The rates of adult neurogenesis and oligodendrogenesis are linked to cell cycle regulation through p27-dependent gene repression of SOX2.

Cell differentiation involves profound changes in global gene expression that often has to occur in coordination with cell cycle exit. Because cyclin-dependent kinase inhibitor p27 reportedly regulates proliferation of neural progenitor cells in the subependymal neurogenic niche of the adult mouse brain, but can also have effects on gene expression, we decided to molecularly analyze its role in adult neurogenesis and oligodendrogenesis. At the cell level, we show that p27 restricts residual cyclin-dependent kinase activity after mitogen withdrawal to antagonize cycling, but it is not essential for cell cycle exit. By integrating genome-wide gene expression and chromatin accessibility data, we find that p27 is coincidentally necessary to repress many genes involved in the transit from multipotentiality to differentiation, including those coding for neural progenitor transcription factors SOX2, OLIG2 and ASCL1. Our data reveal both a direct association of p27 with regulatory sequences in the three genes and an additional hierarchical relationship where p27 repression of Sox2 leads to reduced levels of its downstream targets Olig2 and Ascl1. In vivo, p27 is also required for the regulation of the proper level of SOX2 necessary for neuroblasts and oligodendroglial progenitor cells to timely exit cell cycle in a lineage-dependent manner.

p27Kip1, PCAF and PAX5 cooperate in the transcriptional regulation of specific target genes.

The cyclin-dependent kinase inhibitor p27Kip1 (p27) also behaves as a transcriptional repressor. Data showing that the p300/CBP-associated factor (PCAF) acetylates p27 inducing its degradation suggested that PCAF and p27 could collaborate in the regulation of transcription. However, this possibility remained to be explored. We analyzed here the transcriptional programs regulated by PCAF and p27 in the colon cancer cell line HCT116 by chromatin immunoprecipitation sequencing (ChIP-seq). We identified 269 protein-encoding genes that contain both p27 and PCAF binding sites being the majority of these sites different for PCAF and p27. PCAF or p27 knock down revealed that both regulate the expression of these genes, PCAF as an activator and p27 as a repressor. The double knock down of PCAF and p27 strongly reduced their expression indicating that the activating role of PCAF overrides the repressive effect of p27. We also observed that the transcription factor Pax5 interacts with both p27 and PCAF and that the knock down of Pax5 induces the expression of p27/PCAF target genes indicating that it also participates in the transcriptional regulation mediated by p27/PCAF. In summary, we report here a previously unknown mechanism of transcriptional regulation mediated by p27, Pax5 and PCAF.

p27Kip1 and p21Cip1 collaborate in the regulation of transcription by recruiting cyclin-Cdk complexes on the promoters of target genes.

Transcriptional repressor complexes containing p130 and E2F4 regulate the expression of genes involved in DNA replication. During the G1 phase of the cell cycle, sequential phosphorylation of p130 by cyclin-dependent kinases (Cdks) disrupts these complexes allowing gene expression. The Cdk inhibitor and tumor suppressor p27(Kip1) associates with p130 and E2F4 by its carboxyl domain on the promoters of target genes but its role in the regulation of transcription remains unclear. We report here that p27(Kip1) recruits cyclin D2/D3-Cdk4 complexes on the promoters by its amino terminal domain in early and mid G1. In cells lacking p27(Kip1), cyclin D2/D3-Cdk4 did not associate to the promoters and phosphorylation of p130 and transcription of target genes was increased. In late G1, these complexes were substituted by p21(Cip1)-cyclin D1-Cdk2. In p21(Cip1) null cells cyclin D1-Cdk2 were not found on the promoters and transcription was elevated. In p21/p27 double null cells transcription was higher than in control cells and single knock out cells. Thus, our results clarify the role of p27(Kip1) and p21(Cip1) in transcriptional regulation of genes repressed by p130/E2F4 complexes in which p27(Kip1) and p21(Cip1) play a sequential role by recruiting and regulating the activity of specific cyclin-Cdk complexes on the promoters.

