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The centrosomal E3 ubiquitin ligase FBXO31-SCF regulates neuronal morphogenesis and migration.

Vadhvani M, Schwedhelm-Domeyer N, Mukherjee C, Stegmüller J - PLoS ONE (2013)

Bottom Line: In addition, we identified the polarity protein Par6c as a novel interaction partner and substrate targeted for proteasomal degradation in the control of axon but not dendrite growth.Finally, we ascribe a role for FBXO31 in dendrite growth and neuronal migration in the developing cerebellar cortex.Taken together, we uncovered the centrosomal E3 ligase FBXO31-SCF as a novel regulator of neuronal development.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany.

ABSTRACT
Neuronal development requires proper migration, polarization and establishment of axons and dendrites. Growing evidence identifies the ubiquitin proteasome system (UPS) with its numerous components as an important regulator of various aspects of neuronal development. F-box proteins are interchangeable subunits of the Cullin-1 based E3 ubiquitin ligase, but only a few family members have been studied. Here, we report that the centrosomal E3 ligase FBXO31-SCF (Skp1/Cullin-1/F-box protein) regulates neuronal morphogenesis and axonal identity. In addition, we identified the polarity protein Par6c as a novel interaction partner and substrate targeted for proteasomal degradation in the control of axon but not dendrite growth. Finally, we ascribe a role for FBXO31 in dendrite growth and neuronal migration in the developing cerebellar cortex. Taken together, we uncovered the centrosomal E3 ligase FBXO31-SCF as a novel regulator of neuronal development.

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FBXO31-SCF targets Par6c to proteasomal degradation. A.Transfected HEK 293T cells lysates were subjected to immunoprecipitation with α-Flag antibody and immunoblotted with α-myc antibody. B. Transfected HEK 293T cells lysates were subjected to immunoprecipitation with α-myc antibody and immunoblotted with α-Flag antibody. C. Schematic showing various Par6c deletion mutants and their interaction with FBXO31. D. Granule neurons were transfected with Par6c plasmid at DIV0 and treated with DMSO or lactacystin for 10 hours prior to lysis at DIV 3. Lysates were immunoblotted with α-myc antibody. 14-3-3ß served as a loading control. E. and F. HEK 293T cells (E) and granule neurons (F) were transfected with mycPar6c plasmid together with FBXO31 RNAi plasmids or respective control vectors as indicated. Cell lysates were immunoblotted with α-myc and α-Flag antibodies. 14-3-3ß served as a loading control. G. Cerebellar granule neurons were transfected with mycPar6c plasmid together with FBXO31 RNAi #1 plasmid or respective control vector at DIV 2. Centrosomal purification was performed at DIV 6 using sucrose density gradient centrifugation. The fractions were probed with α-myc antibody. γ-tubulin served as a positive control for centrosomal protein. The histogram shows Par6c levels relative to γ-tubulin at the centrosome (N = 3, mean±SEM, unpaired t-test, *p<0.05). H. HEK 293T cells were co-transfected with mycPar6c and GFP-FBXO31 WT or ΔF plasmids together with respective control vectors. Cell lysates were subjected to immunoprecipitation with α-myc antibody and immunoblotted with α-ubiquitin antibody. I. HEK 293T cells were co-transfected with mycPar6c and GFP-FBXO31 WT plasmids together with respective control vectors. Cell lysates were subjected to immunoprecipitation with α-myc antibody and immunoblotted with K48-specific α-ubiquitin antibody. J. HEK 293T cells were co-transfected with mycPar6c and GFP-FBXO31 WT plasmids together with respective control vectors. Cell lysates were subjected to immunoprecipitation with anti-myc antibody and immunoblotted with K63-specific anti-ubiquitin antibody.
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pone-0057530-g003: FBXO31-SCF targets Par6c to proteasomal degradation. A.Transfected HEK 293T cells lysates were subjected to immunoprecipitation with α-Flag antibody and immunoblotted with α-myc antibody. B. Transfected HEK 293T cells lysates were subjected to immunoprecipitation with α-myc antibody and immunoblotted with α-Flag antibody. C. Schematic showing various Par6c deletion mutants and their interaction with FBXO31. D. Granule neurons were transfected with Par6c plasmid at DIV0 and treated with DMSO or lactacystin for 10 hours prior to lysis at DIV 3. Lysates were immunoblotted with α-myc antibody. 14-3-3ß served as a loading control. E. and F. HEK 293T cells (E) and granule neurons (F) were transfected with mycPar6c plasmid together with FBXO31 RNAi plasmids or respective control vectors as indicated. Cell lysates were immunoblotted with α-myc and α-Flag antibodies. 14-3-3ß served as a loading control. G. Cerebellar granule neurons were transfected with mycPar6c plasmid together with FBXO31 RNAi #1 plasmid or respective control vector at DIV 2. Centrosomal purification was performed at DIV 6 using sucrose density gradient centrifugation. The fractions were probed with α-myc antibody. γ-tubulin served as a positive control for centrosomal protein. The histogram shows Par6c levels relative to γ-tubulin at the centrosome (N = 3, mean±SEM, unpaired t-test, *p<0.05). H. HEK 293T cells were co-transfected with mycPar6c and GFP-FBXO31 WT or ΔF plasmids together with respective control vectors. Cell lysates were subjected to immunoprecipitation with α-myc antibody and immunoblotted with α-ubiquitin antibody. I. HEK 293T cells were co-transfected with mycPar6c and GFP-FBXO31 WT plasmids together with respective control vectors. Cell lysates were subjected to immunoprecipitation with α-myc antibody and immunoblotted with K48-specific α-ubiquitin antibody. J. HEK 293T cells were co-transfected with mycPar6c and GFP-FBXO31 WT plasmids together with respective control vectors. Cell lysates were subjected to immunoprecipitation with anti-myc antibody and immunoblotted with K63-specific anti-ubiquitin antibody.

