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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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BDNF Enhanced Cdk5 Activity(A) Cortical neurons were stimulated with BDNF for different time intervals. Lysates were immunoprecipitated (IP) with p35 antibody and subjected to in vitro kinase assay using histone H1 as substrate. BDNF stimulation for 15 min resulted in a marked increase in Cdk5 activity in cortical neurons. Quantification of the changes in phospho-Histone H1 level following BDNF stimulation was normalized to the value obtained from untreated cultures (time 0) and is shown in the histogram. *, p < 0.05.(B) Addition of Trk inhibitor K252a abolished BDNF-induced increase in Cdk5 activity. Cortical neurons were pretreated with vehicle control (DMSO) or K252a for 30 min before stimulation with BDNF for 15 min. Lysates were immunoprecipitated with p35 antibody and subjected to in vitro kinase assay using histone H1 as substrate. We found that K252a pretreatment markedly reduced the increase in Cdk5 activity triggered by BDNF stimulation, indicating that the induction of Cdk5 activity was dependent on TrkB activation. Quantification of the changes in phospho-Histone H1 level following BDNF stimulation in the presence or absence of K252a treatment was normalized to the value obtained from untreated cultures (time 0) and is shown in the histogram. *, p < 0.05.(C) Cortical neurons were treated with BDNF for 20 min. Lysates were immunoprecipitated with p35 antibody and immunoblotted with TrkB, p35, or Cdk5 antibody. While association between Cdk5 and p35 was not affected by BDNF stimulation, association between p35 and TrkB increased following 20 min of BDNF stimulation.(D) Recombinant TrkB was incubated with GST-Cdk5 in an in vitro kinase assay. TrkB was found to phosphorylate GST-Cdk5 (middle lane).(E) GST-Cdk5 and recombinant TrkB were pretreated with vehicle (DMSO) or K252a for 10 min, subjected to in vitro kinase assay, and immunoblotted with antibodies against phospho-tyrosine (p-Tyr) and the Tyr15 phosphorylated form of Cdk5 (pTyr15 Cdk5). Cdk5 was phosphorylated by TrkB at Tyr15. Addition of K252a abolished phosphorylation of Cdk5 by TrkB, further verifying that Cdk5 was phosphorylated by TrkB.
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pbio-0050063-g004: BDNF Enhanced Cdk5 Activity(A) Cortical neurons were stimulated with BDNF for different time intervals. Lysates were immunoprecipitated (IP) with p35 antibody and subjected to in vitro kinase assay using histone H1 as substrate. BDNF stimulation for 15 min resulted in a marked increase in Cdk5 activity in cortical neurons. Quantification of the changes in phospho-Histone H1 level following BDNF stimulation was normalized to the value obtained from untreated cultures (time 0) and is shown in the histogram. *, p < 0.05.(B) Addition of Trk inhibitor K252a abolished BDNF-induced increase in Cdk5 activity. Cortical neurons were pretreated with vehicle control (DMSO) or K252a for 30 min before stimulation with BDNF for 15 min. Lysates were immunoprecipitated with p35 antibody and subjected to in vitro kinase assay using histone H1 as substrate. We found that K252a pretreatment markedly reduced the increase in Cdk5 activity triggered by BDNF stimulation, indicating that the induction of Cdk5 activity was dependent on TrkB activation. Quantification of the changes in phospho-Histone H1 level following BDNF stimulation in the presence or absence of K252a treatment was normalized to the value obtained from untreated cultures (time 0) and is shown in the histogram. *, p < 0.05.(C) Cortical neurons were treated with BDNF for 20 min. Lysates were immunoprecipitated with p35 antibody and immunoblotted with TrkB, p35, or Cdk5 antibody. While association between Cdk5 and p35 was not affected by BDNF stimulation, association between p35 and TrkB increased following 20 min of BDNF stimulation.(D) Recombinant TrkB was incubated with GST-Cdk5 in an in vitro kinase assay. TrkB was found to phosphorylate GST-Cdk5 (middle lane).(E) GST-Cdk5 and recombinant TrkB were pretreated with vehicle (DMSO) or K252a for 10 min, subjected to in vitro kinase assay, and immunoblotted with antibodies against phospho-tyrosine (p-Tyr) and the Tyr15 phosphorylated form of Cdk5 (pTyr15 Cdk5). Cdk5 was phosphorylated by TrkB at Tyr15. Addition of K252a abolished phosphorylation of Cdk5 by TrkB, further verifying that Cdk5 was phosphorylated by TrkB.

