REPORTS

Biologic and Biochemical Analyses of p16INK4a Mutations From Primary Tumors

Wendell G. Yarbrough, Robert A. Buckmire, Mika Bessho, Edison T. Liu

Affiliations of authors: W. G. Yarbrough (Department of Surgery, Division of Otolaryngology/Lineberger Comprehensive Cancer Center), R. A. Buckmire (Department of Surgery, Division of Otolaryngology), M. Bessho (Lineberger Comprehensive Cancer Center), University of North Carolina at Chapel Hill; E. T. Liu, Division of Clinical Sciences, National Cancer Institute, Bethesda, MD.

Correspondence to: Wendell G. Yarbrough, M.D., University of North Carolina at Chapel Hill, Lineberger Comprehensive Cancer Center, CB#7295, Chapel Hill, NC 27599-7295 (e-mail: wgy{at}med.unc.edu).


    ABSTRACT
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
BACKGROUND: Point mutations in the tumor suppressor gene p16INK4a (also known as p16, CDKN2, MTS1, and INK4a) are found in many tumor types. Because the function of the products of these naturally occurring mutants has not been fully explored, we investigated the functional activities of a wide range of naturally occurring p16 mutant proteins. METHODS: Sixteen cancer-associated p16 mutant proteins, resulting from missense mutations, were characterized for their ability to bind and inhibit the cyclin-dependent kinases (CDK4 and CDK6) and to induce cell cycle arrest in G1 phase. RESULTS/CONCLUSIONS: Among 16 mutants analyzed, nine had detectable functional defects. Three mutants (D84V, D84G, and R87P) had defects in CDK binding, kinase inhibition, and cell cycle arrest. The corresponding mutations are located in the third ankyrin repeat in a highly conserved region believed to form the CDK binding cleft. Three mutants (P48L, D74N, and R87L) had defects in kinase inhibition and cell cycle arrest. Among the 10 mutants with normal CDK binding and inhibitory activity, three mutants (N71S, R80L, and H83Y) had defects only in their ability to induce cell cycle arrest. Thus, p16 mutant proteins that retain CDK4 and CDK6 binding may have more subtle functional defects. All nine mutations leading to functional impairments mapped to the central portion of the p16 protein. Ankyrin repeats II and III appear more critical to p16 function, and mutations in ankyrin repeats I and IV are less likely to disrupt p16 function.



    INTRODUCTION
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Progression of cells through the cell cycle is controlled by the activation and inactivation of cyclin-dependent kinases (CDKs). The phosphorylation status and binding of cyclin partners are common features of CDK regulation. An additional regulatory feature is the binding of G1 CDKs (CDK4 and CDK6) to the p16 family of CDK inhibitors. The founding member (p16INK4a, p16, CDKN2, MTS1, or INK4a; hereafter referred to as p16) of the p16 family plays a central role in arresting cells in the G1 phase of the cell cycle; p16 inhibits phosphorylation of the retinoblastoma (Rb) protein by CDK4 and CDK6, the cyclin D-dependent kinases (1). The somatic inactivation of p16 is frequently observed in many tumor types. This inactivation can occur through gene deletion, promoter methylation, or point nonsense or missense mutations, but the primary means of gene inactivation in tumors is homozygous deletion of the INK4a/ARF gene locus or promoter methylation (2,3). When p16 mutant proteins are made, functional analyses of p16 germline mutants from melanomas, pancreatic cancers, and head and neck cancers show that the p16 mutant proteins are functionally defective (4-7). These data support the role of p16 as a tumor suppressor.

The amino acid composition suggests that p16 is largely composed of four contiguous ankyrin repeats. The secondary structure of wild-type p16, as determined by nuclear magnetic resonance, contains ankyrin repeats that form {alpha}-helices with interposed turns (8). The p16 mutant proteins that carried mutations at positions 26, 66, 84, 92, or 124 had nuclear magnetic resonance spectra that were consistent with the retention of wild-type secondary structure (9). The tertiary structure of wild-type p16 has a putative cleft for CDK binding, with key residues thought to be amino acids 46, 47, 50, 79, 84, 88, and 110.

