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Characterization of the -dependent kinase inhibitory domain of the INK4 family as a model for a synthetic tumour suppressor molecule

Robin FaÊ hraeus, Sonia LaõÂ n, Kathryn L Ball and David P Lane

CRC Cell Transformation Group, Department of Biochemistry, University of Dundee, DD1 4HN Dundee, UK

We have previously shown that a 20 amino acid ylation takes place at a time point in the later part of derived from the third ankyrin-like repeat of the the that coincides with the p16CDKN2/INK4a () tumour suppressor (residues of the in which the cells commit themselves 84 ± 103 of the human p16 protein) can bind to cdk4 and to replicate DNA (Pardee, 1989; Geng and Weinberg, cdk6 and inhibit cdk4- kinase activity in vitro 1993). The induced phosphorylation of pRb as well as block cell cycle progression through G1. by CDK- kinases releases the transcrip- Substitution of two valine residues corresponding to tion factor from pRb which leads to the induction of amino acids 95 and 96 (V95A and V96A) of the p16 products that are essential for the cells to peptide reduces the binding to cdk4 and cdk6 and commence DNA replication (Bandara and La increases its IC0.5 for kinase inhibition approximately Thangue, 1991; Chellappan et al., 1991; Neveins, threefold when linked to the Antennapedia homeodomain 1992; La Thangue, 1994). The release of E2F also carrier sequence. The same mutations increase the IC0.5 ensures that pRb remains in a hyperphosphorylated approximately ®vefold in the p16 protein. Substitution of state by a feed back induction of expression aspartic acid 92 by alanine instead increases the binding which together with cdk2 keeps pRb inactive in an of the peptide to cdk4 and cdk6 and the kinase inhibitory mitogen independent fashion (Weinberg, 1995; Sherr, activity. The p16 peptide blocks S-phase entry in non- 1996). In addition to the regulation of RNA synchronized human HaCaT cells by approximately 90% polymerase II through E2F, pRb also suppresses at a 24 mM concentration. The V95A and V96A double activity of the RNA polymerases I and III which substitution minimizes the cell cycle inhibitory capacity might contribute to its control of the cell cycle (White of the peptide whereas the D92A substitution increases et al., 1996). The CDK-cyclin D complex is further its capacity to block cell cycle progression. A regulated by di€erent phosphorylation events and by series of the p16 derived peptide shows that a 10 residue kinase inhibitory of the p16INK4a/CDKN2 and the peptide still retains cdk4-cyclin D1 kinase and cell cycle p21WAF1/KIP families (Hunter and Pines, 1994; Morgan, inhibitory activity. The p16 peptide inhibited S-phase 1995; Sherr and Roberts, 1995). entry in ®ve cell lines tested, varying between 47 ± 75%, The importance of regulating the pRb phosphoryla- but had only a limited (11%) inhibitory e€ect in the pRb tion pathway in G1 for controlled cell growth is negative Saos-2 cells at a concentration of 24 mM. Like emphasized by the frequency in which alterations of the full length p16 protein, the p16 peptide does not this pathway are detected in human . Gene inhibit cyclin E dependent cdk2 kinase activity in vitro. ampli®cation and rearrangement of regions These data suggest that acute inhibition of CDK- in chromosome 11q13 that involve the cyclin D1 gene cyclin D activity by a peptide derived from the INK4 (also described as PRAD1 or bcl1), or over- family will stop cells in late G1 in a pRb dependent expression of cyclin D1, are common events in various fashion. tumours including those of head and neck, oesophagus, bladder, breast and B-cell origin (Lammie et al., 1991; Keywords: p16INK4a; cyclin dependent kinases 4 and 6; Hall and Peters, 1996). The importance of cyclin D1 in cell cycle; peptide delivery; tumour suppressor the development of neoplastic growth is also stressed by the induction of hyperproliferative growth and carcino- mas in mice carrying a transgenic cyclin D1 (Wang et al., 1994). (Vin-1) has also been reported to Introduction be associated with oncogenic activities but at a much lower frequency (Tremblay et al., 1992; Hanna et al., The D type form kinase active complexes with 1993). the cyclin dependent kinases (CDKs) cdk4 (Matsush- Cdk4 is overexpressed in a subset of glioblastomas ime et al., 1992) and cdk6 (Meyerson et al., 1994) and sarcomas (Kathib et al., 1993; Schmidt et al., 1994; and play a major part in phosphorylation and Reifenberger et al., 1996) and a mutation in cdk4 that inactivation of the (pRb) impairs p16 binding and thereby prevents p16 from (Hinds et al., 1992; Ewen et al., 1993; Sherr, 1993; inhibiting cdk4-cyclin D1 kinase activity in vitro has Kato et al., 1993; Weinberg, 1995). pRb phosphor- been reported (WoÈ lfel et al., 1995). Together with mutations in the Rb gene and loss of p16 function, alterations in regulating the pRb pathway have been reported in more than 90% of glioblastomas Correspondence: R FaÊ hraeus (Ischimura et al., 1996). Received 5 June 1997; revised 18 September 1997; accepted 18 Mutations of p16 are detected in approximately half September 1997 of the tumours derived from oesophagus and the bilary INK4 tumour suppressor RFaÊhraeus et al 588 tract and loss of p16 function through homozygous Results deletions are seen at similar frequency in glioblastomas, nasopharyngeal carcinomas, bladder and acute A p16 derived peptide inhibits cdk4-cyclin D1 but not lymphatic leukaemias whereas certain tumours, like cdk2-cyclin E kinase activity pancreatic cancers frequently carry both types of mutations (Hall and Peters, 1996; Nobori et al., 1994; Our previous results have shown that a 20 aa synthetic Kamb et al., 1994; Caldas et al., 1994; Herbert et al., peptide based on a sequence from the third ankyrin- 1994; Einhorn et al., 1994; Hirama and Koe‚er, 1995). like repeat of p16, corresponding to aa 84 ± 103 of the Silencing of p16 function through methylation of the full length human p16 (p16) protein, has similar promoter region has been reported in renal and lung features as the full length protein including binding cancers (Herman et al., 1994; Otterson, 1995). Taken to the cyclin-dependent kinases cdk4 and ckd6, together, loss of p16 function is next to inactivation of inhibiting cdk4-cyclin D1 