Both cyclin I and p35 are required for maximal survival benefit of cyclin-dependent kinase 5 in kidney podocytes

Cyclin-dependent kinase (Cdk)-5 is activated by both cyclin I and the noncyclin activator p35 in terminally differentiated cells such as kidney podocytes and neurons. Cyclin I and p35 are restricted to podocytes in the kidney, and each limit podocyte apoptosis by activating Cdk5. To determine whether both activators are necessary, or whether they serve backup roles, a double cyclin I-p35 null mouse was generated. Experimental glomerular disease characterized by podocyte apoptosis was then induced by administering an anti-podocyte antibody. The results showed that under nonstressed conditions double mutants had normal kidney structure and function and were indistinguishable from wild-type, cyclin I(-/-), or p35(-/-) mice. In contrast, when stressed with disease, podocyte apoptosis increased fourfold compared with diseased cyclin I(-/-) or p35(-/-) mice. This resulted in a more pronounced decrease in podocyte number, proteinuria, and glomerulosclerosis. Under normal states and nephritic states, levels for the prosurvival protein Bcl-2 were lower in double cyclin I(-/-) p35(-/-) mice than the other mice. Similarly, levels of Bcl-xL, another prosurvival member, were lower in normal and nephritic double cyclin I(-/-) p35(-/-) mice but similar to single-cyclin I(-/-) mice. Moreover, levels of ERK1/2 and MEK1/2 activation were lower in nephritic double cyclin I(-/-) p35(-/-) mice but similar to single-cyclin I(-/-) mice. The results demonstrate that the activators of Cdk5, p35, and cyclin I are not required for normal kidney function. However, they play pivotal coordinated roles in maintaining podocyte survival during stress states in disease.     

Cyclin I protects podocytes from apoptosis

The limited regenerative capacity of the glomerular podocyte following injury underlies the development of glomerulosclerosis and progressive renal failure in a diverse range of kidney diseases. We discovered that, in the kidney, cyclin I is uniquely expressed in the glomerular podocyte, and have constructed cyclin I knock-out mice to explore the biological function of cyclin I in these cells. Cyclin I knock-out (-/-) podocytes showed an increased susceptibility to apoptosis both in vitro and in vivo. Following induction of experimental glomerulonephritis, podocyte apoptosis was increased 4-fold in the cyclin I -/- mice, which was associated with dramatically decreased renal function. Our previous data showed that the Cdk inhibitor p21(Cip1/Waf1) protects podocytes from certain apoptotic stimuli. In cultured cyclin I -/- podocytes, the level of p21(Cip1/Waf1) was lower at base line, had a shorter half-life, and declined more rapidly in response to apoptotic stimuli than in wild-type cells. Enforced expression of p21(Cip1/Waf1) reversed the susceptibility of cyclin I -/- podocytes to apoptosis. Cyclin I protects podocytes from apoptosis, and we provide preliminary data to suggest that this is mediated by stabilization of p21(Cip1/Waf1).     

High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway

Podocytes are highly specialized and terminally differentiated glomerular cells that play a vital role in the development and progression of diabetic nephropathy (DN). Cyclin-dependent kinase 5 (Cdk5), who is an atypical but essential member of the Cdk family of proline-directed serine/threonine kinases, has been shown as a key regulator of podocyte differentiation, proliferation and morphology. Our previous studies demonstrated that the expression of Cdk5 was significantly increased in podocytes of diabetic rats, and was closely related with podocyte injury of DN. However, the mechanisms of how expression and activity of Cdk5 are regulated under the high glucose environment have not yet been fully elucidated. In this study, we showed that high glucose up-regulated the expression of Cdk5 and its co-activator p35 with a concomitant increase in Cdk5 kinase activity in conditionally immortalized mouse podocytes in vitro. When exposed to 30 mM glucose, transforming growth factor-β1 (TGF-β1) was activated. Most importantly, we found that SB431542, the Tgfbr1 inhibitor, significantly decreased the expression of Cdk5 and p35 and Cdk5 kinase activity in high glucose-treated podocytes. Moreover, high glucose increased the expression of early growth response-1 (Egr-1) via TGF-β1-ERK1/2 pathway in podocytes and inhibition of Egr-1 by siRNA decreased p35 expression and Cdk5 kinase activity. Furthermore, inhibition of Cdk5 kinase activity effectively alleviated podocyte apoptosis induced by high glucose or TGF-β1. Thus, the TGF-β1-ERK1/2-Egr-1 signaling pathway may regulate the p35 expression and Cdk5 kinase activity in high glucose-treated podocytes, which contributes to podocyte injury of DN.     

