Distinct versus redundant properties among members of the INK4 family of cyclin-dependent kinase inhibitors

p16(INK4a), p15(INK4b), p18(INK4c) and p19(INK4d) comprise a family of cyclin-dependent kinase inhibitors and tumor suppressors. We report that the INK4 proteins share the ability to arrest cells in G1, and interact with CDK4 or CDK6 with similar avidity. In contrast, only p18 and particularly p19 are phosphorylated in vivo, and each of the human INK4 proteins shows unique expression patterns dependent on cell and tissue type, and differentiation stage. Thus, the INK4 proteins harbor redundant as well as non-overlapping properties, suggesting distinct regulatory modes, and diverse roles for the individual INK4 family members in cell cycle control, cellular differentiation, and multistep oncogenesis.     

Design and characterization of a hyperstable p16INK4a that restores Cdk4 binding activity when combined with oncogenic mutations

Cyclin-dependent kinase inhibitor p16(INK4a) is the founding member of the INK4 family of tumor suppressors capable of arresting mammalian cell division. Missense mutations in the p16(INK4a) gene (INK4a/CDKN2A/MTS1) are strongly linked to several types of human cancer. These mutations are evenly distributed throughout this small, ankyrin repeat protein and the majority of them disrupt the native secondary and/or tertiary structure, leading to protein unfolding, aggregation and loss of function. We report here the use of multiple stabilizing substitutions to increase the stability of p16(INK4a) and furthermore, to restore Cdk4 binding activity of several defective, cancer-related mutant proteins. Stabilizing substitutions were predicted using four different techniques. The three most effective substitutions were combined to create a hyperstable p16(INK4a) variant that is 1.4 kcal/mol more stable than wild-type. This engineered construct is monomeric in solution with wild-type-like secondary and tertiary structure and cyclin-dependent kinase 4 binding activity. Interestingly, these hyperstable substitutions, when combined with oncogenic mutations R24P, P81L or V126D, can significantly restore Cdk4 binding activity, despite the divergent features of each destabilizing mutation. Extensive biophysical studies indicate that the hyperstable substitutions enhance the binding activity of mutant p16 through several different mechanisms, including an increased amount of secondary structure and thermostability, reduction in exposed hydrophobic surface(s) and/or a reduced tendency to aggregate. This apparent global suppressor effect suggests that increasing the thermodynamic stability of p16 can be used as a general strategy to restore the biological activity to defective mutants of this important tumor suppressor protein.     

INK4 proteins, a family of mammalian CDK inhibitors with novel biological functions

The cyclin D-Cdk4-6/INK4/Rb/E2F pathway plays a key role in controlling cell growth by integrating multiple mitogenic and antimitogenic stimuli. The members of INK4 family, comprising p16(INK4a), p15(INK4b), p18(INK4c), and p19(INK4d), block the progression of the cell cycle by binding to either Cdk4 or Cdk6 and inhibiting the action of cyclin D. These INK4 proteins share a similar structure dominated by several ankyrin repeats. Although they appear to be structurally redundant and equally potent as inhibitors, the INK4 family members are differentially expressed during mouse development. The striking diversity in the pattern of expression of INK4 genes suggested that this family of cell cycle inhibitors might have cell lineage-specific or tissue-specific functions. The INK4 proteins are commonly lost or inactivated by mutations in diverse types of cancer, and they represent established or candidate tumor suppressors. Apart from their capacity to arrest cells in the G1-phase of the cell cycle they have been shown to participate in an increasing number of cellular processes. Given their emerging roles in fundamental physiological as well as pathological processes, it is interesting to explore the diverse roles for the individual INK4 family members in different functions other than cell cycle regulation. Extensive studies, over the past few years, uncover the involvement of INK4 proteins in senescence, apoptosis, DNA repair, and multistep oncogenesis. We will focus the discussion here on these unexpected issues.     

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.     