kinase activity in vitro and one of the most frequent events observed in human block cells from entering the S-phase of the cell cycle. tumours. Evidence of p16s role as a tumour suppressor p21WAF1/CIP1 () inhibits cdk2, cdk4 and cdk6 derived protein also comes from observations that families kinase activity by direct interactions with both the carrying p16 mutations are predisposed for malignant cyclin and the kinase components of the enzyme (Hussusain et al., 1994; Ranade et al., complexes (Sherr and Roberts, 1995; Lin et al., 1996) 1995) and that mice lacking p16 develop tumours early and we have previously identi®ed a 20 aa peptide from (Serrano et al., 1996). the C-terminal domain of p21 that inhibits both cdk4 P16INK4a/CDKN2A (Serrano et al., 1994) and its closely and cdk2 kinase activity in vitro (Ball et al., 1997). In related INK4/CDKN2 family members p15INK4b (p15) order to test the speci®city of the p16 peptide we (Hannon and Beach, 1994), p18INK4c (p18) (Guan et compared the kinase inhibitory capacity of 25 mM of al., 1994; Hirai et al., 1995) and p19INK4d (p19) (Hirai the p16 and the p21 peptides using Sf9 insect cell et al., 1995; Chan et al., 1995) interact speci®cally lysates coexpressing either cdk4-cyclin D1 or cdk2- with cdk4 and cdk6 and prevent the phosphorylation cyclin E. As shown in Figure 1, the p16 peptide and of pRb by the cyclin D-CDK complex in vitro. the p21 derived peptide blocks cdk4-cyclin D1 activity, However, only p16 is strongly linked to tumour but only the p21 derived peptide signi®cantly inhibits suppression activity. The exact mechanism of CDK- cdk2-cyclin E activity at this concentration. These cyclin D inhibition by the INK4 proteins and how this results strengthen the speci®city of the p16 peptide in e€ects the CDK-cyclin D interaction remains to be that it mimics the activity of the full length p16 protein elucidated. Di€erent reports suggest that binding of by inducing a G1 arrest through inhibition of cyclin D- p16 to cdk4 will block cyclin D interaction whereas dependent kinase activity. others describe p16 and p19 in a complex together with both CDKs and cyclin Ds (Parry et al., 1995; Characterization of the kinase inhibitory INK4 sequence Hirai et al., 1995). Recent results might shed some light on potential Members of the INK4 (CDKN2) family of kinase physiological processes that requires p16 function in inhibitors block CDK-cyclin D kinase activity in a order to give it tumour suppressor function. The very similar fashion through direct interaction with cdk4 low turnover of p16 mRNA (Parry and Peters, 1996) and cdk6. The p18 (Guan et al., 1994; Hirai et al., and the observations of high levels of p16 in late 1995) and mouse p16 (Quelle et al., 1995) sequences are passage number ®broblasts and keratinocytes are both similar (60 and 90% identity, respectively) to the region in line with the idea that once p16 expression is of p16 corresponding to aa 84 ± 103 of p16 (Figure 2d). induced it will lead to an irreversible exit from the cell Biotin-linked peptides from the same region of p18 and cycle as part of the mechanism (Serrano et al., 1996; Loughran et al., 1996; Alcorta et al., 1996). This hypothesis is supported by data suggesting the existence of a gene involved in senescence mapping to the 9p21 locus (Loughran et al., 1994) and the loss of senescence in p167/7 mouse embryo ®broblasts (Serrano et al., 1996). In this report we have analysed the kinase inhibitory domain of human p16 and the closely related human p18 and mouse p16 (Quelle et al., 1996) in vitro.We have minimized the size of the p16 kinase inhibitory peptide and investigated its capacity to mimic p16 induced cell cycle arrest in the G1 phase of the cell cycle (Lukas et al., 1995; Serrano et al., 1995) in order to explore its potential as a lead molecule for anti proliferative therapeutics targeting cdk4 and cdk6 kinase activity. These results strengthen our previous ®ndings that a small peptide derived from the kinase Figure 1 The p16INK4a and the p21WAF1/CIP1 derived peptides inhibitory domain of the INK4 proteins can be used to were incubated at 25 mM concentration with Sf9 cell lysates block cells from entering the S-phase of the cell cycle coexpressing either cdk4-cyclin D1 or cdk2-cyclin E. The kinase and support the use of a small INK4 derived molecule activity was determined using recombinant pRb as substrate. The values were obtained by densitometer analysis of the pRb band as a CDK-cyclin D kinase inhibitor for anti prolif- on the autoradiographs and the ®gure represents one of two erative purposes. similar experiments INK4 tumour suppressor peptides RFaÊhraeus et al 589 mouse p16 were synthesized and tested for binding to (Figure 2d). Surprisingly, we found that deletion of cdk4 and cdk6 and for inhibition of CDK-cyclin D two residues at either the C-terminus (peptide 5) or the kinase activity in order to understand more about N-terminus (peptide 7) severely reduced both the CDK which residues of the the INK derived peptides that are binding and the kinase inhibitory capacity of the important for the inhibition of cdk4 and cdk6 kinases. peptide and that the kinase inhibitory activity was Figure 2 shows that the p16 peptide (peptide 4) and restored when two more residues were deleted from peptides derived from the corresponding regions of the either terminus (compare peptides 5, 6, 7 and 8 in p18 and mouse p16 proteins (peptides 1 and 2) bind to Figure 2). Peptide 11 contains 10 residues that lie in vitro translated cdk4 (Figure 2a) and cdk6 (Figure within a region that a previous alanine scan (FaÊ hraeus 2b) and inhibit cdk4-cyclin D1 phosphorylation of et al., 1996) identi®ed as important for both CDK recombinant full length pRb protein in a similar binding and kinase inhibition. Since binding of manner (compare ®gures 2a, 2b and 2c). peptides 5, 6, 7 and 11 to cdk4 and cdk6 is similar Next we tested if the 20 aa p16 peptide could be but 5 and 7 are not kinase inhibitory, it can be minimized while still retaining kinase inhibitory speculated that deletion of either the two N- or C- activity. A p16 peptide deletion series was made by terminal residues (peptide 5 and 7) induces an deleting residues from either the N- or the C-terminus infavourable conformation that inhibits these peptides

