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A Cdo-Bnip-2-Cdc42 signaling pathway regulates p38alpha/beta MAPK activity and myogenic differentiation.

Kang JS, Bae GU, Yi MJ, Yang YJ, Oh JE, Takaesu G, Zhou YT, Low BC, Krauss RS - J. Cell Biol. (2008)

Bottom Line: Gain- and loss-of-function experiments with myoblasts indicate that the Cdo-Bnip-2 interaction stimulates Cdc42 activity, which in turn promotes p38alpha/beta activity and cell differentiation.These results reveal a previously unknown linkage between a cell surface receptor and downstream modulation of Cdc42 activity.Furthermore, interaction with multiple scaffold-type proteins is a distinctive mode of cell surface receptor signaling and provides one mechanism for specificity of p38alpha/beta activation during cell differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, New York, NY 10029, USA. jskang@med.skku.ac.kr

ABSTRACT
The p38alpha/beta mitogen-activated protein kinase (MAPK) pathway promotes skeletal myogenesis, but the mechanisms by which it is activated during this process are unclear. During myoblast differentiation, the promyogenic cell surface receptor Cdo binds to the p38alpha/beta pathway scaffold protein JLP and, via JLP, p38alpha/beta itself. We report that Cdo also interacts with Bnip-2, a protein that binds the small guanosine triphosphatase (GTPase) Cdc42 and a negative regulator of Cdc42, Cdc42 GTPase-activating protein (GAP). Moreover, Bnip-2 and JLP are brought together through mutual interaction with Cdo. Gain- and loss-of-function experiments with myoblasts indicate that the Cdo-Bnip-2 interaction stimulates Cdc42 activity, which in turn promotes p38alpha/beta activity and cell differentiation. These results reveal a previously unknown linkage between a cell surface receptor and downstream modulation of Cdc42 activity. Furthermore, interaction with multiple scaffold-type proteins is a distinctive mode of cell surface receptor signaling and provides one mechanism for specificity of p38alpha/beta activation during cell differentiation.

