Cyclin Cyclin-dependent Kinase Research Articles

Phosphorylation of the Pol II CTD by KIN28 Enhances BUR1/BUR2 Recruitment and Ser2 CTD Phosphorylation Near Promoters

Cyclin-dependent kinase BUR1/BUR2 appears to be the yeast ortholog of P-TEFb, which phosphorylates Ser2 of the RNA Pol II CTD, but the importance of BUR1/BUR2 in CTD phosphorylation is unclear. We show that BUR1/BUR2 is cotranscriptionally recruited to the 5' end of ARG1 in a manner stimulated by interaction of the BUR1 C terminus with CTD repeats phosphorylated on Ser5 by KIN28. Impairing BUR1/BUR2 function, or removing the CTD-interaction domain in BUR1, reduces Ser2 phosphorylation in bulk Pol II and eliminates the residual Ser2P in cells lacking the major Ser2 CTD kinase, CTK1. Impairing BUR1/BUR2 or CTK1 evokes a similar reduction of Ser2P in Pol II phosphorylated on Ser5 and in elongating Pol II near the ARG1 promoter. By contrast, CTK1 is responsible for the bulk of Ser2P in total Pol II and at promoter-distal sites. In addition to phosphorylating Ser2 near promoters, BUR1/BUR2 also stimulates Ser2P formation by CTK1 during transcription elongation.

ATP‐Noncompetitive Inhibitors of CDK–Cyclin Complexes

Progression through the cell division cycle is controlled by a family of cyclin-dependent kinases (CDKs), the activity of which depends on their binding to regulatory partners (cyclins A-H). Deregulation of the activity of CDKs has been associated with the development of infectious, neurodegenerative, and proliferative diseases such as Alzheimer's, Parkinson's, or cancer. Most cancer cells contain mutations in the pathways that control the activity of CDKs. This observation led this kinase family to become a central target for the development of new drugs for cancer therapy. A range of structurally diverse molecules has been shown to inhibit the activity of CDKs through their activity as ATP antagonists. Nevertheless, the ATP binding sites on CDKs are highly conserved, limiting the kinase specificity of these inhibitors. Various genetic and crystallographic approaches have provided essential information about the mechanism of formation and activation of CDK-cyclin complexes, providing new ways to implement novel research strategies toward the discovery of new, more effective and selective drugs. Herein we review the progress made in the development of ATP-noncompetitive CDK-cyclin inhibitors.

Clinical significance of cyclin B1 and cyclin-dependent kinase 1 expression in human esophageal squamous cell carcinoma

Clinical significance of cyclin B1 and cyclin-dependent kinase 1 expression in human esophageal squamous cell carcinoma

Morphogenesis signaling components influence cell cycle regulation by cyclin dependent kinase

BackgroundThe yeast cell cycle is largely controlled by the cyclin-dependent kinase (CDK) Cdc28. Recent evidence suggests that both CDK complex stability as well as function during mitosis is determined by precise regulation of Swe1, a CDK inhibitory kinase and cyclin binding partner. A model of mitotic progression has been provided by study of filamentous yeast. When facing nutrient-limited conditions, Ras2-mediated PKA and MAPK signaling cascades induce a switch from round to filamentous morphology resulting in delayed mitotic progression.ResultsTo delineate how the dimorphic switch contributes to cell cycle regulation, temperature sensitive cdc28 mutants exhibiting constitutive filamentation were subjected to epistasis analyses with RAS2 signaling effectors. It was found that Swe1-mediated inhibitory tyrosine phosphorylation of Cdc28 during filamentous growth is in part mediated by Ras2 activation of PKA, but not Kss1-MAPK, signaling. This pathway is further influenced by Cks1, a conserved CDK-binding partner of elusive function with multiple proposed roles in CDK activation, transcriptional regulation and ubiquitin-mediated proteasome degradation.ConclusionThe dynamic balance between Cks1- and Swe1-dependent regulation of Cdc28 and, thereby, the timing of mitosis during yeast dimorphism is regulated in part by Ras2/cAMP-mediated PKA signaling, a key pathway controlling filamentous growth.

