Limits...
Cdk5 phosphorylation of ErbB4 is required for tangential migration of cortical interneurons.

Rakić S, Kanatani S, Hunt D, Faux C, Cariboni A, Chiara F, Khan S, Wansbury O, Howard B, Nakajima K, Nikolić M, Parnavelas JG - Cereb. Cortex (2013)

Bottom Line: Interneuron dysfunction in humans is often associated with neurological and psychiatric disorders, such as epilepsy, schizophrenia, and autism.Some of these disorders are believed to emerge during brain formation, at the time of interneuron specification, migration, and synapse formation.This finding identifies Cdk5 as a crucial signaling factor in cortical interneuron development in mammals.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University College London, London WC1 6BT, UK.

Show MeSH

Related in: MedlinePlus

p35/Cdk5 phosphorylation of ErbB4 in vitro and in vivo. (A) Immunoprecipitation (IP) of ErbB4myc from COS7 cell protein lysates; COS7 cells were co-transfected with ErbB4myc, p35, and Cdk5. Immunoblots of ErbB4 IP protein extracts, revealing p35/Cdk5 phosphorylation of ErbB4 on threonine adjacent to proline (pTP; first and second lanes vs. third lane). Immunoblots of whole-cell protein lysates indicate expression of ErbB4, p35, and Cdk5. βAct serves as a loading control. (B) Schematic, illustrating GST-fusion ErbB4 fragments used in a kinase assay. (C) Kinase assay, showing phosphorylation of ErbB4 on T1152 by p35/Cdk5. GST and histone 1 serve as negative and positive controls, respectively. (D) IP of ErbB4. Immunoblots of ErbB4 IP protein extracts and whole-cell protein lysates from the Cx of E15.5 mice, revealing decreased phosphorylation of ErbB4T1152, ErbB4Y1056, and AktS473 in the Cdk5 KOs compared with littermate controls. ErbB4, Akt, and βAct serve as loading controls. (E, F) Schematic diagrams of ErbB4, showing Cdk5-targeted (T1152; E) and PI3-kinase-binding (Y1056; F) phosphorylation sites recognized by pErbB4T1152 and pErbB4Y1056 antibodies, respectively. (G–R) Forebrain sections of a GAD67GFP mouse embryo, at the indicated time points, immunostained for pErbB4T1152 and pErbB4Y1056 (red). GFPGAD67(+) cells (green) represent GABAergic interneurons. (G–J) Dotted lines indicate the border between the pallium (p) and the subpallium (s). (K–P) show pErbB4T1152 and pErbB4Y1056 in a subset of cortical interneurons (arrows). Higher magnifications are shown in the insets. (Q) and (R) show that pErbB4T1152 and pErbB4Y1056 co-localize with β-III-tubulin (blue) in the proximal leading process (arrows) of cortical interneurons. (S,T) Cdk5-phosphorylation and PI3K-binding sites in ErbB4 are well conserved across species. Bars, 200 μm (F–I), 50 μm (J–O), 20 μm (P,Q). A, alanine; GST, glutathione S-transferase; SVZ, subventricular zone; Y, tyrosine.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4380000&req=5

BHT290F4: p35/Cdk5 phosphorylation of ErbB4 in vitro and in vivo. (A) Immunoprecipitation (IP) of ErbB4myc from COS7 cell protein lysates; COS7 cells were co-transfected with ErbB4myc, p35, and Cdk5. Immunoblots of ErbB4 IP protein extracts, revealing p35/Cdk5 phosphorylation of ErbB4 on threonine adjacent to proline (pTP; first and second lanes vs. third lane). Immunoblots of whole-cell protein lysates indicate expression of ErbB4, p35, and Cdk5. βAct serves as a loading control. (B) Schematic, illustrating GST-fusion ErbB4 fragments used in a kinase assay. (C) Kinase assay, showing phosphorylation of ErbB4 on T1152 by p35/Cdk5. GST and histone 1 serve as negative and positive controls, respectively. (D) IP of ErbB4. Immunoblots of ErbB4 IP protein extracts and whole-cell protein lysates from the Cx of E15.5 mice, revealing decreased phosphorylation of ErbB4T1152, ErbB4Y1056, and AktS473 in the Cdk5 KOs compared with littermate controls. ErbB4, Akt, and βAct serve as loading controls. (E, F) Schematic diagrams of ErbB4, showing Cdk5-targeted (T1152; E) and PI3-kinase-binding (Y1056; F) phosphorylation sites recognized by pErbB4T1152 and pErbB4Y1056 antibodies, respectively. (G–R) Forebrain sections of a GAD67GFP mouse embryo, at the indicated time points, immunostained for pErbB4T1152 and pErbB4Y1056 (red). GFPGAD67(+) cells (green) represent GABAergic interneurons. (G–J) Dotted lines indicate the border between the pallium (p) and the subpallium (s). (K–P) show pErbB4T1152 and pErbB4Y1056 in a subset of cortical interneurons (arrows). Higher magnifications are shown in the insets. (Q) and (R) show that pErbB4T1152 and pErbB4Y1056 co-localize with β-III-tubulin (blue) in the proximal leading process (arrows) of cortical interneurons. (S,T) Cdk5-phosphorylation and PI3K-binding sites in ErbB4 are well conserved across species. Bars, 200 μm (F–I), 50 μm (J–O), 20 μm (P,Q). A, alanine; GST, glutathione S-transferase; SVZ, subventricular zone; Y, tyrosine.

