The COOH Terminus of P18ink4c Distinguishes Function from P16ink4a1

[CANCER RESEARCH 61, 3863–3868, May 15, 2001] Advances in Brief

The COOH Terminus of p18INK4C Distinguishes Function from p16INK4A1

Jay Gump, Saralinda Turner, and James Koh2 Departments of Molecular Physiology and Biophysics [J. G.], Pathology [S. T., J. K.], and Medicine [J. K.], University of Vermont, Burlington, Vermont 05405

Abstract association is less stable than the association of p16 with cdk4 (14). The goal of this study was to determine whether p16 and p18 function The INK4 family of proteins consists of four members which can block differently in CEM cells, and, if so, whether this difference is attrib- progression from the G -to-S phase of the cell cycle by inhibiting the 1 utable to differential association with cdk4. Functional differences activity of cyclin dependent kinases (cdks) 4 and 6. Although the gene between the two proteins could help explain the different mutational encoding p16INK4a is commonly inactivated in human tumors, p18INK4c is rarely altered. We show here that overexpression of p18INK4c does not profiles observed for each CKI in human cancer. block cell cycle progression in a T-cell acute lymphocytic leukemia cell line INK4a Materials and Methods (CEM) sensitive to p16 -mediated G1 arrest. A chimera consisting of INK4a the kinase-binding region of p16 fused to the COOH terminus of Plasmid Construction. The p16/18 chimera was constructed by PCR. The INK4c p18 is active in all known biochemical assays for INK4 function, but 5Ј part of the cDNA was generated using the primers 5Јmycp16 (5Ј-GGGAAT- INK4c it does not arrest CEM cells. These data imply a novel level of p18 TCAAATGGAGCCGGCGGCGGGG-3Ј) with 3Јp16/18 (5Ј-GCACGG- regulation mediated through the COOH terminus and suggest that func- TAGCTGGTCAAGCACACGGCCAGC-3Ј) and the human p16 cDNA as a tional differences might underlie the distinct mutational profiles observed template. PCR conditions were 30 cycles of 94° for 1 min and 72° for 1 min. INK4a INK4c for p16 and p18 in tumors. The 3Ј piece of cDNA was amplified with the primers 5Јp16/18 (5Ј-GCTG- GCCGTGTGCTTGACCAGGTACCGTGC-3Ј) with 3Јmycp18 (5Ј-TGCCTC- Introduction GAGTTATTGAAGATTTGTGGC-3Ј) and the human p18 cDNA as a template. PCR conditions were 30 cycles of 92° for 1 min, 55° for 1 min, and 72° 3 The inactivation of CKIs is an important event in the development for 1 min. The resulting PCR products were gel-purified, combined, and used of human cancer (1, 2). The INK4 family of proteins currently as a template to generate a full-length chimeric cDNA by PCR with the primers consists of four CKIs: p16, p15, p18, and p19. INK4 proteins share a 5Јmycp16 and 3Јmycp18. PCR conditions were 94° for 1 min, 57° for 1 min, common structural organization and all show in vitro binding speci- 72° for 1 min. p16 and p18 were amplified in a similar manner and cloned ficity for only cdk4 and cdk6 among the known cdks. Although each using a 5Ј EcoRI site and a 3Ј XhoI site into a version of pCDNA3 (Invitrogen, member behaves identically in the established in vitro assays for Carlsbad, CA) containing a Myc epitope tag. DNA for transfections was prepared using a cesium chloride gradient or with an EndoFree kit (Qiagen, INK4 function (3–6), only the genes encoding p15 and p16 are Valencia, CA). The integrity of all constructs was verified by sequencing. inactivated in a significant proportion of human cancers (7–9). Inac- Cell Culture and Transfections. U2OS and CEM cells were obtained tivation of p16 appears to be particularly important in the develop- from the American Type Culture Collection and maintained as suggested. ment of melanoma and T-cell ALL (reviewed in Refs. 10 and 11). The U2OS cells were transfected using the Effectene transfection kit (Qiagen) p16 gene is mutated in a high proportion (Ͼ80%) of primary human according to manufacturer’s instructions. CEM cells were transfected by T-cell