Figure 2 Comparison of cdk4 and cdk6 interaction and cdk4-cyclin D1 kinase inhibition of INK4 derived peptides. Biotinylated peptides (see Materials and methods) derived from the human p16 (hp16) kinase inhibitory domain or from the corresponding domain of mouse p16 (mp16) or human p18 (p18) were analysed for binding to in vitro translated cdk4 (a) and cdk6 (b). Peptides bound to streptavidin coated agarose beads were used to precipitate 35S-methionine-labelled cdk4 and cdk6. The relative binding was quanti®ed using densitometer analysis of the autoradiography. The same peptides were tested for their capacity to inhibit cdk4- cyclin D1 phosphorylation of recombinant pRb protein at 25 mM concentration (c). The di€erent values in a, b and c represent one of at least two independent experiments and are given in relation to the data obtained with the human p16 derived peptide. (d) Di€erences in the peptide sequences of human p18, mouse p16 and the R87P mutant compared to the human (hp16) sequence is underlined. This region corresponds to residues 84-103 of the p16 full length protein INK4 tumour suppressor peptides RFaÊhraeus et al 590 from interacting with the CDKs whereas peptides 6 and 11 interaction with cdk4 is sucient for inhibition of kinase activity but not for precipitating the kinases in the assay conditions used. The R87P mutation is derived from a familial and it is inactive as a kinase inhibitor (Koh et al., 1995; Parry et al., 1996). Residue 87 is located outside the 10 amino acid minimal CDK interacting domain and R87P has no e€ect on the kinase inhibitory capacity when inserted in the 20 residue peptide (peptide 3).