Identification of lysosomal Npc1-binding proteins: Cathepsin D activity is regulated by NPC1.

Niemann-Pick type C (NPC) disease is an inherited lysosomal storage disorder, characterized by severe neurodegeneration. It is mostly produced by mutations in the NPC1 gene, encoding for a protein of the late endosomes/lysosomes membrane, involved in cholesterol metabolism. However, the specific role of this protein in NPC disease still remains unknown. We aimed to identify Npc1-binding proteins in order to define new putative NPC1 lysosomal functions. By affinity chromatography using an Npc1 peptide (amino acids 1032-1066 of loop I), as bait, we fished 31 lysosomal proteins subsequently identified by LC-MS/MS. Most of them were involved in proteolysis and lipid catabolism and included the protease cathepsin D. Cathepsin D and NPC1 interaction was validated by immunoprecipitation and the functional relevance of this interaction was studied. We found that fibroblasts from NPC patients with low levels of NPC1 protein have high amounts of procathepsin D but reduced quantities of the mature protein, thus showing a diminished cathepsin D activity. The increase of NPC1 protein levels in NPC cells by treatment with the proteasome inhibitor bortezomib, induced an elevation of cathepsin D activity. All these results suggest a new lysosomal function of NPC1 as a regulator of cathepsin D processing and activity.

Histone deacetylase 3 regulates cyclin A stability.

PCAF and GCN5 acetylate cyclin A at specific lysine residues targeting it for degradation at mitosis. We report here that histone deacetylase 3 (HDAC3) directly interacts with and deacetylates cyclin A. HDAC3 interacts with a domain included in the first 171 aa of cyclin A, a region involved in the regulation of its stability. In cells, overexpression of HDAC3 reduced cyclin A acetylation whereas the knocking down of HDAC3 increased its acetylation. Moreover, reduction of HDAC3 levels induced a decrease of cyclin A that can be reversed by proteasome inhibitors. These results indicate that HDAC3 is able to regulate cyclin A degradation during mitosis via proteasome. Interestingly, HDAC3 is abruptly degraded at mitosis also via proteasome thus facilitating cyclin A acetylation by PCAF/GCN5, which will target cyclin A for degradation. Because cyclin A is crucial for S phase progression and mitosis entry, the knock down of HDAC3 affects cell cycle progression specifically at both, S phase and G2/M transition. In summary we propose here that HDAC3 regulates cyclin A stability by counteracting the action of the acetylases PCAF/GCN5.

PCAF regulates the stability of the transcriptional regulator and cyclin-dependent kinase inhibitor p27 Kip1.

P27(Kip1) (p27) is a member of the Cip/Kip family of cyclin-dependent kinase inhibitors. Recently, a new function of p27 as transcriptional regulator has been reported. It has been shown that p27 regulates the expression of target genes mostly involved in splicing, cell cycle, respiration and translation. We report here that p27 directly binds to the transcriptional coactivator PCAF by a region including amino acids 91-120. PCAF associates with p27 through its catalytic domain and acetylates p27 at lysine 100. Our data showed that overexpression of PCAF induces the degradation of p27 whereas in contrast, the knockdown of PCAF stabilizes the protein. A p27 mutant in which K100 was substituted by arginine (p27-K100R) cannot be acetylated by PCAF and has a half-life much higher than that of p27WT. Moreover, p27-K100R remains stable along cell-cycle progression. Ubiquitylation assays and the use of proteasome inhibitors indicate that PCAF induces p27 degradation via proteasome. We also observed that knockdown of skp2 did not affect the PCAF induced degradation of p27. In conclusion, our data suggest that the p27 acetylation by PCAF regulates its stability.