Mentions: Since BDNF stimulation was observed to increase Ser478 phosphorylation of TrkB, and Cdk5 was required for phosphorylating TrkB at Ser478, we were interested to examine if BDNF stimulation affects Cdk5 activity. BDNF has previously been observed to increase Cdk5 activity after 3 d of BDNF stimulation in cortical neurons [11]. In agreement with this observation, we found that BDNF treatment led to an increase in Cdk5 activity within 15 min of BDNF stimulation (Figure 4A). More importantly, addition of Trk inhibitor K252a essentially abolished BDNF-triggered increase in Cdk5 activity, indicating that the increase in Cdk5 activity was dependent on TrkB activation (Figure 4B). It has previously been demonstrated that Cdk5 activity is enhanced by phosphorylation at Tyr15 [12]. Given the activation of tyrosine kinase activity of TrkB upon ligand stimulation, we were interested to investigate if BDNF treatment leads to phosphorylation of Cdk5 at Tyr15, thereby enhancing its activity. We found that BDNF stimulation enhanced association between Cdk5 and TrkB in cortical neurons (Figure 4C). More importantly, in vitro kinase assay using purified TrkB and Cdk5 revealed that TrkB phosphorylated Cdk5 at Tyr15 (Figure 4D and 4E). TrkB-mediated phosphorylation of Cdk5 was abolished with the addition of Trk inhibitor K252a, further verifying that Tyr15 phosphorylation of Cdk5 was TrkB dependent (Figure 4E). These observations collectively indicate that upon BDNF stimulation, Cdk5 was recruited to TrkB and phosphorylated by TrkB at Tyr15, thus leading to enhanced Cdk5 activity to promote phosphorylation of TrkB at Ser478.