Mentions: To identify targets of FBXO31-SCF and owing to FBXO31’s centrosomal localization, we took a candidate approach and found that FBXO31 interacts with Par6c, a previously identified centrosomal protein [23], [24]. Par6 is known to regulate epithelial cell polarity [25], [26] and neuronal polarity [27], [28]. In the nervous system, Par6c represents the predominantly enriched Par6 family member [29]. We determined the interaction of exogenous FBXO31 and Par6c in heterologous cells by immunoprecipitating FBXO31 followed by immunoblotting for Par6c (Figure 3A). In a reciprocal experiment, we immunoprecipitated Par6c and immunoblotted for FBXO31 (Figure 3B). Mapping analyses of FBXO31 and Par6c revealed that Par6c’s PDZ domain mediates the interaction with FBXO31 (Figure 3C, Figure S5A). These experiments establish the FBXO31-Par6c interaction.


The centrosomal E3 ubiquitin ligase FBXO31-SCF regulates neuronal morphogenesis and migration.

Vadhvani M, Schwedhelm-Domeyer N, Mukherjee C, Stegmüller J - PLoS ONE (2013)

FBXO31-SCF targets Par6c to proteasomal degradation. A.Transfected HEK 293T cells lysates were subjected to immunoprecipitation with α-Flag antibody and immunoblotted with α-myc antibody. B. Transfected HEK 293T cells lysates were subjected to immunoprecipitation with α-myc antibody and immunoblotted with α-Flag antibody. C. Schematic showing various Par6c deletion mutants and their interaction with FBXO31. D. Granule neurons were transfected with Par6c plasmid at DIV0 and treated with DMSO or lactacystin for 10 hours prior to lysis at DIV 3. Lysates were immunoblotted with α-myc antibody. 14-3-3ß served as a loading control. E. and F. HEK 293T cells (E) and granule neurons (F) were transfected with mycPar6c plasmid together with FBXO31 RNAi plasmids or respective control vectors as indicated. Cell lysates were immunoblotted with α-myc and α-Flag antibodies. 14-3-3ß served as a loading control. G. Cerebellar granule neurons were transfected with mycPar6c plasmid together with FBXO31 RNAi #1 plasmid or respective control vector at DIV 2. Centrosomal purification was performed at DIV 6 using sucrose density gradient centrifugation. The fractions were probed with α-myc antibody. γ-tubulin served as a positive control for centrosomal protein. The histogram shows Par6c levels relative to γ-tubulin at the centrosome (N = 3, mean±SEM, unpaired t-test, *p<0.05). H. HEK 293T cells were co-transfected with mycPar6c and GFP-FBXO31 WT or ΔF plasmids together with respective control vectors. Cell lysates were subjected to immunoprecipitation with α-myc antibody and immunoblotted with α-ubiquitin antibody. I. HEK 293T cells were co-transfected with mycPar6c and GFP-FBXO31 WT plasmids together with respective control vectors. Cell lysates were subjected to immunoprecipitation with α-myc antibody and immunoblotted with K48-specific α-ubiquitin antibody. J. HEK 293T cells were co-transfected with mycPar6c and GFP-FBXO31 WT plasmids together with respective control vectors. Cell lysates were subjected to immunoprecipitation with anti-myc antibody and immunoblotted with K63-specific anti-ubiquitin antibody.
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Related In: Results  -  Collection

