Reversible Proteins Phosphorylation

Barford Group

Section: Section of Structural Biology

Reversible protein phosphorylation is a ubiquitous mechanism for the control of signal transduction networks that regulate diverse biological processes including response to extracellular stimuli, DNA damage and cell growth and division. Changes in protein phosphorylation affect the structure and activity of proteins regulating nearly all aspects of cell life including metabolic processes, DNA replication, gene expression, and the cell cycle. In the human genome, 575 protein kinase genes were identified, and these enzymes form the third most populous gene family. The activities of protein kinases and phosphatases are subject to control both by extracellular stimuli and intracellular mechanisms and the molecular basis of this control is complex and varied. To understand the structural basis for the regulation of protein kinases and phosphatases, and their substrate specificities, we are investigating a number of these enzymes. Many protein kinases and phosphatases regulate signal transduction pathways that mediate cell growth and survival. Knowledge of the molecular details of these proteins will provide a framework for the development of novel therapies for the treatment of cancer. This work is carried out in collaboration with colleagues at the CRC Centre for Cancer Therapeutics and elsewhere.

Protein Kinases

We are investigating the structures of protein kinases that stimulate cell growth, proliferation and survival, and whose elevated activities are implicated in tumourigenesis. Protein kinase B (Akt) is stimulated in response to survival and growth factors, whereas Raf kinase is a downstream target of activated Ras, and TOR is subject to control by both growth factors and nutrients. Inhibitors of these kinases will be used for the treatment of cancer.

Sub-Project Protein Kinase B/Akt

Protein kinase B (PKB/Akt) is a component of an intracellular signalling pathway of fundamental importance that functions to exert the effects of growth and survival factors, and which mediates the response to insulin and inflammatory signals. The enzyme is rapidly activated by phosphorylation following stimulation of phosphoinositide 3-kinase, and generation of the lipid second messenger phosphatidylinositol 3,4,5 trisphosphate [PtdIns(3,4,5)P₃]. Activation of PKB occurs by a multi-step mechanism.  PKB is first recruited to the membrane by association with PtdIns(3,4,5)P₃ mediated by its N-terminal pleckstrin homology domain in a process that also induces a conformational change of the protein. In this state, PKB is a substrate for phosphorylation at two regulatory sites by membrane-localised kinases. PDK1 phosphorylates PKB on a Thr residue (Thr-309 of PKBb) within the activation segment, whereas a distinct kinase activity, termed PDK2 which has been shown to be associated with either rictor-TOR or DNA-PK, phosphorylates PKB at Ser-474 of its C-terminal hydrophobic motif. Activated PKB phosphorylates numerous proteins, regulating diverse cellular processes. Phosphorylation of Thr 309 stimulates activity 100-fold, whereas Ser474 phosphorylation augments this activity a further 10 fold.

PKB is responsible for phosphorylating numerous nuclear and cytosolic proteins that regulate cell metabolism and growth. For example, during insulin signalling, the kinase phosphorylates GSK-3, PFK2 and TSC2 to induce glycogenesis and protein synthesis, while the phosphorylation of proteins that regulate apoptosis such as BAD, caspase-9, forkhead transcription factors and IkB kinase, promotes proliferation and survival. PKB stimulates cell cycle progression by phosphorylation of the CDK inhibitors p21^WAF1 and p27^Kip1, causing their retention in the cytoplasm, whereas mdm2 is localised to the nucleus to suppress p53. PKB plays an important role in the generation of human malignancy. The enzyme is the cellular homologue of v-Akt, an oncogene of the transforming murine leukaemia virus AKT8 isolated from a mouse lymphoma. Viral-Akt is a fusion of the viral Gag protein with the PKBa sequence. Myristoylation of the Gag sequence targets v-Akt to the cell membrane, resulting in its constitutive phosphorylation. The genes for the a and b isoforms of PKB are over-expressed and amplified in ovarian, prostate, pancreatic, gastric, and breast tumours. Moreover, the finding that PTEN, one of the most commonly mutated genes in human cancer, encodes a PtdIns(3,4,5)P₃ lipid phosphatase, provided compelling evidence linking PKB to oncogenesis.

In order to investigate the mechanism of activation of PKB by protein phosphorylation of Thr-309 and Ser-474, and to investigate the role of the PH domain in the regulation of PKB activity, we have determined the structures of both the unphosphorylated (inactive state) and also the activated state of the protein in complex with the non-hydrolysable ATP analogue AMP-PNP and a synthetic peptide based on the site of GSK3b phosphorylation. PKB is expressed using Sf9 cells in our cell culture facility.