Given the difficulties of assessing the structure of p16 mutants directly, mutants are more frequently analyzed for functional activity. Pollock et al. (10) analyzed 120 point mutations in p16 obtained from cell lines and primary tumors, but the functional importance of the majority of these mutations is not known. To date, familial mutations involved in the pathogenesis of melanoma have received the most attention. These familial p16 mutants tend to cluster in the central portions of the protein and generally result in nonconservative amino acid substitutions. When we compared tumor-associated missense mutations across the ankyrin repeat structures of p16, we found that more than 50% of the mutations in ankyrin repeats I and IV but fewer than 30% of the mutations in ankyrin repeats II and III result in conservative amino acid changes. Likewise, we noted that tumor-associated mutations in the second, third, and early fourth ankyrin repeats occur more frequently at residues that are invariant in all members of the p16 family characterized. Because highly conserved residues in protein families may identify positions critical to protein structure or function, we hypothesized that mutations of these highly conserved residues would be more likely to alter p16 function. Consequently, those highly conserved amino acids in the second, third, and early fourth ankyrin repeats may be critical for p16 function, and tumor-associated mutations elsewhere may not cause functional abnormalities.

To test the biologic and biochemical activities of p16 mutants, we used three assays to assess the ability of the mutants to 1) bind to CDKs, 2) inhibit the activity of CDKs, and 3) arrest cell proliferation. CDK binding is the most commonly used test of p16 function because all inactivating p16 mutations characterized to date result in mutants that lack CDK binding activity (4,5,7,11-14). In this report, we describe the functional activity of 16 naturally occurring p16 mutants carrying mutations that span the entire coding region of p16. From these analyses, we sought to determine whether mutations in regions of the p16 protein that have not been analyzed alter function and to establish whether mutations that alter function cluster within specific structural domains of the p16 protein.


    MATERIALS AND METHODS
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Identification of p16 Mutants

All p16 mutants analyzed carried single nucleotide substitutions resulting in single amino acid changes that are found in human cancers. We analyzed 16 mutants; 15 mutants were obtained from primary tumors and one mutant (D84V) was obtained from a cell line (UNC7, human squamous cell carcinoma) at passage 2. Mutants carrying point mutations from all four ankyrin repeats of p16 were chosen for analysis. All mutants analyzed, except R87L and D84V, were identified from literature searches (4-6,12,13,15-17). Mutants D84V and R87L were detected by single-strand conformation polymorphism-polymerase chain reaction (SSCP-PCR) with the use of DNA from head and neck squamous cell carcinomas resected at the University of North Carolina at Chapel Hill School of Medicine. Informed written consent from the patient and Institutional Review Board approval were obtained before the tissue was collected and analyzed.

The 20-µL reaction mixture for SSCP-PCR contained 100 ng of genomic DNA, 500 nM of each primer, 1x PCR buffer (The Perkin-Elmer Corp., Foster City, CA; 10 mM Tris-HCl [pH 8.3], 50 mM KCl, 1.5 mM MgCl2, and 0.001% gelatin), all four deoxyribonucleoside 5'-triphosphates (each at 1 mM), 5% dimethyl sulfoxide, 2.5 U of AmpliTaq DNA polymerase (Roche Molecular Biochemicals, Indianapolis, IN), and 100 µCi of deoxycytidine 5'-[{gamma}-32P]triphosphate. PCR cycling conditions were as follows: 94 °C for 30 seconds, 55 °C for 30 seconds, and 72 °C for 30 seconds, with a final extension at 72 °C for 10 minutes. Exon 1 of p16 was amplified as one fragment, and exon 2 of p16 was amplified in three segments (2A, 2B, and 2C) with the oligonucleotide primers described by Hussussian et al. (18). SSCP-PCR products were diluted 1 : 30 with a solution of 1% sodium dodecyl sulfate (SDS) and 10 mM EDTA. The diluted product (3 µL) was mixed with an equal volume of stop buffer (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol) and denatured at 94 °C for 5 minutes. SSCP reaction products were subjected to electrophoresis through nondenaturing 6% polyacrylamide gels containing 5% glycerol, at both room temperature and 4 °C. Negative control samples containing only wild-type p16 PCR products were included in each gel. PCR products with altered electrophoretic mobility were further analyzed as follows: The p16 region of interest was again amplified by PCR. The PCR product was then used as a template for an asymmetric PCR to produce single forward and reverse DNA strands. The asymmetric PCR was carried out under the PCR conditions described above but with primers at 1) 500 nM forward primer and 10 nM reverse primer or at 2) 10 nM forward primer and 500 nM reverse primer. Both forward and reverse single-stranded DNAs were purified and sequenced by the dideoxynucleotide chain-termination method of Sanger et al. (19) and were considered to be positive only when both strands had the same mutation. These mutations were subsequently confirmed by repeating the entire sequencing analysis with separate DNA samples from the same patients.