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pone-0057530-g003: FBXO31-SCF targets Par6c to proteasomal degradation. A.Transfected HEK 293T cells lysates were subjected to immunoprecipitation with α-Flag antibody and immunoblotted with α-myc antibody. B. Transfected HEK 293T cells lysates were subjected to immunoprecipitation with α-myc antibody and immunoblotted with α-Flag antibody. C. Schematic showing various Par6c deletion mutants and their interaction with FBXO31. D. Granule neurons were transfected with Par6c plasmid at DIV0 and treated with DMSO or lactacystin for 10 hours prior to lysis at DIV 3. Lysates were immunoblotted with α-myc antibody. 14-3-3ß served as a loading control. E. and F. HEK 293T cells (E) and granule neurons (F) were transfected with mycPar6c plasmid together with FBXO31 RNAi plasmids or respective control vectors as indicated. Cell lysates were immunoblotted with α-myc and α-Flag antibodies. 14-3-3ß served as a loading control. G. Cerebellar granule neurons were transfected with mycPar6c plasmid together with FBXO31 RNAi #1 plasmid or respective control vector at DIV 2. Centrosomal purification was performed at DIV 6 using sucrose density gradient centrifugation. The fractions were probed with α-myc antibody. γ-tubulin served as a positive control for centrosomal protein. The histogram shows Par6c levels relative to γ-tubulin at the centrosome (N = 3, mean±SEM, unpaired t-test, *p<0.05). H. HEK 293T cells were co-transfected with mycPar6c and GFP-FBXO31 WT or ΔF plasmids together with respective control vectors. Cell lysates were subjected to immunoprecipitation with α-myc antibody and immunoblotted with α-ubiquitin antibody. I. HEK 293T cells were co-transfected with mycPar6c and GFP-FBXO31 WT plasmids together with respective control vectors. Cell lysates were subjected to immunoprecipitation with α-myc antibody and immunoblotted with K48-specific α-ubiquitin antibody. J. HEK 293T cells were co-transfected with mycPar6c and GFP-FBXO31 WT plasmids together with respective control vectors. Cell lysates were subjected to immunoprecipitation with anti-myc antibody and immunoblotted with K63-specific anti-ubiquitin antibody.
Mentions: To identify targets of FBXO31-SCF and owing to FBXO31’s centrosomal localization, we took a candidate approach and found that FBXO31 interacts with Par6c, a previously identified centrosomal protein [23], [24]. Par6 is known to regulate epithelial cell polarity [25], [26] and neuronal polarity [27], [28]. In the nervous system, Par6c represents the predominantly enriched Par6 family member [29]. We determined the interaction of exogenous FBXO31 and Par6c in heterologous cells by immunoprecipitating FBXO31 followed by immunoblotting for Par6c (Figure 3A). In a reciprocal experiment, we immunoprecipitated Par6c and immunoblotted for FBXO31 (Figure 3B). Mapping analyses of FBXO31 and Par6c revealed that Par6c’s PDZ domain mediates the interaction with FBXO31 (Figure 3C, Figure S5A). These experiments establish the FBXO31-Par6c interaction.

Bottom Line: In addition, we identified the polarity protein Par6c as a novel interaction partner and substrate targeted for proteasomal degradation in the control of axon but not dendrite growth.Finally, we ascribe a role for FBXO31 in dendrite growth and neuronal migration in the developing cerebellar cortex.Taken together, we uncovered the centrosomal E3 ligase FBXO31-SCF as a novel regulator of neuronal development.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany.

ABSTRACT
Neuronal development requires proper migration, polarization and establishment of axons and dendrites. Growing evidence identifies the ubiquitin proteasome system (UPS) with its numerous components as an important regulator of various aspects of neuronal development. F-box proteins are interchangeable subunits of the Cullin-1 based E3 ubiquitin ligase, but only a few family members have been studied. Here, we report that the centrosomal E3 ligase FBXO31-SCF (Skp1/Cullin-1/F-box protein) regulates neuronal morphogenesis and axonal identity. In addition, we identified the polarity protein Par6c as a novel interaction partner and substrate targeted for proteasomal degradation in the control of axon but not dendrite growth. Finally, we ascribe a role for FBXO31 in dendrite growth and neuronal migration in the developing cerebellar cortex. Taken together, we uncovered the centrosomal E3 ligase FBXO31-SCF as a novel regulator of neuronal development.

Show MeSH
Related in: MedlinePlus