Novel INK4 proteins, p19 and p18, are specific inhibitors of the cyclin D-dependent kinases CDK4 and CDK6.

Cyclin D-dependent kinases act as mitogen-responsive, rate-limiting controllers of G1 phase progression in mammalian cells. Two novel members of the mouse INK4 gene family, p19 and p18, that specifically inhibit the kinase activities of CDK4 and CDK6, but do not affect those of cyclin E-CDK2, cyclin A-CDK2, or cyclin B-CDC2, were isolated. Like the previously described human INK4 polypeptides, p16INK4a/MTS1 and p15INK4b/MTS2, mouse p19 and p18 are primarily composed of tandemly repeated ankyrin motifs, each ca. 32 amino acids in length, p19 and p18 bind directly to CDK4 and CDK6, whether untethered or in complexes with D cyclins, and can inhibit the activity of cyclin D-bound cyclin-dependent kinases (CDKs). Although neither protein interacts with D cyclins or displaces them from preassembled cyclin D-CDK complexes in vitro, both form complexes with CDKs at the expense of cyclins in vivo, suggesting that they may also interfere with cyclin-CDK assembly. In proliferating macrophages, p19 mRNA and protein are periodically expressed with a nadir in G1 phase and maximal synthesis during S phase, consistent with the possibility that INK4 proteins limit the activities of CDKs once cells exit G1 phase. However, introduction of a vector encoding p19 into mouse NIH 3T3 cells leads to constitutive p19 synthesis, inhibits cyclin D1-CDK4 activity in vivo, and induces G1 phase arrest.     

CDK4 and CDK6 delay senescence by kinase-dependent and p16INK4a-independent mechanisms

Replicative senescence of human diploid fibroblasts (HDFs) is largely implemented by the cyclin-dependent kinase (CDK) inhibitors p16(INK4a) and p21(CIP1). Their accumulation results in a loss of CDK2 activity, and cells arrest with the retinoblastoma protein (pRb) in its hypophosphorylated state. It has become standard practice to bypass the effects of p16(INK4a) by overexpressing CDK4 or a variant form that is unable to bind to INK4 proteins. Although CDK4 and CDK6 and their INK4-insensitive variants can extend the life span of HDFs, they also cause a substantial increase in the levels of endogenous p16(INK4a). Here we show that CDK4 and CDK6 can extend the life span of HDFs that have inactivating mutations in both alleles of INK4a or in which INK4a levels are repressed, indicating that overexpression of CDK4/6 is not equivalent to ablation of p16(INK4a). However, catalytically inactive versions of these kinases are unable to extend the replicative life span, suggesting that the impact of ectopic CDK4/6 depends on their ability to phosphorylate as yet unidentified substrates rather than to sequester CDK inhibitors. Since p16(INK4a) deficiency, CDK4 expression, and p53 or p21(CIP1) ablation have additive effects on replicative life span, our results underscore the idea that senescence is an integrated response to diverse signals.     

Isolation and characterization of p19INK4d, a p16-related inhibitor specific to CDK6 and CDK4.

Cyclin-dependent kinases 4 and 6 are complexed with many small cellular proteins in vivo. We have isolated cDNA sequences, INK4d, encoding a 19-kDa protein that is associated with CDK6 in several hematopoietic cell lines. p19 shares equal similarity and a common ancestor with other identified inhibitors of the p16/INK4 family. p19 interacts with and inhibits the activity of both CDK4 and CDK6 and exhibits no detectable interaction with the other known CDKs. p19 protein is present in both cell nuclei and cytoplasm. The p19 gene has been mapped to chromosome 19p13.2, and the level of its mRNA expression varies widely between different tissues. In contrast to p21 and p27 whose interaction with CDK subunits is dependent on or stimulated by the cyclin subunit, the interaction of p19 and p18 with CDK6 is hindered by the cyclin protein. Binary cyclin D1-p18/p19 or cyclin D1-CDK6 complexes are highly stable and cannot be dissociated by excess amounts of cyclin D1 or p19/p18 proteins, suggesting that p16 inhibitors and D cyclins may interact with CDKs 4 and 6 in a competing or potentially mutually exclusive manner.     

An important role of CDK inhibitor p18(INK4c) in modulating antigen receptor-mediated T cell proliferation.

The inhibitors of cyclin-dependent kinase (CDK) 4 (INK4) bind CDK4/6 to prevent their association with D-cyclins and G(1) cell cycle initiation and progression. We report here that among the seven CDK inhibitors, p18(INK4c) played an important role in modulating TCR-mediated T cell proliferation. Loss of p18(INK4c) in T cells led to hyperproliferation in response to CD3 stimulation. p18(INK4c)-null mice developed lymphoproliferative disorder and T cell lymphomas. Expression of IL-2, IL-2R-alpha, and the major G(1) cell cycle regulatory proteins was not altered in p18-null T cells. Both FK506 and rapamycin efficiently inhibited proliferation of p18-null T cells. In activated T cells, p18(INK4c) remained constant, and preferentially associated with and inhibited CDK6 but not CDK4. We propose that p18(INK4c) sets an inhibitory threshold in T cells and one function of CD28 costimulation is to counteract the p18(INK4c) inhibitory activity on CDK6-cyclin D complexes. The p18(INK4c) protein may provide a novel target to modulate T cell immunity.     

