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Cdk5 is involved in BDNF-stimulated dendritic growth in hippocampal neurons.

Cheung ZH, Chin WH, Chen Y, Ng YP, Ip NY - PLoS Biol. (2007)

Bottom Line: Interestingly, attenuation of Cdk5 activity or overexpression of a TrkB mutant lacking the Cdk5 phosphorylation site essentially abolishes brain-derived neurotrophic factor (BDNF)-triggered dendritic growth in primary hippocampal neurons.In addition, we found that Cdk5 is involved in BDNF-induced activation of Rho GTPase Cdc42, which is essential for BDNF-triggered dendritic growth.Our observations therefore reveal an unanticipated role of Cdk5 in TrkB-mediated regulation of dendritic growth through modulation of BDNF-induced Cdc42 activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Biotechnology Research Institute and Molecular Neuroscience Center, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China.

ABSTRACT
Neurotrophins are key regulators of neuronal survival and differentiation during development. Activation of their cognate receptors, Trk receptors, a family of receptor tyrosine kinases (RTKs), is pivotal for mediating the downstream functions of neurotrophins. Recent studies reveal that cyclin-dependent kinase 5 (Cdk5), a serine/threonine kinase, may modulate RTK signaling through phosphorylation of the receptor. Given the abundant expression of both Cdk5 and Trk receptors in the nervous system, and their mutual involvement in the regulation of neuronal architecture and synaptic functions, it is of interest to investigate if Cdk5 may also modulate Trk signaling. In the current study, we report the identification of TrkB as a Cdk5 substrate. Cdk5 phosphorylates TrkB at Ser478 at the intracellular juxtamembrane region of TrkB. Interestingly, attenuation of Cdk5 activity or overexpression of a TrkB mutant lacking the Cdk5 phosphorylation site essentially abolishes brain-derived neurotrophic factor (BDNF)-triggered dendritic growth in primary hippocampal neurons. In addition, we found that Cdk5 is involved in BDNF-induced activation of Rho GTPase Cdc42, which is essential for BDNF-triggered dendritic growth. Our observations therefore reveal an unanticipated role of Cdk5 in TrkB-mediated regulation of dendritic growth through modulation of BDNF-induced Cdc42 activation.

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Cdk5 Phosphorylated TrkB at Ser478(A) Lysates from COS7 cells overexpressing TrkA, TrkB, and TrkC were immunoprecipitated with pan-Trk antibody and incubated with Cdk5/p25 in an in vitro kinase assay. TrkB and TrkC, but not TrkA, were phosphorylated by Cdk5/p25.(B) GST-TrkB-juxtamembrane fusion protein was incubated with increasing amount of Cdk5/p35 and subjected to an in vitro kinase assay. Histone H1 served as control to verify the activity of the Cdk5 kinase. The TrkB-juxtamembrane region was phosphorylated by Cdk5/p35 in a dose-dependent manner.(C) Purified WT GST-TrkB-juxtamembrane fusion protein and mutants (M1, M2, and DM) were incubated with Cdk5/p25 in an in vitro kinase assay. While WT and M2 were strongly phosphorylated by Cdk5/p25, phosphorylation of M1 and DM were markedly attenuated. Quality of the purified GST and GST-fusion proteins used in the GST pull-down assay was verified by Coomassie blue staining.(D) Characterization of p-Ser TrkB antibody raised against phosphorylated Ser478 of TrkB. TrkB was overexpressed with or without p35/Cdk5 in HEK293T cells. Preincubation of purified p-Ser478 TrkB antibody with blocking peptide completely abolished detection of Ser478 phosphorylation of TrkB.(E) Full-length TrkB WT, M1, M2, and DM were overexpressed with or without Cdk5/p35 in HEK293T cells. In the absence of Cdk5/p35, Ser478-phosphorylated TrkB (p-Ser TrkB) was not detected. Overexpression of Cdk5/p35 resulted in phosphorylation of TrkB WT at Ser478, but phosphorylation at Ser478 was essentially abolished when TrkB M1 and DM were overexpressed. IP, immunoprecipitation.
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pbio-0050063-g002: Cdk5 Phosphorylated TrkB at Ser478(A) Lysates from COS7 cells overexpressing TrkA, TrkB, and TrkC were immunoprecipitated with pan-Trk antibody and incubated with Cdk5/p25 in an in vitro kinase assay. TrkB and TrkC, but not TrkA, were phosphorylated by Cdk5/p25.(B) GST-TrkB-juxtamembrane fusion protein was incubated with increasing amount of Cdk5/p35 and subjected to an in vitro kinase assay. Histone H1 served as control to verify the activity of the Cdk5 kinase. The TrkB-juxtamembrane region was phosphorylated by Cdk5/p35 in a dose-dependent manner.(C) Purified WT GST-TrkB-juxtamembrane fusion protein and mutants (M1, M2, and DM) were incubated with Cdk5/p25 in an in vitro kinase assay. While WT and M2 were strongly phosphorylated by Cdk5/p25, phosphorylation of M1 and DM were markedly attenuated. Quality of the purified GST and GST-fusion proteins used in the GST pull-down assay was verified by Coomassie blue staining.(D) Characterization of p-Ser TrkB antibody raised against phosphorylated Ser478 of TrkB. TrkB was overexpressed with or without p35/Cdk5 in HEK293T cells. Preincubation of purified p-Ser478 TrkB antibody with blocking peptide completely abolished detection of Ser478 phosphorylation of TrkB.(E) Full-length TrkB WT, M1, M2, and DM were overexpressed with or without Cdk5/p35 in HEK293T cells. In the absence of Cdk5/p35, Ser478-phosphorylated TrkB (p-Ser TrkB) was not detected. Overexpression of Cdk5/p35 resulted in phosphorylation of TrkB WT at Ser478, but phosphorylation at Ser478 was essentially abolished when TrkB M1 and DM were overexpressed. IP, immunoprecipitation.

