WO2004035811A2 - Kinase modulation assay - Google Patents

Abstract

The present invention relates to an assay for identifying agents which modulate an AGC kinase activity by interacting with a site other than an ATP binding site. There is also provided a phosphorylated and/or unphosphorylated native and/or mutated AGC kinase derived peptide, polypeptides and/or motifs for use in the assays of the present invention, as well as peptides capable of modulating activity of said AGC kinases by interaction through a site other than an ATP binding site.

Description

KINASE MODULATION ASSAY

Field of the Invention

The present invention relates to an assay for identifying agents which modulate an AGC kinase activity by interacting with a site other than an ATP binding site. There is also provided a phosphorylated and/or unphosphorylated native and/or mutated AGC kinase derived peptide, polypeptides and/or motifs for use in the assays of the present invention, as well as peptides capable of modulating activity of said AGC kinases by interaction through a site other than an ATP binding site.

Background of the Invention

Protein kinases are considered drug targets by pharmaceutical companies. Inhibitors are normally produced by producing inhibitors that bind to the same binding site as ATP, in the kinase domain. A major problem of compounds screened against protein kinases is that, interacting with the ATP binding site, they are usually not very specific towards one protein kinase. In addition, compounds binding to the ATP binding site would be always inhibitors. In AGC kinases, a second hydrophobic site has been described, termed the PIF pocket, which binds a hydrophobic motif comprising the sequence Phe/Tyr-X-X-Phe/Tyr.

The AGC kinase group includes p90 ribosomal S6 kinase (RSK) , p70 ribosomal S6 kinase (S6K) , protein kinase B (PKB) , itogen- and stress-activated protein kinase (MS ) , serum- and glucocorticoid-inducible kinase (SGK) and protein kinase C-related kinase (PR ) . Collectively, these kinases phosphorylate a large array of cellular proteins and thereby regulate cellular division, survival, metabolism, transme brane ion flux, migrative behavior and differentiation. The kinases are activated by partly distinct signalling pathways. PKB, S6K and SGK are downstream mediators of phosphoinositide 3-kinase (PI3-K) (Franke et al . , 1994; Ming et al . , 1994; Kobayashi and Cohen, 1999; Park et al . , 1999), RSK and MSK are effectors of ERK and ERK/p38 MAP kinases, respectively (Sturgill et al . , 1988; Deak et al . , 1998) and PRK2 is subject to control by Rho GTPase (Flynn et al . , 2000) . The differential responsiveness to upstream pathways is due, in part, to the fact that the kinases contain different signalling modules flanking the kinase domain, e .g. a PH-domain in PKB, a Rho-binding domain in PRK, a unique inhibitory domain in S6K and a MAP kinase- activated kinase domain in RSK and MSK.

Despite the divergent regulation, these kinases have two regulatory features in common that are critical for activation (Figure 1) . First, they all require phosphorylation of a serine or threonine residue in the activation loop within the kinase domain. The site is phosphorylated by 3-phosphoinositide-dependent kinase-l

(PDK1) in PKB (Alessi et al . , 1997; Stokoe et al . , 1997),

S6K (Alessi et al . , 1998; Pullen et al . , 1998), RSK

(Jensen et al . , 1999; Richards et al., 1999), SGK

(Kobayashi and Cohen, 1999; Park et al . , 1999) and PRK (Flynn et al . , 2000), whereas MSK may autophosphorylate at this site (Williams et al . , 2000). Phosphorylation of the activation loop augments catalytic activity from absent to typically 10% of maximal activity. Second, this group of kinases all require phosphorylation of a serine or threonine residue in a so-called hydrophobic motif located in a conserved tail region C-terminally to the kinase domain. The motif is characterized by three aromatic amino acids surrounding the Ser/Thr residue that becomes phosphorylated: Phe/Tyr-X-X-Phe-Ser/Thr-Phe/Tyr. In RSK (Vik and Ryder, 1997) and MSK (Deak et al . , 1998), the hydrophobic motif is phosphorylated by their respective C-terminal kinase domains. For PKB, S6K and SGK, the identity of the hydrophobic motif kinase has not yet been firmly established.