Differential regulation of cyclin D1 and D2 in protecting against cardiomyocyte proliferation

Cardiomyocytes withdraw from cell cycle after terminal differentiation due in part to impaired nuclear import of cyclin D1. Thus, we have previously shown that expression of nuclear localization signal-tagged cyclin D1 (D1NLS) and cyclin-dependent kinase 4 promotes cardiomyocyte proliferation both in vitro and in vivo. Here we show that cyclin D2 fails to stimulate cell cycle in cardiomocytes through a mechanism distinct from that of cyclin D1. We demonstrate that cyclin D2 can express in the nucleus much more efficiently than cyclin D1. Cyclin D2, however, was much less effective in activating CDK2 and cell proliferation than cyclin D1 when expressed transiently in the nucleus of cardiomyocytes using nuclear localization signals. Consistent with such an observation, CDK inhibitors p21cip1 and p27kip1 remained bound to CDK2 in cells expressing cyclin D2, whereas p21 and p27 were sequestered to cyclin D1 in cells expressing D1NLS. These data suggest that cyclin D2 has weaker affinities to the CDK inhibitors and therefore is less efficient in activating cell cycle than cyclin D1. According to such a notion, double knockdown of p21 and p27 in cells expressing D2NLS induced activation of CDK2/CDC2 and BrdU incorporation to levels similar to those in cells expressing D1NLS. Taken together, our data suggest that distinct mechanisms keep cyclin D1 and cyclin D2 from activating cell cycle in terminally differentiated cardiomyocytes.

Pathways of Proliferation

See related article, pages 1155–1163 Excessive proliferation of vascular smooth muscle cells (VSMCs) contributes to the pathogenesis of many cardiovascular diseases, including atherosclerosis and pulmonary arterial hypertension (PAH). VSMC proliferation also underlies the failure of many therapies, notable examples being restenosis following coronary angioplasty, vein graft failure in patients with coronary artery bypass grafts, and transplant vasculopathy. Few therapies directly target excess VSMC proliferation, in part, because the underlying pathways have been unknown. Recently, several pathways of VSMC proliferation have been defined, and new therapeutic targets have emerged. An example of the power of preventing VSMC proliferation in reducing human cardiovascular disease is the rapamycin (sirolimus)-coated coronary stent. After dozens of agents failed to prevent the 30% restenosis rate postangioplasty, this VSMC proliferation inhibitor reduced the number to ≈6%.1 However, rapamycin has toxicities, limiting its systemic use. Moreover, the mechanisms of accelerated VSMC proliferation may vary by disease, and, thus, the efficacy of an antiproliferative drug will likely be contextual (ie, disease dependent). Understanding the pathways controlling proliferation offers hope for identifying drugs that selectively target proliferating cells. The contribution by Gizard et al in this issue of Circulation Research identifies such a pathway and suggests new therapeutic targets.2 Members of this group expand on their previous work showing that peroxisome proliferator-activated receptor (PPAR)α activation suppresses G1→S cell cycle progression by increasing the expression of the cyclin dependent kinase (CDK) inhibitor p16INK4a.3 The present study is built on a solid body of knowledge of the cell cycle. Although growth factors are necessary to initiate proliferation in normal cells, cells become independent of these external stimuli during the G1 phase. This “point of no return” is highly regulated by the interaction among cyclins, CDKs, and CDK inhibitors.4 In nonproliferating cells, the …

Expression patterns of cyclins D1, E and cyclin-dependent kinase inhibitors p21waf1/cip1, p27kip1 in colorectal carcinoma: correlation with other cell cycle regulators (pRb, p53 and Ki-67 and PCNA) and clinicopathological features