Construction and Isolation of Glutathione S-Transferase (GST) Fusion Proteins

A full-length, wild-type p16 complementary DNA (a gift from Dr. Yue Xiong, University of North Carolina at Chapel Hill) containing the coding region homologous to the sequence listed in GenBank (accession numbers U12818, U12819, and U12829) with an additional 24 base pairs of coding sequence at the 5' end was cloned into pGEX-KG. Mutant p16 clones were constructed by use of a site-directed mutagenesis procedure (Altered Sites II), as suggested by the manufacturer (Promega Corp., Madison, WI). Mutations were confirmed by sequencing both DNA strands. CDK6-GST and cyclin D1-GST fusion constructs (in pGEX-KG) were also gifts from Dr. Xiong. A GST-Rb fusion plasmid encoding the carboxyl-terminal 148 amino acids (hereafter referred to as GST-Rb) was constructed as previously described (20). Fusion proteins were expressed in Escherichia coli and purified by passage over glutathione-Sepharose as described previously (7). Samples were concentrated with Centricon-30 spin columns (Amicon, Inc., Beverly MA). The GST-p16 protein concentration was estimated by comparison with protein of known concentration after polyacrylamide gel electrophoresis and Coomassie blue staining.

Binding of CDK4 and CDK6

In vitro transcription and translation of CDK4 and CDK6 were performed with [35S]methionine and T7 TNT in vitro translation kit according to the manufacturer's instructions (Promega Corp.). Wild-type and mutant GST-p16 proteins (500 ng) were incubated with 4 µL of the appropriate in vitro-translated CDK for 30 minutes at 25 °C and 37 °C. Binding of CDK4 and CDK6 by wild-type or mutant GST-p16 species was assessed by affinity precipitation with glutathione-Sepharose (Pharmacia LKB Biotechnology, Piscataway, NJ). The precipitated proteins were washed three times with Nonidet P-40 lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5% Nonidet P-40, and 50 mM NaF), followed by SDS-polyacrylamide gel electrophoresis and autoradiography.

Inhibition of CDK6 and CDK-Activating Complex Activities

The CDK-activating complex was immunoprecipitated with a CDK7 polyclonal antibody from a BT20 cell lysate (200 µg of total protein) in Nonidet P-40 lysis buffer as described previously (21). The immunoprecipitated CDK-activating complex was washed twice in Nonidet P-40 lysis buffer and twice in kinase buffer (50 mM HEPES [pH 7.3], 10 mM MgCl2, 5 mM MnCl2, and 1 mM dithiothreitol) and resuspended in the same buffer (adjusted to yield 30 µL). The following proteins then were added sequentially: mutant or wild-type GST-p16 (90 nM, 180 nM, or 360 nM, as described below), 800 ng of GST-CDK6, 800 ng of GST-cyclin D1, and 600 ng of GST-Rb. Finally, 10 µCi of adenosine 5'-[{gamma}-32P]triphosphate was added, and the reaction mixture was incubated at 30 °C for 30 minutes. Reaction products were analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. Mutant or wild-type GST-p16 was initially analyzed for kinase inhibitory activity at a concentration of 180 nM. The p16 mutant fusion proteins that inhibited CDK6 at this concentration were then assayed at 90 nM. The p16 mutant fusion proteins that did not inhibit CDK6 at 180 nM were then assayed for kinase inhibitory activity at 360 nM.