An important feature of the interaction between PDKl and the hydrophobic motif is that the motif must be phosphorylated (or contain a phosphate-mimicking acidic residue) for efficient interaction to occur (Balendran et al . , 2000; Frδdin et al . , 2000; Biondi et al . , 2001). Whether the hydrophobic pocket of PDKl is endowed with the ability to recognize phosphoserine/phosphothreonine or whether the phosphate alters the conformation of the hydrophobic motif so that it can bind the hydrophobic pocket is not known. Moreover, the docking mechanism does not explain why e . g. PKB and MSK require phosphorylation in their hydrophobic motif, since PKB does not appear to use the motif for PDKl docking (Biondi et al . , 2001) and MSK is not a target of PDKl (Williams et al . , 2000 ) .

It is an object of the present invention to provide an assay for identifying agents which modulate AGC kinase activity by interacting with a site on the kinase, other than at an ATP binding site.

The present invention is based in part on observations/findings by the present inventors of residues forming part of a phosphate binding site in close proximity to a hydrophobic PIF pocket in a number of protein kinases from the AGC subfamily. Evidence is provided that this phosphate binding site, not only participates in the binding of phosphates or negatively charged residues next to the hydrophobic motif, but also has a main role in the modulation of protein kinase activity.

Thus, in a first aspect the present invention provides a method of identifying agents which modulate activity of an AGC protein kinase, comprising the steps of: a) providing a polypeptide comprising a kinase domain of an AGC protein kinase, and associated activation loop which may be phosphorylated or unphosphorylated; b) optionally providing a hydrophobic motif polypeptide from a or said AGC protein kinase, comprising a sequence F/Y-X-X-F/Y wherein said hydrophobic motif polypeptide may be phosphorylated or unphosphorylated; c) bring a test agent and said polypeptide(s) into contact under conditions conducive to allow kinase activity to be detected in the absence of said test agent; and d) detecting an effect said test agent has on kinase activity.

The present assay may be conducted on many members of the AGC kinase family (cAMP - dependent protein kinase/protein-kinase G/protein kinase C - see for example www. sdsc. edu/kinases) , including PDKl, RSK, SGK, PKB, MSK and SGK, and is generally applicable to AGC kinases which are shown to use a phosphate-binding pocket as an essential part of their activation mechanism. Typically these will comprise a kinase domain with an activation loop phosphorylation site and a hydrophobic motif or partial hydrophobic motif C-terminal to the kinase domain, comprising the sequence F/Y-X-X-F/Y. It is to be understood that the letters of the foregoing sequence and other sequences used herein represent amino acid residues using standard single letter amino acid nomenclature. Moreover, for example, F/Y is understood to mean that a phenylalanine or tyrosine residue may be present at that particular position of the sequence and X is understood to relate to any amino acid residue.

Depending on the components used in a particular assay according to the present invention, said assay may be used to identify activators or inhibitors of an AGC kinase. It is understood that the term activator means an agent capable of increasing or enhancing a level of activity of a native and/or mutant AGC kinase when compared to a level of activity of said native or mutant AGC kinase in the absence of said agent, and an inhibitor is capable decreasing a level of activity of a native and/or mutant AGC kinase when compared to a level of activity of said AGC kinase in the absence of said agent. The term native AGC kinase is used to refer to a normal naturally occuring AGC kinase and the term mutant refers to an AGC kinase with a different amino acid sequence compared to a native sequence, ie. a mutated sequence. Said mutant AGC kinase may be found in vivo, ie. a mutant form of a native AGC kinase found in a subject, or a synthetically mutated form of an AGC kinase, ie. a form which has been mutated in a laboratory using for example genetic engineering techniques known in the art. They may also be generated as fusion peptides to facilitate their purification.

The polypeptide comprising the kinase domain and phosphorylatable activation loop generally comprises at least about 70-90 amino acids, such as 100-190, eg. 120- 140 amino acids, depending on requirements and the particular AGC kinase. Figure 5a, for example, shows an alignment of the kinase domains of various AGC kinases, with the hydrophobic motif C-terminal to this. The skilled addressee can easily align the sequences of other members of the AGC kinase family in this manner in order to identify the kinase domain, phosphorylatable activation loop and/or hydrophobic motif. The alignment may be carried out using the GAP or PILEUP computer programs of the Universtiy of Wisconsin Genetic Computing Group, or for example the Clustal W Program (Thomson et al (1994) Nucl. Acid. Res. 22, 4673-4680) using appropriate alignment parameters.