Mentions: A number of studies have suggested a strong correlation between Cdk5 activity and the ErbB receptor pathway (Fu et al. 2001, 2005; Li et al. 2003; Xie et al. 2004, 2007). To determine whether p35/Cdk5 phosphorylates ErbB4 in vitro, we carried out experiments using COS7 cells that naturally express Cdk5, but not p35 or ErbB4. Cdk5 phosphorylates proteins containing a [S/T]Px[K/H/R] site (Fig. 3A). We focused on threonine 1152 (T1152PmH/R) that lies in close proximity to Cyt1-specific PI3-kinase-binding site Y1056. To initially test whether ErbB4 is a substrate of Cdk5, we used a phosphospecific ab that recognizes a minimal Cdk consensus site, phosphorylated threonine adjacent to a proline (T(PO4)P), and discovered a stronger ErbB4 phosphorylation in COS7 cells that co-expressed p35/Cdk5 compared with other experimental conditions (Fig. 4A). To further confirm that ErbB4 is indeed phosphorylated by Cdk5, we performed an in vitro p35/Cdk5 kinase assay using recombinant GST-fused ErbB4 fragments containing either the native T1152 residue (GST-T) or an alanine point mutation (T1152A), which was obtained by site-directed mutagenesis and cannot be phosphorylated [GST-T(A); Fig. 4B]. GST and histone 1, an established substrate of Cdk5, were used as negative and positive controls, respectively. This assay revealed a robust phosphorylation by p35/Cdk5 of intact (GST-T), but not mutated [GST-T(A)] ErbB4 fragments (Fig. 4C), identifying ErbB4 as a Cdk5 substrate in vitro.Figure 4.


Cdk5 phosphorylation of ErbB4 is required for tangential migration of cortical interneurons.

Rakić S, Kanatani S, Hunt D, Faux C, Cariboni A, Chiara F, Khan S, Wansbury O, Howard B, Nakajima K, Nikolić M, Parnavelas JG - Cereb. Cortex (2013)

p35/Cdk5 phosphorylation of ErbB4 in vitro and in vivo. (A) Immunoprecipitation (IP) of ErbB4myc from COS7 cell protein lysates; COS7 cells were co-transfected with ErbB4myc, p35, and Cdk5. Immunoblots of ErbB4 IP protein extracts, revealing p35/Cdk5 phosphorylation of ErbB4 on threonine adjacent to proline (pTP; first and second lanes vs. third lane). Immunoblots of whole-cell protein lysates indicate expression of ErbB4, p35, and Cdk5. βAct serves as a loading control. (B) Schematic, illustrating GST-fusion ErbB4 fragments used in a kinase assay. (C) Kinase assay, showing phosphorylation of ErbB4 on T1152 by p35/Cdk5. GST and histone 1 serve as negative and positive controls, respectively. (D) IP of ErbB4. Immunoblots of ErbB4 IP protein extracts and whole-cell protein lysates from the Cx of E15.5 mice, revealing decreased phosphorylation of ErbB4T1152, ErbB4Y1056, and AktS473 in the Cdk5 KOs compared with littermate controls. ErbB4, Akt, and βAct serve as loading controls. (E, F) Schematic diagrams of ErbB4, showing Cdk5-targeted (T1152; E) and PI3-kinase-binding (Y1056; F) phosphorylation sites recognized by pErbB4T1152 and pErbB4Y1056 antibodies, respectively. (G–R) Forebrain sections of a GAD67GFP mouse embryo, at the indicated time points, immunostained for pErbB4T1152 and pErbB4Y1056 (red). GFPGAD67(+) cells (green) represent GABAergic interneurons. (G–J) Dotted lines indicate the border between the pallium (p) and the subpallium (s). (K–P) show pErbB4T1152 and pErbB4Y1056 in a subset of cortical interneurons (arrows). Higher magnifications are shown in the insets. (Q) and (R) show that pErbB4T1152 and pErbB4Y1056 co-localize with β-III-tubulin (blue) in the proximal leading process (arrows) of cortical interneurons. (S,T) Cdk5-phosphorylation and PI3K-binding sites in ErbB4 are well conserved across species. Bars, 200 μm (F–I), 50 μm (J–O), 20 μm (P,Q). A, alanine; GST, glutathione S-transferase; SVZ, subventricular zone; Y, tyrosine.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4380000&req=5