ALLs and is, in fact, the most commonly mutated known gene electroporation in an Electro Square Porator (BTX, San Diego, CA) on the in this tumor type (12, 13). The human p18 protein shares 38% amino low-voltage setting using a single pulse of 65 ms in duration and a field acid identity with p16, binds the same target cdks as p16, and, like strength of 550 V/cm. Just before transfection, the CEM cells were washed ϫ 6 ϩ p16, can provoke cell cycle arrest when exogenously expressed in cell twice in PBS and resuspended at a density of 30 10 cells/ml in RPMI L- glutamine (2 mM). After electroporation, the cells were incubated 15 min at lines retaining wild-type pRb (3, 9). Because of these similarities, it room temperature before plating in complete medium. has generally been assumed that the functions of p18 and p16 are Flow Cytometry. Cells were cotransfected with a plasmid encoding a essentially redundant (1). However, several leukemia cell lines ex- membrane-targeted GFP-F and the construct of interest at a molar ratio of 1:5. press high levels of endogenous p18, a situation that should not be Forty-eight h after transfection, cells were fixed in 80% ethanol for a minimum tolerated in these rapidly dividing cells if p18 and p16 indeed perform of 30 min at Ϫ20°C. Cells were washed once with PBS and once with identical biological functions. CEM cells, which are derived from a PBS ϩ 1% fetal bovine serum and then resuspended at Յ1 ϫ 106 cells/ml in T-cell ALL and are p16 null, express high levels of p18 (3, 14). a PBS ϩ 1% fetal bovine serum solution containing RNase (250 ␮g/ml) and Although p16 and p18 behave identically in in vitro assays, p18 has propidium iodide [diluted from a 50ϫ stock of 0.5 mg/ml in 38 mM sodium been reported to preferentially associate with cdk6 in vivo (3, 4). citrate (pH 7.00)]. The cells were incubated at 37°C for at least 30 min, then stored at 4°C overnight before analysis. Data acquisition and analysis was Others have found that p18 associates with cdk4 in vivo, but that this performed on a Coulter EPICS XL-MCL flow cytometer. Gates were set to restrict analysis to GFP-positive cells, and peak area/peak height ratios were Received 11/17/00; accepted 3/26/01. used to discriminate doublets. Cell cycle distribution was determined using the The costs of publication of this article were defrayed in part by the payment of page Modfit program (Verity Software, Topsham, ME). charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. In Vitro Kinase Assays. Bacterially expressed INK4 proteins were pre- 1 Supported in part by NIH Grant RO1 ES 09673. J. G. is supported by a teaching pared and added to an in vitro kinase assay as described (5). Recombinant, assistantship from the Department of Molecular Physiology and Biophysics at the Uni- active cyclin D1/cdk4 was a generous gift from Dr. Robert Booher (Onyx versity of Vermont. J. K. is a V Foundation Scholar and gratefully acknowledges the Pharmaceuticals, Richmond, CA). support of this organization. 2 To whom requests for reprints should be addressed, at Departments of Pathology and Immunoprecipitation and Immunoblotting. Cell lysates were prepared Medicine, University of Vermont, Burlington, VT 05405. Phone: (802) 656-2043; E-mail: as described (5). Briefly, cells were lysed in EIA lysis buffer ϩ inhibitors [50 [email protected]. mM HEPES (pH 7.0), 250 mM NaCl, 0.1% NP 40, and 5 mM EDTA with 1 mM 3 The abbreviations used are: CKI, cyclin-dependent kinase inhibitor; INK4, inhibitors 4-(2-aminoethyl)benzenesulfonylfluoride, 1 mM DTT, 1 ␮g/ml aprotinin, 1 of cdk4; p16, p16INK4A; p15, p15INK4B; p18, p18INK4C; p19, p19INK4D; cdk, ␮ ␮ cyclin-dependent kinase; ALL, acute lymphocytic leukemia; pRb, retinoblastoma protein; g/ml pepstatin A, 1 g/ml leupeptin, 0.1 mM sodium vanadate, and 1 mM GFP, green fluorescent protein; GFP-F, farnesylated GFP. sodium fluoride], incubated 10 min on ice, and spun 20,000 ϫ g at 4°C for 3863

