CURRENT TOPIC pubs.acs.org/biochemistry Regulatory Mechanisms of Tumor Suppressor P16INK4A and Their Relevance to Cancer † ‡ § || Junan Li,*, , Ming Jye Poi, and Ming-Daw Tsai † ‡ Division of Environmental Health Sciences, College of Public Health, and Comprehensive Cancer Center and § Department of Pharmacy, The Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, The Ohio State University, Columbus, Ohio 43210, United States Genomics) Research Center and Institute of Biological Chemistry, Academia Sinica, Taiwan ABSTRACT: P16INK4A (also known as P16 and MTS1), a protein consisting exclusively of four ankyrin repeats, is recognized as a tumor suppressor mainly because of the prevalence of genetic inactivation of the p16INK4A (or CDKN2A) gene in virtually all types of human cancers. However, it has also been shown that an elevated level of expression (upregulation) of P16 is involved in cellular senescence, aging, and cancer progression, indicating that the regulation of P16 is critical for its function. Here, we discuss the regulatory mechanisms of P16 function at the DNA level, the transcription level, and the posttranscriptional level, as well as their implications for the structureÀfunction relationship of P16 and for human cancers. 16, also designated as MTS1 and P16INK4A, is one of the most ’ BASIC BIOCHEMICAL FEATURES OF P16 Pextensively studied proteins in the past decades because of its In 1993, a truncated version of the p16 cDNA gene was first critical roles in cell cycle progression, cellular senescence, and the fi 1À5 identi ed in a yeast two-hybrid screen for proteins that development of human cancers. At the G1-to-S transition, interact with human CDK4.12,13 The cDNA encoded a P16 specifically inhibits cyclin-dependent kinase 4 and 6 (CDK4 polypeptide of 148 amino acid residues with an estimated and CDK6, respectively)-mediated phosphorylation of pRb, the molecular mass of ∼16 kDa that negatively modulated the retinoblastoma susceptible gene product, thus sequestering E2F kinase activity of CDK4 (Figure 2A). Consequently, it was transcription factors as incompetent pRbÀE2F complexes and designated as P16INK4A (for an inhibitor of CDK4) or consequently blocking cell cycle progression5 (Figure 1). CDKN2(forCDKinhibitor2).Oneyearlater,theMTS1 Furthermore, it has also been demonstrated that an elevated (multiple tumor suppressor 1) locus on human chromosome level of P16 expression induced by oncogenes, DNA damage 9p was described following the discovery that the p16 gene response, or aging can trigger and accelerate cellular sene- was frequently inactivated by homozygous deletion or muta- À scence.1 4 While genetic inactivation of the p16 gene (cyclin- tion in melanomas as well as a broad spectrum of additional 13 dependent kinase inhibitor 2a, CDKN2A) by deletion, methyla- human cancer types. The full-length p16 cDNA gene was tion, and point mutation has been found in nearly 50% of all reported soon thereafter, which encodes a protein with eight 6À9 additional amino acid residues at the N-terminus in compar- human cancers, the overexpression of P16 at both mRNA 14,15 and protein levels is also associated with poor prognosis for ison with the originally reported cDNA sequence (Figure 2A). Further studies have established that P16 is a cancers, including neuroblastoma, cervical, ovarian, and breast pivotal regulator of cell cycle progression with diverse phy- cancers, prostate tumors, and oral cancers.10 Together, these fi siological functions and unique structural and biophysical ndings demonstrate that both the transcriptional level and properties. translational status of P16 are critical for its overall ability to Function. P16 primarily functions in cell cycle control as a mediate cellular activities. However, while numerous studies À 16 fi negative regulator of the prominent pRb E2F pathway. In G0 have focused on de ning the genetic and/or epigenetic status and early G1 phases, pRb is hypophosphorylated and forms ff of p16 in di erent cancers in investigating the molecular complexes with members of the E2F family of transcription 6,11 mechanism(s) of carcinogenesis, the critical regulation of factors. These complexes sequester E2Fs and prevent their access P16 itself has been understudied until recently. Here we review recent findings in P16 regulation and discuss their significance Received: April 27, 2011 in understanding the roles of P16 in cancer from a biochemical Revised: May 26, 2011 perspective. Published: May 27, 2011 r 2011 American Chemical Society 5566 dx.doi.org/10.1021/bi200642e | Biochemistry 2011, 50, 5566–5582 Biochemistry CURRENT TOPIC resulting in entry into the S phase. Binding of P16 directly downregulates the kinase activities of CDK4 and CDK6, keeping pRb in a