BHT290F4: p35/Cdk5 phosphorylation of ErbB4 in vitro and in vivo. (A) Immunoprecipitation (IP) of ErbB4myc from COS7 cell protein lysates; COS7 cells were co-transfected with ErbB4myc, p35, and Cdk5. Immunoblots of ErbB4 IP protein extracts, revealing p35/Cdk5 phosphorylation of ErbB4 on threonine adjacent to proline (pTP; first and second lanes vs. third lane). Immunoblots of whole-cell protein lysates indicate expression of ErbB4, p35, and Cdk5. βAct serves as a loading control. (B) Schematic, illustrating GST-fusion ErbB4 fragments used in a kinase assay. (C) Kinase assay, showing phosphorylation of ErbB4 on T1152 by p35/Cdk5. GST and histone 1 serve as negative and positive controls, respectively. (D) IP of ErbB4. Immunoblots of ErbB4 IP protein extracts and whole-cell protein lysates from the Cx of E15.5 mice, revealing decreased phosphorylation of ErbB4T1152, ErbB4Y1056, and AktS473 in the Cdk5 KOs compared with littermate controls. ErbB4, Akt, and βAct serve as loading controls. (E, F) Schematic diagrams of ErbB4, showing Cdk5-targeted (T1152; E) and PI3-kinase-binding (Y1056; F) phosphorylation sites recognized by pErbB4T1152 and pErbB4Y1056 antibodies, respectively. (G–R) Forebrain sections of a GAD67GFP mouse embryo, at the indicated time points, immunostained for pErbB4T1152 and pErbB4Y1056 (red). GFPGAD67(+) cells (green) represent GABAergic interneurons. (G–J) Dotted lines indicate the border between the pallium (p) and the subpallium (s). (K–P) show pErbB4T1152 and pErbB4Y1056 in a subset of cortical interneurons (arrows). Higher magnifications are shown in the insets. (Q) and (R) show that pErbB4T1152 and pErbB4Y1056 co-localize with β-III-tubulin (blue) in the proximal leading process (arrows) of cortical interneurons. (S,T) Cdk5-phosphorylation and PI3K-binding sites in ErbB4 are well conserved across species. Bars, 200 μm (F–I), 50 μm (J–O), 20 μm (P,Q). A, alanine; GST, glutathione S-transferase; SVZ, subventricular zone; Y, tyrosine.
Mentions: A number of studies have suggested a strong correlation between Cdk5 activity and the ErbB receptor pathway (Fu et al. 2001, 2005; Li et al. 2003; Xie et al. 2004, 2007). To determine whether p35/Cdk5 phosphorylates ErbB4 in vitro, we carried out experiments using COS7 cells that naturally express Cdk5, but not p35 or ErbB4. Cdk5 phosphorylates proteins containing a [S/T]Px[K/H/R] site (Fig. 3A). We focused on threonine 1152 (T1152PmH/R) that lies in close proximity to Cyt1-specific PI3-kinase-binding site Y1056. To initially test whether ErbB4 is a substrate of Cdk5, we used a phosphospecific ab that recognizes a minimal Cdk consensus site, phosphorylated threonine adjacent to a proline (T(PO4)P), and discovered a stronger ErbB4 phosphorylation in COS7 cells that co-expressed p35/Cdk5 compared with other experimental conditions (Fig. 4A). To further confirm that ErbB4 is indeed phosphorylated by Cdk5, we performed an in vitro p35/Cdk5 kinase assay using recombinant GST-fused ErbB4 fragments containing either the native T1152 residue (GST-T) or an alanine point mutation (T1152A), which was obtained by site-directed mutagenesis and cannot be phosphorylated [GST-T(A); Fig. 4B]. GST and histone 1, an established substrate of Cdk5, were used as negative and positive controls, respectively. This assay revealed a robust phosphorylation by p35/Cdk5 of intact (GST-T), but not mutated [GST-T(A)] ErbB4 fragments (Fig. 4C), identifying ErbB4 as a Cdk5 substrate in vitro.Figure 4.

Bottom Line: Interneuron dysfunction in humans is often associated with neurological and psychiatric disorders, such as epilepsy, schizophrenia, and autism.Some of these disorders are believed to emerge during brain formation, at the time of interneuron specification, migration, and synapse formation.This finding identifies Cdk5 as a crucial signaling factor in cortical interneuron development in mammals.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University College London, London WC1 6BT, UK.

Show MeSH
Related in: MedlinePlus