10 min before the supernatant was transferred to a clean tube. Samples were verified by Western blotting (Fig. 2C and 3A and data not shown). The mixed with an equal volume of 2ϫ Laemmli sample buffer, boiled 5 min, and intrinsic activity of p18 is clearly not limiting, as it is capable of separated by SDS-PAGE before immunoblotting with antibodies versus the arresting cell cycle progression to the same extent as p16 in U2OS myc tag (9E10; purified from hybridoma supernatant), cdk4 (Santa Cruz cells (Fig. 1). These data demonstrate that p16 and p18 are not Biotechnology H-22, Santa Cruz, CA), or pRB (PharMingen, San Diego, CA). functionally redundant and that their respective activities are cell For immunoprecipitation, lysates were incubated 1 h with primary antibody on ice. Immune complexes were captured by the addition of protein G- type-dependent. This result is in agreement with reports that adeno- Sepharose (Amersham-Pharmacia Biotech, Inc., Piscataway, NJ) and then 30 viral expression of p16 and p18 produced differing results with min of rocking at 4°C. The beads were washed four times with lysis buffer and respect to cell cycle arrest according to the cell type used (15). then boiled in sample buffer before electrophoresis. A Functional Chimera with the p16 cdk-binding Domain Does Pulse Chase Analysis. Twenty-four h after transfection, cells were washed Not Arrest CEM Cells. The INK4 family members consist of a twice in PBS and incubated for 20 min in medium free of methionine and series of ankyrin repeats. p15 and p16 contain four ankyrin repeats, 35 cysteine. S-methionine-labeling mix (New England Nuclear, Boston, MA) whereas p18 and p19 each consist of five ( Ref. 9 and Fig. 2A). The was added to the medium and the cells were incubated for4hat37°C. The crystal structures of p16 bound to cdk6 and of p19 bound to cdk6 have cells were then washed twice with PBS and fresh, unlabeled medium was been solved, and they suggest that the residues necessary for kinase added for the indicated times. The cells were lysed as above. The lysates were precleared by the addition of protein G-Sepharose 4 gel beads which were binding are contained in the first four ankyrin repeats of the INK4 prebound to normal rabbit serum. Immunoprecipitation was carried out as proteins (16). Nuclear magnetic resonance data suggest that the fifth above. Immunoprecipitates were separated by SDS-PAGE and visualized by ankyrin repeat of p18 is not necessary for kinase binding, but that it autoradiography. might be important for stabilizing the protein (17). Moreover, the Isolation of Transfected Cells. The pHOOK system (Invitrogen, Carlsbad, crystal structure of an inactive ternary structure containing cyclin K (a CA) was used to separate transfected cells from nontransfected cells. Briefly, viral D-type cyclin), p18, and cdk6 confirms that the fourth and fifth pHOOK encodes a single chain antibody which recognizes the hapten antigen ankyrin repeats of p18 do not participate in association with the kinase phOx. Cells expressing the pHOOK antibody are captured by binding to the (18). Consistent with these observations, the domains of greatest phOX hapten conjugated to magnetic beads. Cells were cotransfected with the pHOOK plasmid and the DNA of interest at a ratio of 2:1. Twenty-four h after divergence between INK4 family members lie in the COOH-terminal transfection, the cells were resuspended in 1 ml of medium, to which 15 ␮lof regions of the proteins beyond the fourth ankyrin repeat (Ref. 9 and the bead solution were added. The cells were rocked gently for 30 min at 37°C. Fig. 2A). A magnet was used to collect cells bound to the magnetic beads. The bound To determine whether differential association with cdk4 mediates cells were washed three times in whole media before replating. Sixteen h later, differences between p16 and p18 in CEM cells, we constructed the cells were harvested and lysates were prepared as described above. chimeras in which the COOH-terminus of p18, including its fifth ankyrin repeat, was fused to the first four ankyrin repeats of p16 Results and Discussion (p16/18; see Fig. 2A for the site of exchange). Because this chimera p16 and p18 Are Functionally Distinct. The T-cell ALL cell line contains the p16 residues important in kinase association, we pre- CEM has been reported to express high levels of the protein p18 (3, dicted that the protein would mimic p16 in its kinase preference. The 14). Because the vast majority of T-cell ALLs are p16-null and reverse chimera was also constructed in which the COOH terminus of sensitive to p16-mediated cell cycle arrest (11), we wanted to deter- p16 was fused to p18 after the fourth ankyrin repeat, as was a mine whether the CEM cell line responded differently to p16 and p18. truncated version of p18 consisting of only its first four ankyrin CEM cells were transiently transfected with myc-tagged versions of repeats. p18 constructs lacking the fifth ankyrin repeat were not either p16 or p18 under control of the powerful CMV promoter. The recovered from mammalian cell extracts in quantities large enough to cell cycle distribution of the transfected cells was subsequently ana- be detected by Western blot, in agreement with the assertion that this lyzed by flow cytometry. A plasmid encoding the GFP was cotrans- part of the protein contributes to the stability of p18 (17). Addition- fected as a marker to allow for identification of successfully trans- ally, the p18/16 chimera was recovered only in minute quantities from fected cells. As shown in Fig. 1, wild-type p16 induced a reproducible bacterial expression lysates, leading us to believe that the instability of accumulation of CEM cells in the G1 phase of the cell cycle under this protein is not limited to mammalian cells (data not shown). these conditions. In contrast, p18 failed to cause a G1 arrest and We verified the functional integrity of the p16/18 chimera using the behaved similarly to the nonfunctional, tumor-derived p16 mutant three established assays for INK4 function: (a) binding to cdk4 and P114L (5). Equivalent expression of the proteins in each cell line was cdk6; (b) inhibition of cdk4/6 activity, and (c) the ability to arrest cell cycle progression in certain cell lines (19). To test the ability of each construct to inhibit cyclin D1/cdk4 complexes in vitro, His-tagged INK4 proteins were expressed in bacteria and purified to apparent homogeneity. These proteins were added to an in vitro kinase reaction containing active cyclin D1/cdk4 complexes and a COOH-terminal fragment of the pRb as a substrate. p16, p18, and p16/18 all inhibited 32P incorporation onto pRb in a dose-dependent manner, with each CKI requiring a similar molar excess relative to cdk4 for full inhibition (Fig. 2B). This effect is specific, because the addition of equivalent amounts of p16 P114L did not inhibit cyclin D1/cdk4 activity. To determine whether the p16/18 chimera was capable of binding cdk4 in mammalian cells, U2OS cells were transfected with myc- tagged versions of p16, p18, p16/18, and p16 P114L. As shown in Fig. Fig. 1. p16 and p18 are not functionally redundant. The indicated inhibitors were 2C, immunoprecipitation through the myc tag recovered roughly Ⅺ f transfected into either CEM cells ( ) or U2OS cells ( ). The change in G1 population is equivalent amounts of each of the transfected inhibitors. Western expressed relative to vector-transfected controls. Bars, SD of at least three experiments for each condition. Approximately equivalent expression of each construct was verified by blotting the immunoprecipitated complexes for cdk4 confirmed that Western blotting for an N NH2-terminal myc-tag (Fig. 2C,3A, and data not shown). p16, p18, and p16/18 were each associated with cdk4 in U2OS cells 3864