Cell Cycle Analysis

U2OS human osteosarcoma cells were cotransfected by electroporation (Gene Pulser with capacitance extender at 350 mV/960 mF; Bio-Rad Laboratories, Hercules, CA) with 1 µg of pCMVCD20 (provided by Dr. Sander van den Heuvel, Massachusetts General Hospital, Boston) and 10 µg of pCI-neo (Promega Corp.) alone or carrying a wild-type or mutant p16 sequence. Growth arrest was similarly assayed with 0.3 µg of pCI-neo alone or carrying wild-type or mutant p16INK4a sequences and 0.3 µg of pCMVCD20. After 48-60 hours, the U2OS cells were stained with a fluorescein isothiocyanate-conjugated anti-CD20 antibody (Becton-Dickinson Immunocytometry Systems, San Jose, CA) as described previously (22). Cells were then fixed and stained with propidium iodide for flow cytometry by use of a Becton-Dickinson FACSort. Data were analyzed with Cell Quest software (Becton-Dickinson Immunocytometry Systems). Propidium iodide intensity of CD20-positive cell populations was used to determine their DNA content and their cell cycle distribution. The data are the average of at least two experiments, in which more than 5000 CD20-positive cells were analyzed per point. Polyacrylamide gel electrophoresis and immunoblotting with p16 antibodies (provided by Dr. Xiong) confirmed expression of wild-type or mutant p16 in cells during cell cycle arrest.


    RESULTS
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Identification of Tumor-Associated p16 Missense Mutations Used for Functional Analysis

We collected 16 mutations in p16, 14 tumor-associated somatic and germline missense mutations identified in a literature search and two mutations (D84G and R87L) that we isolated from head and neck squamous cell carcinomas. We detected functional defects in nine of the corresponding mutants and observed normal CDK binding and kinase inhibitory activity in 10 of these mutants. A graphic representation of all p16 mutations in relation to the ankyrin repeat structure of p16 is shown in Fig. 1,Go A. We functionally analyzed p16 mutants that carried missense mutations that altered amino acids in each of the ankyrin repeats, as well as in the region carboxyl-terminal to the fourth ankyrin motif (Fig. 1Go, B). The distribution of mutants analyzed mimicked the distribution of tumor-associated mutations described in the literature. Of the mutants selected, eight have been previously characterized to various degrees. We examined these mutants again because of inconsistencies in the literature derived from the use of a variety of assays and because of the fact that not all functions of p16 had been characterized in most mutants. In addition, the possibility remained that assays of directed kinase inhibition and growth arrest could detect subtle functional defects that may have been overlooked with other assay systems.



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Fig. 1. Schematic representation of tumor-associated p16 missense mutations and alignment of sequences from members of the p16 superfamily. A) Schematic representation of the p16 amino acid sequence, with relative position of ankyrin repeats I, II, III, and IV shown. Somatic (arrows) and germline (carets) tumor-associated mutations are indicated. B) Relative location and amino acid substitutions encoded by p16 tumor-associated missense mutations in the p16 mutants chosen for functional analysis. C) Alignment of sequences of p16 family members. Invariant amino acids are shaded.

 
Amino acids within a protein conserved evolutionarily across species are often critical for function. To determine which amino acid residues are invariant in the p16 family members, we aligned the amino acid sequences of wildtype p16 family members from various species (Fig. 1Go, C). The frequency of invariant amino across the structure of p16 varied, with ankyrin repeats II and III having the most invariant residues. The frequency of point mutations in human p16 that altered invariant amino acid positions also varied widely. Of seven tumor-associated mutations in the ankyrin repeat I, none were at invariant amino acid positions. In the ankyrin repeat IV, only three of the 12 mutations occurred at highly conserved amino acids. In contrast, mutational alteration of invariant amino acids occurred more frequently in ankyrin repeats II and III (four of 10 and 12 of 23 residues, respectively).