E€ect of alanine substitutions at position 95, 96 and 92 Previous results have suggested that the two valines at position 95 and 96 are important for the binding of the p16 peptide to cdk4 and cdk6 and for its CDK-cyclin D1 kinase inhibitory function and that substitution of aspartic acid 92 to alanine increases both the binding to CDK and its kinase inhibitory activity (FaÊ hraeus et al., 1996). These residues lie within the 10 amino acid minimal inhibitory region of the p16 peptide that is shown in Figure 2. V95A and V96A double substitution and a D92A substitu- tion were introduced into highly puri®ed p16 peptides linked to a 16 amino acid sequence derived from the third domain of the Antennapedia homeodomain allowing the peptides to be transported into cells directly from the tissue culture medium (Derossi et al., 1994). This allows comparison of in vitro activity Figure 3 Comparison of the cdk4-cyclin D1 kinase inhibitory with cell cycle inhibitory capacity using the same capacity of human p16 derived peptides linked to the peptides (see further in next section). We also Antennapedia carrier sequence (a) and full length His-tagged p16 protein (b) following alanine substitutions of the two valines introduced the V95A and V96A mutation into the corresponding to residues 95 and 96 of the p16 protein and the full length His-tagged p16 protein in order to D92A substitution. Peptides, or p16 proteins, were incubated compare the e€ect of these amino acid substitutions together with Sf9 insect cell lysates containing cdk4 and cyclin D1 in p16 full length protein and in the peptide with before recombinant pRb protein and [g32P]ATP was added. regard to kinase inhibition in vitro. Figure 3a shows Alanine substitution of valines 95 and 96 increases the IC0.5 of the full length p16 protein less than ®vefold and the carrier linked that the IC0.5 of the carrier linked p16 peptide with peptide approximately threefold. The values show one of three

the V95A and V96A mutations increased the IC0.5 indpendent and similar experiments approximately threefold (1.5 ± 4.5 mM) whereas the same substitutions in the His-tagged p16 protein

increased the IC0.5 less than ®vefold (6 ± 25 nM)of In order to compare peptides 20 and 21 with the

(Figure 3b). It should be noticed that the IC0.5 of the `wild type' carrier linked p16 peptide in terms of their

full length protein is signi®cantly lower than the IC0.5 ability to block cell cycle progression we added them of the carrier linked p16 peptide and, interestingly, directly to tissue culture medium containing 10% FCS

the IC0.5 of a non-carrier linked p16 peptide is more using non-synchronized HaCaT cells or HaCaT cells than tenfold higher than of the carrier linked peptide that had been deprived of serum for 72 h. Cell cycle (data not shown). distribution was determined 24 h after peptide addition by studying BrdU incorporation by FACS analysis. Figure 5 shows that 12 m of the p16 carrier linked Cell cycle arrest by p16 derived peptides M peptide eciently blocks serum deprived HaCaT cells The Antennapedia homeodomain sequence linked to the from entering S-phase after these cells have been p16 peptide allows the chimeric peptides to be treated with 10% FCS. The V95A and V96A double transported across the cell membranes directly from substitution has only a marginal e€ect on the cell cycle the tissue culture medium to both the cytoplasm and progression compared to the non-peptide treated cells, the nuclear compartments (Derossi et al., 1994). In whereas the same concentration of the peptide carrying Figure 4, biotinylated carrier-linked p16 peptides the D92A substitution induces a near complete G1 (pep 20 carries D92A substitution and pep 21 carries block. Table 1 shows a dose dependent inhibition of V95A and V96A substitutions) were added to the tissue cells entering S-phase in the presence of p16 carrier culture medium 15 min and 2 h prior to ®xation and linked peptides and that the concentration of peptides staining with FITC conjugated streptavidin. These required to induce an equivalent G1 block in cells that results show that both peptides are delivered into the have been serum deprived is approximately 50% less cells and distributed in the nucleus in a similar way, than for non-synchronized cells. The reason for this is suggesting that any di€erences in cell behaviour after not clear but it suggests that the amount of the adding these peptides are not likely due to di€erences protein/s that is target by the p16 peptide and/or the in peptide uptake. binding of the peptide to its target is reduced after INK4 tumour suppressor peptides RFaÊhraeus et al 591

Figure 4 Non-synchronized HaCaT cells were treated with 10 mM biotinylated p16 peptides linked to the Antennapedia carrier sequence. Peptide 20 (pep 20) carries the D92A substitution and peptide 21 (pep 21) the V95A and V96A substitutions. Cells were ®xed 15 min or 2 h after peptide addition and stained with FITC coupled avidin. Both peptides give a similar nuclear staining pattern. Non-peptide treated cells show a negative staining and results are not shown

