Subject Review p 57 KIP 2 : “ Kip ” ing the Cell under Control

p57 is an imprinted gene located at the chromosomal locus 11p15.5. It is a cyclin-dependent kinase inhibitor belonging to the CIP/KIP family, which includes additionally p21 and p27. It is the least studied CIP/KIP member and has a unique role in embryogenesis. p57 regulates the cell cycle, although novel functions have been attributed to this protein including cytoskeletal organization. Molecular analysis of animal models and patients with Beckwith-Wiedemann Syndrome have shown its nodal implication in the pathogenesis of this syndrome. p57 is frequently down-regulated in many common human malignancies through several mechanisms, denoting its anti-oncogenic function. This review is a thorough analysis of data available on p57, in relation to p21 and p27, on gene and protein structure, its transcriptional and translational regulation, and its role in human physiology and pathology, focusing on cancer development. (Mol Cancer Res 2009;7(12):1902–19) Introduction Throughout the evolutionary process, cells have developed sophisticated molecular machinery in order to elicit, at any moment, the proper decisionwith respect to cell fate. For this reason, external and internal signals are continuously monitored, assimilated, processed, and/or integrated, via complex biochemical-signaling networks (1). At the heart of this complex, decision-making molecular machinery, lies a cell nucleus-located core that resembles, in its operation, a clock apparatus (1). The determination of the cell's fate toward proliferation, arrest, differentiation, quiescence, or apoptosis relies upon this cell clock (1). The decision for cell cycle progression to occur, which is synonymous with proliferation, requires timely activation, followed by subsequent ordered inactivation of cyclin-dependent kinases (CDK; ref. 1). CDKs are activated through the formation of heterodimers with cyclins, whose concentration alters periodically Received 7/20/09; revised 9/14/09; accepted 9/16/09; published OnlineFirst 11/24/09. Grant support: European Social Fund and National Resources-(EPEAEK-II), PYTHAGORAS II (grant number 7953), the European Commission funded FP7-projects GENICA and INFLA-CARE and the SARG-NKUA grant numbers: 70/3/1703, 70/4/9913, 70/4/4281. Request for reprints Vassilis G. Gorgoulis, Antaiou 53 Str. Lamprini, Ano Patissia, GR-11146 Athens, Greece. Phone: 30-210-7462352; Fax: 30-210-6535894. E-mail: [email protected] Copyright © 2009 American Association for Cancer Research. doi:10.1158/1541-7786.MCR-09-0317 1902 on June 21, 2017. mcr.aacrjournals.org Downloaded from during the cell cycle. Sequentially coordinated stimulation of different CDK-cyclin complexes ensures proper cell cycle progression (1). On the other hand, inhibitory effects over the activity of CDK-cyclin complexes may lead to cell cycle arrest (temporary or permanent), differentiation, senescence, quiescence, or apoptosis (1). Important negative regulators of CDKs are the CDK inhibitors (CKI). There are two families of CKIs, the INK4 and the CIP/KIP family. The INK4 family includes p16 (2), p15 (3), p18 (4), and p19 (5, 6). The INK4 members are structurally related, having four ankyrin repeats. They cause G1 arrest through competition with cyclin D, thus preventing formation of active complexes of CDK4/6-cyclin D. The CIP/KIP family includes p21/CDKN1A (7, 8), p27/CDKN1B (9, 10), and p57/CDKN1C (11, 12). The CIP/KIP family has a broader specificity for CDKs than the INK4 members. At low levels they bind to the CDK-cyclin heterodimer and promote their assembly, whereas at high levels they abrogate CDK activity. In this review we focus on the younger and least studied member of the CIP/KIP family, p57, presenting up-to-date evidence about the regulation of p57, its physiologic functions, and its impact in human pathology, focusing on carcinogenesis, in comparison with p21 and p27. Structure of the CDKN1C Gene and mRNA The human CDKN1C gene resides in the telomeric end of chromosome 11, at the 11p15.5 locus (Fig. 1A; ref. 12). Mouse and rat p57 reside in the distal region of chromosomes 7 and 1, respectively (13, 14). The coding region of CDKN1C consists of four exons separated by three introns (Fig. 1A; ref. 15). p57 is rich in CpG islands both upstream and downstream of the putative transcriptional start site (16). Analysis of the CDKN1C promoter revealed the following consensus binding sites: (i) several Sp-1 (Stimulatory protein-1), (ii) ETS (erythroblastosis virus E26 