Differential post-transcriptional regulation of two Ink4 proteins,p18Ink4c and p19Ink4d

Cyclin(-D-)-dependent kinase (Cdk) inhibitors of the Ink4 family specifically bind to Cdk4 and Cdk6, but not to other Cdks. Ink4c and Ink4d mRNAs are maximally and periodically expressed during the G2/M phase of the cell division cycle, but the abundance of their encoded proteins is regulated through distinct mechanisms. Both proteins undergo polyubiquitination, but the half life of p18Ink4c (~10 hours) is much longer than that of p19Ink4d (~2.5 hours). Lysines 46 and 112 are preferred sites of ubiquitin conjugation in p18Ink4c, although substitution of these and other lysine residues with arginine, particularly in combination, triggers protein misfolding and accelerates p18Ink4c degradation. When tethered to either catalytically active or inactive Cdk4 or Cdk6, polyubiquitination of p18Ink4c is inhibited, and the protein is further stabilized. Conversely, in competing with p18Ink4c for binding to Cdks, cyclin D1 accelerates p18Ink4c turnover. In direct contrast, polyubiquitination of p19Ink4d is induced by its association with Cdks, whereas cyclin D1 overexpression retards p19Ink4d degradation. Although it has been generally assumed that p18Ink4c and p19Ink4d are biochemically similar Cdk inhibitors, the major differences in their stability and turnover are likely key to understanding their distinct biological functions.     

The CDK Inhibitor p18Ink4c is a Tumor Suppressor in Medulloblastoma

Medulloblastoma (MB) is the most common malignant pediatric brain tumor which is thought to originate from cerebellar granule cell precursors (CGNPs) that fail to properly exit the cell cycle and differentiate. Although mutations in the Sonic Hedgehog (Shh) signaling pathway occur in ~30% of cases, genetic alterations that account for MB formation in most patients have not yet been identified. We recently determined that the cyclin D-dependent kinase inhibitor, p18Ink4c, is expressed as CGNPs exit the cell cycle, suggesting that this protein might play a central role in arresting the proliferation of these cells and in timing their subsequent migration and differentiation. In mice, disruption of Ink4c collaborates independently with loss of p53 or with inactivation of the gene (Ptc1) encoding the Shh receptor, Patched, to induce MB formation. Whereas loss of both Ink4c alleles is required for MB formation in a p53-null background, Ink4c is haplo-insufficient for tumor suppression in a Ptc1+/- background. Moreover, MBs derived from Ptc1+/- mice that lack one or two Ink4c alleles retain wild-type p53. Methylation of the INK4C (CDKN2C) promoter and complete loss of p18INK4C protein expression were detected in a significant fraction of human MBs again pointing toward a role for INK4C in suppression of MB formation.     

Control of spermatogenesis in mice by the cyclin D-dependent kinase inhibitors p18(Ink4c) and p19(Ink4d)

Male mice lacking both the Ink4c and Ink4d genes, which encode two inhibitors of D-type cyclin-dependent kinases (Cdks), are infertile, whereas female fecundity is unaffected. Both p18(Ink4c) and p19(Ink4d) are expressed in the seminiferous tubules of postnatal wild-type mice, being largely confined to postmitotic spermatocytes undergoing meiosis. Their combined loss is associated with the delayed exit of spermatogonia from the mitotic cell cycle, leading to the retarded appearance of meiotic cells that do not properly differentiate and instead undergo apoptosis at an increased frequency. As a result, mice lacking both Ink4c and Ink4d produce few mature sperm, and the residual spermatozoa have reduced motility and decreased viability. Whether or not Ink4d is present, animals lacking Ink4c develop hyperplasia of interstitial testicular Leydig cells, which produce reduced levels of testosterone. The anterior pituitary of fertile mice lacking Ink4c or infertile mice doubly deficient for Ink4c and Ink4d produces normal levels of luteinizing hormone (LH). Therefore, the failure of Leydig cells to produce testosterone is not secondary to defects in LH production, and reduced testosterone levels do not account for infertility in the doubly deficient strain. By contrast, Ink4d-null or double-null mice produce elevated levels of follicle-stimulating hormone (FSH). Because Ink4d-null mice are fertile, increased FSH production by the anterior pituitary is also unlikely to contribute to the sterility observed in Ink4c/Ink4d double-null males. Our data indicate that p18(Ink4c) and p19(Ink4d) are essential for male fertility. These two Cdk inhibitors collaborate in regulating spermatogenesis, helping to ensure mitotic exit and the normal meiotic maturation of spermatocytes.     