Nestin protects mouse podocytes against high glucose-induced apoptosis by a Cdk5-dependent mechanism

Podocyte apoptosis contributes to the pathogenesis of diabetic nephropathy (DN). However, the mechanisms that mediate hyperglycemia‐induced podocyte apoptosis remain poorly understood. Recent findings indicate that the disruption of the cytoskeleton is related to the podocyte apoptosis. In the present study, we investigated the involvement of nestin, an important cytoskeleton‐associated class VI intermediate filament (IF) protein, in the high glucose (HG)‐induced podocyte apoptosis. Our data showed that HG decreased the expression level of nestin, either mRNA or protein, in a time‐dependent manner in cultured podocytes. Also, through knockdown of nestin expression by miRNA interference, the HG‐induced podocyte apoptotic rate was significantly increased. The expression of cleaved caspase‐3 was also markedly elevated. Considering that nestin is a substrate of cyclin‐dependent kinase 5 (Cdk5), we further assessed the expression of Cdk5 in HG‐treated podocytes. The results showed that HG stimulation increased the protein and mRNA expression of Cdk5 in a time‐dependent manner in cultured mouse podocytes. The protein activator of Cdk5, p35, was also increased in a time‐dependent manner by HG stimulation, and downregulation of Cdk5 by miRNA interference attenuated the nestin reduction in HG‐treated podocytes; the HG‐induced podocyte apoptosis, the increased cleaved caspase‐3 expression and the Bax/Bcl‐2 ratio were all effectively attenuated. These data suggested that nestin, which is dependent on Cdk5 regulation, plays a cytoprotective role in HG‐induced podocyte apoptosis. J. Cell. Biochem. 113: 3186–3196, 2012. © 2012 Wiley Periodicals, Inc.     

DNA binding protein dbpA binds Cdk5 and inhibits its activity

Progress in the cell cycle is governed by the activity of cyclin dependent kinases (Cdks). Unlike other Cdks, the Cdk5 catalytic subunit is found mostly in differentiated neurons. Interestingly, the only known protein that activates Cdk5 (i.e. p35) is expressed solely in the brain. It has been suggested that, besides its requirement in neuronal differentiation, Cdk5 activity is induced during myogenesis. However, it is not clear how this activity is regulated in the pathway that leads proliferative cells to differentiation. In order to find if there exists any Cdk5-interacting protein, the yeast two-hybrid system was used to screen a HeLa cDNA library. We have determined that a C-terminal 172 amino acid domain of the DNA binding protein, dbpA, binds to Cdk5. Biochemical analyses reveal that this fragment (dbpA(Cdelta)) strongly inhibits p35-activated Cdk5 kinase. The protein also interacts with Cdk4 and inhibits the Cdk4/cyclin D1 enzyme. Surprisingly, dbpA(Cdelta) does not bind Cdk2 in the two-hybrid assay nor does it inhibit Cdk2 activated by cyclin A. It could be that dbpA's ability to inhibit Cdk5 and Cdk4 reflects an apparent cross-talk between distinct signal transduction pathways controlled by dbpA on the one hand and Cdk5 or Cdk4 on the other.     

Proteolytic cleavage of cyclin E leads to inactivation of associated kinase activity and amplification of apoptosis in hematopoietic cells