INK4d-deficient mice are fertile despite testicular atrophy

The INK4 family of cyclin-dependent kinase (CDK) inhibitors includes four 15- to 19-kDa polypeptides (p16(INK4a), p15(INK4b), p18(INK4c), and p19(INK4d)) that bind to CDK4 and CDK6. By disrupting cyclin D-dependent holoenzymes, INK4 proteins prevent phosphorylation of the retinoblastoma protein and block entry into the DNA-synthetic phase of the cell division cycle. The founding family member, p16(INK4a), is a potent tumor suppressor in humans, whereas involvement, if any, of other INK4 proteins in tumor surveillance is less well documented. INK4c and INK4d are expressed during mouse embryogenesis in stereotypic tissue-specific patterns and are also detected, together with INK4b, in tissues of young mice. INK4a is expressed neither before birth nor at readily appreciable levels in young animals, but its increased expression later in life suggests that it plays some checkpoint function in response to cell stress, genotoxic damage, or aging per se. We used targeted gene disruption to generate mice lacking INK4d. These animals developed into adulthood, had a normal life span, and did not spontaneously develop tumors. Tumors did not arise at increased frequency in animals neonatally exposed to ionizing radiation or the carcinogen dimethylbenzanthrene. Mouse embryo fibroblasts, bone marrow-derived macrophages, and lymphoid T and B cells isolated from these animals proliferated normally and displayed typical lineage-specific differentiation markers. Males exhibited marked testicular atrophy associated with increased apoptosis of germ cells, although they remained fertile. The absence of tumors in INK4d-deficient animals demonstrates that, unlike INK4a, INK4d is not a tumor suppressor but is instead involved in spermatogenesis.     

Contribution of salt bridges near the surface of a protein to the conformational stability

Salt bridges play important roles in the conformational stability of proteins. However, the effect of a surface salt bridge on the stability remains controversial even today; some reports have shown little contribution of a surface salt bridge to stability, whereas others have shown a favorable contribution. In this study, to elucidate the net contribution of a surface salt bridge to the conformational stability of a protein, systematic mutant human lysozymes, containing one Glu to Gln (E7Q) and five Asp to Asn mutations (D18N, D49N, D67N, D102N, and D120N) at residues where a salt bridge is formed near the surface in the wild-type structure, were examined. The thermodynamic parameters for denaturation between pH 2.0 and 4.8 were determined by use of a differential scanning calorimeter, and the crystal structures were analyzed by X-ray crystallography. The denaturation Gibbs energy (DeltaG) of all mutant proteins was lower than that of the wild-type protein at pH 4, whereas there was little difference between them near pH 2. This is caused by the fact that the Glu and Asp residues are ionized at pH 4 but protonated at pH 2, indicating a favorable contribution of salt bridges to the wild-type structure at pH 4. Each contribution was not equivalent, but we found that the contributions correlate with the solvent inaccessibility of the salt bridges; the salt bridge contribution was small when 100% accessible, while it was about 9 kJ/mol if 100% inaccessible. This conclusion indicates how to reconcile a number of conflicting reports about role of surface salt bridges in protein stability. Furthermore, the effect of salts on surface salt bridges was also examined. In the presence of 0.2 M KCl, the stability at pH 4 decreased, and the differences in stability between the wild-type and mutant proteins were smaller than those in the absence of salts, indicating the compensation to the contribution of salt bridges with salts. Salt bridges with more than 50% accessibility did not contribute to the stability in the presence of 0.2 M KCl.     

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.     

A hyperthermophilic protein acquires function at the cost of stability

Active-site residues are not often optimized for conformational stability (activity-stability trade-offs) in proteins from organisms that grow at moderate temperature. It is unknown if the activity-stability trade-offs can be applied to proteins from hyperthermophiles. Because enzymatic activity usually increases at higher temperature and hyperthermophilic proteins need high conformational stability, they might not sacrifice the stability for their activity. This study attempts to clarify the contribution of active-site residues to the conformational stability of a hyperthermophilic protein. We therefore examined the thermodynamic stability and enzymatic activity of wild-type and active-site mutant proteins (D7N, E8A, E8Q, D105A, and D135A) of ribonuclease HII from Thermococcus kodakaraensis (Tk-RNase HII). Guanidine hydrochloride (GdnHCl)-induced denaturation was measured with circular dichroism at 220 nm, and heat-induced denaturation was studied with differential scanning calorimetry. Both GdnHCl- and heat-induced denaturation were highly reversible in these proteins. All the mutations of these active-site residues, except that of Glu8 to Gln, reduced the enzymatic activity dramatically but increased the protein stability by 7.0 to 11.1 kJ mol(-1) at 50 degrees C. The mutation of Glu8 to Gln did not seriously affect the enzymatic activity and increased the stability only by 2.5 kJ mol(-1) at 50 degrees C. These results indicate that hyperthermophilic proteins also exhibit the activity-stability trade-offs. Therefore, the architectural mechanism for hyperthermophilic proteins is equivalent to that for proteins at normal temperature.     