Nucleolar disruption ensures nuclear accumulation of p21 upon DNA damage.

p21(cip1) is a protein with a dual function in oncogenesis depending mainly on its intracellular localization: tumor suppressor in the nucleus and oncogenic in the cytoplasm. After DNA damage, p21(cip1) increases and accumulates in the nucleus to ensure cell cycle arrest. We show here that the nuclear accumulation of p21(cip1) is not only a consequence of its increased levels but to a DNA damage cellular response, which is ataxia telangiectasia and Rad3 related (ATR)/ataxia telangiectasia mutated (ATM) and p53 independent. Furthermore, after DNA damage, p21(cip1) not only accumulates in the nucleoplasm but also in the disrupted nucleolus. Inside the nucleolus, it is found in spherical structures, which are not a protrusion of the nucleoplasm. The steady-state distribution of p21(cip1) in the nucleolus resulted from a highly dynamic equilibrium between nucleoplasmic and nucleolar p21(cip1) and correlated with the inhibition of p21(cip1) nuclear export. Most interestingly, inhibition of ribosomal export after expressing a dominant-negative mutant of nucleophosmin induced p21(cip1) accumulation in the nucleus and the nucleolus in the absence of DNA damage. This proved the existence of a nucleolar export route to the cytoplasm for p21(cip1) in control conditions that would be inhibited upon DNA damage leading to nuclear and nucleolar accumulation of p21(cip1).

The transcriptional co-activator PCAF regulates cdk2 activity.

Cyclin dependent kinases (cdks) regulate cell cycle progression and transcription. We report here that the transcriptional co-activator PCAF directly interacts with cdk2. This interaction is mainly produced during S and G(2)/M phases of the cell cycle. As a consequence of this association, PCAF inhibits the activity of cyclin/cdk2 complexes. This effect is specific for cdk2 because PCAF does not inhibit either cyclin D3/cdk6 or cyclin B/cdk1 activities. The inhibition is neither competitive with ATP, nor with the substrate histone H1 suggesting that somehow PCAF disturbs cyclin/cdk2 complexes. We also demonstrate that overexpression of PCAF in the cells inhibits cdk2 activity and arrests cell cycle progression at S and G(2)/M. This blockade is dependent on cdk2 because it is rescued by the simultaneous overexpression of this kinase. Moreover, we also observed that PCAF acetylates cdk2 at lysine 33. As this lysine is essential for the interaction with ATP, acetylation of this residue inhibits cdk2 activity. Thus, we report here that PCAF inhibits cyclin/cdk2 activity by two different mechanisms: (i) by somehow affecting cyclin/cdk2 interaction and (ii) by acetylating K33 at the catalytic pocket of cdk2. These findings identify a previously unknown mechanism that regulates cdk2 activity.

Acetylation of cyclin A: a new cell cycle regulatory mechanism.

Cyclin A must be degraded at prometaphase in order to allow mitosis progression. Nevertheless, the signals that trigger cyclin A degradation at mitosis have been largely elusive. In the present paper, we review the status of cyclin A degradation in the light of recent evidence indicating that acetylation plays a role in cyclin A stability. The emerging model proposes that the acetyltransferase PCAF [p300/CREB (cAMP-response-element-binding protein)-binding protein-associated factor] [perhaps also its homologue GCN5 (general control non-derepressible 5)] acetylates cyclin A at Lys(54), Lys(68), Lys(95) and Lys(112) during mitosis, leading to its ubiquitylation by the anaphase-promoting factor/cyclosome and its subsequent degradation via proteasome. Interestingly, these four lysine residues in cyclin A also participate in the regulation of cyclin A-Cdk (cyclin-dependent kinase) activity by modulating its interaction with Cdks.

CCAAT/enhancer binding protein δ is a transcriptional repressor of α-synuclein.

