Kathryn L. Ball

Inhibition of pRb phosphorylation and cell-cycle progression by a 20-residue peptide from p16CDKN2/INK4A

Abstract Background: The CDKN2/INK4A tumour suppressor gene is deleted or mutated in a large number of human cancers. Overexpression of its product, p16, has been shown to block the transition through the G 1 /S phase of the cell cycle in a pRb-dependent fashion by inhibiting the cyclin D-dependent kinases cdk4 and cdk6. Reconstitution of p16 function in transformed cells is therefore an attractive target for anti-cancer drug design. Results We have identified a 20-residue synthetic peptide — corresponding to amino acids 84–103 of p16 – that interacts with cdk4 and cdk6, and inhibits the in vitro phosphorylation of pRb mediated by cdk4–cyclin D1. The amino-acid residues of p16 important for its interaction with cdk4 and cdk6 and for the inhibition of pRb phosphorylation were defined by an alanine substitution series of peptides. In normal proliferating human HaCaT cells and in cells released from serum starvation, entry into S phase was blocked by the p16-derived peptide when it was coupled to a small peptide carrier molecule and applied directly to the tissue culture medium. This cell-cycle block was associated with an inhibition of pRb phosphorylation in vivo . Conclusion These results demonstrate that a p16-derived peptide can mediate three of the known functions of p16: firstly, it interacts with cdk4 and cdk6; secondly, it inhibits pRb phosphorylation in vitro and in vivo ; and thirdly, it blocks entry into S phase. The fact that one small synthetic peptide can enter the cells directly from the tissue culture medium to inhibit pRb phosphorylation and block cell-cycle progression makes this an attractive approach for future peptidometic drug design. Our results suggest a novel and exciting means by which the function of the p16 suppressor gene can be restored in human tumours.

Cell-cycle arrest and inhibition of Cdk4 activity by small peptides based on the carboxy-terminal domain of p21WAF1

BACKGROUNDnA common event in the development of human neoplasia is the inactivation of a damage-inducible cell-cycle checkpoint pathway regulated by p53. One approach to the restoration of this pathway is to mimic the activity of key downstream effectors. The cyclin-dependent kinase (Cdk) inhibitor p21(WAF1) is one such molecule, as it is a major mediator of the p53-dependent growth-arrest pathway, and can, by itself, mediate growth suppression. The primary function of the p21(WAF1) protein appears to be the inhibition of G1 cyclin-Cdk complexes. Thus, if we can identify the region(s) of p21(WAF1) that contain its inhibitor activity they may provide a template from which to develop novel anti-proliferative drugs for use in tumours with a defective p53 pathway.nnnRESULTSnWe report on the discovery of small synthetic peptides based on the sequence of p21(WAF1) that bind to and inhibit cyclin D1-Cdk4. The peptides and the full-length protein are inhibitory at similar concentrations. A 20 amino-acid peptide based on the carboxy-terminal domain of p21(WAF1) inhibits Cdk4 activity with a concentration for half-maximal inhibition (l0.5) of 46 nM, and it is only four-fold less active than the full-length protein. The length of the peptide has been minimized and key hydrophobic residues forming the inhibitory domain have been defined. When introduced into cells, both a 20 amino-acid and truncated eight amino-acid peptide blocked phosphorylation of the retinoblastoma protein (pRb) and induced a potent G1/S growth arrest. These data support a physiological role for the carboxyl terminus of p21(WAF1) in the inhibition of Cdk4 activity.nnnCONCLUSIONSnWe have discovered that a small peptide is sufficient to mimic p21(WAF1) function and inhibit the activity of a critical G1 cyclin-Cdk complex, preventing pRb phosphorylation and producing a G1 cell-cycle arrest in tissue culture cell systems. This makes cyclin D1-Cdk4 a realistic and exciting target for the design of novel synthetic compounds that can act as anti-proliferative agents in human cells.

The conformationally flexible S9-S10 linker region in the core domain of p53 contains a novel MDM2 binding site whose mutation increases ubiquitination of p53 in vivo.