Aberrations in the cell cycle regulators are common features of many tumours and several have been shown to have prognostic significant in colorectal cancer. The expression patterns of cyclins D1 and E as well as cyclin-dependent kinase (CDK) inhibitors p21waf1/cip1 and p27kip1 and their interrelationship with other cell cycle checkpoint proteins [p53, pRb, Ki-67 and proliferative cell nuclear antigen (PCNA)] were investigated in colorectal cancer in order to ascertain coregulation and influence on tumour behaviour or survival. These molecular markers were localisated immunohistochemically using the monoclonal antibodies anticyclin D1 (DCS-6), anticyclin E (13A3), anti-p21 (4D10), anti-p27 (1B4), anti-p53 (DO7), anti-Rb (AB-5), MIB1 and PC10 in colorectal cancer tissue from 97 patients. Data were analysed statistically using the spss software program. Overexpression of cyclin D1, cyclin E and p21waf1/cip1 proteins (>5% positive neoplastic cells) was observed in 5.9%, 30% and 7.2% of the cases respectively. Increased levels of cyclin D1 (p = 0.0001) and p21waf1/cip1 protein (p = 0.03) in tumours with mucous differentiation were observed. Overexpression of cyclin D1 was correlated with tumour stage (p = 0.03), the lymph node involvement (p = 0.02), as well as p21waf1/cip1 protein expression (p < 0.0001). Cyclin E was positively correlated with p21waf1/cip1 (p = 0.014), as well as with the cell proliferation as measured by PCNA-labelling index (p = 0.011) and Ki-67 score (p = 0.007). A positive relationship of p21waf1/cip1 expression with the proliferative-associated index Ki-67 was noted (p = 0.005). Downregulation of p27kip1 was observed in 47.4% of the cases and was correlated with downregulation of pRb (p = 0.002) and PCNA score (p = 0.004). The prognostic significance of cyclins D1, E and CDK inhibitors p21waf1/cip1, p27kip1 in determining the risk of recurrence and overall survival with both univariate (long-rang test) and multivariate (Cox regression) methods of analysis showed no statistically significance differences. In conclusion, these findings suggest that, the levels of the cell cycle regulators studied, do not seems to have a prognostic value, in terms of predicting the risk of early recurrence and overall survival. In addition, the interrelationships, probably means their contribution to the regulation of cell growth, through different pathways in colorectal carcinogenesis.

Post-translational regulation of the tumor suppressor p27(KIP1).

Mitogenic signals stimulate cell division by activating cyclin/cyclin-dependent kinase (CDK) complexes. Their timely regulation ensures proper cell cycle progression. It is therefore not surprising that cyclin/CDK complexes are integrators of multiple signals from both the extracellular environment and intracellular cues. Important regulators of cyclin/CDKs are the CDK inhibitors that have attracted attention due to their association with disease. p27(KIP1) is a CDK inhibitor that controls CDK activity throughout the cell cycle. As a CDK inhibitor, p27(KIP1) has tumor suppressor activity. Besides CDKs, p27(KIP1) regulates additional cellular processes, including cell motility, some of which seem to mediate oncogenic activities of p27(KIP1). These activities of p27(KIP1) are regulated through multiple phosphorylation sites, targeted by several signal transduction pathways. Understanding functions and regulation of p27(KIP1) will be important to determine which isoform of p27(KIP1) has anti- or pro-tumorigenic activities. Such knowledge might be of prognostic value and may offer novel therapeutic windows.

Nucleocytoplasmic Trafficking of G2/M Regulators in Yeast

Nucleocytoplasmic shuttling is prevalent among many cell cycle regulators controlling the G2/M transition. Shuttling of cyclin/cyclin-dependent kinase (CDK) complexes is thought to provide access to substrates stably located in either compartment. Because cyclin/CDK shuttles between cellular compartments, an upstream regulator that is fixed in one compartment could in principle affect the entire cyclin/CDK pool. Alternatively, the regulators themselves may need to shuttle to effectively regulate their moving target. Here, we identify localization motifs in the budding yeast Swe1p (Wee1) and Mih1p (Cdc25) cell cycle regulators. Replacement of endogenous Swe1p or Mih1p with mutants impaired in nuclear import or export revealed that the nuclear pools of Swe1p and Mih1p were more effective in CDK regulation than were the cytoplasmic pools. Nevertheless, shuttling of cyclin/CDK complexes was sufficiently rapid to coordinate nuclear and cytoplasmic events even when Swe1p or Mih1p were restricted to one compartment. Additionally, we found that Swe1p nuclear export was important for its degradation. Because Swe1p degradation is regulated by cytoskeletal stress, shuttling of Swe1p between nucleus and cytoplasm serves to couple cytoplasmic stress to nuclear cyclin/CDK inhibition.