Cyclin E/Cdk2 is a critical regulator of cell cycle progression from G(1) to S in mammalian cells and has an established role in oncogenesis. Here we examined the role of deregulated cyclin E expression in apoptosis. The levels of p50-cyclin E initially increased, and this was followed by a decrease starting at 8 h after treatment with genotoxic stress agents, such as ionizing radiation. This pattern was mirrored by the cyclin E-Cdk2-associated kinase activity and a time-dependent expression of a novel p18-cyclin E. p18-cyclin E was induced during apoptosis triggered by multiple genotoxic stress agents in all hematopoietic tumor cell lines we have examined. The p18-cyclin E expression was prevented by Bcl-2 overexpression and by the general caspase and specific caspase 3 pharmacologic inhibitors zVAD-fluoromethyl ketone (zVAD-fmk) and N-acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO), indicating that it was linked to apoptosis. A p18-cyclin E(276-395) (where cyclin E(276-395) is the cyclin E fragment containing residues 276 to 395) was reconstituted in vitro, with mutagenesis experiments, indicating that the caspase-dependent cleavage was at amino acid residues 272 to 275. Immunoprecipitation analyses of the ectopically expressed cyclin E(1-275), cyclin E(276-395) deletion mutants, and native p50-cyclin E demonstrated that caspase-mediated cyclin E cleavage eliminated interaction with Cdk2 and therefore inactivated the associated kinase activity. Overexpression of cyclin E(276-395), but not of several other cyclin E mutants, specifically induced phosphatidylserine exposure and caspase activation in a dose-dependent manner, which were inhibited in Bcl-2-overexpressing cells or in the presence of zVAD-fmk. Apoptosis and generation of p18-cyclin E were significantly inhibited by overexpressing the cleavage-resistant cyclin E mutant, indicating a functional role for caspase-dependent proteolysis of cyclin E for apoptosis of hematopoietic tumor cells.     

A Jekyll and Hyde Role of Cyclin E in the Genotoxic Stress Response: Switching from Cell Cycle Control to Apoptosis Regulation

Cyclin E protein levels and associated kinase activity rise in late G1 phase, reach a peak at the G1/S transition, and quickly decline during S phase. The Cyclin E /Cdk2 complex has a well-established function in regulating two fundamental biological processes: cell cycle progression and DNA replication. However, Cyclin E expression is deregulated in a wide range of tumors. Our recent reports have uncovered a critical role for Cyclin E, independent of Cdk2, in the cell death of hematopoietic tumor cells exposed to genotoxic stress. An 18-kD C-terminal fragment of Cyclin E, p18-Cyclin E, which is generated by caspase-mediated cleavage in hematopoietic cells during genotoxic stress-induced apoptosis has a critical role in the amplification of the intrinsic apoptotic pathway. By interacting with Ku70, p18-Cyclin E liberates Bax, which participates in the amplification of apoptosis by sustaining a positive feedback loop targeting mitochondria. This process is independent of p53 function and new RNA or protein synthesis. Therefore, Cyclin E emerges as an arbiter of the genotoxic stress response by regulating a finite physiological balance between cell proliferation and death in hematopoietic cells.     

Cyclin-dependent kinase-5 prevents neuronal apoptosis through ERK-mediated upregulation of Bcl-2

Cyclin-dependent kinase-5 (Cdk5) is required for neuronal survival, but its targets in the apoptotic pathways remain unknown. Here, we show that Cdk5 kinase activity prevents neuronal apoptosis through the upregulation of Bcl-2. Treatment of SH-SY5Y cells with retinoid acid (RA) and brain-derived neurotrophic factor (BDNF) generates differentiated neuron-like cells. DNA damage triggers apoptosis in the undifferentiated cells through mitochondrial pathway; however, RA/BDNF treatment results in Bcl-2 upregulation and inhibition of the mitochondrial pathway in the differentiated cells. RA/BDNF treatment activates Cdk5-mediated PI3K/Akt and ERK pathways. Inhibition of Cdk5 inhibits PI3K/Akt and ERK phosphorylation and Bcl-2 expression, and thus sensitizes the differentiated cells to DNA-damage. Inhibition of ERK, but not PI3K/Akt, abrogates Cdk5-medidated Bcl-2 upregulation and the protection of the differentiated cells. This study suggests that ERK-mediated Bcl-2 upregulation contributes to BDNF-induced Cdk5-mediated neuronal survival.     