oncogene), (iii) a TATAbox [TBP (TATA box binding protein)], (iv) EGR1 (early growth response 1), (v) OCT1 (octamer-binding transcription factor 1), (vi) NF1 (neurofibromin 1), (vii) two CAT elements, (viii) a GRE (glucocorticoid response element), (ix) a binding site for the p63 isoform ΔNp63α, and (x) binding sites for the Hes1 (hairy and enhancer of split 1) and Herp2 (hes-related repressor protein 2) notch effectors (Fig. 1A; refs. 15, 17-21). Alternative splicing of p57 intron 1 at different 3′ splice acceptor sites produces three transcripts (Fig. 1A). The major transcript corresponds to the one with the whole intron 1 spliced out (15). The splicing and expression pattern is conserved among humans and rodents (14). Mol Cancer Res 2009;7(12). December 2009 © 2009 American Association for Cancer Research. A p57 Review 1903 Published OnlineFirst November 24, 2009; DOI: 10.1158/1541-7786.MCR-09-0317 p57 Protein Structure of p57 Protein Human and mouse p57 encode a 316and a 335-amino acid protein, respectively, both migrating as 57 kD by SDSPAGE electrophoresis (12). Mouse p57 protein consists of four distinct domains: (i) domain I (located in the aminoMol Cancer Res 2009;7(12). December 2009 on June 21, 2017. mcr.aacrjournals.org Downloaded from terminal region), a CDK inhibitory domain, sharing significant homology with the respective CDK inhibitory domain of p21 and p27; (ii) domain II, a proline rich region; (iii) domain III, comprising an exceptional acidic repeat, in which every fourth amino acid is glutamic or aspartic acid; and (iv) domain IV (located in the carboxy-terminal region), FIGURE 1. Structure of p57 gene and p57 protein; comparison with p27 and p21 proteins.Ai. Ideogram of human chromosome 11. Aii. Schematic of the 11p15.5 subchromosomal region. The human p57 gene is located in the centromeric region of 11p15.5. It is transcribed from the maternal allele (large arrow), because of imprinting of the paternal allele. However, there is a leaky expression from the paternal allele (small arrow). Aiii. Structure of the human p57gene. Transcribed regions (exons,Ex) are denoted in green boxes, with the coding regions hatched. Introns are depicted with red boxes. The human promoter encompasses several consensus binding sites for a series of transcription factors. Owing to a space limitation in the figure, see text for a full description of the transcription factors binding to the p57 gene promoter.Aiv.Splicing pattern of p57mRNA. Three transcripts are produced because of alternative splicing of intron 1 at 3′ different acceptor sites. A3 is themajor transcript produced from whole intron-1 splicing out.B.Mouse and human p57 proteins are conserved in the aminoand carboxy-terminal domains. The internal regions inmousehavebeensubstituted in humanwith thePAPAdomain,which is uniqueamong theCIP/KIP members. The amino-terminal region is highly homologous between all human CIP/KIP members encompassing the CDK inhibitory domain. The CDK inhibitory domain possesses the cyclin-binding domain, the CDK-binding domain, and the 310 helix (orange boxes). The carboxy-terminal domain of human p57 KIP2 has similarities with both p21 (PCNA-binding domain) and p27 (QT box). The PCNA-binding domain is not depicted in the mouse p57 carboxy-terminal domain, because, according to Watanabe et al. (28), mouse p57 exhibits a much weaker PCNA binding activity than human p57. © 2009 American Association for Cancer Research. Pateras et al. 1904 Published OnlineFirst November 24, 2009; DOI: 10.1158/1541-7786.MCR-09-0317 containing a conserved motif, named QT box (Fig. 1B). The QT box domain is significantly homologous with the carboxyterminal domain of p27. The human and mouse p57 aminoand carboxy-terminal domains are conserved, whereas the internal domains in the mouse p57 have been substituted in human p57 with a distinct domain rich in prolinealanine repeats, named the PAPA region (Fig. 1B; ref. 12). A putative nuclear localization signal (NLS) has been recognized in the QT box domain of the mouse p57, similar to the NLS in the carboxy-terminal of mouse p21 and mouse p27 (11). Spectroscopic and hydrodynamic analysis indicate that human p57 lacks a stable helical and β-sheet structure, although the formation of a secondary structure cannot be precluded (22). p57 as well as p21 and p27 are considered to be highly unfolded molecules (23, 24). Proteins that are fully or partially unfolded show a binding plasticity at the expense of thermodynamic stability, are degraded faster, and have an