Figure 5 FACS analysis of HaCaT cells treated with p16 derived peptides linked to the Antennapedia carrier sequence. Cells were culture in DMEM without FCS for 72 h before 10% FCS, 10% FCS and 12 mM of peptides, or fresh DMEM with no FCS was added. Cells were treated with 10 mM of BrdU for 20 min 24 h after peptide addition INK4 tumour suppressor peptides RFaÊhraeus et al 592 serum deprivation. The data also shows that the V95A treated cells is increased after 36 h due to colony and V96A substitutions gives a slight inhibition of cell density. These results also show that the p16 peptide cycle progression which is in line with the e€ect of this has little or no e€ect on the G2/M transition of the cell peptide on cdk4-cyclin D1 activity in vitro (Figure 3b). cycle. Table 2 shows that the maximal e€ect on cell cycle We also compared the e€ect of the carrier linked p16 inhibition in HaCaT cell is obtained after 36 ± 48 h if peptides on six di€erent cell lines at approximately the peptide is left in the medium. The decrease of cells 50% con¯uence using 20 mM peptide concentration. in G1 after 36 ± 48 h suggests that the a€ect of the Table 3 shows that the p16 peptide blocks S-phase peptide is reversible, presumably due to degradation, or entry in four of these cell lines in a fashion similar to that a small population of cells is left una€ected. The that observed in the HaCaT cells, varying between 47 ± time course on serum deprived cells shows a similar 75%. We also tested the pRb negative cell line Saos-2 pattern even though it has to be considered that BrdU and could show that the peptide only reduced the uptake cannot be estimated until 24 h after serum percentage of cells in S-phase from 36 to 32%, or addition and that the number of cells in G1 in the non- reduced the entry of S-phase by 11%. Since p16

Table 1 % Cell cycle distribution of HaCaT cells treated with mutated carrier-linked p16 peptides Peptide conc. Cycling cells mM G1 S G2/M Inhib. of S-phase entry % D92A 24.0 84.7 3.7 11.3 92.6 12.0 68.7 21.8 8.4 56.7 6.0 50.7 37.4 9.9 25.2 3.0 40.5 43.7 11.8 12.6 1.5 37.5 45.2 12.2 9.6 0.7 35.7 48.9 11.7 2.2 V95A and V96A 24.0 55.5 32.5 10.0 35.0 12.0 45.9 39.8 11.9 20.4 6.0 40.7 45.5 11.1 9.0 3.0 38.6 46.9 11.1 6.2 1.5 36.4 48.1 11.4 3.8 0.7 36.1 50.2 10.8 0 Serum deprived cells D92A 12.0 93.3 2.5 4.2 94.7 6.0 75.5 17.9 6.5 61.4 3.0 52.3 39.9 7.8 14.8 1.5 44.1 43.1 7.3 7.0 0.75 45.8 47.6 6.6 0 V95A and V96A 12.0 63.4 28.3 9.1 38.8 10% FCS 47.1 46.3 8.5 No FCS 88.2 6.9 4.6

The Antennapedia carrier-linked p16 peptide was added to normal cycling HaCaT cells at 20 mM in the presence of 10% FCS 24 h before harvest. 72 h serum deprived HaCaT cells were treated with 10% FCS and 8 mM peptides 24 h before harvest. % cell cycle distribution was determined by FACS analysis