Binding of CDK4 and CDK6

The ability of p16 mutant proteins to bind stably to CDK4 and CDK6 was tested by affinity co-precipitation of in vitro-translated CDK4 or CDK6 with GST-p16 fusion proteins as previously described (7). The p16 mutants D84V and R87P had no detectable CDK binding (Fig. 2Go), and mutant D84G had reduced binding to in vitro-translated CDK4 and CDK6. All of the other mutants tested had binding activity equivalent to that of wild-type p16. A recent report (23) of p16 mutants that had temperature-sensitive CDK binding activity led us to test CDK4 and CDK6 binding at 37 °C, but we found no p16 mutant with temperature-conditional binding to CDKs (data not shown).



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Fig. 2. Binding of p16 mutant proteins to the G1-phase cyclin-dependent kinases (CDKs). 35S-labeled in vitro-translated CDK4 or CDK6 was affinity precipitated with the purified bacterially produced fusion protein GST-p16. Bound CDK4 and CDK6 were detected by autoradiography after gel electrophoresis. GST = glutathione S-transferase; WT = wild type.

 
Inhibition of CDK6 Activity

The CDK inhibitory activity of the panel of p16 mutants was tested in an in vitro kinase inhibition assay. Wild-type or mutant GST-p16 fusion protein at a final concentration of 360 nM, 180 nM, or 90 nM was added to activated complexes of GST-CDK6 and GST-cyclin D1, and the resulting phosphorylation of a GST-Rb carboxyl-terminal (Rb-CT) fusion protein was assessed (7). To determine whether p16 mutants that failed to inhibit CDK activity at 180 nM retained partial activity, we then assayed these mutants at 360 nM. Likewise, to determine whether p16 mutants that inhibited CDK activity fully at 180 nM had more subtle defects, we assayed the mutant at 90 nM. As expected, mutants D84V and R87P, with no detectable binding to CDKs, did not inhibit the activity of CDK6 at any concentration (Fig. 3).Go Mutant D84G, which had decreased binding to CDKs, also failed to inhibit kinase activity in our standard assay (180 nM); however, when added at a twofold greater concentration (360 nM), it partially inhibited the activity. Of the 13 mutants that retained normal CDK4/6 binding, two (P48L and R87L) failed to inhibit CDK6 kinase activity at 180 nM but did inhibit it at 360 nM. Mutant D74N partially inhibited CDK6 activity under the more stringent assay conditions (90 nM), but all other mutants inhibited equally well at either 180 nM or 90 nM. In summary, of 16 mutants, six (P48L, D74N, D84G, D84V, R87L, and R87P) had detectable defects in kinase inhibition. Each mutant with defective binding of CDKs also had defective kinase inhibition.



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Fig. 3. Inhibition of cyclin-dependent kinase 6 (CDK6) activity by p16 mutant proteins. The ability of the complex of CDK6 and cyclin D1 to phosphorylate a retinoblastoma carboxyl-terminal fusion protein (Rb-CT) was assessed in the presence of 180 nM (A), 360 nM (B), or 90 nM (C) purified GST-p16 fusion protein. Phosphorylated Rb-CT was detected by autoradiography after gel electrophoresis. GST = glutathione S-transferase; WT = wild type.

 
G1-Phase Cell Cycle Arrest

When introduced into cells containing functional Rb protein, wild-type p16 causes growth arrest in the G1 phase of the cell cycle. To determine whether p16 mutant proteins could function similarly, we ectopically expressed all mutants in U2OS cells and then analyzed the cell cycle status of these cells by flow cytometry. The G1-phase arrest caused by the p16 mutants was then compared with the arrest caused by wild-type p16. To standardize results from multiple experiments, we used the magnitude of the G1 arrest after transfection with wild-type p16 as the 100% reference. If no proliferative arrest was observed (i.e., the G1 fraction in mutant p16-transfected cells was equal to the G1 fraction in cells transfected with vector alone), a value of 0% was assigned. Negative numbers indicate that cells transfected with the p16 expression vector had fewer cells in G1 phase than cells transfected with the vector control. At least 5000 transfected cells were analyzed for each data point, and results of multiple experiments were combined to determine the mean and standard deviation. Expression of p16 proteins after transfection was confirmed by gel electrophoresis of a cell lysate followed by immunoblotting with a p16-specific antibody.

