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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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RNAi-mediated depletion of Bnip-2 reduces myogenic differentiation. (A) Lysates of C2C12 cells stably transfected with pSilencer containing one of three independent Bnip-2 siRNA sequences (designated 1, 3, and 4) or pSilencer containing an irrelevant sequence (−) were Western blotted with Bnip-2 or, as a control, pan-cadherin antibodies. (B) Photomicrographs of C2C12 cells that express Bnip-2 siRNA sequences or an irrelevant sequence (pSilencer) as indicated, cultured in DM, fixed, and stained with an antibody to MHC. Bar, 0.5 mm. (C) Quantification of myotube formation. Values represent means of triplicate determinations ±1 SD. The experiment was repeated three times with similar results. Asterisks indicate difference from pSilencer control, P < 0.01. A level of myotube formation by control cells (∼80% nuclei in MHC+ cells) was selected so as to permit visualization of diminished differentiation by Bnip-2 siRNA. (D) Western blot analysis of C2C12 cells that express Bnip-2 siRNA sequences (+) or an irrelevant sequence (−) cultured in GM (G) or in DM for the indicated times.
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fig4: RNAi-mediated depletion of Bnip-2 reduces myogenic differentiation. (A) Lysates of C2C12 cells stably transfected with pSilencer containing one of three independent Bnip-2 siRNA sequences (designated 1, 3, and 4) or pSilencer containing an irrelevant sequence (−) were Western blotted with Bnip-2 or, as a control, pan-cadherin antibodies. (B) Photomicrographs of C2C12 cells that express Bnip-2 siRNA sequences or an irrelevant sequence (pSilencer) as indicated, cultured in DM, fixed, and stained with an antibody to MHC. Bar, 0.5 mm. (C) Quantification of myotube formation. Values represent means of triplicate determinations ±1 SD. The experiment was repeated three times with similar results. Asterisks indicate difference from pSilencer control, P < 0.01. A level of myotube formation by control cells (∼80% nuclei in MHC+ cells) was selected so as to permit visualization of diminished differentiation by Bnip-2 siRNA. (D) Western blot analysis of C2C12 cells that express Bnip-2 siRNA sequences (+) or an irrelevant sequence (−) cultured in GM (G) or in DM for the indicated times.

Mentions: To assess the effects of diminished Bnip-2 levels on differentiation, three independent Bnip-2 sequences placed into the pSilencer siRNA vector were individually and stably expressed in C2C12 cells. Each sequence substantially reduced Bnip-2 protein levels (Fig. 4 A). Note that the most slowly migrating band was more variably affected than the faster migrating forms in Fig. 4 A and in later figures. This could represent a more stable species of Bnip-2 that is relatively resistant to RNAi-mediated knockdown or revelation of a protein nonspecifically recognized by the Bnip-2 antibody that comigrates with the top Bnip-2 band. C2C12 cells that expressed each siRNA sequence displayed a smaller percentage of cell nuclei in MHC+ myotubes, and the myotubes that formed were shorter and thinner, than control cells that expressed an irrelevant siRNA sequence (Fig. 4, B and C). Expression of MHC was delayed and reduced in Bnip-2 siRNA-expressing cells, whereas expression of myogenin was not substantially altered, perhaps because of incomplete knockdown of Bnip-2 (Fig. 4 D). These latter results suggest that myotube formation may be more sensitive to partial loss of Bnip-2 than is expression of muscle-specific proteins.


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)

RNAi-mediated depletion of Bnip-2 reduces myogenic differentiation. (A) Lysates of C2C12 cells stably transfected with pSilencer containing one of three independent Bnip-2 siRNA sequences (designated 1, 3, and 4) or pSilencer containing an irrelevant sequence (−) were Western blotted with Bnip-2 or, as a control, pan-cadherin antibodies. (B) Photomicrographs of C2C12 cells that express Bnip-2 siRNA sequences or an irrelevant sequence (pSilencer) as indicated, cultured in DM, fixed, and stained with an antibody to MHC. Bar, 0.5 mm. (C) Quantification of myotube formation. Values represent means of triplicate determinations ±1 SD. The experiment was repeated three times with similar results. Asterisks indicate difference from pSilencer control, P < 0.01. A level of myotube formation by control cells (∼80% nuclei in MHC+ cells) was selected so as to permit visualization of diminished differentiation by Bnip-2 siRNA. (D) Western blot analysis of C2C12 cells that express Bnip-2 siRNA sequences (+) or an irrelevant sequence (−) cultured in GM (G) or in DM for the indicated times.
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fig4: RNAi-mediated depletion of Bnip-2 reduces myogenic differentiation. (A) Lysates of C2C12 cells stably transfected with pSilencer containing one of three independent Bnip-2 siRNA sequences (designated 1, 3, and 4) or pSilencer containing an irrelevant sequence (−) were Western blotted with Bnip-2 or, as a control, pan-cadherin antibodies. (B) Photomicrographs of C2C12 cells that express Bnip-2 siRNA sequences or an irrelevant sequence (pSilencer) as indicated, cultured in DM, fixed, and stained with an antibody to MHC. Bar, 0.5 mm. (C) Quantification of myotube formation. Values represent means of triplicate determinations ±1 SD. The experiment was repeated three times with similar results. Asterisks indicate difference from pSilencer control, P < 0.01. A level of myotube formation by control cells (∼80% nuclei in MHC+ cells) was selected so as to permit visualization of diminished differentiation by Bnip-2 siRNA. (D) Western blot analysis of C2C12 cells that express Bnip-2 siRNA sequences (+) or an irrelevant sequence (−) cultured in GM (G) or in DM for the indicated times.
Mentions: To assess the effects of diminished Bnip-2 levels on differentiation, three independent Bnip-2 sequences placed into the pSilencer siRNA vector were individually and stably expressed in C2C12 cells. Each sequence substantially reduced Bnip-2 protein levels (Fig. 4 A). Note that the most slowly migrating band was more variably affected than the faster migrating forms in Fig. 4 A and in later figures. This could represent a more stable species of Bnip-2 that is relatively resistant to RNAi-mediated knockdown or revelation of a protein nonspecifically recognized by the Bnip-2 antibody that comigrates with the top Bnip-2 band. C2C12 cells that expressed each siRNA sequence displayed a smaller percentage of cell nuclei in MHC+ myotubes, and the myotubes that formed were shorter and thinner, than control cells that expressed an irrelevant siRNA sequence (Fig. 4, B and C). Expression of MHC was delayed and reduced in Bnip-2 siRNA-expressing cells, whereas expression of myogenin was not substantially altered, perhaps because of incomplete knockdown of Bnip-2 (Fig. 4 D). These latter results suggest that myotube formation may be more sensitive to partial loss of Bnip-2 than is expression of muscle-specific proteins.

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