p18Ink4c, but not p27Kip1, collaborates with Men1 to suppress neuroendocrine organ tumors

Mutant mice lacking both cyclin-dependent kinase (CDK) inhibitors p18(Ink4c) and p27(Kip1) develop a tumor spectrum reminiscent of human multiple endocrine neoplasia (MEN) syndromes. To determine how p18 and p27 genetically interact with Men1, the tumor suppressor gene mutated in familial MEN1, we characterized p18-Men1 and p27-Men1 double mutant mice. Compared with their corresponding single mutant littermates, the p18(-/-); Men1(+/-) mice develop tumors at an accelerated rate and with an increased incidence in the pituitary, thyroid, parathyroid, and pancreas. In the pituitary and pancreatic islets, phosphorylation of the retinoblastoma (Rb) protein at both CDK2 and CDK4/6 sites was increased in p18(-/-) and Men1(+/-) cells and was further increased in p18(-/-); Men1(+/-) cells. The remaining wild-type Men1 allele was lost in most tumors from Men1(+/-) mice but was retained in most tumors from p18(-/-); Men1(+/-) mice. Combined mutations of p27(-/-) and Men1(+/-), in contrast, did not exhibit noticeable synergistic stimulation of Rb kinase activity, cell proliferation, and tumor growth. These results demonstrate that functional collaboration exists between p18 and Men1 and suggest that Men1 may regulate additional factor(s) that interact with p18 and p27 differently.     

p18 Ink4c and Pten constrain a positive regulatory loop between cell growth and cell cycle control

Inactivation of the Rb-mediated G1 control pathway is a common event found in many types of human tumors. To test how the Rb pathway interacts with other pathways in tumor suppression, we characterized mice with mutations in both the cyclin-dependent kinase (CDK) inhibitor p18 Ink4c and the lipid phosphatase Pten, which regulates cell growth. The double mutant mice develop a wider spectrum of tumors, including prostate cancer in the anterior and dorsolateral lobes, with nearly complete penetrance and at an accelerated rate. The remaining wild-type allele of Pten was lost at a high frequency in Pten+/- cells but not in p18+/- Pten+/- or p18-/- Pten+/- prostate tumor cells, nor in other Pten+/- tumor cells, suggesting a tissue- and genetic background-dependent haploinsufficiency of Pten in tumor suppression. p18 deletion, CDK4 overexpression, or oncoviral inactivation of Rb family proteins caused activation of Akt/PKB that was recessive to the reduction of PTEN activity. We suggest that p18 and Pten cooperate in tumor suppression by constraining a positive regulatory loop between cell growth and cell cycle control pathways.     

Interplay between p53 and Ink4c in spermatogenesis and fertility

The tumor suppressor p53, and the cyclin-dependent kinase inhibitor Ink4c, have been both implicated in spermatogenesis control. Both p53-/- and Ink4c-/- single knockout male mice are fertile, despite testicular hypertrophy, Leydig cell differentiation defect, and increased sperm count in Ink4c-/- males. To investigate their collaborative roles, we studied p53-/- Ink4c-/- dual knockout animals, and found that male p53-/- Ink4c-/- mice have profoundly reduced fertility. Dual knockout male mice show a marked decrease in sperm count, abnormal sperm morphology and motility, prolongation of spermatozoa proliferation and delay of meiosis entry, and accumulation of DNA damage. Genetic studies showed that the effects of p53 loss on fertility are independent of its downstream effector Cdkn1a. Absence of p53 also partially reverses the hyperplasia seen upon Ink4c loss, and normalizes the Leydig cell differentiation defect. These results implicate p53 in mitigating both the delayed entry into meiosis and the secondary apoptotic response that occur in the absence of Ink4c. We conclude that the cell cycle genes p53 and Ink4c collaborate in sperm cell development and differentiation, and may be important candidates to investigate in human male infertility conditions.     

p18INK4c and p27KIP1 are required for cell cycle arrest of differentiated myotubes