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

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

BDNF Enhanced Cdk5 Activity(A) Cortical neurons were stimulated with BDNF for different time intervals. Lysates were immunoprecipitated (IP) with p35 antibody and subjected to in vitro kinase assay using histone H1 as substrate. BDNF stimulation for 15 min resulted in a marked increase in Cdk5 activity in cortical neurons. Quantification of the changes in phospho-Histone H1 level following BDNF stimulation was normalized to the value obtained from untreated cultures (time 0) and is shown in the histogram. *, p < 0.05.(B) Addition of Trk inhibitor K252a abolished BDNF-induced increase in Cdk5 activity. Cortical neurons were pretreated with vehicle control (DMSO) or K252a for 30 min before stimulation with BDNF for 15 min. Lysates were immunoprecipitated with p35 antibody and subjected to in vitro kinase assay using histone H1 as substrate. We found that K252a pretreatment markedly reduced the increase in Cdk5 activity triggered by BDNF stimulation, indicating that the induction of Cdk5 activity was dependent on TrkB activation. Quantification of the changes in phospho-Histone H1 level following BDNF stimulation in the presence or absence of K252a treatment was normalized to the value obtained from untreated cultures (time 0) and is shown in the histogram. *, p < 0.05.(C) Cortical neurons were treated with BDNF for 20 min. Lysates were immunoprecipitated with p35 antibody and immunoblotted with TrkB, p35, or Cdk5 antibody. While association between Cdk5 and p35 was not affected by BDNF stimulation, association between p35 and TrkB increased following 20 min of BDNF stimulation.(D) Recombinant TrkB was incubated with GST-Cdk5 in an in vitro kinase assay. TrkB was found to phosphorylate GST-Cdk5 (middle lane).(E) GST-Cdk5 and recombinant TrkB were pretreated with vehicle (DMSO) or K252a for 10 min, subjected to in vitro kinase assay, and immunoblotted with antibodies against phospho-tyrosine (p-Tyr) and the Tyr15 phosphorylated form of Cdk5 (pTyr15 Cdk5). Cdk5 was phosphorylated by TrkB at Tyr15. Addition of K252a abolished phosphorylation of Cdk5 by TrkB, further verifying that Cdk5 was phosphorylated by TrkB.
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pbio-0050063-g004: BDNF Enhanced Cdk5 Activity(A) Cortical neurons were stimulated with BDNF for different time intervals. Lysates were immunoprecipitated (IP) with p35 antibody and subjected to in vitro kinase assay using histone H1 as substrate. BDNF stimulation for 15 min resulted in a marked increase in Cdk5 activity in cortical neurons. Quantification of the changes in phospho-Histone H1 level following BDNF stimulation was normalized to the value obtained from untreated cultures (time 0) and is shown in the histogram. *, p < 0.05.(B) Addition of Trk inhibitor K252a abolished BDNF-induced increase in Cdk5 activity. Cortical neurons were pretreated with vehicle control (DMSO) or K252a for 30 min before stimulation with BDNF for 15 min. Lysates were immunoprecipitated with p35 antibody and subjected to in vitro kinase assay using histone H1 as substrate. We found that K252a pretreatment markedly reduced the increase in Cdk5 activity triggered by BDNF stimulation, indicating that the induction of Cdk5 activity was dependent on TrkB activation. Quantification of the changes in phospho-Histone H1 level following BDNF stimulation in the presence or absence of K252a treatment was normalized to the value obtained from untreated cultures (time 0) and is shown in the histogram. *, p < 0.05.(C) Cortical neurons were treated with BDNF for 20 min. Lysates were immunoprecipitated with p35 antibody and immunoblotted with TrkB, p35, or Cdk5 antibody. While association between Cdk5 and p35 was not affected by BDNF stimulation, association between p35 and TrkB increased following 20 min of BDNF stimulation.(D) Recombinant TrkB was incubated with GST-Cdk5 in an in vitro kinase assay. TrkB was found to phosphorylate GST-Cdk5 (middle lane).(E) GST-Cdk5 and recombinant TrkB were pretreated with vehicle (DMSO) or K252a for 10 min, subjected to in vitro kinase assay, and immunoblotted with antibodies against phospho-tyrosine (p-Tyr) and the Tyr15 phosphorylated form of Cdk5 (pTyr15 Cdk5). Cdk5 was phosphorylated by TrkB at Tyr15. Addition of K252a abolished phosphorylation of Cdk5 by TrkB, further verifying that Cdk5 was phosphorylated by TrkB.
Mentions: Since BDNF stimulation was observed to increase Ser478 phosphorylation of TrkB, and Cdk5 was required for phosphorylating TrkB at Ser478, we were interested to examine if BDNF stimulation affects Cdk5 activity. BDNF has previously been observed to increase Cdk5 activity after 3 d of BDNF stimulation in cortical neurons [11]. In agreement with this observation, we found that BDNF treatment led to an increase in Cdk5 activity within 15 min of BDNF stimulation (Figure 4A). More importantly, addition of Trk inhibitor K252a essentially abolished BDNF-triggered increase in Cdk5 activity, indicating that the increase in Cdk5 activity was dependent on TrkB activation (Figure 4B). It has previously been demonstrated that Cdk5 activity is enhanced by phosphorylation at Tyr15 [12]. Given the activation of tyrosine kinase activity of TrkB upon ligand stimulation, we were interested to investigate if BDNF treatment leads to phosphorylation of Cdk5 at Tyr15, thereby enhancing its activity. We found that BDNF stimulation enhanced association between Cdk5 and TrkB in cortical neurons (Figure 4C). More importantly, in vitro kinase assay using purified TrkB and Cdk5 revealed that TrkB phosphorylated Cdk5 at Tyr15 (Figure 4D and 4E). TrkB-mediated phosphorylation of Cdk5 was abolished with the addition of Trk inhibitor K252a, further verifying that Tyr15 phosphorylation of Cdk5 was TrkB dependent (Figure 4E). These observations collectively indicate that upon BDNF stimulation, Cdk5 was recruited to TrkB and phosphorylated by TrkB at Tyr15, thus leading to enhanced Cdk5 activity to promote phosphorylation of TrkB at Ser478.

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