An Opposite Effect of the CDK Inhibitor,p18INK4c on Embryonic Stem Cells Compared with Tumor and Adult Stem Cells

Self-renewal is a feature common to both adult and embryonic stem (ES) cells, as well as tumor stem cells (TSCs). The cyclin-dependent kinase inhibitor, p18^INK4c, is a known tumor suppressor that can inhibit self-renewal of tumor cells or adult stem cells. Here, we demonstrate an opposite effect of p18 on ES cells in comparison with teratoma cells. Our results unexpectedly showed that overexpression of p18 accelerated the growth of mouse ES cells and embryonic bodies (EB); on the contrary, inhibited the growth of late stage teratoma. Up-regulation of ES cell markers (i.e., Oct4, Nanog, Sox2, and Rex1) were detected in both ES and EB cells, while concomitant down-regulation of various differentiation markers was observed in EB cells. These results demonstrate that p18 has an opposite effect on ES cells as compared with tumor cells and adult stem cells. Mechanistically, expression of CDK4 was significantly increased with overexpression of p18 in ES cells, likely leading to a release of CDK2 from the inhibition by p21 and p27. As a result, self-renewal of ES cells was enhanced. Our current study suggests that targeting p18 in different cell types may yield different outcomes, thereby having implications for therapeutic manipulations of cell cycle machinery in stem cells.     

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.     

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.     

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.     

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.     

Structure-based design of p18INK4c proteins with increased thermodynamic stability and cell cycle inhibitory activity

p18(INK4c) is a member of the INK4 family of proteins that regulate the G(1) to S cell cycle transition by binding to and inhibiting the pRb kinase activity of cyclin-dependent kinases 4 and 6. The p16(INK4a) member of the INK4 protein family is altered in a variety of cancers and structure-function studies of the INK4 proteins reveal that the vast majority of missense tumor-derived p16(INK4a) mutations reduce protein thermodynamic stability. Based on this observation, we used p18(INK4c) as a model to test the proposal that INK4 proteins with increased stability might have enhanced cell cycle inhibitory activity. Structure-based mutagenesis was used to prepare p18(INK4c) mutant proteins with a predicted increase in stability. Using this approach, we report the generation of three mutant p18(INK4C) proteins, F71N, F82Q, and F92N, with increased stability toward thermal denaturation of which the F71N mutant also showed an increased stability to chemical denaturation. The x-ray crystal structures of the F71N, F82Q, and F92N p18INK4C mutant proteins were determined to reveal the structural basis for their increased stability properties. Significantly, the F71N mutant also showed enhanced CDK6 interaction and cell cycle inhibitory activity in vivo, as measured using co-immunoprecipitation and transient transfection assays, respectively. These studies show that a structure-based approach to increase the thermodynamic stability of INK4 proteins can be exploited to prepare more biologically active molecules with potential applications for the development of molecules to treat p16(INK4a)-mediated cancers.     

The expression of p18INK4 and p27kip1 cyclin-dependent kinase inhibitors is regulated differently during human B cell differentiation

Cell cycle progression is under the control of cyclin-dependent kinases (cdks), the activity of which is dependent on the expression of specific cdk inhibitors. In this paper we report that the two cdk inhibitors, p27(Kip1) and p18(INK4c), are differently expressed and control different steps of human B lymphocyte activation. Resting B cells contain large amounts of p27(Kip1) and no p18(INK4c). In vitro stimulation by Staphylococcus aureus Cowan 1 strain or CD40 ligand associated with IL-10 and IL-2 induces a rapid decrease in p27(Kip1) expression combined with cell cycle entry and progression. In contrast, in vitro Ig production correlates with specific expression of p18(INK4c) and early G(1) arrest. This G(1) arrest is associated with inhibition of cyclin D3/cdk6-mediated retinoblastoma protein phosphorylation by p18(INK4c). A similar contrasting pattern of p18(INK4c) and p27(Kip1) expression is observed both in B cells activated in vivo and in various leukemic cells. Expression of p18(INK4c) was also detected in various Ig-secreting cell lines in which both maximum Ig secretion and specific p18(INK4c) expression were observed during the G(1) phase. Our study shows that p27(Kip1) and p18(INK4c) have different roles in B cell activation; p27(Kip1) is involved in the control of cell cycle entry, and p18(INK4c) is involved in the subsequent early G(1) arrest necessary for terminal B lymphocyte differentiation.     