Myogenic differentiation is characterized by permanent and irreversible cell cycle withdrawal and increased resistance to apoptosis. These functions correlate with changes in expression and activity of several cyclin-dependent kinase inhibitors, including p18, p21, and p27. In this study, we examined the requirements for p18, p21, and p27 in initiating growth arrest in multinucleated myotubes under differentiation conditions and in maintaining terminal arrest upon restimulation of differentiated myotubes with mitogenic signals. Under differentiation conditions, only p27(-/-) or p18(-/-)p27(-/-) myotubes are capable of reentering the cell cycle and synthesizing DNA at a very low frequency. Escape from cell cycle arrest was significantly greater in p18(-/-)p27(-/-) myotubes than in p27(-/-) myotubes. Stimulation of differentiated cultures with a mitogen-rich growth medium enhances p18(-/-)p27(-/-) myotube proliferation to encompass approximately half of the nuclei. p18(-/-)p21(-/-) and p21(-/-)p27(-/-) myotubes remain terminally arrested. Nuclei within individual restimulated p18(-/-)p27(-/-) myotubes can be found in all phases of the cell cycle, and a myotube can be multiphasic without any obvious deleterious effects. Increasing the time of differentiation or serum stimulation of p18(-/-)p27(-/-) myotubes neither increases the proliferation index of the myotube nuclei, nor does it alter the percentage of nuclei in each of the cell cycle phases. During the first 24 h of serum stimulation, the p18(-/-)p27(-/-) myotube nuclei that escape G0 arrest will rearrest in either S or G2 phase, without either mitosis or endoreplication. Apoptosis is increased in restimulated p18(-/-)p27(-/-) myotube nuclei, but is not specific for any cell cycle phase. These results suggest a collaborative role for p18 and p27 in initiating and maintaining G0 arrest during myogenic differentiation. While p18 and p27 appear to be essential in initiating G0 arrest in a proportion of postmitotic myotube nuclei, there must be another cell cycle inhibitor protein that functions with p18 and p27 in maintaining terminal arrest. We propose that the combined rate-limiting expressions of p18, p27, and this other inhibitor determine whether the myotube nuclei will remain postmitotic, or reenter the cell cycle, and if the nuclei escape G0 arrest, in which phase of the cell cycle the nuclei will ultimately rearrest.     

Phosphorylation of p16INK4A correlates with Cdk4 association

Progression through the eukaryotic cell cycle is driven by the activity of cyclin-dependent kinases. The cyclin D-dependent kinase Cdk4 promotes progression through the G(1) phase of the cell cycle and is deregulated in many human tumors. The tumor suppressor protein p16(INK4A) (p16) forms a complex with Cdk4 and inhibits kinase activity. Here we report that p16 is phosphorylated, and the phosphorylated form of p16 is preferentially associated with Cdk4 in normal human fibroblasts. We mapped phosphorylation sites on exogenously overexpressed p16 to serines 7, 8, 140, and 152 and found that endogenous p16 associated with Cdk4 is phosphorylated at serine 152. All mapped phosphorylation sites lie outside of the conserved kinase-binding domain of p16 but in regions of the protein affected by mutations in familial and sporadic cancer. Our results suggest a novel regulation of p16 activity.     

Genetic evidence for 18S rRNA binding and an Rps19p assembly function of yeast nucleolar protein Nep1p

The nucleolar protein Nep1 and its human homologue were previously shown to be involved in the maturation of 18S rRNA and to interfere directly or indirectly with a methylation reaction. Here, we report that the loss-of-function mutation Δsnr57 and multicopy expression of the ribosomal 40S subunit protein 19 (Rps19p) can partially suppress the Saccharomyces cerevisiae Δnep1 growth defect. SnR57 mediates 2′-O-ribose-methylation of G₁₅₇₀ in the 18S rRNA. By performing a three-hybrid screen, we isolated several short RNA sequences with strong binding affinity to Nep1p. All isolated RNAs shared a six-nucleotide consensus motif C/UUCAAC. Furthermore, one of the isolated RNAs exactly corresponded to nucleotides 1553–1577 of the 18S rRNA, which includes G₁₅₇₀, the site of snR57-dependent 18S rRNA methylation. From protein–protein crosslink data and the cryo-EM map of the S. cerevisiae small ribosomal subunit, we suggest that Rps19p is localized in close vicinity to the Nep1p 18S rRNA binding site. Our results suggest that Nep1p binds adjacent to helix 47 of the 18S rRNA and possibly supports the association of Rps19p to pre-ribosomal particles.     