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.     

Requirements for cell cycle arrest by p16INK4a

Analysis of tumor-derived mutations has led to the suggestion that p16INK4a, cyclin D1, cdk4, and the retinoblastoma protein (pRB) are components of a regulatory pathway that is inactivated in most tumor cells. Cell cycle arrest induced by p16INK4a, an inhibitor of cyclin D-dependent kinases, requires pRB, and it has been proposed that this G1 arrest is mediated by pRB-E2F repressor complexes. By comparing the properties of primary mouse embryonic fibroblasts specifically lacking pRB-family members, we find that pRB is insufficient for a p16INK4a-induced arrest. In addition to pRB, a second function provided by either p107 or p130, two pRB-related proteins, is required for p16INK4a to block DNA synthesis. We infer that p16INK4a-induced arrest is not mediated exclusively by pRB, but depends on the nonredundant functions of at least two pRB-family members.     

Direct binding of the N-terminus of HTLV-1 tax oncoprotein to cyclin-dependent kinase 4 is a dominant path to stimulate the kinase activity

The involvement of Tax oncoprotein in the INK4-CDK4/6-Rb pathway has been regarded as a key factor for immortalization and transformation of human T-cell leukemia virus 1 (HTLV-1) infected cells. In both p16 -/- and +/+ cells, expression of Tax has been correlated with an increase in CDK4 activity, which subsequently increases the phosphorylation of Rb and drives the infected cells into cell cycle progression. In relation to these effects, Tax has been shown to interact with two components of the INK4-CDK4/6-Rb pathway, p16 and cyclin D(s). While Tax competes with CDK4 for p16 binding, thus suppressing p16 inhibition of CDK4, Tax also binds to cyclin D(s) with concomitant increases in both CDK4 activity and the phosphorylation of cyclin D(s). Here we show that both Tax and residues 1-40 of the N-terminus of Tax, Tax40N, bind to and activate CDK4 in vitro. In the presence of INK4 proteins, binding of Tax and Tax40N to CDK4 counteracts against the inhibition of p16 and p18 and acts as the major path to regulate Tax-mediated activation of CDK4. We also report that Tax40N retains the transactivation ability. These results of in vitro studies demonstrate a potentially novel, p16-independent route to regulate CDK4 activity by the Tax oncoprotein in HTLV-1 infected cells.     

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.     

Human papillomavirus type 16 E6 promotes retinoblastoma protein phosphorylation and cell cycle progression

We show that E6 proteins from benign human papillomavirus type 1 (HPV1) and oncogenic HPV16 have the ability to alter the regulation of the G(1)/S transition of the cell cycle in primary human fibroblasts. Overexpression of both viral proteins induces cellular proliferation, retinoblastoma (pRb) phosphorylation, and accumulation of products of genes that are negatively regulated by pRb, such as p16(INK4a), CDC2, E2F-1, and cyclin A. Hyperphosphorylated forms of pRb are present in E6-expressing cells even in the presence of ectopic levels of p16(INK4a). The E6 proteins strongly increased the cyclin A/cyclin-dependent kinase 2 (CDK2) activity, which is involved in pRb phosphorylation. In addition, mRNA and protein levels of the CDK2 inhibitor p21(WAF1/CIP1) were strongly down-regulated in cells expressing E6 proteins. The down-regulation of the p21(WAF1/CIP1) gene appears to be independent of p53 inactivation, since HPV1 E6 and an HPV16 E6 mutant unable to target p53 were fully competent in decreasing p21(WAF1/CIP1) levels. E6 from HPV1 and HPV16 also enabled cells to overcome the G(1) arrest imposed by oncogenic ras. Immunofluorescence staining of cells coexpressing ras and E6 from either HPV16 or HPV1 revealed that antiproliferative (p16(INK4a)) and proliferative (Ki67) markers were coexpressed in the same cells. Together, these data underline a novel activity of E6 that is not mediated by inactivation of p53.     