α-Synuclein is the main component of Lewy bodies, the intracellular protein aggregates representing the histological hallmark of Parkinson's disease. Elevated α-synuclein levels and mutations in SNCA gene are associated with increased risk for Parkinson's disease. Despite this, little is known about the molecular mechanisms regulating SNCA transcription. CCAAT/enhancer binding protein (C/EBP) ß and δ are b-zip transcription factors that play distinct roles in neurons and glial cells. C/EBPß overexpression increases SNCA expression in neuroblastoma cells and putative C/EBPß and δ binding sites are present in the SNCA genomic region suggesting that these proteins could regulate SNCA transcription. Based on these premises, the goal of this study was to determine if C/EBPß and δ regulate the expression of SNCA. We first observed that α-synuclein CNS expression was not affected by C/EBPß deficiency but it was markedly increased in C/EBPδ-deficient mice. This prompted us to characterize further the role of C/EBPδ in SNCA transcription. C/EBPδ absence led to the in vivo increase of α-synuclein in all brain regions analyzed, both at mRNA and protein level, and in primary neuronal cultures. In agreement with this, CEBPD overexpression in neuroblastoma cells and in primary neuronal cultures markedly reduced SNCA expression. ChIP experiments demonstrated C/EBPδ binding to the SNCA genomic region of mice and humans and luciferase experiments showed decreased expression of a reporter gene attributable to C/EBPδ binding to the SNCA promoter. Finally, decreased CEBPD expression was observed in the substantia nigra and in iPSC-derived dopaminergic neurons from Parkinson patients resulting in a significant negative correlation between SNCA and CEBPD levels. This study points to C/EBPδ as an important repressor of SNCA transcription and suggests that reduced C/EBPδ neuronal levels could be a pathogenic factor in Parkinson's disease and other synucleinopathies and C/EBPδ activity a potential pharmacological target for these neurological disorders.

Functional characterization of the novel APC N1026S variant associated with attenuated familial adenomatous polyposis.

BACKGROUND & AIMS: We identified the APC N1026S variant of unknown malignant potential in the adenomatous polyposis coli (APC) gene in a Spanish attenuated familial adenomatous polyposis (AFAP) family. The variant was located in the first of the 4 highly conserved 15-amino acid (AA) repeats within the beta-catenin union domain. Our aim was to determine its functional relevance to establish its pathogenicity. METHODS: N1026S variant was analyzed in 22 members of the AFAP family studied, in 236 sporadic colorectal cancer cases, 203 matched controls, and 205 unrelated familial colorectal cancer cases. To assess its effects on beta-catenin binding, beta-catenin/Tcf-4-mediated transcription and beta-catenin subcellular distribution we performed affinity chromatography experiments, BIAcore 1000 (BIAcore AB, Uppsala, Sweden) assays, luciferase reporter assays, assessment of c-myc messenger RNA levels, and cell fractionation. RESULTS: N1026S variant cosegregated with the disease in the AFAP family studied. None of the sporadic or familial cases as well as the controls analyzed was positive for the variant. N1026S variant completely precluded beta-catenin binding to the first 15-AA repeat and diminished it when all four 15-AA repeats were present. Expression of APC N1026S in SW480 and DLD-1 cells did not diminish beta-catenin/Tcf-4-mediated transcription as effectively as APC wild-type. N1026S did not decrease c-myc transcription in DLD1 cells and nuclear beta-catenin in SW480 cells as effectively as WT. CONCLUSIONS: These findings strongly support a pathogenic role of the APC N1026S variant in the AFAP phenotype, reinforcing the importance of functional characterization of APC variants for genetic counseling.

Binding of calmodulin to the carboxy-terminal region of p21 induces nuclear accumulation via inhibition of protein kinase C-mediated phosphorylation of Ser153.