Activated PKB was obtained by phosphorylation of the activation segment Thr (Thr309) with PDK1, whereas a mimic of Ser474 phosphorylation was provided by either substituting the hydrophobic motif of PRK2 for the PKB HM, or substituting Asp for Ser474. Our data suggest that a phosphorylated hydrophobic motif has a greatly increased affinity for a hydrophobic motif binding channel within the small lobe of the kinase. By interacting with this channel, the phosphorylated hydrophobic motif orders the aC-helix, leading to concomitant ordering of the activation segment with associated increased kinase activity.

We have recently solved the crystal structures of both the inactive and activated states of protein kinase B (Yang et al., 2002a; 2002b), providing a molecular explanation of how multi-site phosphorylation stimulates the kinase activity of this enzyme. This work provides the framework for initiating structure-based drug discovery programmes that will be conducted in collaboration with Astex Technologies.

PKB publications:

Yang J, Cron P, Good VM, Thompson V, Hemmings BA, Barford D. (2002). Crystal structure of an activated Akt/protein kinase B ternary complex with GSK3-peptide and AMP-PNP.  Nat Struct Biol., 9, 940-944.

Yang J, Cron P, Thompson V, Good VM, Hess D, Hemmings BA, Barford D. (2002). Molecular mechanism for the regulation of protein kinase B/Akt by hydrophobic motif phosphorylation. Mol Cell, 9, 1227-1240.

(NCBI Medline link to ‘akt OR protein kinase B AND review’)

Sub-project: B-Raf Kinase

Raf kinase is a proto-oncogene that links activated cell surface receptors to ERK1/2 by MEK phosphorylation. The activity of Raf-kinase is subject to intricate regulatory mechanisms, that includes control mediated by Ras, reversible Ser/Thr and Tyr phosphorylation, and 14-3-3 interactions. Furthermore, Hsp90 and p50^CDC37 are important for maintaining Raf activity. Current models for Raf regulation suggest that differential phosphorylation and 14-3-3 association allows for intrasteric inhibition of the C-terminal active kinase domain by the conserved N-terminal regulatory domain of Raf-1. Membrane localised interactions with these domains and Ras-GTP with further phosphorylation immediately N-terminal to the protein kinase domain alleviates inhibition and triggers partial Raf kinase activity. Full activity is achieved in concert with activated Src. B-Raf activation differs from Raf-1 in that the former has a higher level of constitutive phosphorylation and the presence of an Asp residue equivalent to phospho-Tyr residues of Raf-1.

Constitutive activation of the RAS-ERK signaling pathway is common to numerous cancers. Approximately 15% of human cancers have activating RAS mutations, and recently mutations in B-RAF were identified in a large-scale screen for genes mutated in human cancer (Davies et al., 2002). Somatic mutations of B-RAF are associated with 60% of malignant melanomas, and occur with moderate to high frequency in colorectal, ovarian and papillary thyroid carcinomas, implicating activating oncogenic mutations of B-RAF as critical promoters of malignancy. Significantly, B-RAF and RAS mutations are restricted to the same tumour types, usually in a mutually exclusive fashion, suggesting that these genes are on the same oncogenic signaling pathway, and that RAS acts to activate B-RAF in these tumours.                                         

Sequence analysis of the B-RAF gene associated with human cancers has identified more than 30 single site mis-sense mutations, mostly within the kinase domain. The mechanism of oncogenic activation of B-RAF therefore differs fundamentally from that of v-Raf, a retroviral oncogene derived from C-RAF. The constitutive activity and high transforming potential of v-Raf most likely results from loss of the auto-inhibitory N-terminal region combined with targeting to the plasma membrane. Most of the mutations of B-RAF are clustered to two regions; the glycine-rich P-loop of the N-lobe, and the activation segment and flanking regions. A Glu for Val substitution at residue 599 in the activation segment, adjacent to the conserved DFG motif, accounts for 90% of B-RAF mutations in human cancers. The V599E mutant of B-RAF possesses the hallmarks of a conventional oncogene. The kinase activity of this mutant protein is greatly elevated, it constitutively stimulates ERK activity in vivo independent of RAS, and potently transforms NIH3T3 cells. Interestingly, the conserved regulatory phosphorylation sites within the activation segment of B-RAF, Thr 598 and Ser 601, flank Val 599, leading to the suggestion that the Glu substitution at this position functions as a phospho-mimetic (Davies et al., 2002). Analysis of three other oncogenic mutants of B-RAF showed that they stimulate kinase activity in a manner similar to ^V599EB-RAF (Davies et al., 2002). Intriguingly however, extensive analysis of B-RAF mutations in cancer shows that seven of the mutations involve highly conserved or invariant residues in the catalytic domain (Davies et al., 2002) that in other kinases are known to be required for optimal catalytic activity, raising the question of how these mutants promote tumourogenesis.