Table 2 FACS analysis of cycling and serum deprived HaCaT cells treated with carrier-linked p16 peptide G1 S G2/M Peptide + 7 + 7 + 7 Cycling cellsa 12 h 70.4 41.0 16.4 48.9 12.2 8.9 24 h 88.6 41.9 7.2 49.0 3.8 7.4 36 h 93.4 62.8 4.2 30.5 2.2 5.7 48 h 90.9 79.0 6.0 17.6 2.9 2.8 60 h 71.4 81.2 21.9 13.8 5.4 3.9 Washb 24 h 75.4 44.2 17.3 42.2 3.7 9.3 36 h 67.5 57.2 21.7 33.5 5.8 7.3 Serum deprived cellsc 24 h 69.2 29.7 24.0 63.5 5.4 4.6 36 h 40.3 45.3 40.0 39.5 11.4 13.1 48 h 52.4 39.3 38.9 51.7 7.1 14.1 60 h 63.2 67.0 27.8 27.8 7.5 4.6 Washd 36 h 28.2 44.6 50.0 39.8 13.4 14.0 44 h 43.3 45.1 7.5 a 20 mM of Antennapedia carrier-linked p16 peptide was added at 0 h to cycling cells and harvested at indicated time points orb washed with fresh medium 12 h after peptide was added. cCells were depleted for serum for 72 h before d serum and 8 mM peptide was added or washed with fresh medium 24 h after peptide was added INK4 tumour suppressor peptides RFaÊhraeus et al 593 induced cell cycle arrest has been shown to depend on p16 protein that is included in the minimal kinase the presence of a functional pRb protein (Lukas et al., inhibitory p16 peptide (residues 89 ± 98). We have 1995) these data strengthens the speci®city of the previously suggested that these mutations would peptide. induce conformational changes in the protein that The in vitro data demonstrate that the kinase would e€ect the presentation of the kinase inhibitory inhibitory p16 derived sequence can be reduced to 10 domain of the p16 protein. Recent results obtained by amino acids. We also wanted to know if this sequence NMR support this hypothesis since two mutations would be sucient to mediate cell cycle arrest. Two associated with familial melanomas, P114L and chimeric peptides were synthesized linking the Anten- G101W, were both shown to induce global conforma- napedia carrier sequence to 14 and 10 residues of the tional changes of the p16 protein (Tevelev et al., 1996). p16 kinase inhibitory sequence and the cell cycle This is further supported by data demonstrating that inhibitory capacity was compared with that of the the G101W mutation and another mutation associated p16 peptide carrying the D92A substitution (Table 4). with familial melanoma, V126D, are both temperature At 12 mM concentration these truncated peptides were sensitive (Parry and Peters, 1996). The R87P mutation as e€ective in blocking S-phase entry of serum deprived is, however, an inactive mutation that is not HaCaT cells as the longer peptide, demonstrating that temperature sensitive and the R87P substitution a 10 amino acid peptide sequence linked to the carrier peptide was synthesized in order to see if it would peptide is sucient to block cell cycle progression. e€ect the kinase inhibitory activity of the peptide. We found that it did not e€ect either kinase inhibition or cdk4 and cdk6 binding of and since the R87 residue is Discussion not located in the minimal sequence required for direct interaction with cdk4 and cdk6 it is likely that the R87 There is mounting genetic data supporting the mutation will only a€ect the kinase inhibitory domain importance of controlled CDK-cyclin D kinase activ- when introduced in the full length protein. ity for normal cell growth. Deregulated expression of The results obtained with the carrier linked p16 cyclin D1 and cdk4 are frequently seen in various peptides show that alanine substitution for the two human tumours and loss of p16 function is one of the valine residues at positions corresponding to resi- most common events associated with human cancers dues 95 and 96 of the full length protein increase the

(Sherr, 1996; Hall and Peters, 1996; Ichimura et al., IC0.5 of the CDK-cyclin inhibition in vitro approxi- 1996; Hirama and Koe‚er, 1995). This work has mately threefold in the p16 peptide linked to the focused on characterization of the kinase inhibitory Antennapedia carrier sequence and approximately domain of the INK4 family as a model for the ®vefold in the full length protein. These data indicate development of small molecules in order to inhibit that the V95A and V96A substitutions have a similar CDK-cyclin D activity in tumour cells that are e€ect on cdk4-cyclin D1 kinase inhibition in both the defective in the regulation of the G1 checkpoint but carrier linked peptide and in the full length protein. In carry a functional pRb. this context it is also interesting to note that the carrier Single amino acid substitutions that results in loss of linked peptides has gained approximately tenfold function can be found throughout p16 (Ranade et al., higher activity in vitro compared to the non-carrier 1995; Koh et al., 1995; Parry and Peters, 1996). These linked peptide suggesting that the carrier sequence mutations are often located outside the sequence of the confers stability to the p16 inhibitory peptide conformation. Importantly, the carrier-linked peptide containing Table 3 Cell type dependent G1 arrest by the hp16 derived peptide the V95A and V96A substitutions is less active as a cell G1 S G2/M % inhibition cycle inhibitor compared to the wild type p16 peptide Peptide 7 + 7 + 7 + of entry into S and the D92A substitution peptide is a more potent HaCaT 42 79 49 12 12 9 75 inhibitor. This suggests a strong correlation between MCF-7 40 65 35 15 12 11 57 the kinase inhibition in vitro and cell cycle inhibition. MRC-5 37 62.5 38 20 16 12 47 In order to evaluate the possibility of using the p16 3T3 48 72 39 19 8 6 52 peptide as a lead for anti proliferative drug develop- HT-29 54 83 37 11 5 4 70 ment we wanted to see if we could reduce the size of Saos-2 37 36 36 32 16 15 11 the p16 peptide from the original 20 residues to a size Non-synchronized cells were treated with 20 mM of p16 peptide linked to the Antennapedia carrier sequence for 24 h before being harvested that could be possible to mimic with a small molecule. and analysed by FACS. The percentage of cells in each stage of the The deletion series presented in Figure 2 shows that the cell cycle is given with or without peptide treatment minimal sequence of the peptide can be reduced by at