The atr protein kinase controls UV-dependent upregulation of p16INK4A through inhibition of Skp2-related polyubiquitination/degradation

The tumor suppressor p16(INK4A), a phosphoprotein that exists in human cells under both phosphorylated and nonphosphorylated forms, plays crucial roles during the cellular response to UV light. However, it is still unclear how this protein is activated in response to this carcinogenic agent. We have shown here that UVC upregulates p16(INK4A) and the phosphorylated form of the protein at the 4 serine sites; Ser-7, Ser-8, Ser-140, and Ser-152. This accumulation of p16(INK4A) occurred through increasing the stability of both forms of the protein. Importantly, phospho-p16(INK4A) showed much higher stability, and UV treatment strongly increased its level in absence of de novo protein synthesis. Furthermore, we have shown that the UV-dependent upregulation of both forms of p16(INK4A) is under the control of the protein kinase Atr, which suppresses their UVC-dependent proteasomal degradation. Interestingly, although this degradation is ubiquitin-related for p16(INK4A) through the Skp2 ubiquitin ligase protein, it is ubiquitin-independent for the phosphorylated form. In addition, we present clear evidence that Skp2 is upregulated in ATR-deficient cells, leading to the downregulation of the p27(Kip1) protein in response to UVC light. Moreover, we have shown a preferential association of endogeneous phospho-p16(INK4A) with Cdk4. This association increased following UV-treatment mainly for p16(INK4A) phosphorylated at Ser-140 and Ser-152. Besides, we have shown that Atr regulates UV-related p16/Cdk4-dependent and -independent phosphorylation of pRB and G1 cell cycle delay. Together, these results indicate that p16(INK4A) and p27(Kip1) are key targets in the Atr-dependent signaling pathway in response to UV damage.     

Rb independent inhibition of cell growth by p15(INK4B).

The INK4 cyclin dependent kinase inhibitors (CDKI), such as p15(INK4B) and p16(INK4A), block cell cycle progression from G to S phase. This is mediated by inhibition of phosphorylation of proteins, including the retinoblastoma susceptibility protein (Rb), by cyclin dependent kinases. Ectopic over-expression of the p16(INK4A) CDKI can inhibit growth of cell lines depending on Rb status. Cell lines lacking Rb, with few exceptions, are resistant to growth inhibition by p16(INK4A). The effects of ectopic over-expression of p15(INK4B) in cell lines with and without wild type Rb were examined by measuring cell recovery. Proliferation was inhibited in cells lacking Rb as well as in cells with wild type Rb expression. Experiments analyzing the effectiveness of chimeric p15(INK4B)/p16(INK4A) proteins indicated that the Rb independent growth inhibition required N-terminal residues of p15(INK4B). Linker insertion mutation of p15(INK4B) showed that the inhibition was dependent on intact ankyrin structures. Double staining flow cytometry found that the growth inhibition correlated with a decrease in cells in G2/M phases of the cell cycle. These findings are consistent with Rb independent inhibition of the progression from G1 to S caused by overexpression of p15(INK4B).     

Role of non-glycine residues in left-handed helical conformation for the conformational stability of human lysozyme.