Fig. 2. Characterization of the p16/18 chimera. A, alignment of p16 and p18. Regions of amino acid identity are shaded. Labeled boxes above the primary sequence, the first and second helices of each ankyrin repeat (e.g., the first helix of the third ankyrin repeat is labeled 3A). Arrow, the site of exchange for the p16/18 chimera. B, inhibition of recombinant cyclin D1/cdk4. Purified INK4 proteins were added at the indicated molar excess relative to cdk4 in in vitro kinase assays using a COOH-terminal fragment of pRb as a substrate. The kinase reactions were resolved on an 8% polyacrylamide gel, and the phosphorylated substrate was visualized by autoradiography. C, association with cdk4. Constructs encoding the indicated myc-tagged INK4 proteins were cotransfected with GFP into U2OS cells. Cell lysates prepared 48 h after transfection were subjected to immunoprecipitation using the anti-myc antibody 9E10. Immunoprecipitates were resolved on Western blots and probed with antibodies against either the myc-tag (9E10) or cdk4. D, cell cycle distribution of cells expressing INK4 proteins. U2OS cells harvested 48 h after transfection were fixed and stained with propidium iodide for flow cytometry. Gates were set to restrict analysis to cells expressing the cotransfected GFP marker. Each histogram profile depicts data from at least 5000 GFP-positive cells. The proportion of cells in the G1 phase of the cell cycle was calculated using the ModFit flow cytometry analysis software.