To investigate the mechanisms by which mutant oncogenic forms of B-RAF promote cancer, we have examined a panel of 22 mutants. We showed that eighteen mutants activate B-RAF in vitro and stimulate ERK signalling in vivo, conforming to the conventional model of an activating oncogene. However, four mutants have reduced kinase activity in vitro, but surprisingly, three of these can activate wild-type C-RAF and thereby signal to ERK. We have determined the structures of the ^WTB-RAF and ^V599EB-RAF kinase domains in complex with the C-RAF inhibitor BAY43-9006. These structures suggest that many of the residues that are mutated in cancer contribute to stabilization of an inactive conformation of the B-RAF kinase domain. Mutation of these residues destabilizes this inactive conformation, promoting the active state. For most mutants this stimulates enhanced B-RAF kinase activity towards MEK. However, a few mutants act through a different mechanism, because although their activity towards MEK is reduced, they adopt a conformation that activates wild-type C-RAF which then signals to ERK.

BRAF Kinase publications:

Davies et al., (2002). Mutations of the BRAF gene in human cancer. Nature, 417, 949-954.

Wan PT, Garnett MJ, Roe SM, Lee S, Niculescu-Duvaz D, Good VM, Jones CM, Marshall CJ, Cancer Genome Project, Springer CJ, Barford D, Marais R (2004). Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell, 116, 55-867.

Sub-project: TOR (Target Of Rapamycin)

The TOR signalling pathway functions to activate protein synthesis by induced the stimulatory phosphorylation of S6K and 4E-BP1, and its activity is promoted both by growth factor induced PI(3)K activation and by intracellular nutrient levels (Abraham, 2002). Inhibition of TOR by the anti-fungal agent rapamycin reduces S6K phosphorylation, inhibiting cell growth and division. Rapamycin functions as an immunosuppressant, and its ability to inhibit cell growth and division suggests the exciting potential to control the proliferation of cancer cells.

The molecular details by which TOR mediates its effects, and the mechanisms by which TOR is activated in response to growth factor stimulation and cellular nutrient levels, are not fully understood. Moreover, various models including direct or indirect phosphorylation, or control of protein phosphatases, have been proposed to explain the ability of TOR to control the extent of S6K and 4E-BP1 phosphorylation. Since 2001, however, there has been significant progress towards understanding the function and regulation of TOR, and how TOR is integrated within upstream and downstream signalling pathways, including suggestions that the tumour suppressor complex TSC1/TSC2 functions in the same or in a parallel pathway to TOR (reviewed by McManus and Alessi, 2002; Abraham, 2002). TOR is a large protein (~3000 kDa) comprising a C-terminal protein kinase domain belonging to the phosphoinositide kinase related protein kinase (PIKK) family. The PIKK family comprises a functionally diverse set of molecules. Other members of the family are implicated in signal transduction events triggered by damage to DNA and RNA (ATM, ATR, DNA-PK and SMG-1). All PIKK proteins are encoded by large polypeptides. In the case of TOR, 40 repetitive HEAT motif sequences of ~40 amino acids were recognised at the N-terminus of the protein. Such repeats are also found in huntington protein, an elongation factor, A-subunit of PP2A, in addition to TOR and, as is now known, numerous other proteins, being in fact related to the ARM repeat found in the importins and armadillo. HEAT motifs form a super-secondary scaffolding structure first observed in the A-subunit of PP2A (Groves et al., 1999) that functions to mediate protein-protein interactions, and the formation of large multi-protein complexes. Recent sequence analysis indicates that the N-termini of PIKK family members are constituted entirely from multiple HEAT repeats, with approximately 40 repeats predicted for TOR (Perry and Kleckner, 2003). This region of TOR probably functions to mediate the formation of a multi-protein complex that is important for the ability of TOR to sense nutrient levels and to respond to extracellular signals. The genes for TOR were first discovered in budding yeast (TOR1 and TOR2) as the target of rapamycin, which binds to and inhibits TOR in association with its intacellular receptor FKPB12. In vivo TOR associates with other subunits to form TOR complexes (TORC). Raptor (KOG1) is an adapter protein that couples mTOR to 4E-BP1 and S6 kinase for their phosphorylation and mediates nutrient sensing. In yeast, Loewith et al., (2002) identified five proteins that coprecipitate with S. cerevisiae TOR1 and/or TOR2, including the yeast homolog of raptor, KOG1. Two distinct TOR complexes TORC1 and TORC2 were identified that function in nutrient signally and actin cytoskeletal reorganisation, respectively. Yeast TORC1 is probably the homolog of the mammalian TOR-raptor complex. Our studies are focused on cryo-electron microscopy studies of S. cerevisiae TORC1 and its component subunits.

