Cyclin-dependent kinase 5 supports neuronal survival through phosphorylation of Bcl-2

Abstract

Accumulating evidence indicates that deregulation of cyclin-dependent kinase 5 (Cdk5) activity is associated with apoptosis in various neurodegenerative disease models. Interestingly, recent studies suggest that Cdk5 may also favor neuronal survival. Nonetheless, whether Cdk5 is directly required for neuronal survival during development remains enigmatic. In the current study, we established the pivotal role of Cdk5 in neuronal survival during development by demonstrating that reduction or absence of Cdk5 activity markedly exacerbated neuronal death in cultures and in vivo. Interestingly, the antiapoptotic protein Bcl-2 (B-cell lymphoma protein 2) was identified as a novel substrate of Cdk5. We found that Cdk5-mediated phosphorylation of Bcl-2 at Ser70 was required for the neuroprotective effect of Bcl-2. Intriguingly, inhibition of this phosphorylation conferred proapoptotic property to Bcl-2. Furthermore, overexpression of a Bcl-2 mutant lacking the Cdk5 phosphorylation site abolished the protective effect of Cdk5 re-expression in Cdk5(-/-) neurons, suggesting that Ser70 phosphorylation of Bcl-2 contributed to Cdk5-mediated neuronal survival. Our observations revealed that Cdk5-mediated Bcl-2 phosphorylation is pivotal for the antiapoptotic effect of Bcl-2 and contributes to the maintenance of neuronal survival by Cdk5. Our study has also identified Cdk5 as a regulator of Bcl-2 function in neuronal apoptosis.

Figure 1.

Cdk5 was required for neuronal survival. A, Treatment with Cdk5-selective inhibitor Ros (25 μm) induced nuclear fragmentation and caspase-3 activation in pure RGC culture. B, Overexpression of dnCdk5 led to a marked increase in nuclear fragmentation in retinal neurons. C, Western blot analysis demonstrated that transfection of Cdk5 siRNA, but not scrambled siRNA (control), markedly reduced Cdk5 expression in retinal neurons. Transfection of Cdk5 siRNA increased the percentage of retinal neurons exhibiting nuclear fragmentation. D, Retinal neurons taken from Cdk5 ^−/− mice displayed a higher incidence of nuclear fragmentation, caspase-3 activation, and cytochrome c (Cyt c) release (arrows), as indicated by diffuse cytochrome c staining. E, Intravitreal injection of Ros significantly increased the incidence of nuclear fragmentation in the ganglion cell layer of adult rat retina. Scale bar, 20 μm. *p < 0.05.

Figure 2.

Bcl-2 interacted with Cdk5 in adult brain. A, Bcl-2 and p35 were concentrated in the mitochondrial fraction of cortical neurons, whereas Cdk5 was ubiquitously expressed. COX4 serves as a marker for the mitochondrial fraction, and α-tubulin serves as an equal loading control. B, p35 colocalized with both Bcl-2 and COX4 in developing cortical neurons. Scale bar, 10 μm. C, GST–Bcl-2, but not GST, pulled down p35 and to a lesser extent Cdk5 in HEK293T cell lysates overexpressing p35 or Cdk5. D, p35 (bottom), but not Cdk5 (top), associated with Bcl-2 when overexpressed in HEK293T cells. IgG was included as a control. E, Bcl-2 associated endogenously with Cdk5 and p35 in the membrane fraction of P7 rat brain. IP, Immunoprecipitation.

Figure 3.

Cdk5 phosphorylated Bcl-2 at Ser70. A, Potential Cdk5 phosphorylation sites are found at Thr55, Ser70, and Ser84 of the loop region of mouse Bcl-2. B, GST–Bcl-2 was phosphorylated by Cdk5/p35 in a dose-dependent manner in in vitro kinase assay. Histone H1 served as a control to demonstrate activity of the Cdk5/p35 complex. C, Wild-type (WT) Bcl-2, Bcl-2–S70A, Bcl-2–T55A, or Bcl-2–S84A were subjected to in vitro kinase assay. Mutation of Ser70, but not Thr55 or Ser84, to alanine abolished phosphorylation of Bcl-2 by Cdk5/p35. D, Bcl-2, but not Bcl-2–S70A, was serine phosphorylated (pSer–Bcl-2) in the presence of Cdk5/p35. The serine-phosphorylated band corresponded to an upward shift in mobility (pBcl-2) when the blot was reprobed with Bcl-2 antibody. E, Bcl-2 phosphorylation (p-Bcl-2) was markedly reduced in Cdk5 ^−/− brain. Activation of Bcl-2 kinases, such as Erk1/2, JNK, and p38 MAPK, was not reduced in Cdk5 ^−/− brain.

Figure 4.

Phosphorylation of Bcl-2 at Ser70 determined whether Bcl-2 exhibited proapoptotic or antiapoptotic function and was essential for the maintenance of neuronal survival by Cdk5. A, Overexpression of Bcl-2 markedly reduced nuclear fragmentation in P8 retinal neurons, but no protection was offered by Bcl-2–S70A. B, Incidence of nuclear fragmentation was markedly higher in Cdk5 ^−/− neurons. Overexpression of Bcl-2–S70A induced nuclear fragmentation in Cdk5 ^+/+ neurons, but overexpression of Bcl-2–S70A failed to further exacerbate neuronal death in Cdk5 ^−/− neurons. C, Overexpression of p35 attenuated Bcl-2 induced apoptosis in HEK293T cells. Overexpression of Bcl-2 and Bcl-2–S70A induced massive nuclear fragmentation. Overexpression of p35 attenuated cell death triggered by Bcl-2 but not Bcl-2–S70A. D, Nuclear fragmentation was markedly reduced by Cdk5 overexpression in Cdk5 ^−/− neurons. Coexpression of Bcl-2–S70A abolished the neuroprotective effect of Cdk5 and further enhanced nuclear fragmentation in Cdk5 ^−/− neurons. *p < 0.05.