increased rate of evolution compared with folded on June 21, 2017. mcr.aacrjournals.org Downloaded from proteins (25-27). CIP/KIP family members have a broad specificity for CDKs and are short-lived proteins that may be attributed to their internal regional structure. The p57 Protein Interactions: Comparison with p21 and p27 p57 binds with CDKs in a cyclin-dependent manner (12). Association of p57, with cyclins only, is very weak. p57 inhibits in vitro the activity of the following CDKcyclin complexes: CDK2-cyclin E, CDK2-cyclin A, CDK3cyclin E, CDK4-cyclin D1, CDK4-cyclin D2, and to a lesser extent CDK1(CDC2)-cyclin B and CDK6-cyclin D2 (11, 12, 28). At low concentrations p57 can form active complexes with CDK2-cyclin A, enhancing their assembly, whereas at higher levels p57 inhibits the kinase activity of CDK2 (29). Similarly, CDK4-cyclin D1-heterodimer formation and activity is regulated in a p57 dose-dependent manner (28). This property is in accordance with the ability of both p21 and p27, at low concentrations, to promote FIGURE 2. Imprinting control regions of 11p15.5. The human chromosomal region 11p15.5 encompasses the H19-ICR (ICR-1) and the KCNQ1/KvDMRICR (ICR-2). H19-ICR is responsible for the imprinting status of several genes located in the telomeric end of 11p15.5 including H19 and IGF2. It is imprinted in the paternal allele (orange-dotted box), leading to the activation of the paternal IGF2 and to the suppression of the maternal H19. KCNQ1/KvDMR-ICR is located within intron 10 of the KCNQ1 gene in the promoter region of a noncoding antisense transcript named KCNQ1OT1/KvLQT1AS/LIT1. KCNQ1/KvDMRICR affects the imprinting status of the centromeric region of 11p15.5. The imprinting of the maternal ICR-2 (orange-dotted box) is associated with the suppression of the maternal LIT1 and the expression of the maternal genes p57 and KCNQ1. The arrows and the green boxes depict the active alleles, whereas the red boxes show the silent alleles. Mol Cancer Res 2009;7(12). December 2009 © 2009 American Association for Cancer Research. A p57 Review 1905 Published OnlineFirst November 24, 2009; DOI: 10.1158/1541-7786.MCR-09-0317 the assembly of CDK4-cyclin D complex, whereas at higher levels to abrogate the kinase activity of the CDK-cyclin heterodimers (30-32). The amino-terminal region of CIP/KIP members is necessary and sufficient for the inhibition of CDK-cyclin activity. It consists of three sites that are involved in the suppression of CDK-cyclin activity, a cyclin binding region, a CDK binding site, and a 310 helix (Fig. 1B). The 310 helix contains a phenylalanine-tyrosine pair of amino acids that is bound to the aminoterminal of CDK2, inside the catalytic cleft, mimicking the purine base of adenosine triphosphate (24). The 310 helix is indispensable for the in vivo inhibition of CDK2-cyclin A and CDK2-cyclin E by p57, but not for p21 and p27 (29). It has been proposed that the central region of p57 may serve as an important domain for protein interactions implicated in functions other than the CDK-inhibitory role (15). Indeed, the central region of mouse p57 interacts with the N-terminal of LIMK-1, a downstream effector of Rho (analyzed in the Cytoskeletal Organization section; ref. 33). The carboxy-terminus of p57 binds in vitro with proliferating cell nuclear antigen (PCNA) with a much lower affinity than p21 (34). In addition, p57 interacts with and inhibits the kinase activity of c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) through its QT domain (35). Interestingly, Mol Cancer Res 2009;7(12). December 2009 on June 21, 2017. mcr.aacrjournals.org Downloaded from p27 does not inhibit JNK/SAPK activity, although it contains a QT domain (35). p21 also has the ability to bind and suppress the activity of JNK/SAPK via the amino-terminal domain (36). p57, but not p21 or p27, interacts both in vivo and in vitro through its amino-terminal domain with the transcription factor B-Myb (37). B-Myb competes with Cyclin A2 for the association with p57. Overexpression of B-Myb partially overwhelms the G1 cell cycle arrest induced by p57 in Saos-2 cells. Expression and Tissue Distribution Pattern of p57 A highly cell-specific, temporal and spatial p57 expression profile has been found during the fetal, the postnatal, up to puberty, and the adult life cycle. Most of the data have been obtained from rodents (14, 38-44) and only sporadically in humans (11, 12, 44, 45). Specifically, the highest levels and most widespread tissue localization were observed during organogenesis. p57 