Intracellular localization plays an important role in the functional regulation of the cell cycle inhibitor p21. We have previously shown that calmodulin binds to p21 and that calmodulin is essential for the nuclear accumulation of p21. Here, we analyze the mechanism of this regulation. We show that calmodulin inhibits in vitro phosphorylation of p21 by protein kinase C (PKC) and that this inhibition is dependent upon calmodulin binding to p21. Two-dimensional electrophoresis analysis of cells expressing the p21 wild type or p21S153A, a nonphosphorylatable mutant of p21 at position 153, indicates that Ser153 of p21 is a phosphorylable residue in vivo. Furthermore, Western blot analysis using phospho-Ser153-specific antibodies indicates that Ser153 phosphorylation in vivo is induced when PKC is activated and calmodulin is inhibited. The mutation of Ser153 to aspartate, a pseudophosphorylated residue, inhibits the nuclear accumulation of p21. Finally, whereas wild-type p21 translocates to the cytoplasm after PKC activation in the presence of calmodulin inhibitors, p21 carrying a nonphosphorylatable residue at position 153 remains in the nucleus. We propose that calmodulin binding to p21 prevents its phosphorylation by PKC at Ser153 and consequently allows its nuclear localization. When phosphorylated at Ser153, p21 is located at the cytoplasm and disrupts stress fibers.

Cmk2 kinase is essential for survival in arsenite by modulating translation together with RACK1 orthologue Cpc2 in Schizosaccharomyces pombe.

Different studies have demonstrated multiple effects of arsenite on human physiology. However, there are many open questions concerning the mechanism of response to arsenite. Schizosaccharomyces pombe activates the Sty1 MAPK pathway as a common response to several stress conditions. The specificity of the response is due to the activation of different transcription factors and specific targets such the Cmk2 MAPKAP kinase. We have previously shown that Cmk2 is phosphorylated and activated by the MAPK Sty1 in response to oxidative stress. Here, we report that Cmk2 kinase is specifically necessary to overcome the stress caused by metalloid agents, in particular arsenite. Deletion of cmk2 increases the protein level of various components of the MAPK pathway. Moreover, Cmk2 negatively regulates translation through the Cpc2 kinase: the RACK1 orthologue in fission yeast. RACK1 is a receptor for activated C-kinase. Interestingly, RACK1 is a constituent of the eukaryotic ribosome specifically localized in the head region of the 40 S subunit. Cmk2 controls arsenite response through Cpc2 and it does so through Cpc2 ribosomal function, as observed in genetic analysis using a Cpc2 mutant unable to bind to ribosome. These findings suggest a role for Cmk2 in regulating translation and facilitating adaptation to arsenite stress in the ribosome.

Role of p27Kip1 as a transcriptional regulator.

The protein p27Kip1 is a member of the Cip/Kip family of cyclin-dependent kinase (Cdk) inhibitors. It interacts with both the catalytic and the regulatory subunit (cyclin) and introduces a region into the catalytic cleave of the Cdk inducing its inactivation. Its inhibitory capacity can be modulated by specific tyrosine phosphorylations. p27Kip1 also behaves as a transcriptional regulator. It associates with specific chromatin domains through different transcription factors. ChIP on chip, ChIP-seq and expression microarray analysis allowed the identification of the transcriptional programs regulated by p27Kip1. Thus, important cellular functions as cell division cycle, respiration, RNA processing, translation and cell adhesion, are under p27Kip1 regulation. Moreover, genes involved in pathologies as cancer and neurodegeneration are also regulated by p27Kip1, suggesting its implication in these pathologies. The carboxyl moiety of p27Kip1 can associate with different proteins, including transcriptional regulators. In contrast, its NH2-terminal region specifically interacts with cyclin-Cdk complexes. The general mechanistic model of how p27Kip1 regulates transcription is that it associates by its COOH region to the transcriptional regulators on the chromatin and by the NH2-domain to cyclin-Cdk complexes. After Cdk activation it would phosphorylate the specific targets on the chromatin leading to gene expression. This model has been demonstrated to apply in the transcriptional regulation of p130/E2F4 repressed genes involved in cell cycle progression. We summarize in this review our current knowledge on the role of p27Kip1 in the regulation of transcription, on the transcriptional programs under its regulation and on its relevance in pathologies as cancer and neurodegeneration.

p27Kip1 regulates alpha-synuclein expression.