Oxidative stress regulates the interaction of p16 with Cdk4

Oxidative stress can have a myriad of effects on many different cell types. The mechanisms by which these effects occur are not completely known. Chimeric proteins of the GAL4 DNA binding domain and Cdk4, or the GAL4 activation domain with p16, were expressed in the yeast two-hybrid system. Cells expressing these chimeric proteins were cultured with hydrogen peroxide and decreases in beta-galactosidase activity were observed when compared to cells incubated without hydrogen peroxide. When cells, which expressed the intact GAL4 binding protein, were cultured in the presence of hydrogen peroxide the opposite was observed. Incubation of cells with buthionine sulfoximine augmented these responses to hydrogen peroxide. These data suggest that one of the mechanisms by which oxidative stress acts is via the modulation of protein-protein interactions and demonstrate that the yeast two-hybrid system may be a model by which to study protein interactions due to oxidative stress.     

Haploinsufficiency of p18(INK4c) sensitizes mice to carcinogen-induced tumorigenesis

The INK4 family of cyclin-dependent kinase (CDK) inhibitors negatively regulates cyclin D-dependent CDK4 and CDK6 and thereby retains the growth-suppressive function of Rb family proteins. Mutations in the CDK4 gene conferring INK4 resistance are associated with familial and sporadic melanoma in humans and result in a wide spectrum of tumors in mice. Whereas loss of function of other INK4 genes in mice leads to little or no tumor development, targeted deletion of p18(INK4c) causes spontaneous pituitary tumors and lymphoma late in life. Here we show that treatment of p18 null and heterozygous mice with a chemical carcinogen resulted in tumor development at an accelerated rate. The remaining wild-type allele of p18 was neither mutated nor silenced in tumors derived from heterozygotes. Hence, p18 is a haploinsufficient tumor suppressor in mice.     

Structural Analysis of the Inhibition of Cdk4 and Cdk6 by p16INK4a through Molecular Dynamics Simulations

Abstract

Cyclin-dependent kinases 4, 6 and 2 (Cdk4/6/2), are proteins that lead progression through the G1-S transition, a step strictly regulated in the process of cell proliferation. The p16^INK4a tumor suppressor, whose expression is inhibited in a high number of cancers, binds to Cdk4/6 and inhibits phosphorylation of the retinoblastoma protein, forcing cells to remain in the G1 phase and therefore, arresting cell division. Accordingly, the design of small compounds mimicking the inhibition of p16^INK4a appears to be a promising way to treat cancer. In order to get some insight into the key interactions governing recognition between different cyclin-dependent kinases and the p16^INK4a tumor suppressor, the present work reports the results of molecular dynamics simulations of both, the Cdk6-p16^INK4a complex and the Cdk4-p16^INK4a complex, respectively at 300 K. Most of the key interactions observed, were already anticipated in the analysis of the crystal structure of Cdk6-p16^INK4a. However, a few different features found out from the analysis of these calculations provide a better understanding of the role of the T-loop conformation, a fragment of Cdks, and the way the ATP binding-site is distorted upon binding of p16^INK4a.     

Tumor suppressor INK4: determination of the solution structure of p18INK4C and demonstration of the functional significance of loops in p18INK4C and p16INK4A

Since the structures of several ankyrin-repeat proteins including the INK4 (inhibitor of cyclin-dependent kinase 4) family have been reported recently, the detailed structures and the functional roles of the loops have drawn considerable interest. This paper addresses the potential importance of the loops of ankyrin-repeat proteins in three aspects. First, the solution structure of p18INK4C was determined by NMR, and the loop structures were analyzed in detail. The loops adapt nascent antiparallel beta-sheet structures, but the positions are slightly different from those in the crystal structure. A detailed comparison between the solution structures of p16 and p18 has also been presented. The determination of the p18 solution structure made such detailed comparisons possible for the first time. Second, the [1H,15N]HSQC NMR experiment was used to probe the interactions between p18INK4C and other proteins. The results suggest that p18INK4C interacts very weakly with dna K and glutathione S-transferase via the loops. The third aspect employed site-specific mutagenesis and functional assays. Three mutants of p18 and 11 mutants of p16 were constructed to test functional importance of loops and helices. The results suggest that loop 2 is likely to be part of the recognition surface of p18INK4C or p16INK4A for CDK4, and they provide quantitative functional contributions of specific residues. Overall, our results enhance understanding of the structural and functional roles of the loops in INK4 tumor suppressors in particular and in ankyrin-repeat proteins in general.     