Intrinsic and Cyclin-dependent Kinase-dependent Control of Spindle Pole Body Duplication in Budding Yeast

Centrosome duplication must be tightly controlled so that duplication occurs only once each cell cycle. Accumulation of multiple centrosomes can result in the assembly of a multipolar spindle and lead to chromosome mis-segregation and genomic instability. In metazoans, a centrosome-intrinsic mechanism prevents reduplication until centriole disengagement. Mitotic cyclin/cyclin-dependent kinases (CDKs) prevent reduplication of the budding yeast centrosome, called a spindle pole body (SPB), in late S-phase and G2/M, but the mechanism remains unclear. How SPB reduplication is prevented early in the cell cycle is also not understood. Here we show that, similar to metazoans, an SPB-intrinsic mechanism prevents reduplication early in the cell cycle. We also show that mitotic cyclins can inhibit SPB duplication when expressed before satellite assembly in early G1, but not later in G1, after the satellite had assembled. Moreover, electron microscopy revealed that SPBs do not assemble a satellite in cells expressing Clb2 in early G1. Finally, we demonstrate that Clb2 must localize to the cytoplasm in order to inhibit SPB duplication, suggesting the possibility for direct CDK inhibition of satellite components. These two mechanisms, intrinsic and extrinsic control by CDK, evoke two-step system that prevents SPB reduplication throughout the cell cycle.

Phosphorylation of Retinoblastoma Protein by Viral Protein with Cyclin-Dependent Kinase Function

As obligate intracellular parasites, viruses expertly modify cellular processes to facilitate their replication and spread, often by encoding genes that mimic the functions of cellular proteins while lacking regulatory features that modify their activity. We show that the human cytomegalovirus UL97 protein has activities similar to cellular cyclin-cyclin-dependent kinase (CDK) complexes. UL97 phosphorylated and inactivated the retinoblastoma tumor suppressor, stimulated cell cycle progression in mammalian cells, and rescued proliferation of Saccharomyces cerevisiae lacking CDK activity. UL97 is not inhibited by the CDK inhibitor p21 and lacks amino acid residues conserved in the CDKs that permit the attenuation of kinase activity. Thus, UL97 represents a functional ortholog of cellular CDKs that is immune from normal CDK control mechanisms.

Global control of cell-cycle transcription by coupled CDK and network oscillators

A significant fraction of the Saccharomyces cerevisiae genome is transcribed periodically during the cell division cycle, indicating that properly timed gene expression is important for regulating cell-cycle events. Genomic analyses of the localization and expression dynamics of transcription factors suggest that a network of sequentially expressed transcription factors could control the temporal programme of transcription during the cell cycle. However, directed studies interrogating small numbers of genes indicate that their periodic transcription is governed by the activity of cyclin-dependent kinases (CDKs). To determine the extent to which the global cell-cycle transcription programme is controlled by cyclin-CDK complexes, we examined genome-wide transcription dynamics in budding yeast mutant cells that do not express S-phase and mitotic cyclins. Here we show that a significant fraction of periodic genes are aberrantly expressed in the cyclin mutant. Although cells lacking cyclins are blocked at the G1/S border, nearly 70% of periodic genes continued to be expressed periodically and on schedule. Our findings reveal that although CDKs have a function in the regulation of cell-cycle transcription, they are not solely responsible for establishing the global periodic transcription programme. We propose that periodic transcription is an emergent property of a transcription factor network that can function as a cell-cycle oscillator independently of, and in tandem with, the CDK oscillator.