Table 4 G1 S G2/M % S inhibition Chimeric Peptide DAAREGFLATLVVLHRAGAR- 91.4 3.5 5.0 90 EGFLDTLVVLHRAGAR- 93.1 2.2 4.6 94 FLDTLVVLHR- 95.6 1.8 2.7 95 No peptide 55.3 36.1 8.4

Serum deprived HaCaT cells were treated with 12 mM of p16 derived peptides linked to the Antennapedia carrier sequence at the C-terminus. Cell cycle distribution was determined by FACS analysis 24 h after addition of 10% FCS and peptides INK4 tumour suppressor peptides RFaÊhraeus et al 594 least 10 residues without loosing activity. The 10 amino expression is induced after the cells have been treated acid sequence (peptide 11) has lost its capacity to form with TGF-b (Hannon and Beach 1995) and increased a stable complex to cdk4 but is still e€ective as a kinase levels of p16 and p18 are observed in cells of late inhibitor and, most importantly, when linked to the passage numbers (Hara et al., 1996; Alcorta et al., Antennapedia carrier sequence this chimeric peptide 1996; Loughran et al., 1996) and in skeletal muscle blocks cell cycle progression. This deletion series, as cells undergoing di€erentiation (Franklin and Xiong, well as a previous alanine scan of the p16 peptide, fails 1996), respectively. This might suggest that in spite of to identify peptides where there is clear binding to the similar biochemical activity, it is possible that the e€ect CDKs but no kinase inhibition and, apart from the of CDK inhibition in the cell by the INK proteins can shorter peptides 6 and 11, there is a strong correlation vary depending on the cell phenotype. This would between CDK binding and kinase inhibition. imply that acute inhibition of CDK-cyclin D kinase The sequence of the 10 aa minimal inhibitory p16 activity by substances introduced directly into the cells, peptide show similarities with the consensus of central like the carrier linked INK4 peptides, will induce a G1 which has been suggested to form an arrest which would be associated with di€erent cellular amphipatic helical wheel (Bork, 1993). The p18 peptide events depending on cell phenotype and growth carries eight substitutions of which three are located in conditions. the 10 aa minimal binding sequence compared to the The data presented here extend our previous p16 peptide. Since we previously identi®ed the observation that a small peptide that blocks cdk4- hydrophobic residues 94 ± 97 as important for the p16 cyclin D1 kinase activity in vitro can be used to mimic peptide function the glutamine at positions 95 and the the tumour suppressing function of the p16 protein. threonine at position 96 in p18 would at ®rst seem to The peptide, like the p16 protein, can inhibit cyclin D1 disrupt the consensus pattern of the binding domain. interaction with cdk4 if added to cdk4 prior to However, the p18 peptide carries two hydrophobic cyclin D1 (Parry et al., 1995; FaÊ hraeus et al., 1996), leucine residues at position 97 and 98 which allows it suggesting that a possible mean of action is to to form an amphipatic structure in the same helical antagonize the formation of the holoenzyme complex. wheel. Both the p16 and the p18 peptides have the However, the p16 peptide, as well as the full length p16 GFLDTL sequence intact suggesting that this motif protein, inhibits CDK-cyclin D1 activity when added has an important role for the peptide interaction with to enzyme complexes that have already been formed cdk4 and cdk6. without dissociating cdk4-cyclin D1, demonstrating The deletion series shows that when two residues are that more work is needed in order to determine the deleted from the N- or the C-terminal part of the mechanism/s of action of the p16 peptide as well as of peptide it loses its CDK binding and its kinase the p16 protein. inhibitory activity and interestingly, both these proper- ties are restored when two more residues are deleted from either end. This demonstrates that the conforma- Materials and methods tion of the domain of the peptide that interacts with cdk4 and cdk6 can be, like in the full length protein, Peptides altered by residues located outside those directly involved in binding. Peptides were synthesized by Chiron Mimotopes and by Dr The p16 peptide has only marginal e€ect on Weng Chan at the Department of Pharmacological blocking cell cycle progression through G1 in the Sciences at the University of Nottingham. The peptides used for the cdk4 and cdk6 precipitation and for in vitro pRb negative cell line Saos-2 suggesting that the cell kinase inhibitory assays carry a biotin group on a SGSG cycle block depends largely on an intact pRb function linker at the N-terminus of the peptide. The Antennapedia which is in line with what would be predicted by a p16 carrier sequence consists of 16 amino acids and was mimicking CDK-cyclin D speci®c inhibitor (Lukas et synthesized as a fusion peptide linked to the C-terminus al., 1995). The in vitro results showing that the p16 of the p16 peptides. peptide does not