Western blot analysis on total cell lysates (TCL) using an anti-GFP–specific antibody was used to control that comparable amounts of total GFP-Cdc42 were used for the pull down. (C) Schematic representation of the different GST-Mig6 deletion mutants. 14-3-3, 14-3-3 binding region; AH, Ack homology domain; PDZ, PDZ target site; SH3BM, SH3 binding site. (D, left blot) GST-Mig6FL, GST-Mig6ΔCRIB, GST-Mig6NT, and GST-Mig6CRIB were transfected in MLP29 cells and pulled down using glutathione-Sepharose beads (Glut.). The proteins were eluted from the beads and analyzed by SDS-PAGE. Western blot (W.B.) analysis using an α-GST antibody shows that the Mig6 fusion proteins were expressed at similar levels. Transfection with YFP expression plasmid was used as a negative control for the α-GST antibody. (D, right blot) GST-Mig6FL plus GFP-Cdc42*, GFP-Cdc42*, or GST-Mig6FL plus GFP expression plasmids were transfected in MLP29 cells. The cells were lysed, and total cell lysates analyzed by SDS-PAGE. Western blot analysis using an anti-GST– (top panel) and an anti-GFP (bottom panel) specific–antibody shows that the exogenous Mig6 and Cdc42* (arrowheads) are expressed at equal levels. Transfection of GFP expression plasmid was used as a negative control for the anti-GST and anti-GFP antibodies. Asterisk in panel D denotes an unspecific cross-reactive protein that is detected with anti-GST antibodies. Quantification of cell migration expressed as fold of induction over unstimulated cells in the presence of 10% FBS (E and G) or HGF (F and H). MLP29 cells were left untransfected or were transfected with the indicated GST-Mig6 expression plasmids (E and F) or cotransfected with a combination of GFP-Cdc42* and GST-Mig6FL expression plasmids as indicated (G and H) and seeded onto the upper compartment of the Boyden chamber. The NH2-terminal half of Mig6 (Mig6NT) and its CRIB domain (Mig6CRIB) were able to inhibit HGF-mediated migration significantly compared with control cells (for both P = 0.001, t test) (panel F). **, P < 0.01. In contrast, Mig6ΔCRIB was not able to reduce HGF-mediated migration significantly compared with untransfected control (P = 0.88, t test). The inhibition of HGF-mediated migration due to GST-Mig6FL expression was rescued significantly by the coexpression of GFP-Cdc42* (P = 0.76), but not of GFP expression plasmids (average of three separate experiments; P < 0.0001). 10% FBS-induced cell migration was not affected by any of the Mig6-expressing constructs (E and G).
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fig7: The CRIB domain of Mig6 is necessary and sufficient to inhibit HGF-induced cell migration. (A) Mig6 binds Grb2. GST-Mig6FL and GST-Mig6ΔCRIB purified fusion proteins were used to pull down endogenous Grb2 from MLP29 total cell lysate. The fusion proteins were incubated for 1 h with total (Tot.) cell lysate and pulled down using glutathione-sepharose beads. Pulled-down proteins were eluted from the beads and analyzed by SDS-PAGE. Western blot (W.B.) analysis using an α-Grb2 antibody shows that Grb2 associates with Mig6, and that the association is independent of Mig6's CRIB domain. A GST-PAKBD fusion protein was used as a negative control. (B) Mig6 reduces the levels of Cdc42-GTP. The GST-Ack-CRIB–purified fusion protein was used to pull down (P.D.) the active form (GTP-bound) of transfected GFP-Cdc42 at different time points of HGF stimulation in the presence or the absence of Mig6 expression plasmid. MLP29 cells were transfected with GFP-Cdc42 plus GFP (left half) or GFP-Cdc42 plus Mig6FL-V5 expression plasmids (right half), starved for 48 h in 0.1% FBS, stimulated with 40 ng/ml of HGF for the indicated times, and lysed. The GST-Ack-CRIB protein was incubated with the cell lysates, pulled down using glutathione-sepharose beads, eluted from the beads, and analyzed by SDS-PAGE. Western blot (W.B.) analysis using an anti-GFP–specific antibody showed that exogenous GFP-Cdc42 is activated upon 15 and 30 min (′) of HGF stimulation. The expression of Mig6 significantly reduced exogenous GFP-Cdc42 activation upon HGF stimulation (compare 15- and 30-min time points, left and right part of the blot). Western blot analysis on total cell lysates (TCL) using an anti-GFP–specific antibody was used to control that comparable amounts of total GFP-Cdc42 were used for the pull down. (C) Schematic representation of the different GST-Mig6 deletion mutants. 14-3-3, 14-3-3 binding region; AH, Ack homology domain; PDZ, PDZ target site; SH3BM, SH3 binding site. (D, left blot) GST-Mig6FL, GST-Mig6ΔCRIB, GST-Mig6NT, and GST-Mig6CRIB were transfected in MLP29 cells and pulled down using glutathione-Sepharose beads (Glut.). The proteins were eluted from the beads and analyzed by SDS-PAGE. Western blot (W.B.) analysis using an α-GST antibody shows that the Mig6 fusion proteins were expressed at similar levels. Transfection with YFP expression plasmid was used as a negative control for the α-GST antibody. (D, right blot) GST-Mig6FL plus GFP-Cdc42*, GFP-Cdc42*, or GST-Mig6FL plus GFP expression plasmids were transfected in MLP29 cells. The cells were lysed, and total cell lysates analyzed by SDS-PAGE. Western blot analysis using an anti-GST– (top panel) and an anti-GFP (bottom panel) specific–antibody shows that the exogenous Mig6 and Cdc42* (arrowheads) are expressed at equal levels. Transfection of GFP expression plasmid was used as a negative control for the anti-GST and anti-GFP antibodies. Asterisk in panel D denotes an unspecific cross-reactive protein that is detected with anti-GST antibodies. Quantification of cell migration expressed as fold of induction over unstimulated cells in the presence of 10% FBS (E and G) or HGF (F and H). MLP29 cells were left untransfected or were transfected with the indicated GST-Mig6 expression plasmids (E and F) or cotransfected with a combination of GFP-Cdc42* and GST-Mig6FL expression plasmids as indicated (G and H) and seeded onto the upper compartment of the Boyden chamber. The NH2-terminal half of Mig6 (Mig6NT) and its CRIB domain (Mig6CRIB) were able to inhibit HGF-mediated migration significantly compared with control cells (for both P = 0.001, t test) (panel F). **, P < 0.01. In contrast, Mig6ΔCRIB was not able to reduce HGF-mediated migration significantly compared with untransfected control (P = 0.88, t test). The inhibition of HGF-mediated migration due to GST-Mig6FL expression was rescued significantly by the coexpression of GFP-Cdc42* (P = 0.76), but not of GFP expression plasmids (average of three separate experiments; P < 0.0001). 10% FBS-induced cell migration was not affected by any of the Mig6-expressing constructs (E and G).
Mentions: Mig6 was proposed to inhibit EGFR signaling by direct binding to EGFR and ErbB2—by suppressing the EGFR kinase activity—and by a receptor distal mechanism (Anastasi et al., 2003). To begin dissecting the mechanism of Mig6-mediated inhibition of cell migration, we asked whether Mig6 would directly bind Met in MLP29 cells that were stimulated with HGF. In pull-down experiments that used bacterially expressed, GST-tagged, purified, full-length Mig6, we were unable to demonstrate a direct association between Mig6 and Met (unpublished data). Moreover, the minimal region that was mapped in EGFR to be essential for the binding of Mig6 is not found in the amino acid sequence of mouse Met. However, pull-down experiments with GST-tagged Mig6 confirmed the association with growth factor receptor–bound protein 2 (Grb2) (Fig. 7 A), which had been observed previously (Fiorentino et al., 2000). Control pull-downs with the GST-tagged CRIB domain of P21-associated serine/threonine kinase failed to pull down Grb2 (Fig. 7 A). This suggested the possibility that Mig6 may bind Met indirectly by way of Grb2, thereby inhibiting Met in a receptor-proximal fashion.