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Bnip-2 and JLP bind the same Cdo complexes, and Bnip-2 regulates p38α/β activity to promote myogenesis. (A) Lysates of COS7 cells transiently transfected with JLP-S, Bnip-2, Cdo, or control (−) expression vectors as indicated were immunoprecipitated (IP) with anti–S agarose and then Western blotted with JLP, Bnip-2, or Cdo antibodies. (B) Lysates of myoblasts of the indicated Cdo genotype cultured in DM for 48 h were immunoprecipitated with Cdo antibodies and then Western blotted with Cdo, Bnip-2, or JLP antibodies. Total cell lysates were also Western blotted with Bnip-2 or JLP antibodies. (C) Lysates of myoblasts of the indicated Cdo genotype cultured in DM for 48 h were immunoprecipitated with Bnip-2 antibodies and then Western blotted with Cdo, Bnip-2, or JLP antibodies. Total cell lysates were also Western blotted with Cdo or JLP antibodies. The Cdo band below the one indicated by the arrow is a partial degradation product. (D) Lysates of C2C12/Bnip-2 (+) or vector control (−) cells in GM or cultured for 48 h in DM were Western blotted with anti–phospho p38α/β (pp38) or p38α/β (p38) antibodies. (E) Lysates of C2C12 cells stably expressing a Bnip-2 siRNA sequence (+) or an irrelevant sequence (−) in GM or cultured for 48 h in DM were Western blotted with pp38 or p38 antibodies. (F) Lysates of C2C12 cells stably expressing a Cdc42Gap siRNA sequence (+) or an irrelevant sequence (−) in GM or cultured for 48 h in DM were Western blotted with pp38 or p38 antibodies. As the lanes were somewhat unevenly loaded in F, the pp38 and p38 signals were quantified by densitometry, and the pp38/p38 ratio reported under each lane in arbitrary units with the control transfectants in GM set to 1. (G) C2C12 cells were cotransfected with control, Bnip-2 siRNA, or Bnip-2 deletion mutant expression vectors, control or MKK6(EE) expression vectors, and pQ-lacZ (a vector that drives expression of nuclear-localized β-gal to mark transfectants). Differentiated cultures were double-stained for β-gal activity (blue) and for MHC expression (brown). Bar, 0.1 mm. (H) Quantification of C2C12 cell differentiation shown in G. Cultures were scored as MHC− or MHC+, with MHC+ cells further scored as having a single (1) nucleus, two to five nuclei, or greater than five nuclei. Values represent means of triplicate determinations ±1 SD. The experiment was repeated three times with similar results.
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fig9: Bnip-2 and JLP bind the same Cdo complexes, and Bnip-2 regulates p38α/β activity to promote myogenesis. (A) Lysates of COS7 cells transiently transfected with JLP-S, Bnip-2, Cdo, or control (−) expression vectors as indicated were immunoprecipitated (IP) with anti–S agarose and then Western blotted with JLP, Bnip-2, or Cdo antibodies. (B) Lysates of myoblasts of the indicated Cdo genotype cultured in DM for 48 h were immunoprecipitated with Cdo antibodies and then Western blotted with Cdo, Bnip-2, or JLP antibodies. Total cell lysates were also Western blotted with Bnip-2 or JLP antibodies. (C) Lysates of myoblasts of the indicated Cdo genotype cultured in DM for 48 h were immunoprecipitated with Bnip-2 antibodies and then Western blotted with Cdo, Bnip-2, or JLP antibodies. Total cell lysates were also Western blotted with Cdo or JLP antibodies. The Cdo band below the one indicated by the arrow is a partial degradation product. (D) Lysates of C2C12/Bnip-2 (+) or vector control (−) cells in GM or cultured for 48 h in DM were Western blotted with anti–phospho p38α/β (pp38) or p38α/β (p38) antibodies. (E) Lysates of C2C12 cells stably expressing a Bnip-2 siRNA sequence (+) or an irrelevant sequence (−) in GM or cultured for 48 h in DM were Western blotted with pp38 or p38 antibodies. (F) Lysates of C2C12 cells stably expressing a Cdc42Gap siRNA sequence (+) or an irrelevant sequence (−) in GM or cultured for 48 h in DM were Western blotted with pp38 or p38 antibodies. As the lanes were somewhat unevenly loaded in F, the pp38 and p38 signals were quantified by densitometry, and the pp38/p38 ratio reported under each lane in arbitrary units with the control transfectants in GM set to 1. (G) C2C12 cells were cotransfected with control, Bnip-2 siRNA, or Bnip-2 deletion mutant expression vectors, control or MKK6(EE) expression vectors, and pQ-lacZ (a vector that drives expression of nuclear-localized β-gal to mark transfectants). Differentiated cultures were double-stained for β-gal activity (blue) and for MHC expression (brown). Bar, 0.1 mm. (H) Quantification of C2C12 cell differentiation shown in G. Cultures were scored as MHC− or MHC+, with MHC+ cells further scored as having a single (1) nucleus, two to five nuclei, or greater than five nuclei. Values represent means of triplicate determinations ±1 SD. The experiment was repeated three times with similar results.

Mentions: Cdc42 signaling activates p38α/β MAPK in several cell systems (Coso et al., 1995; Minden et al., 1995; Molnár et al., 1997; Bourdoulous et al., 1998). The ability of Cdo to bind Bnip-2 and JLP, which in turn bind Cdc42 and p38α/β, respectively, suggests that Cdo might coordinate Cdc42→p38α/β signaling. The possibility that Bnip-2 and JLP bind the same Cdo complexes was tested initially. COS7 cells were transfected with expression vectors encoding S epitope–tagged JLP (JLP-S), JLP-S and Bnip-2, or JLP-S, Bnip-2, and Cdo. Cell lysates were then precipitated with anti–S agarose and blotted with antibodies to each protein (Fig. 9 A). JLP coprecipitated Bnip-2 in the presence, but not the absence, of coexpressed Cdo. Therefore, JLP and Bnip-2 did not directly interact but were brought together by a mutual interaction with Cdo. To assess whether such complexes form endogenously, lysates of Cdo+/+ and Cdo−/− myoblasts were immunoprecipitated with antibodies to Cdo or to Bnip-2 and then Western blotted with antibodies to Cdo, Bnip-2, and JLP. As expected, Bnip-2 and JLP both coprecipitated with Cdo (Fig. 9 B; Takaesu et al., 2006). Furthermore, JLP coprecipitated with Bnip-2 from Cdo+/+ cell lysates but not Cdo−/− cell lysates, indicating that Bnip-2 and JLP associate in myoblasts in a Cdo-dependent manner (Fig. 9 C).