To understand the role of non-Gly residues in the left-handed helical conformation for the conformational stability of a protein, the non-Gly to Gly and Ala mutations at six left-handed residues (R21, Y38, R50, Q58, H78, and N118) of the human lysozyme were examined. The thermodynamic parameters for denaturation were determined using a differential scanning calorimeter, and the crystal structures were analyzed by X-ray crystallography. If a left-handed non-Gly had an unfavorable steric interaction between the side-chain Cbeta and backbone, the Gly mutation would be expected to stabilize more than the Ala mutation at the same position. For the mutant human lysozymes, however, there were few differences in the denaturation Gibbs energy (DeltaG) between the Gly and Ala mutants, except for the substitution at position 58. Analysis of the changes in stability (DeltaDeltaG) based on the structures of the wild-type and mutant proteins showed that the experimental DeltaDeltaG value of Q58G was approximately 7 kJ/mol higher than the estimated value without consideration of any local steric interaction. These results indicate that only Q58G increased the stability by elimination of local constraints. The residue 58 is located at the most rigid position in the left-handed non-Gly residues and is involved in its enzymatic function. It can be concluded that the left-handed non-Gly residues do not always have unfavorable strain energies as compared with Gly at the same position.     

EB病毒感染人胎鼻咽上皮细胞逃避老化期过程中抑制p16INK4a表达

细胞周期调控紊乱是细胞永生化进程中一个重要的分子事件,本文利用Western blotting和S-P法分别检测p16~(INK4a)、p53、p21~(WAF1/CIP1)和E2F1的蛋白表达,试图从细胞周期调控的角度,探讨EB病毒诱导人胎鼻咽上皮细胞逃避老化期的分子机制。结果表明,EB病毒通过抑制p16~(INK4a)表达而阻断p16~(INK4a)/Rb途径,上调转录因子E2F1,而对p53、p21~(WAF1/CIP1)表达无明显的影响。结果初步揭示,EB病毒介导的p16~(INK4a)/Rb/E2F1细胞周期调控紊乱参与了人胎鼻咽上皮细胞逃避老化期过程,为进一步探讨鼻咽癌发病机制提供了科学依据。     

The p16(INK4a) tumour suppressor protein inhibits alphavbeta3 integrin-mediated cell spreading on vitronectin by blocking PKC-dependent localization of alphavbeta3 to focal contacts

Expression of full-length p16(INK4a) blocks alphavbeta3 integrin-dependent cell spreading on vitronectin but not collagen IV. Similarly, G1-associated cell cycle kinases (CDK) inhibitory (CKI) synthetic peptides derived from p16(INK4a), p18(INK4c) and p21(Cip1/Waf1), which can be delivered directly into cells from the tissue culture medium, do not affect non-alphavbeta3-dependent spreading on collagen IV, laminin and fibronectin at concentrations that inhibit cell cycle progression in late G1. The alphavbeta3 heterodimer remains intact after CKI peptide treatment but is immediately dissociated from the focal adhesion contacts. Treatment with phorbol 12-myristate 13-acetate (PMA) allows alphavbeta3 to locate to the focal adhesion contacts and the cells to spread on vitronectin in the presence of CKI peptides. The cdk6 protein is found to suppress p16(INK4a)-mediated inhibition of spreading and is also shown to localize to the ruffling edge of spreading cells, indicating a function for cdk6 in controlling matrix-dependent cell spreading. These results demonstrate a novel G1 CDK-associated integrin regulatory pathway that acts upstream of alphavbeta3-dependent activation of PKC as well as a novel function for the p16(INK4a) tumour suppressor protein in regulating matrix-dependent cell migration.     

EB病毒感染人胎鼻咽上皮细胞逃避老化期过程中抑制p16^INK4a表达

细胞周期调控紊乱是细胞永生化进程中一个重要的分子事件,本文利用Western blotting和S-P法分别检测p16^INK4a、p53、p21^WAF1／CIP1和E2F1的蛋白表达,试图从细胞周期调控的角度,探讨EB病毒诱导人胎鼻咽上皮细胞逃避老化期的分子机制。结果表明,EB病毒通过抑制p16^INK4a表达而阻断p16^INK4a／Rb途径,上调转录因子E2F1,而对p53、p21^WAF1／CIP表达无明显的影响。结果初步揭示,EB病毒介导的p16^INK4a／Rb／E2F1细胞周期调控紊乱参与了人胎鼻咽上皮细胞逃避老化期过程,为进一步探讨鼻咽癌发病机制提供了科学依据。