Alpha-synuclein (α-SYN) is the main component of anomalous protein aggregates (Lewy bodies) that play a crucial role in several neurodegenerative diseases (synucleinopathies) like Parkinson's disease and multiple system atrophy. However, the mechanisms involved in its transcriptional regulation are poorly understood. We investigated here the role of the cyclin-dependent kinase (Cdk) inhibitor and transcriptional regulator p27Kip1 (p27) in the regulation of α-SYN expression. We observed that selective deletion of p27 by CRISPR/Cas9 technology in neural cells resulted in increased levels of α-SYN. Knock-down of the member of the same family p21Cip1 (p21) also led to increased α-SYN levels, indicating that p27 and p21 collaborate in the repression of α-SYN transcription. We demonstrated that this repression is mediated by the transcription factor E2F4 and the member of the retinoblastoma protein family p130 and that it is dependent of Cdk activity. Chromatin immunoprecipitation analysis revealed specific binding sites for p27, p21 and E2F4 in the proximal α-SYN gene promoter. Finally, luciferase assays revealed a direct action of p27, p21 and E2F4 in α-SYN gene expression. Our findings reveal for the first time a negative regulatory mechanism of α-SYN expression, suggesting a putative role for cell cycle regulators in the etiology of synucleinopathies.

ChIP-Seq analysis identifies p27(Kip1)-target genes involved in cell adhesion and cell signalling in mouse embryonic fibroblasts.

The protein p27Kip1 (p27), a member of the Cip-Kip family of cyclin-dependent kinase inhibitors, is involved in tumorigenesis and a correlation between reduced levels of this protein in human tumours and a worse prognosis has been established. Recent reports revealed that p27 also behaves as a transcriptional regulator. Thus, it has been postulated that the development of tumours with low amounts of p27 could be propitiated by deregulation of transcriptional programs under the control of p27. However, these programs still remain mostly unknown. The aim of this study has been to define the transcriptional programs regulated by p27 by first identifying the p27-binding sites (p27-BSs) on the whole chromatin of quiescent mouse embryonic fibroblasts. The chromatin regions associated to p27 have been annotated to the most proximal genes and it has been considered that the expression of these genes could by regulated by p27. The identification of the chromatin p27-BSs has been performed by Chromatin Immunoprecipitation Sequencing (ChIP-seq). Results revealed that p27 associated with 1839 sites that were annotated to 1417 different genes being 852 of them protein coding genes. Interestingly, most of the p27-BSs were in distal intergenic regions and introns whereas, in contrast, its association with promoter regions was very low. Gene ontology analysis of the protein coding genes revealed a number of relevant transcriptional programs regulated by p27 as cell adhesion, intracellular signalling and neuron differentiation among others. We validated the interaction of p27 with different chromatin regions by ChIP followed by qPCR and demonstrated that the expressions of several genes belonging to these programs are actually regulated by p27. Finally, cell adhesion assays revealed that the adhesion of p27-/- cells to the plates was much higher that controls, revealing a role of p27 in the regulation of a transcriptional program involved in cell adhesion.

Immunoprecipitation of Cdk-Cyclin Complexes for Determination of Kinase Activity.

Cyclin-dependent kinases (Cdks) belong to a family of key regulators of cell division cycle and transcription. The activity of some of them is deregulated in tumor cells and to find specific inhibitors is an important goal to be achieved. We report here the current methods to determine their in vitro activity in order to facilitate the identification of specific inhibitors. Mainly, the activity can be determined by using immunoprecipitates from cell samples with antibodies against specific Cdks as a source of the enzymes.

Expression and Purification of Recombinant Cyclins and CDKs for Activity Evaluation.