Mentions: We next proceeded to examine if Trk receptors, TrkB in particular, served as Cdk5 substrates using in vitro kinase assay. TrkA, TrkB, and TrkC were overexpressed in COS7 cells and immunoprecipitated by pan-Trk antibody. Incubation with Cdk5/p25 revealed that TrkB and TrkC, but not TrkA, were phosphorylated by Cdk5/p25 in vitro (Figure 2A). This is in agreement with the lack of Cdk5 consensus sites in TrkA, and points to the possibility that Cdk5 may phosphorylate TrkB and TrkC at the Cdk5 consensus sites at the juxtamembrane region (Figure 1A). To examine this possibility, a GST fusion protein containing only the juxtamembrane region of TrkB was prepared. In vitro kinase assay verified that Cdk5/p35 phosphorylated TrkB at the juxtamembrane region (Figure 2B). It has previously been proposed that p25 and p35 may confer different substrate specificities. Results from our in vitro kinase assay suggested that Cdk5 phosphorylated TrkB regardless of whether it was activated by p25 or p35, although further studies will be required to delineate the relative contributions of p25 and p35 to endogenous phosphorylation of TrkB by Cdk5.


Cdk5 is involved in BDNF-stimulated dendritic growth in hippocampal neurons.

Cheung ZH, Chin WH, Chen Y, Ng YP, Ip NY - PLoS Biol. (2007)