(Fig. 2C). Again, the specificity of this interaction was confirmed arrest in CEM cells, we repeated this experiment in the presence of the using the p16 P114L variant as a negative control. mitotic spindle inhibitor nocodazole. Nocodazole was added to the A third assay for INK4 function is the ability to arrest certain culture media 16 h before harvest to impose a secondary block at the pRb-positive cell lines. We chose to analyze the activity of the G2-M boundary. Cells that are not arrested at the G1-S transition will chimera in the osteosarcoma cell line U2OS because it is an environ- proceed through the cell cycle and accumulate at the nocodazole block ment in which p16 and p18 have been shown to behave similarly (3, point in G2. As shown in Fig. 3B, only wild-type p16 was able to 5). As expected, the expression of both p16 and p18 caused a pro- impose a G1 block in these cells, despite the equivalent protein nounced accumulation of U20S cells in the G0/G1 phase of the cell expression levels of each test construct. These data clearly indicate cycle (Fig. 2D). p16/18 is capable of arresting these cells to the same that expression of the p16/18 chimera does not block cell cycle extent as either p16 or p18. Immunoprecipitation followed by Western progression in CEM cells. The plasmid DNA amounts used in this blotting of lysates from this experiment verified equivalent expression experiment were adjusted as before to produce equivalent levels of of each construct (Fig. 2C). In all of these assays, which collectively protein expression, and were as follows: vector, 25 ␮g; p16, 30 ␮g; represent the currently accepted definitive proof of INK4 activity, p18, 25 ␮g; p16/18, 75 ␮g; and p16 P114L, 75 ␮g. both p18 and the p16/18 chimera are indistinguishable from p16. It has been proposed that differences between p16 and p18 are When transfected into CEM cells, however, the p16/18 chimera did attributable to either the relatively decreased in vivo affinity of p18 for not arrest cell cycle progression. As shown in Fig. 3A, expression of cdk4 (3, 4) or to a decreased in vivo stability of p18/cdk4 interactions p16 resulted in a marked depletion of the proportion of cells in (14). Because our chimera contains all of the residues of p16 thought S-phase, whereas p18, the p16/18 chimera, and the p16 mutant P114L to participate in kinase binding (16, 20), we reasoned that the chimera had little effect on cell cycle distribution. Equivalent expression of the should associate with cdks just as p16 does. Therefore, the functional constructs was verified by Western blotting lysates prepared from an differences we observed should not be attributable to an intrinsic aliquot of the same cells used for flow cytometry (Fig. 3A, bottom preference of p16/18 for cdk6 over cdk4. However, because the loop right). The input amount of each plasmid construct required to yield connecting the fourth and fifth ankyrin repeats of p18 might play a approximately equivalent protein expression was determined empiri- role in p18 function (21), and because this loop is part of the chimera, cally and was as follows: vector, 25 ␮g; p16, 25 ␮g; p18, 25 ␮g; we cannot definitively rule out a difference in kinase preference. p16/18, 75 ␮g; and p16 P114L, 50 ␮g. In all experiments where equal To determine whether the p16/18 chimera associates stably with amounts of DNA were introduced into CEM cells, transfection with cdk4 in CEM cells, we performed a pulse chase analysis (14). CEM the p16 construct resulted in G1 arrest, whereas the expression of p18 cells were transfected with either an empty vector or with constructs and p16/18 did not. The experiment shown here was performed encoding the myc-tagged inhibitor of interest. Twenty h after trans- exactly as those depicted in Fig. 1, and the data are representative of fection, the cells were pulse-labeled with 35S-methionine for4hand at least four independent replicates. chased for 0, 2, 4, or 19 h with cold medium. At each time point,