Cyclin-dependent kinases (Cdks) belong to a family of key regulators of cell division cycle and transcription. Their activity is mainly regulated by association with regulatory subunits named cyclins but their activities are also regulated by phosphorylation, acetylation, and the association with specific inhibitory proteins (CKIs). The activity of different Cdks is deregulated in many different type of tumors, and thus, Cdks are considered targets for antitumoral therapy. For large screenings of inhibitors the use of purified recombinant Cdks and cyclins is recommended. We report here the current methods to determine their in vitro activity for large screenings of inhibitors.

ATM/ATR-independent inhibition of cyclin B accumulation in response to hydroxyurea in nontransformed cell lines is altered in tumour cell lines.

The DNA replication checkpoint is an inhibitory pathway ensuring that mitosis occurs only after completion of DNA synthesis. Its function may be relevant to the stability of the genome. The essential elements of this checkpoint are ATM/ATR kinases that indirectly lead to the phosphorylation and inhibition of the mitosis-promoting factor (Cdc2/cyclin B1). The function of this checkpoint was analysed in diverse nontransformed and tumour-derived cell lines. All cell lines tested arrested mitosis entry when DNA synthesis was inhibited by hydroxyurea (HU) treatment. But, unlike what has been described in yeast and Xenopus, in normal rat kidney (NRK) cells and NIH 3T3 fibroblasts, the arrest induced by HU treatment was not abrogated by caffeine, an ATM and ATR inhibitor. This indicated the presence of an ATM/ATR-independent response to DNA synthesis inhibition in these nontransformed mammalian cell lines. Interestingly, the behaviour of different tumour cell lines after caffeine treatment varied. While SW480, NP29, NP18 and HeLa cells did not enter mitosis in the presence of caffeine after HU treatment, in CaCo2, DLD1, HCT116 and HT29 caffeine abrogated the checkpoint response. In nontransformed cell lines, lack of cyclin B1 accumulation was observed when DNA synthesis was inhibited. This response was not abrogated by caffeine. In the tumour cell lines, a good correlation between the ability to arrest cell cycle when DNA synthesis was inhibited in the presence of caffeine and the lack of cyclin B1 accumulation was observed. Thus, there is an ATM/ATR-independent checkpoint response that leads to a decrease in cyclin B1 accumulation. However, this response is not functional in some tumour cell lines. Using inhibitors of p38alpha and beta, Mek1, 2 and p53-/- knocked-out fibroblasts, we showed that these proteins were also not involved in this particular checkpoint response. Lack of cyclin B1 accumulation after DNA synthesis inhibition in NRK cells was not due to increased degradation of the protein, but correlated with a decrease in mRNA accumulation.

P38SAPK2 phosphorylates cyclin D3 at Thr-283 and targets it for proteasomal degradation.

Cyclin D3 plays a critical role in maturation of precursor T cells and their levels are tightly regulated during this process. Alteration of cyclin D3 levels has been proposed to be important in the development of different human cancers, including malignancies of the lymphoid system. Thus, we have analysed the mechanisms involved in the regulation of cyclin D3 levels. Our results indicate that cyclin D3 is degraded via proteasome and that Thr-283 is essential for its degradation. Wild-type cyclin D3 but not the Thr-283A mutant accumulated ubiquitylated forms after treatment with proteasome inhibitors. We also observed that different type of stresses promote the Thr-283-dependent in vivo degradation of cyclin D3. The analysis of the kinases involved in Thr-283 phosphorylation indicates that all the members of the p38SAPK family of serine-threonine kinases are able to phosphorylate cyclin D3 at this specific site. Moreover, we found that the overexpression of p38alphaSAPK2 induce the decrease of cyclin D3 in vivo. These results indicate that p38SAPK might be involved in the regulation of cyclin D3 levels and suggest that this mechanism is involved in the maturation of precursor T-cells. Alterations of this mechanism might be important for oncogenesis.