Although the N-terminal BOX-I domain of the tumor suppressor protein p53 contains the primary docking site for MDM2, previous studies demonstrated that RNA stabilizes the MDM2·p53 complex using a p53 mutant lacking theBOX-I motif. In vitro assays measuring the specific activity of MDM2 in the ligand-free and RNA-bound state identified a novel MDM2 interaction site in the core domain of p53. As defined using phage-peptide display, the RNA·MDM2 isoform exhibited a notable switch in peptide binding specificity, with enhanced affinity for novel peptide sequences in either p53 or small nuclear ribonucleoprotein-U (snRNP-U) and substantially reduced affinity for the primary p53 binding site in the BOX-I domain. The consensus binding site for the RNA·MDM2 complex within p53 is SGXLLGESXF, which links the S9–S10 β-sheets flanking the BOX-IV and BOX-V motifs in the core domain and which is a site of reversible conformational flexibility in p53. Mutation of conserved amino acids in the linker at Ser261 and Leu264, which bridges the S9–S10 β-sheets, stimulated p53 activity from reporter templates and increased MDM2-dependent ubiquitination of p53. Furthermore, mutation of the conserved Phe270 within the S10 β-sheet resulted in a mutant p53, which binds more stably to RNA·MDM2 complexes in vitro and which is strikingly hyper-ubiquitinated in vivo. Introducing an Ala19 mutation into the p53F270A protein abolished both RNA·MDM2 complex binding and hyper-ubiquitinationin vivo, thus indicating that p53F270A protein hyper-ubiquitination depends upon MDM2 binding to its primary site in the BOX-I domain. Together, these data identify a novel MDM2 binding interface within the S9–S10 β-sheet region of p53 that plays a regulatory role in modulating the rate of MDM2-dependent ubiquitination of p53 in cells.

Specificity determinants for the AMP-activated protein kinase and its plant homologue analysed using synthetic peptides

Inspection of sequences around sites phosphorylated by the AMP‐activated protein kinase (AMP‐PK), and homologous sequences from other species, indicates conserved features. There are hydrophobic residues (M, V, L, I) at P‐5 and P+4, and at least one basic residue (R, K, H) at P‐2, P‐3 or P‐4. The importance of these residues has been established for AMP‐PK and its putative plant homologue using a series of synthetic peptides. These results confirm the functional similarity of the animal and plant kinases, and suggest that the required motif for recognition of substrate by either kinase is M/V/L/I‐(R/K/H,X,X)‐X‐S/T‐X‐X‐X‐M/V/L/I.

Resistance of human nucleotide excision repair synthesis in vitro to p21Cdn1

The p21Cdn1 protein (cip1/waf1/sdi1) plays an important role as an inhibitor of mammalian cell proliferation in response to DNA damage. By interacting with and inhibiting the function of cyclin-Cdk complexes, p21 can block entry into S phase. p21 can also directly inhibit replicative DNA synthesis by binding to the DNA polymerase sliding clamp factor PCNA. When cells are damaged and p21 is induced, DNA nucleotide excision repair (NER) continues, even though this pathway is PCNA-dependent. We investigated features of p21-resistant NER using human cell extracts. A direct end-labelling approach was used to measure the excision of damaged oligonucleotides by NER and no inhibition by p21 was found. By contrast, filling of the ∼30u2009nt gaps created by NER could be inhibited by pre-binding p21 to PCNA, but only when gap filling was uncoupled from incision. Binding p21 to PCNA could also inhibit filling of model 30u2009nt gaps by both purified DNA polymerases δ and ε. When p21 was incubated in a cell extract before addition of PCNA, inhibition of repair synthesis was gradually relieved with time. This incubation gives p21 the opportunity to associate with other targets. As p21 blocks association of DNA polymerases with PCNA but does not prevent loading of PCNA onto DNA, repair gap filling can occur rapidly as soon as p21 dissociates from PCNA. A synthetic PCNA-binding p21 peptide was an efficient inhibitor of NER synthesis in cell extracts.