Cdk5 Phosphorylated TrkB at Ser478(A) Lysates from COS7 cells overexpressing TrkA, TrkB, and TrkC were immunoprecipitated with pan-Trk antibody and incubated with Cdk5/p25 in an in vitro kinase assay. TrkB and TrkC, but not TrkA, were phosphorylated by Cdk5/p25.(B) GST-TrkB-juxtamembrane fusion protein was incubated with increasing amount of Cdk5/p35 and subjected to an in vitro kinase assay. Histone H1 served as control to verify the activity of the Cdk5 kinase. The TrkB-juxtamembrane region was phosphorylated by Cdk5/p35 in a dose-dependent manner.(C) Purified WT GST-TrkB-juxtamembrane fusion protein and mutants (M1, M2, and DM) were incubated with Cdk5/p25 in an in vitro kinase assay. While WT and M2 were strongly phosphorylated by Cdk5/p25, phosphorylation of M1 and DM were markedly attenuated. Quality of the purified GST and GST-fusion proteins used in the GST pull-down assay was verified by Coomassie blue staining.(D) Characterization of p-Ser TrkB antibody raised against phosphorylated Ser478 of TrkB. TrkB was overexpressed with or without p35/Cdk5 in HEK293T cells. Preincubation of purified p-Ser478 TrkB antibody with blocking peptide completely abolished detection of Ser478 phosphorylation of TrkB.(E) Full-length TrkB WT, M1, M2, and DM were overexpressed with or without Cdk5/p35 in HEK293T cells. In the absence of Cdk5/p35, Ser478-phosphorylated TrkB (p-Ser TrkB) was not detected. Overexpression of Cdk5/p35 resulted in phosphorylation of TrkB WT at Ser478, but phosphorylation at Ser478 was essentially abolished when TrkB M1 and DM were overexpressed. IP, immunoprecipitation.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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pbio-0050063-g002: Cdk5 Phosphorylated TrkB at Ser478(A) Lysates from COS7 cells overexpressing TrkA, TrkB, and TrkC were immunoprecipitated with pan-Trk antibody and incubated with Cdk5/p25 in an in vitro kinase assay. TrkB and TrkC, but not TrkA, were phosphorylated by Cdk5/p25.(B) GST-TrkB-juxtamembrane fusion protein was incubated with increasing amount of Cdk5/p35 and subjected to an in vitro kinase assay. Histone H1 served as control to verify the activity of the Cdk5 kinase. The TrkB-juxtamembrane region was phosphorylated by Cdk5/p35 in a dose-dependent manner.(C) Purified WT GST-TrkB-juxtamembrane fusion protein and mutants (M1, M2, and DM) were incubated with Cdk5/p25 in an in vitro kinase assay. While WT and M2 were strongly phosphorylated by Cdk5/p25, phosphorylation of M1 and DM were markedly attenuated. Quality of the purified GST and GST-fusion proteins used in the GST pull-down assay was verified by Coomassie blue staining.(D) Characterization of p-Ser TrkB antibody raised against phosphorylated Ser478 of TrkB. TrkB was overexpressed with or without p35/Cdk5 in HEK293T cells. Preincubation of purified p-Ser478 TrkB antibody with blocking peptide completely abolished detection of Ser478 phosphorylation of TrkB.(E) Full-length TrkB WT, M1, M2, and DM were overexpressed with or without Cdk5/p35 in HEK293T cells. In the absence of Cdk5/p35, Ser478-phosphorylated TrkB (p-Ser TrkB) was not detected. Overexpression of Cdk5/p35 resulted in phosphorylation of TrkB WT at Ser478, but phosphorylation at Ser478 was essentially abolished when TrkB M1 and DM were overexpressed. IP, immunoprecipitation.
Mentions: We next proceeded to examine if Trk receptors, TrkB in particular, served as Cdk5 substrates using in vitro kinase assay. TrkA, TrkB, and TrkC were overexpressed in COS7 cells and immunoprecipitated by pan-Trk antibody. Incubation with Cdk5/p25 revealed that TrkB and TrkC, but not TrkA, were phosphorylated by Cdk5/p25 in vitro (Figure 2A). This is in agreement with the lack of Cdk5 consensus sites in TrkA, and points to the possibility that Cdk5 may phosphorylate TrkB and TrkC at the Cdk5 consensus sites at the juxtamembrane region (Figure 1A). To examine this possibility, a GST fusion protein containing only the juxtamembrane region of TrkB was prepared. In vitro kinase assay verified that Cdk5/p35 phosphorylated TrkB at the juxtamembrane region (Figure 2B). It has previously been proposed that p25 and p35 may confer different substrate specificities. Results from our in vitro kinase assay suggested that Cdk5 phosphorylated TrkB regardless of whether it was activated by p25 or p35, although further studies will be required to delineate the relative contributions of p25 and p35 to endogenous phosphorylation of TrkB by Cdk5.

Bottom Line: Interestingly, attenuation of Cdk5 activity or overexpression of a TrkB mutant lacking the Cdk5 phosphorylation site essentially abolishes brain-derived neurotrophic factor (BDNF)-triggered dendritic growth in primary hippocampal neurons.In addition, we found that Cdk5 is involved in BDNF-induced activation of Rho GTPase Cdc42, which is essential for BDNF-triggered dendritic growth.Our observations therefore reveal an unanticipated role of Cdk5 in TrkB-mediated regulation of dendritic growth through modulation of BDNF-induced Cdc42 activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Biotechnology Research Institute and Molecular Neuroscience Center, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China.

ABSTRACT
Neurotrophins are key regulators of neuronal survival and differentiation during development. Activation of their cognate receptors, Trk receptors, a family of receptor tyrosine kinases (RTKs), is pivotal for mediating the downstream functions of neurotrophins. Recent studies reveal that cyclin-dependent kinase 5 (Cdk5), a serine/threonine kinase, may modulate RTK signaling through phosphorylation of the receptor. Given the abundant expression of both Cdk5 and Trk receptors in the nervous system, and their mutual involvement in the regulation of neuronal architecture and synaptic functions, it is of interest to investigate if Cdk5 may also modulate Trk signaling. In the current study, we report the identification of TrkB as a Cdk5 substrate. Cdk5 phosphorylates TrkB at Ser478 at the intracellular juxtamembrane region of TrkB. Interestingly, attenuation of Cdk5 activity or overexpression of a TrkB mutant lacking the Cdk5 phosphorylation site essentially abolishes brain-derived neurotrophic factor (BDNF)-triggered dendritic growth in primary hippocampal neurons. In addition, we found that Cdk5 is involved in BDNF-induced activation of Rho GTPase Cdc42, which is essential for BDNF-triggered dendritic growth. Our observations therefore reveal an unanticipated role of Cdk5 in TrkB-mediated regulation of dendritic growth through modulation of BDNF-induced Cdc42 activation.

Show MeSH
Related in: MedlinePlus