is present in all major organs during embryonic development, and its levels of expression peaks at key stages of differentiation for each specific organ (14, 39, 41). Afterward, its expression declines to low or undetectable levels in several tissues (14, 39, 41), and only organs that mature after birth, such as testis (44), exhibit strong p57 staining in FIGURE 3. Methylation analysis of p57 promoter in normal and tumor human lung specimens. A. Methylation-specific PCR analysis shows promoter methylation in both normal (N) and tumor (T) lung tissue in a representative case. M+ and Mdenote the methylation-specific PCR analysis with primers designed to bind specifically to methylated and nonmethylated CpG islands, correspondingly. B. Pyrosequencing analysis of the same representative case (depicted in A), showing decreased promoter methylation [assayed as methylation index (MtI)] in the tumor (T) versus the corresponding normal (N) tissue. Note the different scale in the y-axis of the pyrosequencing diagram. © 2009 American Association for Cancer Research. Pateras et al. 1906 Published OnlineFirst November 24, 2009; DOI: 10.1158/1541-7786.MCR-09-0317 certain tissue compartments. Comparatively to prenatal tissues, fewer adult tissues continue to abundantly express p57 (11, 12). Nevertheless, an age-dependent p57 decline in some of these tissues has also been reported (42). Human adult tissues expressing significant p57 mRNA levels are mainly skeletal muscle, brain, heart, lung, kidney, pancreas, testis, and placenta (11, 12); this is in contrast with the wider tissue distribution of higher levels of human p21 and p27 mRNA (11, 12). In rodents, p57 expression correlates, on average, with the human distribution pattern (12, 14, 39, 41), but a few species-specific exceptions do exist (14, 39, 41, 44). Regulation of p57 Transcriptional Regulation of p57 Epigenetic control has emerged as an important mechanism for the transcriptional regulation of p57. The genetic locus of human p57, 11p15.5, harbors several imprinted genes (Fig. 2). Genomic imprinting refers to the differential epigenetic modulation of a gene leading to a parent-of-origin specific expression. This term means that the maternal and paternal allele of an imprinted gene display different properties, despite the fact that they have identical sequences. Epigenetic mechanisms include modulations of DNA methylation within CpG islands, chromatin remodeling (covalent modifications of histones), and silencing from various types of noncoding RNA transcripts (46). Methylation of DNA within CpG doublets, as well as methylation and deacetylation, in the aminoterminal tails of histones H3 and H4 are related with an inactive on June 21, 2017. mcr.aacrjournals.org Downloaded from gene state, whereas the opposite is associated with gene activation. The cluster of imprinted genes in specific chromosomal regions, suggests that they share a common regulatory mechanism. The imprinting of such clusters is regulated by specific sequences acting in cis, known as imprinted control regions (ICR). ICRs encompass the following features: (i) a differentially methylated region (DMR) and chromatin conformation status between the maternal and the paternal genome and (ii) the establishment of the imprinting marks during gametogenesis, because this is the only developmental period in which the maternal and paternal genome are separated (47, 48). The linkage of genes within the 11p15.5 region and the mouse homolog on distal chromosome 7 is conserved, showing the significance of the integrity of this region for the proper imprinting of the genes located therein (49-51). The regulation of imprinted genes in 11p15.5 is controlled by two ICRs, H19-ICR (ICR1) and KCNQ1/KvDMR-ICR (ICR2; Fig. 2). H19-ICR is located in the telomeric end of 11p15.5 and influences the imprinting of H19 and IGF2 by functioning as a chromatin insulator, restricting the access to shared enhancers (52). H19 is maternally expressed, whereas IGF2 is paternally transcribed. The KCNQ1-ICR coordinates the imprinting status of the more centromeric domain of 11p15.5 encompassing p57 and KCNQ1/KvLQT1 (53-56). p57 is maternally expressed, however there is a leaky expression from the paternal allele in most human tissues (57). The imprinted status of p57 is conserved between mice and human (13). p57 is hypomethylated in normal human kidneys (58), whereas we showed promoter methylation in normal human bronchial tissue (Fig. 3; ref. 59), suggesting the involvement of tissue-specific