Extracellular regulated kinase-2 immunoreactivity increases in parallel with cervical intraepithelial neoplasia grade in cervical neoplasia

The cell cycle control system includes cyclins, cyclin-dependent kinases (CDK), and their inhibitors (CDK1). Extracellular regulated kinase (ERK1/2) (p44 and p42 mitogen-activated protein kinases [MAPKs]) is a component of the MAPK pathway, which is associated with cyclin D1 and CDK. It is a critical signaling system for the induction of cell proliferation, differentiation, and cell survival. The aim of this study was to investigate the usefulness of ERK2 expression as a marker of biological aggressiveness complementary to cervical intraepithelial neoplasia (CIN) grade as well as to compare its expression in preinvasive lesions with that in invasive carcinoma. Paraffin-embedded sections of 146 CIN lesions (32 CIN I, 49 CIN II, and 43 CIN III) and 22 invasive cervical carcinomas (13 squamous and 9 adenocarcinomas) were used for the standard immunohistochemical procedure with the application of the ERK2 monoclonal antibody. ERK2 staining displayed a cytoplasmic and nuclear pattern. The staining intensity was gradually increased according to the severity of the dysplastic lesions; ERK2 immunoreactivity was significantly increased in high-grade dysplastic lesions (CIN II and CIN III) and invasive carcinomas by comparison to low-grade dysplastic lesions (CIN I) (P < 0.001). When high-grade lesions were separately assessed, the differences between each one of them and CIN I retained their statistical significance: CIN II versus CIN I (P < 0.001) and CIN III versus CIN I (P < 0.001). In conclusion, our study found a direct relationship between the increasing grade of the dysplastic cervical lesions and the intensity of ERK2 staining, thus implying a role of ERK2 as an early event in cervical carcinogenesis.

Distinct Modes of Activation of Phosphatidylinositol 3-Kinase in Response to Cyclic Adenosine 3′, 5′-Monophosphate or Insulin-Like Growth Factor I Play Different Roles in Regulation of Cyclin D1 and p27Kip1 in FRTL-5 Cells

Bioactivities of IGFs in various cells are often potentiated in the presence of other hormones. In previous studies we showed that pretreatment of rat FRTL-5 thyroid cells with TSH or other cAMP-generating agents markedly potentiated DNA synthesis induced by IGF-I. Under these conditions we found that phosphatidylinositol (PI) 3-kinase was activated in response to either cAMP or IGF stimulus, and both activation modes were indispensable for the potentiation of DNA synthesis. The present studies were undertaken to elucidate how cAMP and/or IGF-I stimulus regulated the G1 cyclin-cyclin dependent kinase (CDK)-inhibitor system, and to determine the roles of PI 3-kinase activation by cAMP or IGF-I stimulus in this system. We found that cAMP pretreatment enhanced IGF-I-dependent increases in cyclin D1, due to synergistic increases in mRNA and elevation of translation rates. Furthermore, cAMP pretreatment enhanced IGF-I-induced protein degradation of the CDK inhibitor, p27(Kip1). These changes well explained an increase in cyclin E, leading to marked activation of G1 CDKs, followed by retinoblastoma protein phosphorylation. Our results using a PI 3-kinase inhibitor showed that cAMP-dependent PI 3-kinase activation plays an important role in the increase in cyclin D1 translation. In contrast, IGF-I-dependent PI 3-kinase activation was required for the increase in cyclin D1 mRNA levels and degradation of p27(Kip1). Together, the present study elucidates the role of cAMP and IGF-I in differentially activating PI 3-kinase as a mediator of multiple molecular events. These events converge in the regulation of cyclin D1 and p27(Kip1), leading to cAMP-dependent potentiation of IGF-I-dependent CDK activation and DNA synthesis.