Furano-1,2-naphthoquinone inhibits EGFR signaling associated with G2/M cell cycle arrest and apoptosis in A549 cells

Furano-1,2-naphthoquinone (FNQ), prepared from 2-hydroxy-1,4-naphthoquinone and chloroacetaldehyde in an efficient one-pot reaction, exhibits an anti-carcinogenic effect. FNQ exerted anti-proliferative activity with the G(2)/M cell cycle arrest and apoptosis in A549 cells. FNQ-induced G(2)/M arrest was correlated with a marked decrease in the expression levels of cyclin A and cyclin B, and their activating partner cyclin-dependent kinases (Cdk) 1 and 2 with concomitant induction of p53, p21, and p27. FNQ-induced apoptosis was accompanied with Bax up-regulation and the down-regulation of Bcl-2, X-linked inhibitor of apoptosis (XIAP), and survivin, resulting in cytochrome c release and sequential activation of caspase-9 and caspase-3. Western blot analysis revealed that FNQ suppressed EGFR phosphorylation and JAK2, STAT3, and STAT5 activation, but increased in activation of p38 MAPK and c-Jun NH2-terminal kinase (JNK) stress signal. The combined treatment of FNQ with AG1478 (a specific EGFR inhibitor) significantly enhanced the G(2)/M arrest and apoptosis, and also led to up-regulation in Bax, p53, p21, p27, release of mitochondrial cytochrome c, and down-regulation of Bcl-2, XIAP, survivin, cyclin A, cyclin B, Cdk1, and Cdk2 in A549 cells. These findings suggest that FNQ-mediated cytotoxicity of A549 cell related with the G(2)/M cell cycle arrest and apoptosis via inactivation of EGFR-mediated signaling pathway.     

Cytokine-driven cell cycling is mediated through Cdc25A

Lymphocytes are the central mediators of the immune response, requiring cytokines for survival and proliferation. Survival signaling targets the Bcl-2 family of apoptotic mediators, however, the pathway for the cytokine-driven proliferation of lymphocytes is poorly understood. Here we show that cytokine-induced cell cycle progression is not solely dependent on the synthesis of cyclin-dependent kinases (Cdks) or cyclins. Rather, we observe that in lymphocyte cell lines dependent on interleukin-3 or interleukin-7, or primary lymphocytes dependent on interleukin 7, the phosphatase Cdc25A is the critical mediator of proliferation. Withdrawal of IL-7 or IL-3 from dependent lymphocytes activates the stress kinase, p38 MAPK, which phosphorylates Cdc25A, inducing its degradation. As a result, Cdk/cyclin complexes remain phosphorylated and inactive and cells arrest before the induction of apoptosis. Inhibiting p38 MAPK or expressing a mutant Cdc25A, in which the two p38 MAPK target sites, S75 and S123, are altered, renders cells resistant to cytokine withdrawal, restoring the activity of Cdk/cyclin complexes and driving the cell cycle independent of a growth stimulus.     

Construction of a cyclin D1-Cdk2 fusion protein to model the biological functions of cyclin D1-Cdk2 complexes

Cyclin D1 is frequently overexpressed in human breast cancers, and cyclin D1 overexpression correlates with poor prognosis. Cyclin D1-Cdk2 complexes were previously observed in human breast cancer cell lines, but their role in cell cycle regulation and transformation was not investigated. This report demonstrates that Cdk2 in cyclin D1-Cdk2 complexes from mammary epithelial cells is phosphorylated on the activating phosphorylation site, Thr(160). Furthermore, cyclin D1-Cdk2 complexes catalyze Rb phosphorylation on multiple sites in vitro. As a model to investigate the biological and biochemical functions of cyclin D1-Cdk2 complexes, and the mechanisms by which cyclin D1 activates Cdk2, a cyclin D1-Cdk2 fusion gene was constructed. The cyclin D1-Cdk2 fusion protein expressed in epithelial cells was phosphorylated on Thr(160) and catalyzed the phosphorylation of Rb on multiple sites in vitro and in vivo. Kinase activity was not observed if either the cyclin D1 or Cdk2 domain was mutationally inactivated. Mutational inactivation of the cyclin D1 domain prevented activating phosphorylation of the Cdk2 domain on Thr(160). These results indicate that the cyclin D1 domain of the fusion protein activated the Cdk2 domain through an intramolecular mechanism. Cells stably expressing the cyclin D1-Cdk2 fusion protein exhibited several hallmarks of transformation including hyperphosphorylation of Rb, resistance to TGFbeta-induced growth arrest, and anchorage-independent proliferation in soft agar. We propose that cyclin D1-Cdk2 complexes mediate some of the transforming effects of cyclin D1 and demonstrate that the cyclin D1-Cdk2 fusion protein is a useful model to investigate the biological functions of cyclin D1-Cdk2 complexes.     