A Cdo-Bnip-2-Cdc42 signaling pathway regulates p38alpha/beta MAPK activity and myogenic differentiation.

Kang JS, Bae GU, Yi MJ, Yang YJ, Oh JE, Takaesu G, Zhou YT, Low BC, Krauss RS - J. Cell Biol. (2008)

Bnip-2 and JLP bind the same Cdo complexes, and Bnip-2 regulates p38α/β activity to promote myogenesis. (A) Lysates of COS7 cells transiently transfected with JLP-S, Bnip-2, Cdo, or control (−) expression vectors as indicated were immunoprecipitated (IP) with anti–S agarose and then Western blotted with JLP, Bnip-2, or Cdo antibodies. (B) Lysates of myoblasts of the indicated Cdo genotype cultured in DM for 48 h were immunoprecipitated with Cdo antibodies and then Western blotted with Cdo, Bnip-2, or JLP antibodies. Total cell lysates were also Western blotted with Bnip-2 or JLP antibodies. (C) Lysates of myoblasts of the indicated Cdo genotype cultured in DM for 48 h were immunoprecipitated with Bnip-2 antibodies and then Western blotted with Cdo, Bnip-2, or JLP antibodies. Total cell lysates were also Western blotted with Cdo or JLP antibodies. The Cdo band below the one indicated by the arrow is a partial degradation product. (D) Lysates of C2C12/Bnip-2 (+) or vector control (−) cells in GM or cultured for 48 h in DM were Western blotted with anti–phospho p38α/β (pp38) or p38α/β (p38) antibodies. (E) Lysates of C2C12 cells stably expressing a Bnip-2 siRNA sequence (+) or an irrelevant sequence (−) in GM or cultured for 48 h in DM were Western blotted with pp38 or p38 antibodies. (F) Lysates of C2C12 cells stably expressing a Cdc42Gap siRNA sequence (+) or an irrelevant sequence (−) in GM or cultured for 48 h in DM were Western blotted with pp38 or p38 antibodies. As the lanes were somewhat unevenly loaded in F, the pp38 and p38 signals were quantified by densitometry, and the pp38/p38 ratio reported under each lane in arbitrary units with the control transfectants in GM set to 1. (G) C2C12 cells were cotransfected with control, Bnip-2 siRNA, or Bnip-2 deletion mutant expression vectors, control or MKK6(EE) expression vectors, and pQ-lacZ (a vector that drives expression of nuclear-localized β-gal to mark transfectants). Differentiated cultures were double-stained for β-gal activity (blue) and for MHC expression (brown). Bar, 0.1 mm. (H) Quantification of C2C12 cell differentiation shown in G. Cultures were scored as MHC− or MHC+, with MHC+ cells further scored as having a single (1) nucleus, two to five nuclei, or greater than five nuclei. Values represent means of triplicate determinations ±1 SD. The experiment was repeated three times with similar results.
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fig9: Bnip-2 and JLP bind the same Cdo complexes, and Bnip-2 regulates p38α/β activity to promote myogenesis. (A) Lysates of COS7 cells transiently transfected with JLP-S, Bnip-2, Cdo, or control (−) expression vectors as indicated were immunoprecipitated (IP) with anti–S agarose and then Western blotted with JLP, Bnip-2, or Cdo antibodies. (B) Lysates of myoblasts of the indicated Cdo genotype cultured in DM for 48 h were immunoprecipitated with Cdo antibodies and then Western blotted with Cdo, Bnip-2, or JLP antibodies. Total cell lysates were also Western blotted with Bnip-2 or JLP antibodies. (C) Lysates of myoblasts of the indicated Cdo genotype cultured in DM for 48 h were immunoprecipitated with Bnip-2 antibodies and then Western blotted with Cdo, Bnip-2, or JLP antibodies. Total cell lysates were also Western blotted with Cdo or JLP antibodies. The Cdo band below the one indicated by the arrow is a partial degradation product. (D) Lysates of C2C12/Bnip-2 (+) or vector control (−) cells in GM or cultured for 48 h in DM were Western blotted with anti–phospho p38α/β (pp38) or p38α/β (p38) antibodies. (E) Lysates of C2C12 cells stably expressing a Bnip-2 siRNA sequence (+) or an irrelevant sequence (−) in GM or cultured for 48 h in DM were Western blotted with pp38 or p38 antibodies. (F) Lysates of C2C12 cells stably expressing a Cdc42Gap siRNA sequence (+) or an irrelevant sequence (−) in GM or cultured for 48 h in DM were Western blotted with pp38 or p38 antibodies. As the lanes were somewhat unevenly loaded in F, the pp38 and p38 signals were quantified by densitometry, and the pp38/p38 ratio reported under each lane in arbitrary units with the control transfectants in GM set to 1. (G) C2C12 cells were cotransfected with control, Bnip-2 siRNA, or Bnip-2 deletion mutant expression vectors, control or MKK6(EE) expression vectors, and pQ-lacZ (a vector that drives expression of nuclear-localized β-gal to mark transfectants). Differentiated cultures were double-stained for β-gal activity (blue) and for MHC expression (brown). Bar, 0.1 mm. (H) Quantification of C2C12 cell differentiation shown in G. Cultures were scored as MHC− or MHC+, with MHC+ cells further scored as having a single (1) nucleus, two to five nuclei, or greater than five nuclei. Values represent means of triplicate determinations ±1 SD. The experiment was repeated three times with similar results.
Mentions: Cdc42 signaling activates p38α/β MAPK in several cell systems (Coso et al., 1995; Minden et al., 1995; Molnár et al., 1997; Bourdoulous et al., 1998). The ability of Cdo to bind Bnip-2 and JLP, which in turn bind Cdc42 and p38α/β, respectively, suggests that Cdo might coordinate Cdc42→p38α/β signaling. The possibility that Bnip-2 and JLP bind the same Cdo complexes was tested initially. COS7 cells were transfected with expression vectors encoding S epitope–tagged JLP (JLP-S), JLP-S and Bnip-2, or JLP-S, Bnip-2, and Cdo. Cell lysates were then precipitated with anti–S agarose and blotted with antibodies to each protein (Fig. 9 A). JLP coprecipitated Bnip-2 in the presence, but not the absence, of coexpressed Cdo. Therefore, JLP and Bnip-2 did not directly interact but were brought together by a mutual interaction with Cdo. To assess whether such complexes form endogenously, lysates of Cdo+/+ and Cdo−/− myoblasts were immunoprecipitated with antibodies to Cdo or to Bnip-2 and then Western blotted with antibodies to Cdo, Bnip-2, and JLP. As expected, Bnip-2 and JLP both coprecipitated with Cdo (Fig. 9 B; Takaesu et al., 2006). Furthermore, JLP coprecipitated with Bnip-2 from Cdo+/+ cell lysates but not Cdo−/− cell lysates, indicating that Bnip-2 and JLP associate in myoblasts in a Cdo-dependent manner (Fig. 9 C).