A representative experiment showing expression of p16 mutants after transfection with 0.3 µg of expression plasmid is depicted in Fig. 4.Go The ability of p16 mutants to cause growth arrest in G1 phase was initially analyzed by transfecting cells with 10 µg of the expression vector carrying p16 sequences. As expected, mutants that had defective CDK binding (D84G, D84V, and R87P) failed to inhibit growth (Fig. 4Go). The mutant that had a decreased ability to inhibit CDK6 kinase activity (P48L) also did not cause growth arrest in the G1 phase under these conditions. The cell cycle arrest caused by the remaining 12 mutants was roughly equivalent to that caused by wild-type p16.



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Fig. 4. Relative G1-phase arrest caused by p16 mutants. U2OS human osteosarcoma cells were transfected with either 10 µg or 0.3 µg of plasmids encoding wild-type (WT) or mutant p16 sequences. The cell cycle distribution of cells was analyzed by flow cytometry. The G1-phase arrest caused by transfection with wild-type p16 was chosen as 100% arrest. The G1 arrest caused by the p16 mutants is expressed relative to wild-type p16. Two to four experiments were combined to allow calculation of standard deviation (shown as error bars). Cell lysates derived from a portion of the cells transfected with 0.3 µg of plasmid and analyzed for cell cycle distribution were separated by gel electrophoresis and examined by immunoblotting with p16-specific antibodies. Control lane shows a lysate from cells transfected with plasmid containing no p16 insert.

 
To determine whether p16 functional defects were being masked by overexpression of mutant proteins, we repeated the growth arrest assay with cells transfected with 30-fold less expression vector. The magnitude of the growth arrest caused by wild-type p16 was equivalent in cells transfected with 10 µg or 0.3 µg of expression vector (data not shown). Three mutants (D84G, D84V, and R87P) that had CDK binding defects did not arrest cell proliferation when they were transfected with either 10 µg or 0.3 µg of p16 expression construct (Fig. 4Go). Mutant P48L that bound CDKs normally but had partial defects in CDK inhibition also could not arrest cell growth at either 10 µg or 0.3 µg of transfected plasmid. Mutant plasmids that encoded a protein with partial defects in CDK6 kinase inhibition (D74N and R87L) were unable to cause G1-phase arrest when they were transfected at lower concentrations. Furthermore, three mutants that had no detectable binding or kinase inhibition defects (N71S, R80L, and H83Y) were unable to cause a growth arrest under the more stringent assay conditions.

Thus, all of the p16 mutants containing amino acid alterations in the second or third ankyrin repeat had defective growth arrest properties. Reciprocally, wild-type p16 and all mutants carrying a mutations outside ankyrin repeats II and III caused equivalent cell cycle arrests in the G1 phase (Fig. 4Go).


    DISCUSSION
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Our analysis of mutant p16 proteins identified an association between mutations in the ankyrin repeat structure and the biochemical and biologic activities of p16. All mutations located in the central ankyrin repeats (ankyrin repeats II and III) had detectable alterations of function. Reciprocally, all mutations located outside ankyrin repeats II and III had binding, kinase inhibitory, and growth arrest activities equivalent to those of the wild-type protein. These functional data are in agreement with the recently reported tertiary structure of p16 that identifies amino acids in the second, third, and early forth ankyrin repeats that are important for the formation or stabilization of the putative CDK binding pocket (9). Truncation of the carboxyl-terminal portion of p16, including that of six amino acids in ankyrin repeat IV, conveys no functional defect, implying that this region is dispensable for p16 function (13). These data support our findings suggesting that areas of the p16 protein outside ankyrin repeats II and III are less critical for p16 activity.