Cyclin A1/Cdk2 is Sufficient but not Required for the Induction of Apoptosis in Early Xenopus laevis Embryos

The role of cyclin-dependent kinases in cell proliferation is well characterized, whereas their somewhat paradoxical role in catalyzing apoptosis is less understood. One Cdk complex implicated in both cell proliferation and cell death is cyclin A/Cdk2. During early embryonic development of Xenopus laevis, distinct isoforms of cyclin A are expressed at different times. From fertilization through gastrulation, cyclin A1 is the predominant isoform. Cyclin A1 dimerizes with Cdk2 but not Cdk1. In contrast, cyclin A2 is expressed at a low level until gastrulation, when it becomes the major A-type cyclin and associates with both Cdk1 and Cdk2. When Xenopus embryos are treated with ionizing radiation (IR) prior to the midblastula transition (MBT), cyclin A1 protein persists beyond the MBT and forms an active complex with Cdk2. During this window of cyclin A1/Cdk2 activity, the embryo undergoes apoptosis. To test the hypothesis that cyclin A1-associated activity is a mediator of apoptosis, cyclin A1 protein level and associated kinase activity were measured in embryos treated with aphidicolin to induce apoptosis. Both cyclin A1 content and associated kinase activity were sustained after the MBT as embryos underwent apoptosis. To determine whether cyclin A1/Cdk2 was sufficient to induce apoptosis, recombinant cyclin A1/Cdk2 complex was injected into single-celled embryos, which induced apoptosis after the MBT. However, morpholinos targeting translation of cyclins A1 and A2 did not block apoptosis in embryos treated with X-rays or aphidicolin. These data indicate that cyclin A1/Cdk2 is sufficient, but not required for apoptosis during early development.     

Identification of ribosomal protein L34 as a novel Cdk5 inhibitor

The cell cycle is regulated by sequential activation, inactivation of cyclin dependent kinases (Cdk-s). Like all other Cdk-s, the catalytic subunit of Cdk5 is present in cycling cells. However, its highest concentration is found in differentiated neurons, and the only known protein that activates Cdk5 (i.e., p35) is expressed solely in the brain. Active Cdk5 is thought to be involved in the in vivo phosphorylation of the neurofilament proteins and tau which are hyperphosphorylated in neurodegenerative diseases. Recent reports suggest that Cdk5 may also contribute to cellular differentiation. Therefore, it would not be unusual to surmise that there exist specific proteins that regulate Cdk5 activity in cycling cells. In order to find if this was true, a cDNA library prepared from HeLa cells was screened using the yeast-two-hybrid system. The 60S ribosomal protein, L34, was identified as a Cdk5-interacting protein. Biochemical analyses reveal that L34 cannot activate Cdk5 but potently inhibits the p35-activated kinase. L34 also interacts with Cdk4 and, in parallel, inhibits the Cdk4/cyclin D1 activity. Interestingly, L34 does not interact with Cdk2 in the two-hybrid assay nor does it inhibit the Cdk2/cyclin A enzyme. The fact that a ribosomal protein inhibits Cdk5 and Cdk4 may suggest that these two kinases have a cellular role in translational regulation.     

Phosphorylation of Cyclin-dependent Kinase 5 (Cdk5) at Tyr-15 Is Inhibited by Cdk5 Activators and Does Not Contribute to the Activation of Cdk5