hypophosphorylated status. Furthermore, P16 can disrupt complexes of CDK4/6 and non-P16 CDK inhibitors such as P27 (CDKN1B), thus leading to the release of these non- P16 inhibitors, suppression of CDK2 activity, and increases in the level of expression of hypophosphorylated pRb.5 Consequently, these P16-dependent events culminate in cell arrest at the G1ÀS boundary. Interestingly, it has been reported recently that the suppressive impact of P16 on E2F-mediated gene expression can be enhanced by the physical association between P16 and GRIM- 19 (gene-associated retinoid-IFN-induced mortality-19), a proa- poptotic protein functioning in the IFN-β/RA-induced cell death pathway.19 A more detailed account of these findings is presented below. Figure 1. P16ÀCDK4/6ÀpRb pathway. Arrows and minus signs In addition to the pRbÀE2F pathway, P16 also contributes to represent positive and negative regulatory effects, respectively. P denotes cell cycle progression through alternate and independent reg- À phosphorylated. This figure was modified from ref 56. ulatory pathways.20 22 First, phosphorylation of the carboxyl- terminal domain (CTD) of the large subunit of RNA polymerase II by the CDK7 subunit of general transcription factor TFIIH is an essential regulatory event in transcription.20,21 P16 is able to interact with TFIIH in the preinitiation complex, inhibit phos- phorylation of the CTD, and contribute to the capacity of this pathway to induce cell cycle arrest. Second, it has been reported that P16 contacts the glycine-rich loop of c-jun N-terminal kinases 1 and 3 (JNK1 and JNK3, respectively) and suppresses their kinase activities.22 The loss of JNK activity negatively regulates UV-induced c-Jun phosphorylation in melanoma cells and consequently interferes with cell transformation promoted by the H-Ras-JNK-c-Jun-AP1 signaling axis.22 Additionally, accumulating evidence has shown that P16 is involved in cellular senescence and aging through molecular mechanisms yet to be explicitly elucidated.4,23,24 The level of expression of P16 increases remarkably with aging in a large number of rodent and human tissues in both healthy and disease states.4 This elevated level of P16 expression has been shown to induce cellular senescence and aging in various progenitor cells and premalignant tumor cells,3 including neural progenitor cells,25 pancreas islet progenitor cells,26 and hematopoietic stem cells.27 These findings suggest that an aging-associated increase in the level of P16 expression can contribute to the decline in replicative potential for certain self-renewing compartments, a characteristic of aging.4 Moreover, a number of recent studies have demonstrated that P16 could be involved in the cellular response to genotoxic agents.4,12 P16-compromised or P16- deficient cells demonstrated sensitivity to ultraviolet (UV) light-induced apoptosis, suggesting that the absence of functional P16 allows propagation of proapoptotic signaling.28 Conversely, Figure 2. Primary, secondary, and tertiary structures of P16. (A) following genotoxically induced DNA damage, an elevated level Sequence and secondary structure of P16. Italic residues at the N-ter- minus represent missing residues in the cDNA gene first reported. Red of expression of P16 in tumor cells resulted in cell cycle arrest and and dashed lines represent helical and loop regions, respectively. inhibition of apoptotic events such as cytochrome c release, Residues with asterisks are those with mutations in human cancers. mitochondrial membrane depolarization, and activation of the AR is the ankyrin repeat. (B) Tertiary structure and domains of P16. The caspase cascade. These findings demonstrate that P16 is able to solution structure of P16 (Protein Data Bank entry 2A5E) is presented mitigate the sensitivity of mitochondria to proapoptotic signals in here,30 in which D84, the residue critical for CDK4 inhibition, is cancer cells with damaged DNA.29 highlighted. JNK denotes c-Jun N-terminal kinases. Structure. P16 is exclusively composed of four ankyrin repeat (AR) motifs30 (Figure 2B). Ankyrin repeats are relatively con- to the promoters of proliferation-associated genes, such as cyclin served motifs of approximately 31À34 residues.30,31 They are B1 (CCNB), dihydrofolate reductase (DHFR), jun B proto- abundantly present in proteins of plants, prokaryotes, viruses, oncogene (JUNB), and thymidine kinase 1 (TK1)17,18 (Figure 1). yeast, invertebrates, and vertebrates and are involved in numer- Once committed to cell proliferation, pRb is progressively hyper- ous physiological processes through the mediation of proteinÀ phosphorylated by CDK4 and CDK6 in the late G1 phase, protein interactions.31 Like in other AR proteins, each AR motif 5567 dx.doi.org/10.1021/bi200642e |Biochemistry