Cellular response to oncogenic ras involves induction of the Cdk4 and Cdk6 inhibitor p15(INK4b)

The cell cycle inhibitor p15(INK4b) is frequently inactivated by homozygous deletion together with p16(INK4a) and p19(ARF) in some types of tumors. Although the tumor suppressor capability of p15(INK4b) is still questioned, it has been found to be specifically inactivated by hypermethylation in hematopoietic malignancies in the absence of p16(INK4a) alterations. Here we show that, in vitro, p15(INK4b) is a strong inhibitor of cellular transformation by Ras. Surprisingly, p15(INK4b) is induced in cultured cells by oncogenic Ras to an extent similar to that of p16(INK4a), and their expression is associated with premature G(1) arrest and senescence. Ras-dependent induction of these two INK4 genes is mediated mainly by the Raf-Mek-Erk pathway. Studies with activated and dominant negative forms of Ras effectors indicate that the Raf-Mek-Erk pathway is essential for induction of both the p15(INK4b) and p16(INK4a) promoters, although other Ras effector pathways can collaborate, giving rise to a stronger response. Our results indicate that p15(INK4b), by itself, is able to stop cell transformation by Ras and other oncogenes such as Rgr (a new oncogene member of the Ral-GDS family, whose action is mediated through Ras). In fact, embryonic fibroblasts isolated from p15(INK4b) knockout mice are susceptible to transformation by the Ras or Rgr oncogene whereas wild-type embryonic fibroblasts are not. Similarly, p15(INK4b)-deficient mouse embryo fibroblasts are more sensitive than wild-type cells to transformation by a combination of the Rgr and E1A oncogenes. The cell cycle inhibitor p15(INK4b) is therefore involved, at least in some cell types, in the tumor suppressor activity triggered after inappropriate oncogenic Ras activation in the cell.     

Structural analysis of the inhibition of Cdk4 and Cdk6 by p16(INK4a) through molecular dynamics simulations

Cyclin-dependent kinases 4, 6 and 2 (Cdk4/6/2), are proteins that lead progression through the G1-S transition, a step strictly regulated in the process of cell proliferation. The p16(INK4a) tumor suppressor, whose expression is inhibited in a high number of cancers, binds to Cdk4/6 and inhibits phosphorylation of the retinoblastoma protein, forcing cells to remain in the G1 phase and therefore, arresting cell division. Accordingly, the design of small compounds mimicking the inhibition of p16(INK4a) appears to be a promising way to treat cancer. In order to get some insight into the key interactions governing recognition between different cyclin-dependent kinases and the p16(INK4a) tumor suppressor, the present work reports the results of molecular dynamics simulations of both, the Cdk6-p16(INK4a) complex and the Cdk4-p16(INK4a) complex, respectively at 300 K. Most of the key interactions observed, were already anticipated in the analysis of the crystal structure of Cdk6-p16(INK4a). However, a few different features found out from the analysis of these calculations provide a better understanding of the role of the T-loop conformation, a fragment of Cdks, and the way the ATP binding-site is distorted upon binding of p16(INK4a).     

Genetic evidence for functional specificity of the yeast GCN2 kinase

In yeast the GCN2 kinase mediates translational control ofGCN4 by phosphorylating the subunit of eIF-2 in response to extracellular amino acid limitation. Although phosphorylation of eIF-2 has been shown to inhibit global protein synthesis, amino acid starvation results in a specific activation effect onGCN4 mRNA translation. Under the same conditions, translation of other mRNAs appears only slightly affected. The mechanism responsible for the observed selectivity of the GCN2 kinase is not clear. Here, we present genetic evidence that suggests that locally restricted action of the GCN2 kinase facilitatesGCN4-specific translational regulation.