Bottom Line: Here we report a mechanism by which mitogen-inducible gene 6 (Mig6; also called Gene 33 and receptor-associated late transducer) negatively regulates HGF/Met-induced cell migration.The effect is observed by Mig6 overexpression and is reversed by Mig6 small interfering RNA knock-down experiments; this indicates that endogenous Mig6 is part of a mechanism that inhibits Met signaling.Because Mig6 also is induced by HGF stimulation, our results suggest that Mig6 is part of a negative feedback loop that attenuates Met functions in different contexts and cell types.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Neurobiology, Max Planck Institute of Neurobiology, 82152 Munich-Martinsried, Germany.

ABSTRACT
Hepatocyte growth factor (HGF)/Met signaling controls cell migration, growth and differentiation in several embryonic organs and is implicated in human cancer. The physiologic mechanisms that attenuate Met signaling are not well understood. Here we report a mechanism by which mitogen-inducible gene 6 (Mig6; also called Gene 33 and receptor-associated late transducer) negatively regulates HGF/Met-induced cell migration. The effect is observed by Mig6 overexpression and is reversed by Mig6 small interfering RNA knock-down experiments; this indicates that endogenous Mig6 is part of a mechanism that inhibits Met signaling. Mig6 functions in cells of hepatic origin and in neurons, which suggests a role for Mig6 in different cell lineages. Mechanistically, Mig6 requires an intact Cdc42/Rac interactive binding site to exert its inhibitory action, which suggests that Mig6 acts, at least in part, distally from Met, possibly by inhibiting Rho-like GTPases. Because Mig6 also is induced by HGF stimulation, our results suggest that Mig6 is part of a negative feedback loop that attenuates Met functions in different contexts and cell types.

Show MeSH
Related in: MedlinePlus