inhibit cdk2-cyclin E activity further indicate that the e€ect of the peptide is linked to the Peptide precipitation cyclin D dependent kinase activity at the G1 phase of the cell cycle. Peptide precipitation assays were carried out essentially as The biochemical properties of p16 suggest that it described previously (FaÊ hraeus et al., 1996; Ball et al., 1997) by coupling the biotinylated peptides to streptavidin mediates its tumour suppressor function by controlling coated agarose beads for 1 h at room temperature. After CDK-cyclin D activity at the time of the restriction extensive washing the peptide-coupled beads were incu- point in the late part of G1. It is, however, unclear why bated with 3 ml of reticulocyte lysates containing either 35S- p16 and not its closely related INK4 family members methionine-labelled cdk4 or cdk6 in a total volume of 20 ml are associated with tumour suppressor activity since of PBS and protease inhibitors at 48C for 1 h. The beads the di€erent INK4 proteins are thought to carry out were then washed with 1.56PBS containing 0.02% Triton similar CDK-cyclin D inhibitory function. This lack of X-100 before the samples were analysed by SDS ± PAGE redundancy might be due to post-translation modifica- followed by autoradiography. The bands were quanti®ed tions of the INK4 proteins that allows a higher degree using a Bio-Imager densitometer analyser using whole of speci®city in the cells that is not observed in in vitro band analyser software. assays. In addition, the regulatory mechanism that distinguishes p16 function from the other INK4 Kinase assays proteins could be at the level of . Kinase activity was determined as described (FaÊ hraeus et Di€erent signalling pathways have been suggested to al., 1996; Ball et al., 1997). Sf9 insect cells were infected control the levels of INK4a proteins in the cells. p15 with baculovirus constructs expressing cdk4 and cyclin D1. INK4 tumour suppressor peptides RFaÊhraeus et al 595 Cells coexpressing cdk4 and cyclin D1 were lysed in 10 mM FACS analysis HEPES pH 7.4 bu€er containing 10 mM NaCl, 2 mM DTT, HaCaT cells were either serum deprived for 72 h or 1mM EDTA and 0.1 mM PMSF. The kinase reaction was cultured in 10% FCS before peptides and DMEM medium performedina15ml20mM HEPES pH 7.5 containing containing 10% FCS were added. Cells were incubated for 50 mM ATP (1000 c.p.m. pmol71 [g32P]ATP), 10 mM MgCl , 2 24 h in the presence of peptide before 10 mM BrdU was 2.5 mM EGTA, 10 mM beta-glycerphosphate, 1 mM NaF, added for 20 min. Cells were harvested and ®xed in 75% 1mM DTT and 1 mM Benzamidine. The assays were ethanol before treatment with pepsin (1 mg/ml) for 30 min started by addition of 0.3 mg of recombinant full length and 2 M HCl for 15 min. After extensive washings in PBS, pRb and incubated for 10 min at 308C before the reaction anti-BrdU mAb was added for 1 h in room temperature was stopped by addition of SDS loading bu€er. The before they were washed once and FITC conjugated samples were then run on a SDS gel followed by a secondary was added. The cells were ®nally autoradiography and the bands were analysed using a Bio- resuspended in PBS containing propidium iodine and Imager densitometer. analysed by single cell ¯ow cytometry. p16 Cell lines p16 full length protein was obtained by expressing a Cells were cultured in DMEM with 10% FCS. HaCaT is a pRSET His-tagged fusion construct (kindly provided by G human derived keratinocyte line, MCF-7 is derived from a Peters) in E. coli and the following puri®cations over a human breast carcinoma, MRC-5 cells are human nickel chelating column according to the manufacturers ®broblasts, 3T3 cells are mouse ®broblast, HT-29 is a manual (Invitrogen). The alanine substitutions were human colon carcinoma derived cell line and Saos-2 is an introduced by site directed mutagenesis (Promega) and Rb negative human sarcoma derived cell line. the sequence was con®rmed by DNA sequence analysis.

Peptide uptake Peptide uptake into the cells was determined by adding biotinylated Antennapedia carrier-linked human derived p16 peptides directly into the tissue culture medium. After Acknowledgements 15 min and 2 h the cells were washed in medium and ®xed We are thankful to A Prescott for help with confocal in ethanol : acetic acid (9 : 1) and blocked in medium microscopy, to C Sherr for baculovirus constructs and to G containing 10% FCS and 0.02% Tween-20 before being Peters for p16 and CDK in vitro translation constructs as incubated with FITC conjugated streptavidin. The cells well as to W Chan for peptide synthesis. This work is were mounted in Mowiol containing Hoescht nuclear stain supported by the Cancer Research Campaign. DPL is a for viewing by confocal microscopy. Non-peptide treated Gibb Fellow of the CRC and a Howard Hughes cells did not stain using this protocol. International Scholar.

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