TOR publications:

Abraham, R.T. (2002). Identification of TOR signaling complexes: more TORC for the cell growth engine. Cell, 111, 9-12.

Protein Phosphatases

Protein phosphatases are a diverse group of proteins that are encoded by three distinct gene families. Two of these, the PPP and PPM families are protein serine/threonine specific phosphatases, whereas the PTP super-family encodes tyrosine specific and dual specificity phosphatases. We are studying a number of protein phosphatases that regulate insulin signalling, cell growth, the cell cycle and DNA repair, that have direct links to the treatment of diabetes and cancer.

Sub-project: Protein Phosphatase 1B

Protein tyrosine phosphatase 1B was the first protein tyrosine phosphatase to be isolated in 1988 by Nicholas Tonks, and the first protein phosphatase whose structure was determined in 1994 (Barford et al., 1994). The enzyme is now known to play an important role to regulate signalling from the insulin receptor in muscle and liver tissues and from the leptin receptors of the hypothalamus. Inhibitors of PTP1B activity would provide the means to treat diabetes and obesity.

Recently, we have addressed the regulation of PTP1B by redox-based regulatory mechanisms, mediated by reactive oxygen species such as hydrogen peroxide and super oxide. Hydrogen peroxide (H2O2) is a cellular second messenger that is required for the optimal activation of numerous signal transduction pathways, particularly those mediated by protein tyrosine kinases. One mechanism by which H2O2 regulates cellular processes is to transiently inhibit protein tyrosine phosphatases (PTPs) by reversibly oxidising their catalytic cysteine, thereby suppressing protein dephosphorylation.

We have described a structural analysis of the redox-dependent regulation of PTP1B, a phosphatase that is reversibly inhibited by oxidation after insulin and EGFstimulation of cells. Unexpectedly, the sulfenic acid intermediate produced in response to PTP1B oxidation is rapidly converted into a novel sulfenyl-amide species where the sulphur atom of the catalytic Cys is covalently linked to the main-chain nitrogen of an adjacent residue. Oxidation of PTP1B to the sulfenyl-amide form was accompanied by large conformational changes in the catalytic site that inhibited substrate binding. We propose that this unusual protein modification both protects the active site Cys residue of PTP1B from irreversible oxidation to sulfonic acid, and permits redox-regulation of the enzyme by promoting its reversible reduction by thiols.

PTP1B publications:

Barford D. (2004).  The role of cysteine residues as redox-sensitive regulatory switches.  Curr Opin Struct Biol., 14, 679-686.

Salmeen A, Andersen JN, Myers MP, Meng TC, Hinks JA, Tonks NK, Barford D. (2003).  Redox regulation of protein tyrosine phosphatase 1B involves a sulphenyl-amide intermediate.  Nature, 423, 769-773.

A. Salmeen, J.N. Andersen, M.P. Myers, N.K. Tonks and D. Barford. (2000). Molecular basis for the dephosphorylation of the activation segment of the insulin receptor by protein tyrosine phosphatase 1B.  Mol. Cell 6, 1401-1412.

A.D.B. Pannifer, A.J. Flint, N.K. Tonks and D. Barford. (1998). Visualisation of the catalytic phosphoryl-enzyme intermediate of PTP1B using X-ray crystallography - Implications for catalytic mechanisms.  J. Biol. Chem., 273, 10454-10462.

A.J. Flint, T. Tiganis, D. Barford and N.K. Tonks. (1997). Development of "substrate trapping" mutants to identify physiological substrates of protein tyrosine phosphatases. Proc. Natl. Acad. Sci. (USA) 94, 1680-1685.

M.R. Groves, Z.-J. Yao, P.R. Roller, T.R. Burke, Jr. and D. Barford. (1998). Structural basis for inhibition of the protein tyrosine phosphatase 1B by phosphotyrosine peptide mimetics.  Biochemistry, 37, 17773-17783.