Plugging the GAP between cell polarity and cell cycle

The establishment and maintenance of cell polarity—that is, the asymmetrical partitioning of cellular materials—is fundamental to stem‐cell renewal and development. The budding yeast Saccharomyces cerevisiae is a leading model organism for studying cell polarity. Yeast cell polarity is intimately connected to the organization of the actin cytoskeleton, which guides secretion towards the budding site or tip of the growing bud, resulting in polarized cell growth (Pruyne et al , 2004). Pioneering work by Lew & Reed has established roles for cyclin‐dependent kinases (CDKs) and their cyclin partners in cell polarity; at the time of bud emergence, G1 cyclins trigger polarized growth, whereas mitotic cyclins reverse this effect during mitosis. Inactivation of CDK following mitosis triggers redistribution of the actin cytoskeleton to the neck region for cytokinesis (Fig 1; Lew & Reed, 1993). The role of G1 cyclins in cell polarity has been known for more than ten years but the lingering problem has been the identity of their targets. Three recent studies published in The EMBO Journal (Knaus et al , 2007; Sopko et al , 2007; Zheng et al , 2007) and one in Nature Cell Biology (McCusker et al , 2007) have significantly advanced our understanding of the important substrates of G1 cyclin–CDK and their interesting mechanisms of action. Figure 1. Cell‐cycle regulation of cell polarity in budding yeast. Polarized growth sites (red), where Cdc42 is presumably active, change during the cell cycle. These changes are brought about by the action of G1 cyclin bound to cyclin‐dependent kinase (CDK) and mitotic cyclin–CDK activity. In some environments, Candida albicans keeps its polarized status and develops highly polarized hyphae. For cell polarization, cell division cycle (Cdc) 42 is the ‘Rome to which all roads lead’. Cdc42 is a Rho‐type small GTPase, the activity of which is regulated in budding yeast by a single guanine‐nucleotide …

Expression of Cyclin D1, P53 and CDK4 in pancreatic carcinoma

AIM: To study the expression of Cyclin D1, P53 and Cyclin-dependent kinase (CDK4) in pancreatic carcinoma, and to explore the relationship of Cyclin D1, P53 and CDK4 with pancreatic carcinoma. METHODS: A total of 48 cases with pancreatic cancer were collected from the First Hospital of Lanzhou University and Dingxi district hospital from July 1997 to January 2004. Immunohisto-chemical staining (S-P method) was used to detect the expressions of Cyclin D1, P53 and CDK4 in 48 cases of pancreatic carcinoma. RESULTS: No significant difference between Cyelin D1, P53 and COK4 expression was detected in different gender, age groups of pancreatic cancer. Positive expression rates of Cyclin D1 and COK4 were significantly higher in the poorly differentiated, lymph node metastasis, clinical stage Ⅲ, Ⅳ phase groups than in well-differentiated, no lymph node metastasis, clinical stage Ⅰ, Ⅱ phase groups (Cyclin D1: χ^2=10.540, 14.225, 5.043, P＜0.01 or 0.05; CD4: χ^2=7.619, 4.006, 5.581, P＜0.05). The P53 positive expression rate was significantly higher in lymphatic metastasis group than the non-lymph node metastasis group (χ^2=6.146, P＜0.05). However, no significant difference in P53 expression was detected between different differentiation groups or between different clinical stage groups. CONCLUSION: As Cyclin D1 is over expressed in pancreatic carcinoma, it can be used as a biomarker for early diagnosis of pancreatic carcinoma. However, P53 and CDK4 play important synergistic roles in pathogenesis and development of pancreatic carcinoma.

A Discrete Signaling Function for an Inositol Pyrophosphate

Inositol pyrophosphates were, until recently, without clearly defined functions. Two recent papers in Science have now clearly defined a function for an IP(7) pyrophosphate (inositol hexaphosphate with one pyrophosphate) that is the product of the enzyme encoded by the Vip1 gene in Saccharomyces cerevisiae. This IP(7) with a pyrophosphate tentatively assigned to be on either the 4 or 6 position is a cofactor that is required for inactivating the cyclin-cyclin-dependent kinase complex of Pho80, Pho81, and Pho85. Inhibition of the kinase results in the nuclear translocation of Pho4, which is a transcription factor that promotes expression of genes required for phosphate assimilation under conditions of low phosphate. When grown in low-phosphate media, IP(7) accumulates, which leads to the expression of genes involved in the acquisition of phosphate.