Bottom Line: Gain- and loss-of-function experiments with myoblasts indicate that the Cdo-Bnip-2 interaction stimulates Cdc42 activity, which in turn promotes p38alpha/beta activity and cell differentiation.These results reveal a previously unknown linkage between a cell surface receptor and downstream modulation of Cdc42 activity.Furthermore, interaction with multiple scaffold-type proteins is a distinctive mode of cell surface receptor signaling and provides one mechanism for specificity of p38alpha/beta activation during cell differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, New York, NY 10029, USA. jskang@med.skku.ac.kr

ABSTRACT
The p38alpha/beta mitogen-activated protein kinase (MAPK) pathway promotes skeletal myogenesis, but the mechanisms by which it is activated during this process are unclear. During myoblast differentiation, the promyogenic cell surface receptor Cdo binds to the p38alpha/beta pathway scaffold protein JLP and, via JLP, p38alpha/beta itself. We report that Cdo also interacts with Bnip-2, a protein that binds the small guanosine triphosphatase (GTPase) Cdc42 and a negative regulator of Cdc42, Cdc42 GTPase-activating protein (GAP). Moreover, Bnip-2 and JLP are brought together through mutual interaction with Cdo. Gain- and loss-of-function experiments with myoblasts indicate that the Cdo-Bnip-2 interaction stimulates Cdc42 activity, which in turn promotes p38alpha/beta activity and cell differentiation. These results reveal a previously unknown linkage between a cell surface receptor and downstream modulation of Cdc42 activity. Furthermore, interaction with multiple scaffold-type proteins is a distinctive mode of cell surface receptor signaling and provides one mechanism for specificity of p38alpha/beta activation during cell differentiation.

Show MeSH