Z. Jia, D. Barford, A.J. Flint and N.K. Tonks. (1995). Structural basis for phosphotyrosine peptide recognition by protein tyrosine phosphatase 1B.  Science, 268, 1754-1758.

D. Barford, A.J. Flint and N.K. Tonks. (1994). The crystal structure of human protein tyrosine phosphatase 1B.  Science, 263, 1397-1404.

Sub-project: Protein Phosphatase 5

Protein phosphatase 5 (Ppp5), a ubiquitous member of the PPP family of serine/threonine protein phosphatases, participates in several stress activated cellular signalling pathways that regulate growth arrest, apoptosis, and response to ionising radiation induced DNA damage. Similar to other PPP family members (PP1, PP2A, Ppp4), Ppp5 is potently inhibited by the tumour promoters okadaic acid, microcystin, cantharadin, calyculin A and tautomycin.  However, whereas PP1, PP2A and Ppp4 exists as dimers or trimers with a catalytic subunit bound to regulatory subunit(s), Ppp5 uniquely comprises a regulatory N-terminal tetratricopeptide repeat (TPR) domain fused to a C-terminal phosphatase catalytic domain.

The discoveries that Ppp5 forms complexes with glucocorticoid receptors and the Ask1 protein kinase implicated the phosphatase as a regulator of signalling networks initiated by glucocorticoids and oxidative stress. Ppp5 interacts with the TPR acceptor site of the heat shock protein Hsp90 and competes with TPR containing immunophilins in binding to some glucocorticoid receptor complexes.  Suppression of Ppp5 expression with antisense oligonucleotides enhances glucocorticoid-mediated phosphorylation of p53 on Ser15 with consequent expression of p21^WAF1/Cip1 and concomitant G₁ growth arrest in a lung carcinoma cell line.  Oxidative stress mediated activation of apoptosis signal-regulating kinase 1 (Ask1), which promotes apoptosis via the JNK and p38 MAP kinase cascades, was inhibited by overexpression of Ppp5.  This negative regulation of Ask1 by Ppp5 is likely to involve dephosphorylation of a phospho-threonine residue within the activation loop of Ask1.  Ppp5 also suppresses the transient increase in Ask1 activity produced by hypoxia. In a further link between Ppp5 and Ask1, rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), induces sustained activation of Ask1, associated with inhibition of Ppp5 activity.

Recently two important studies have demonstrated roles for Ppp5 in the DNA-damage response. Ppp5 associates with both ATM (ataxia telangiectasia mutated) and DNA-PK, two PI3K-related kinases whose activation in response to genotoxic stress initiates DNA damage-induced checkpoints and DNA repair respectively. Ppp5 regulates the phosphorylation states of these kinases, and their downstream signalling events. Significantly, a fragment of DNA-PK containing an auto-phosphorylation site (Thr2609) interacts with the TPR domain of Ppp5, suggesting that DNA-PK-Ppp5 interactions may be dependent on DNA-PK phosphorylation states. A variety of other proteins have also been reported to associate with Ppp5. These include the atrial natriuretic peptide receptor, the TPR containing Cdc16 and Cdc27 subunits of the anaphase promoting complex, the human homologue of Arabidopsis thaliana blue-light photoreceptor, cryptochrome 2, the A subunit of PP2A and the Hsp90-dependent heme-regulated eIF2a kinase. The Ga₁₂/Ga₁₃ subunits of heterotrimeric G proteins also interact with Ppp5, suggesting that Ppp5 is a downstream effector of Ga₁₂/Ga₁₃ signalling to ion channels.

Unlike PP1 and PP2A, the in vitro basal activity of Ppp5 is extremely low. Numerous studies have demonstrated that the TPR domain and C-terminal 13 residues cooperate to maintain the phosphatase in an auto-inhibited state, with suppressed phosphatase activity. Removal of either the TPR domain or C-terminal 13 residues by limited proteolysis or deletion mutagenesis potently activates the enzyme some 10-50-fold. These findings indicate that the isolated phosphatase domain is constitutively active, and that the TPR domain and C-terminal segment co-ordinately suppress catalytic activity. How Ppp5 is stimulated in vivo remains unclear, although a number of Ppp5 activators have been identified from in vitro studies. For example, polyunsaturated fatty acids, such as arachidonic acid, and saturated and unsaturated fatty acyl CoA esters stimulate the phosphatase activity of Ppp5 through interaction with its TPR domain. Furthermore, proteins that interact with the TPR domain also stimulate phosphatase activity, notably the C-terminal domain of Hsp90 and Ga₁₂/Ga₁₃ subunits.