To verify that the p16/18 chimera is incapable of causing a G1 lysates were prepared and immunoprecipitated with the 9E10 anti- 3865

data were obtained when this experiment was repeated in U2OS cells (data not shown), with the exception that these cells contain signifi- cantly less cdk6 (14). Because it appears that both p18 and p16/18 associate readily with cdk4 in CEM cells, we assessed whether phosphorylation of the endogenous pRb was affected under these conditions. Hyperphospho- rylated pRb can be distinguished from hypophosphorylated pRb on a Western blot by the slower relative mobility of the phosphorylated form (22). CEM cells were transfected with the construct of interest along with a plasmid encoding a single chain antibody against the hapten phOx. Transfected cells were separated from nontransfected cells using magnetic beads conjugated to the phOx antigen. Lysates prepared from the separated cells were probed with an antibody that recognizes pRb in all of its various phospho-forms. Expression of p16 caused an accumulation of pRb in its hypophosphorylated, active form (Fig. 4B). The expression of p18 did not completely prevent the phosphorylation of pRb, although there seems to be an accumulation of the lower band relative to vector-transfected cells. The expression of the p16/18 chimera had no effect on the phosphorylation status of pRb. These results suggest that the failure of p18 and the p16/18 chimera to arrest CEM cells is attributable to an inability to inhibit phosphorylation of pRb. Taken together, our results are consistent with three principal conclusions: (a) p16 and p18 are not functionally equivalent; (b) p18 is subject to a cell type-specific regulation that prevents this inhibitor from arresting CEM cells despite the intrinsic capability of p18 to inhibit the same target kinases as p16; and (c) this regulatory influence can be conferred onto p16 by transferral of the fifth ankyrin repeat of p18. Previous reports suggest that p16 and p18 might be distinguished by their relative affinities for cdk4 and cdk6 (3, 4). Our results argue against this explanation for functional differences between the inhibitors, because the p16/18 chimera should have the same affinity for cdk4 as does p16. If the proposed in vivo destabilization of p18/cdk4 complexes (14) is the cause of phenotypic differences between p16 and p18, our results would indicate that: (a) very transient dissociation of p18 and cdk4 is sufficient to allow kinase activity and to promote

Fig. 3. The p16/18 chimera does not arrest CEM cells. A, CEM cells were cotransfected by electroporation with a GFP marker and the indicated constructs. Plasmid DNA quantities for the electroporation of the indicated constructs were determined empirically to give approximately equivalent expression by Western blotting (lower right). B, cells were transfected as in A, except that nocodazole was added to the cultures for 16 h before collecting the cells for flow cytometry analysis. The proportion of cells in G1 phase is as follows: vector, 2%; p16, 45%; p18, 18%; p16/18, 18%; and p16P114 L, 15%. Each histogram profile depicts data from at least 5000 GFP-positive cells.

body against the myc-tag. Proteins recovered in the immunocom- plexes were visualized by autoradiography. Two bands corresponding to cdk4 and cdk6 were immunoprecipitated with p16, p18, and p16/ 18, but were not present in the vector control (Fig. 4). In contrast to a previous report we found no gross difference between p16 and p18 with respect to the stability of the complexes formed with cdk4 (14). Subtle differences in the stability of kinase/inhibitor complexes cannot be ruled out, as evidenced by examination of time points later than those reported in the previous study (see the 19-h time points). A Fig. 4. p18 and p16/18 associate with cdk4 but do not prevent pRb phosphorylation. A, CEM cells transfected with an empty vector control or with the indicated myc-tagged likely explanation for the difference in results lies in our use of an INK4 constructs were pulse-labeled with 35S-methionine and then switched to unlabeled overexpression system versus the examination of endogenous pro- media for 0, 2, 4, or 19 h before lysates were prepared. Lysates from each time point were teins. An enforced excess of p18 protein could drive the equilibrium incubated with the anti-myc antibody 9E10, and the immunoprecipitated material was resolved on a 12% SDS-PAGE gel and visualized by autoradiography. Upper panel, cdk4 in favor of the formation of complexes with cdk4 regardless of the and cdk6; lower panel, the various INK4 proteins. B, p18 and the p16/18 chimera do not inherent stability of the association. Still, it is difficult to understand block pRb phosphorylation in CEM cells. CEM cells transfected with the indicated INK4 constructs were separated from nontransfected cells using the pHOOK system. Cell why CEM cells continue to divide in the presence of overexpressed lysates prepared from the purified transfected cells were probed by Western blotting with p18 when the protein readily associates with cdk4. Similar binding an antibody capable of detecting pRb in all phosphorylation states. 3866