Molecular basis of cyclin-CDK-CKI regulation by reversible binding of an inositol pyrophosphate

When Saccharomyces cerevisiae cells are starved of inorganic phosphate, the Pho80-Pho85 cyclin-cyclin-dependent kinase (CDK) is inactivated by the Pho81 CDK inhibitor (CKI). The regulation of Pho80-Pho85 is distinct from previously characterized mechanisms of CDK regulation: the Pho81 CKI is constitutively associated with Pho80-Pho85, and a small-molecule ligand, inositol heptakisphosphate (IP7), is required for kinase inactivation. We investigated the molecular basis of the IP7- and Pho81-dependent Pho80-Pho85 inactivation using electrophoretic mobility shift assays, enzyme kinetics and fluorescence spectroscopy. We found that IP7 interacts noncovalently with Pho80-Pho85-Pho81 and induces additional interactions between Pho81 and Pho80-Pho85 that prevent substrates from accessing the kinase active site. Using synthetic peptides corresponding to Pho81, we define regions of Pho81 responsible for constitutive Pho80-Pho85 binding and IP7-regulated interaction and inhibition. These findings expand our understanding of the mechanisms of cyclin-CDK regulation and of the biochemical mechanisms of IP7 action.

Ddi1p and Rad23p play a cooperative role as negative regulators in the PHO pathway in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the PHO pathway regulates expression of phosphate-responsive genes such as PHO5, which encodes repressible acid phosphatase (rAPase). In this pathway, Pho81p functions as an inhibitor of the cyclin–cyclin-dependent kinase (CDK) complex Pho80p–Pho85p. However, the mechanism regulating the inhibitory activity of Pho81p is poorly understood. Through use of the yeast two-hybrid system, we identified the UbL–UbA protein Ddi1p as a Pho81p-binding protein. Further, Pho81p levels were found to be low under high-phosphate condition and high during phosphate starvation, indicating that Pho81p is regulated by phosphate concentration. However, our results revealed that Ddi1p and its associated protein Rad23p are not involved in the decrease in Pho81p level under high-phosphate condition. Significantly, the Δddi1Δrad23 strain exhibited a remarkable increase in rAPase activity at an intermediate-phosphate concentration of 0.4 mM, suggesting that Ddi1p and Rad23p play a cooperative role as negative regulators in the PHO pathway.

Deacetylation of the retinoblastoma tumour suppressor protein by SIRT1

The activity of Rb (retinoblastoma protein) is regulated by phosphorylation and acetylation events. Active Rb is hypophosphorylated and acetylated on multiple residues. Inactivation of Rb involves concerted hyper-phosphorylation by cyclin-CDK (cyclin-dependent kinase) complexes combined with deacetylation of appropriate lysine residues within Rb. In the present study, using in vivo co-immunoprecipitation experiments, we identified mammalian SIRT1 (sirtuin 1) as a binding partner for Rb and its family members p107 and p130. Formation of Rb-SIRT1 complexes required the pocket domain of Rb. p300 catalysed the acetylation of Rb, and SIRT1 was a potent deacetylase for Rb. The ability of SIRT1 to catalyse the deacetylation of Rb was dependent on NAD and was inhibited by the SIRT1 inhibitor nicotinamide. Deacetylated lysine residues within Rb formed a domain similar to the SIRT1-targeted domain of the p53 tumour suppressor protein. Cultures of arrested cells, via contact inhibition or DNA damage, exhibited decreased Rb phosphorylation and increased Rb acetylation. Overexpression of SIRT1 in either confluent or etoposide-treated cells resulted in a significant reduction in Rb acetylation, which was restored with nicotinamide. Gene knockdown of SIRT1 by siRNA (short interfering RNA) produced an accumulation of acetylated Rb. This increase was augmented further when siRNA against SIRT1 was used in conjunction with nicotinamide. In conclusion, our results demonstrate that SIRT1 is an in vitro and in vivo deacetylase for the Rb tumour suppressor protein.