The crystal structure of the TPR domain of Ppp5 revealed how the three TPR motifs, each comprising a pair of antiparallel a-helices, are organised to form a super-helical structure presenting an amphipathic groove that provides a binding site for interacting proteins.  Basic residues within this groove interact with acidic residues in the TPR acceptor site at the C-terminus of Hsp90. More recently, the crystal structure of the catalytic domain of Ppp5 (residues 169-499) was reported. The tertiary structure of the catalytic core and proposed catalytic mechanism are very similar to those of PP1 and calcineurin/PP2B with which Ppp5 shares approximately 40% identity.

An understanding of how the TPR domain and C-terminal segment of Ppp5 cooperate to suppress catalytic activity requires structural information of the full-length protein. Here we describe the crystal structure of the auto-inhibited conformation of human Ppp5. The structure reveals an extensive interface between the TPR and phosphatase domains, augmented by the C-terminus of the protein, that blocks access to the catalytic site, thereby inhibiting activity. The TPR-phosphatase domain interface includes a region of the Hsp90 binding groove of the TPR domain, suggesting that TPR-Hsp90 interactions would dissociate the TPR domain from the phosphatase catalytic site, activating the phosphatase. In contrast, long chain fatty acids activate Ppp5 by stabilising an alternate conformation of the TPR domain that disrupts contacts with the phosphatase domain.

Insert figure /home/dbarford/webpage/tpr-1.jpg and tpr-2.jpg Structure of the tetratricopeptide repeats (TPRs) of PP5

Insert figure /home/dbarford/webpage/pp5-1.jpg Structure of the auto-inhibited state of human PP5

Protein Phosphatase 5 Publications:

Yang J, Roe SM, Cliff MJ, Williams MA, Ladbury JE, Cohen PT, Barford D. (2005).  Molecular basis for TPR domain-mediated regulation of protein phosphatase 5.  EMBO J., 24, 1-10.

Cliff MJ, Williams MA, Brooke-Smith J, Barford D, Ladbury JE. (2005). Molecular recognition via coupled folding and binding in a TPR domain. J Mol Biol., 346, 717-732.

Das AK, Cohen PW, Barford D. (1998).  The structure of the tetratricopeptide repeats of protein phosphatase 5: implications for TPR-mediated protein-protein interactions.  EMBO J., 17, 1192-1199.

(NCBI Medline link to ‘protein phosphatase 5’)

Sub-project: Cdc25

Reversible protein phosphorylation is a ubiquitous mechanism for the control of signal transduction networks that regulate diverse biological processes including response to extracellular stimuli, DNA damage and cell growth and division. Changes in protein phosphorylation affect the structure and activity of proteins regulating nearly all aspects of cell life including metabolic processes, DNA replication, gene expression, and the cell cycle. Progression through the cell cycle is coordinated by a family of cyclin dependent protein kinases (CDKs) composed of a protein kinase catalytic subunit and a regulatory cyclin subunit. The principle transitions of the cell cycle are triggered by oscillations of CDK activity, a complex process regulated by reversible phosphorylation and controlled synthesis and degradation of activator and inhibitor complexes. Activation of the CDKs requires dephosphorylation of the inhibitory pThr-14 and pTyr-15 sites by the dual specificity protein phosphatase Cdc25. The activity of Cdc25 therefore plays a vital role in the regulation of cell cycle progression, and inhibition of Cdc25 activity is responsible for arresting the cell cycle.  Regulated cell cycle arrest occurs in response to DNA damage, caused for example by ionising radiation. Such an arrest allows cells to repair their DNA, before commitment to DNA replication and mitosis, or to undergo apoptosis. Recent data indicate that damage to DNA is detected in the cell, triggering a signal transduction pathways that leads to the inactivation of Cdc25, and consequent inhibition of the CDKs, and cell cycle arrest.

Currently, our studies are aimed at understanding mechanisms of reversible regulation of Cdc25A by redox processes and via 14-3-3 interactions.

Sub-project: PPM1D

PPM1D is a PP2C-like protein phosphatase that positively regulates cell proliferation and suppresses DNA-repair pathways. The enzyme is encoded by an oncogene, whose expression is elevated in numerous tumours, particularly breast cancers.  Ppm1D activates cell growth by dephosphorylating the activation segment Thr residue of p38 MAP kinase, thereby suppressing the expression of p53 and CDK inhibitors p21 and p16.