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2001 American Association for Cancer Research. FUNCTIONAL DIFFERENCES BETWEEN p16 AND p18 the inactivation of pRb, and (b) that destabilization of the p18/cdk shown to be phosphorylated on serine residues in vivo (24). Because complex is mediated by the COOH-terminal region of p18 (2). This an incipient cancer cell may thus have alternative means for bypassing seems the most likely explanation in light of the fact that p18 exists p18 function, there would be minimal selective pressure for direct primarily as a monomer or in a heterodimeric complex with cdk4 or mutation of the p18 gene. Conversely, there would be significant cdk6 in CEM cells (14), and these complexes are catalytically inactive selective pressure for direct mutational inactivation of p16 (or com- in vitro. ponents in the p16 pathway) during tumorigenesis, because p16 does One vexing issue regarding p18 has been how to resolve the not contain the regulatory fifth ankyrin repeat, and therefore the cell apparent contradiction between the complexes this inhibitor forms in has no alternative means for bypassing the p16-imposed cell cycle cells and the measured biochemical affinities observed in vitro. In arrest. Currently, studies are under way to test this model and to vitro, p18 can form stable complexes with cdk4 with affinity equal to elucidate further the mechanisms by which p16 differs from p18 in that of p16, but in vivo p18 is found to associate preferentially with vivo. Future experiments will be required to determine more precisely cdk6 (3, 4, 23, 24). Moreover, endogenous p18/cdk4 complexes the role these inhibitors play in the transformed environment and to appear to be unstable, implying that currently undefined cellular define the biological activities that are targeted for inactivation in components may influence the turnover rate of the complex (14). In human tumors. the current study, we have provided additional evidence that p18 must be subject to in vivo posttranslational regulatory mechanisms that Acknowledgments modulate its activity. Our data implicate a region of p18 that does not participate in kinase binding. We thank Dr. Robert Booher of Onyx Pharmaceuticals (Richmond, CA) for Our results and those of others cited above highlight a fundamental graciously providing recombinant cyclin D1/cdk4 and Peter Burch (University discrepancy between the behavior of p18 under in vitro and in vivo of Vermont) for thoughtful comments and discussions. We also thank Dr. Jeff conditions. Because cellular INK4 complexes contain only an INK4 Bond (University of Vermont) for his assistance with molecular modeling. protein and a partner cdk (14), we conclude that some property resident in the fifth ankyrin repeat of p18 allows for modulation of the References stability of the INK4/cdk complex through a transient interaction with 1. Sherr, C. J., and Roberts, J. M. CDK inhibitors: positive and negative regulators of additional cellular factors. We believe that the transferrable nature of G1-phase progression. Genes Dev., 13: 1501–1512, 1999. 2. Hanahan, D., and Weinberg, R. A. The hallmarks of cancer. Cell, 100: 57–70, 2000. the difference between p18 and p16 strongly suggests the existence of 3. Guan, K. L., Jenkins, C. W., Li, Y., Nichols, M. A., Wu, X., O’Keefe, C. L., Matera, an active regulatory process mediated through the COOH terminus of A. G., and Xiong, Y. Growth suppression by p18, a p16INK4/MTS1- and p14INK4B/ p18. 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3868

Jay Gump, Saralinda Turner and James Koh

Cancer Res 2001;61:3863-3868.

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