PDK1-dependent activation of atypical PKC leads to degradation of the p21 tumour modifier protein.

p21WAF1/CIP1 contributes to positive and negative growth control on multiple levels. We previously mapped phosphorylation sites within the C‐terminal domain of p21 that regulate proliferating cell nucear antigen binding. In the current study, a kinase has been fractionated from mammalian cells that stoichiometrically phosphorylates p21 at the Ser146 site, and the enzyme has been identified as an insulin‐responsive atypical protein kinase C (aPKC). Expression of PKCζ or activation of the endogenous kinase by 3‐phosphoinositide dependent protein kinase‐1 (PDK1) decreased the half‐life of p21. Conversely, dnPKCζ or dnPDK1 increased p21 protein half‐life, and a PDK1‐dependent increase in the rate of p21 degradation was mediated by aPKC. Insulin stimulation gave a biphasic response with a rapid transient decrease in p21 protein levels during the initial signalling phase that was dependent on phosphatidylinositol 3‐ kinase, PKC and proteasome activity. Thus, aPKC provides a physiological signal for the degradation of p21. The rapid degradation of p21 protein during the signalling phase of insulin stimulation identifies a novel link between energy metabolism and a key modulator of cell cycle progression.

Allosteric effects mediate CHK2 phosphorylation of the p53 transactivation domain.

The tumour suppressor p53 is a tetrameric protein that is phosphorylated in its BOX‐I transactivation domain by checkpoint kinase 2 (CHK2) in response to DNA damage. CHK2 cannot phosphorylate small peptide fragments of p53 containing the BOX‐I motif, indicating that undefined determinants in the p53 tetramer mediate CHK2 recognition. Two peptides derived from the DNA‐binding domain of p53 bind to CHK2 and stimulate phosphorylation of full‐length p53 at Thr 18 and Ser 20, thus identifying CHK2‐docking sites. CHK2 can be fully activated in trans by the two p53 DNA‐binding‐domain peptides, and can phosphorylate BOX‐I transactivation‐domain fragments of p53 at Thr 18 and Ser 20. Although CHK2 has a basal Ser 20 kinase activity that is predominantly activated towards Thr 18, CHK1 has constitutive Thr 18 kinase activity that is predominantly activated in trans towards Ser 20. Cell division cycle 25C (CDC25C) phosphorylation by CHK2 is unaffected by the p53 DNA‐binding‐domain peptides. The CHK2‐docking site in the BOX‐V motif is the smallest of the two CHK2 binding sites, and mutating certain amino acids in the BOX‐V peptide prevents CHK2 activation. A database search identified a p53 BOX‐I‐homology motif in p21WAF1 and although CHK2 is inactive towards this protein, the p53 DNA‐binding‐domain peptides induce phosphorylation of p21WAF1 at Ser 146. This provides evidence that CHK2 can be activated allosterically towards some substrates by a novel docking interaction, and identify a potential regulatory switch that may channel CHK2 into distinct signalling pathways in vivo.

p21: structure and functions associated with cyclin-CDK binding

The cyclin dependent kinase inhibitor, p21, is a multifunctional protein involved in coordinating the cellular response to negative growth signals. Induced by cellular damage under the transcriptional control of the p53 tumour suppressor protein, p21 interfaces with a number of cellular proteins involved in growth control. Although p21 has a diverse range of activities, from assembly factor to transcriptional modulator, its ability to interact with and regulate the activity of the cyclin dependent protein kinases is paramount to many of these functions.

Immunological evidence that HMG-CoA reductase kinase-A is the cauliflower homologue of the RKIN1 subfamily of plant protein kinases

Three different antibodies against the RKIN1 and BKIN12 gene products from rye and barley recognized the 58 kDa subunit of HMG‐CoA reductase kinase‐A (HRK‐A) from Brassica oleracea on Western blots. HRK‐A was also detected by an antipeptide antibody in enzyme‐linked immunoassays, and this was competed by the peptide antigen. HRK‐A was not recognized by antibodies against plant, mammalian and Saccharomyces cereviseae relatives of RKIN1, i.e. wheat PKABA1, rat AMP‐activated protein kinase and S. cerevisiae Snf1p. RKIN1/HMG‐CoA reductase kinase‐A are now among the first protein kinases in plants to be well characterized at both the molecular and biochemical levels.