The present inventors have for example utilised essentially all of an AGC kinase sequence up to the hydrophobic motif (eg. bases 1-373 of RSK2) , as this comprises the kinase domain and activation loop, although shorter polypeptides may be utilised providing such polypeptides have a detectable kinase activity. As mentioned herein, mutant sequences may be used, based on a native AGC kinase domain/ ctivation loop. In this manner said mutant polypeptide which may comprise a mutation in the kinase and/or activation domain is likely to display reduced, significantly reduced or substantially no kinase activity when compared to a corresponding unmutated native sequence. Such mutant sequences may find particular utility in identifying agents capable of activating/restoring kinase activity to said mutant. All types of mutation are envisaged including inversions, transversions, substitutions and/or deletions.

Typically the kinase domain and associated activation loop will be provided by recombinant means. That is, the nucleic acid encoding these motifs may be cloned and expressed in eukaryotic cells, typically mammalian cells such as a COS cell, or other suitable cell. The activation loop may be phosphorylated if required, by for example a kinase such as PDKl, or other means. Alternatively the activation loop phosphorylation site can be mutated for example to a non-phorylatable residue such as an acidic residue, e.g. Glu or Asp which may mimic the native phosphorylated residue.

The kinase domain may be native ie. comprise the a non-mutated sequence corresponding with wild-type. Alternatively a mutated kinase domain may be provided which has been for example, point mutated at the phosphate-binding site. Such mutants may assist in the identification of agents that target the phosphate- binding site versus the ATP binding site.

In one embodiment two assays may be carried out, one utilising a wild-type kinase and the other a mutant kinase. Agents which are identified as inhibiting or activating the wild-type kinase, but not the mutant kinase are likely to influence kinase activity by binding the phosphate binding pocket.

In another embodiment a polypeptide comprising the kinase domain may infact be the full-length AGC kinase. In this instance the polypeptides as described in parts a) and b) are in fact provided as a single polypeptide comprising both the kinase domain/activation loop and hydrophobic motif. Agents could be tested for their effect against the full length kinase and compared against a truncated kinase, that is a kinase which lacks the hydrophic motif. Agents which inhibit or activate a truncated kinase better than full-length kinase are likely to bind the kinase at the same site as the hydrophobic motif, ie. either the PIF-pocket, the phosphate-binding site or the hydrophobic site for the last F/Y of the hydrophobic motif. The hydrophobic motif may comprise the sequence F/Y- X-X-F/Y-S/T-F/Y, preferably F-X-X-F-S/T-F/Y in which the S/T residue is phosphorylatable. However, certain members of the AGC kinase family do not appear to possess phosphorylatable hydrophobic domains, such as PRK, which contains negatively charged amino acids, aspartic acid or glutamic acid, that mimics the phosphoserine/ phosphothreonine, or PKA which possesses a partial hydrophobic motif F-X-X-F-COOH. Such hydrophobic motifs may also be utilised. In a preferred embodiment however the hyrophobic motif utilised comprises the sequence F-X- X-F-S/T-F/Y where the S/T is phosphorylated. As mentioned above phosphoylation may be carried out by another kinase, or in the presence of a kinase such as PDKl or by synthetic means known to those skilled in the art..

In a further aspect the present invention provides truncated AGC kinase polypeptides for use in assays according to the present invention for detecting agents which effect AGC kinase activity. Said truncated mutants may lack, for example, the hydrophobic motif.

The present invention also provides mutant AGC kinases which have been mutated in the kinase domain and/or activation loop in order to reduce binding ability to the hydrophobic motif for use in assays according to the present invention.

The present invention also provides a kit comprising any one or more of the components of the described assays, for use in conducting said assays. Typically the components may be designed so that the assays may be conducted as high-throughput screening assays. Alernatively an antibody able to specifically bind to the phosphorylated substrate and not the un-phosphorylated substrate may be utilised.