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.     

Identification of human and mouse p19, a novel CDK4 and CDK6 inhibitor with homology to p16ink4.

The cell cycle in mammalian cells is regulated by a series of cyclins and cyclin-dependent kinases (CDKs). The G1/S checkpoint is mainly dictated by the kinase activities of the cyclin D-CDK4 and/or cyclin D-CDK6 complex and the cyclin E-CDK2 complex. These G1 kinases can in turn be regulated by cell cycle inhibitors, which may cause the cells to arrest at the G1 phase. In T-cell hybridomas, addition of anti-T-cell receptor antibody results not only in G1 arrest but also in apoptosis. In searching for a protein(s) which might interact with Nur77, an orphan steroid receptor required for activation-induced apoptosis of T-cell hybridomas, we have cloned a novel human and mouse CDK inhibitor, p19. The deduced p19 amino acid sequence consists of four ankyrin repeats with 48% identity to p16. The human p19 gene is located on chromosome 19p13, distinct from the positions of p18, p16, and p15. Its mRNA is expressed in all cell types examined. The p19 fusion protein can associate in vitro with CDK4 but not with CDK2, CDC2, or cyclin A, B, E, or D1 to D3. Addition of p19 protein can lead to inhibition of the in vitro kinase activity of cyclin D-CDK4 but not that of cyclin E-CDK2. In T-cell hybridoma DO11.10, p19 was found in association with CDK4 and CDK6 in vivo, although its association with Nur77 is not clear at this point. Thus, p19 is a novel CDK inhibitor which may play a role in the cell cycle regulation of T cells.     

p21(WAF1) is associated with CDK2 and CDK4 protein during HL-60 cell differentiation by TPA treatment

TPA-treated HL-60 cells are mainly arrested in G1 by p21(WAF1) accumulation. We investigate the downstream changes following such accumulation. Increased p21(WAF1) is associated with CDK2 and CDK4. pRb is dephosphorylated in the presence of p21-CDK2/4 complexes, and the Rb-E2F1 complex increases after TPA treatment, whereas the Rb-HDAC1 complex decreases slightly. Our results suggest that increased p21(WAF1) is associated with CDK2/4, and that these complexes induce pRb dephosphorylation. In turn, hypophosphorylated pRb are mainly complexed with E2F1, but HDAC1 appears not to be a key component in this process.     

Induction of apoptosis in p16INK4A mutant cell lines by adenovirus-mediated overexpression of p16INK4A protein

The tumor suppressor gene p16INK4A is a cyclin-dependent kinase inhibitor (CDKI) and an important cell cycle regulator. We have previously constructed a recombinant adenovirus which expresses p16 (Adp16) and shown that infection in a variety of human tumor cell lines with this recombinant virus results in high levels of p16INK4A protein expression resulting in cell cycle arrest and loss of cyclin-cdk activity. Furthermore, adenoviral-mediated overexpression of wild-type p16INK4A is more toxic in cancer cells which express mutant forms of p16INK4A compared to cancer cell lines containing endogenous wild-type p16. TUNEL assay and DAPI staining following infection of MDA-MB 231 breast cancer cells with Adp16 indicate that p16INK4A-mediated cytotoxicity was associated with apoptosis. This is supported by studies demonstrating a decrease in cpp32 and cyclinB1 protein levels and induction of poly (ADP-ribose) polymerase (PARP) cleavage following infection of MDA-MB-231 cells with Adp16. These results suggest that gene therapy using Adp16 may be a promising treatment option for human cancers containing alterations in p16 expression.     

Tumor suppressor INK4: quantitative structure-function analyses of p18INK4C as an inhibitor of cyclin-dependent kinase 4

We report the first detailed structure-function analyses of p18INK4C (p18), which is a homologue of the important tumor suppressor p16INK4A (p16). Twenty-four mutants were designed rationally. The global conformations of the mutants were characterized by NMR, while the function was assayed by inhibition of cyclin-dependent kinase 4 (CDK4). Most of these mutants have unperturbed global structures, thus the changes in their inhibitory abilities can be attributed to the mutated residues. The important results are summarized as follows: (a) some residues at loops 1 and 2, but not 3, are important for the inhibitory function of p18, similar to the results for p16; (b) two residues at the first helix-turn-helix motif and two at the third are important for inhibition; (c) while the results generally agree with the prediction based on the crystal structures of p16-CDK6 and p19-CDK6 binary complexes, there are significant differences in a few residues, suggesting that the interactions in the binary complexes may not accurately represent the interactions in the ternary complexes (in the presence of cyclin D2); (d) most importantly, the extra loop of p18 appears to contribute to the function of p18, even though the crystal structure of the p19INK4D-CDK6 complex indicates no interactions involving this loop; (e) detailed analyses of the crystal structures and the functional results suggest that there are notable differences in the interactions between different members of the INK4 family and CDKs.