The three p16 mutations that confer defective CDK binding (D84G, D84V, and R87P) were located at or immediately adjacent to amino acid positions thought to be critical for the direct interaction with CDKs (9). Our binding assay mimics other assays commonly described in the literature; however, on the basis of our results, we cannot rule out the possibility that some nonfunctional mutants have subtle binding defects that would not be detected with this binding assay. Our functional data are in agreement with and strengthen the structural data, in that amino acid alterations outside the putative CDK binding cleft did not destabilize the binding between p16 mutants and CDKs. All p16 mutants with defective CDK binding were inactive in each of the known functions of p16, confirming that CDK binding is necessary for p16 function. Several mutants retained CDK binding, yet they had other functional defects, suggesting that CDK binding, although necessary for p16 activity, is insufficient for full inhibition of CDK or for growth arrest. Mutations outside the CDK binding cleft could theoretically alter p16 function by decreasing protein stability or subcellular localization. Protein stability, localization, or an as yet undetermined defect resulting from p16 missense mutations would not be detected by in vitro assays. Despite the use of identical expression constructs and transfection conditions, protein levels of p16 mutants showed some variation (Fig. 4Go). For instance, mutant H83Y, which had binding and kinase inhibitory functions equivalent to those of wild-type p16, was consistently expressed at lower levels, possibly as a consequence of decreased protein stability. Perhaps because of the assay's ability to detect changes in protein stability or defects dependent on unknown cellular or biochemical functions, we found that the in vivo growth arrest assay was the most sensitive test of p16 activity. As a confirmation of accuracy, the growth arrest assay detected defects in all mutants that had abnormal CDK binding or kinase inhibition activity. In addition, the growth arrest assay was able to define functional defects not detected by the in vitro binding or kinase inhibitory assays. Kinase inhibition data for one mutant carrying a mutation in ankyrin repeat I and three mutants carrying mutations in ankyrin repeat IV have been published (9). Although the authors (9) did not test binding or growth arrest, their data suggest that each mutant tested had decreased kinase inhibitory activity, including the two mutants E26D and R124H that we found to function normally in all of our assays. The amino acid substitutions in both proteins are conservative in nature, in that acidic amino acids are replaced with acidic amino acids and basic amino acids are replaced with basic amino acids. By chance, one of these amino acid substitutions, E26D, that functioned normally in our battery of assays occurs naturally in murine p16 (Fig. 1Go, C). Although it is possible that a Glu-26 to Asp substitution could affect functional activities of p16, it is less likely given that Asp-26 occurs naturally in the murine p16 homologue.

Our analysis does not suggest that all p16 mutations in ankyrin repeats I and IV will confer wild-type activity. Tertiary structure analysis suggests that some residues located early in ankyrin repeat IV may contribute directly to the CDK binding cleft. Support for this model comes from the observation that mutant P114L carrying a mutation in ankyrin repeat IV lacks CDK binding and growth arrest activities (5). Conversely, of nine p16 mutations occurring in ankyrin repeats II and III, mutants resulting from six of these mutations retained partial functional activity. All mutants capable of stable CDK binding could also at least partially inhibit their kinase activity. Likewise, all mutants that could bind and inhibit CDKs in the in vitro assays, excluding P48L, could also partially arrest growing cells. Mutants that retained partial function were found in primary tumors, suggesting that incomplete loss of p16 function may be sufficient for carcinogenesis. However, gene deletion and promoter methylation, both of which totally eliminate expression of the p16 protein, appear to be the methods of p16 gene inactivation most often observed in tumors.


    NOTES
 
Supported by Public Health Service grant K08CA72968 (to W. G. Yarbrough) from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services.

We are particularly grateful to Dr. Yue Xiong for his support and reagents. We also thank Dr. Tona Gilmer and her laboratory personnel for assistance with flow cytometric analysis and for helpful discussion.


    REFERENCES
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 

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Manuscript received March 1, 1999; revised July 12, 1999; accepted July 28, 1999.


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