Cdk5 is a member of the cyclin-dependent kinase (Cdk) family. In contrast to other Cdks that promote cell proliferation, Cdk5 plays a role in regulating various neuronal functions, including neuronal migration, synaptic activity, and neuron death. Cdks responsible for cell proliferation need phosphorylation in the activation loop for activation in addition to binding a regulatory subunit cyclin. Cdk5, however, is activated only by binding to its activator, p35 or p39. Furthermore, in contrast to Cdk1 and Cdk2, which are inhibited by phosphorylation at Tyr-15, the kinase activity of Cdk5 is reported to be stimulated when phosphorylated at Tyr-15 by Src family kinases or receptor-type tyrosine kinases. We investigated the activation mechanism of Cdk5 by phosphorylation at Tyr-15. Unexpectedly, however, it was found that Tyr-15 phosphorylation occurred only on monomeric Cdk5, and the coexpression of activators, p35/p25, p39, or Cyclin I, inhibited the phosphorylation. In neuron cultures, too, the activation of Fyn tyrosine kinase did not increase Tyr-15 phosphorylation of Cdk5. Further, phospho-Cdk5 at Tyr-15 was not detected in the p35-bound Cdk5. In contrast, expression of active Fyn increased p35 in neurons. These results indicate that phosphorylation at Tyr-15 is not an activation mechanism of Cdk5 but, rather, indicate that tyrosine kinases could activate Cdk5 by increasing the protein amount of p35. These results call for reinvestigation of how Cdk5 is regulated downstream of Src family kinases or receptor tyrosine kinases in neurons, which is an important signaling cascade in a variety of neuronal activities.     

Cdk5 activator-binding protein C53 regulates apoptosis induced by genotoxic stress via modulating the G2/M DNA damage checkpoint

In response to DNA damage, the cellular decision of life versus death involves an intricate network of multiple factors that play critical roles in regulation of DNA repair, cell cycle, and cell death. DNA damage checkpoint proteins are crucial for maintaining DNA integrity and normal cellular functions, but they may also reduce the effectiveness of cancer treatment. Here we report the involvement of Cdk5 activator p35-binding protein C53 in regulation of apoptosis induced by genotoxic stress through modulating Cdk1-cyclin B1 function. C53 was originally identified as a Cdk5 activator p35-binding protein and a caspase substrate. Importantly, our results demonstrated that C53 deficiency conferred partial resistance to genotoxic agents such as etoposide and x-ray irradiation, whereas ectopic expression of C53 rendered cells susceptible to multiple genotoxins that usually trigger G(2)/M arrest. Furthermore, we found that Cdk1 activity was required for etoposide-induced apoptosis of HeLa cells. Overexpression of C53 promoted Cdk1 activity and nuclear accumulation of cyclin B1, whereas C53 deficiency led to more cytoplasmic retention of cyclin B1, suggesting that C53 acts as a pivotal player in modulating the G(2)/M DNA damage checkpoint. Finally, C53 and cyclin B1 co-localize and associate in vivo, indicating a direct role of C53 in regulating the Cdk1-cyclin B1 complex. Taken together, our results strongly indicate that in response to genotoxic stress, C53 serves as an important regulatory component of the G(2)/M DNA damage checkpoint. By overriding the G(2)/M checkpoint-mediated inhibition of Cdk1-cyclin B1 function, ectopic expression of C53 may represent a novel approach for chemo- and radio-sensitization of cancer cells.     

Cdc2 and Cdk2 kinase activated by transforming growth factor-beta1 trigger apoptosis through the phosphorylation of retinoblastoma protein in FaO hepatoma cells.

The signaling pathway leading to TGF-beta1-induced apoptosis was investigated using a TGF-beta1-sensitive hepatoma cell line, FaO. Cell cycle analysis demonstrated that the accumulation of apoptotic cells was preceded by a progressive decrease of the cell population in the G(1) phase concomitant with a slight increase of the cell population in the G(2)/M phase in response to TGF-beta1. TGF-beta1 induced a transient increase in the expression of Cdc2, cyclin A, cyclin B, and cyclin D1 at an early phase of apoptosis. During TGF-beta1-induced apoptosis, the transient increase in cyclin-dependent kinase (Cdk) activities coincides with a dramatic increase in the hyperphosphorylated forms of RB. Treatment with roscovitine or olomoucine, inhibitors of Cdc2 and Cdk2, blocked TGF-beta1-induced apoptosis by inhibiting RB phosphorylation. Overexpression of Bcl-2 or adenovirus E1B 19K suppressed TGF-beta1-induced apoptosis by blocking the induction of Cdc2 mRNA and the subsequent activation of Cdc2 kinase, whereas activation of Cdk2 was not affected, suggesting that Cdc2 plays a more critical role in TGF-beta1-induced apoptosis. In conclusion, we present the evidence that Cdc2 and Cdk2 kinase activity transiently induced by TGF-beta1 phosphorylates RB as a physiological target in FaO cells and that RB hyperphosphorylation may trigger abrupt cell cycle progression, leading to irreversible cell death.