Detection of kinase activity may be carried out using any suitable means. For example, the ability of the kinase to phosphorylate its natural substrate may be detected using for example radio/chemical/photochemical labelling of a phosphate and detection of its incorporation into the substrate.

An agent identified by an assay according to the present invention as being able to modulate an AGC kinase activity through action on a site other than an ATP binding site is envisaged to find use in therapy. Thus, in a further aspect the present invention provides a compound/agent identified by an assay according to the present invention, as being able to modulate AGC kinase activity, for use in therapy.

In a further aspect the present invention provides use of an AGC kinase modulation agent as defined herein for the manufacture of a medicament for the treatment of a subject, such as a mammal, eg. human, in need of modulating said AGC kinase activity.

A compound that is capable of reducing or inhibiting said AGC kinase activity may be useful for example in the treatment of diseases associated with abnormal cell proliferation or apoptosis, such as cancer, or undesirable inflamation. A compound which is capable of activating or increasing AGC kinase activity may be useful in treating for example diabetes or obesity, or may be useful in inhibiting apoptosis.

In a further aspect the present invention provides a method of treating a subject by administering to a subject in need of modulation of an AGC kinase, an effective amount an agent, identified by an assay according to the present invention as being capable of modulating said AGC kinase activity.

Moreover, the present invention provides compounds which are able to interact with AGC kinases and mimmic the binding of the phosphate, in the native hydrophobic motif, to the phosphate binding site in the kinase domain which may be used as activators of AGC kinase activities when used in "trans", and compounds that bind without mimmicking the effects of the phosphate may be useful as agents or as lead agents which may be useful for the development of specific non ATP inhibitors of AGC kinase activities.

Based on the inventors observations suitable activation agents may be based on phosphorylated peptides designed against the hydrophobic domain of a particular AGC kinase, as these have been shown to activate said AGC kinase. Such peptides may comprise the sequence F/Y-X-X- F/Y-S/T-F wherein the S/T residue has been phosphorylated. Thus, in a further aspect, the present inventors provide phophorylated peptides comprising the sequence F/Y-X-X-F/Y-S/T-F, more preferably F-X-X-F-S/T- F, in which the S/T residue is phosphorylated, for use in modulating an AGC kinase activity. Also peptides based on such a sequence which possess an alternative amino acid(s) in place of the phosphorylated S/T residue, but designed to mimic the residue may be used as an alternative e.g. aspartic acid or glutamic acid. It may be envisaged that a useful phosphorylated peptide, or mimic would be similar or substantially identical in sequence to the native hydrophobic sequence of the AGC kinase which is to be activated.

The present invenotrs have also found that use of an unphosphorylated hydrophobic motif peptide is capable of inhibiting AGC kinase activity. Such peptides may be used as agents or may be useful in designing new inhibitory agents. Peptides identical to a particular native AGC hydrophobic motif may not be the best candidate agents as they could be phosphorylated by a kinase when used in vivo. However, peptides in which the S/T residue has been replaced with an amino acid(s) which is not capable of being phosphorylated and does not mimic such a phosphorlyated residue, may be useful. It is understood that such peptides will generally be less than 50, 25, 15 or 10 amino acids in length.

The present invention will now be further described by way of Example and with reference to the Figures which show:

Figure 1 - Shows a structural alignment of PKA and major growth factor-activated AGC kinases. The alignment illustrates that the growth factor-activated AGC kinases share two regulatory features: phosphorylation of the activation loop (stippled area) and phosphorylation of a hydrophobic motif (blue box) , located in a tail region (red box) C-terminal to the kinase domain. Note that PRK2 contains a phosphate-mimicking Asp residue and that PKA lacks a phosphorylation site in the hydrophobic motif.

Figure 2 - Shows a model of the docking interaction between PDKl and the phosphorylated hydrophobic motif of RSK2. (A) Electrostatic surface potential model of the hydrophobic pocket of PDKl with positive potential in blue and negative potential in red. The RSK2 hydrophobic motif peptide (FRGFpSFV) is shown in white with the phosphogroup on Ser386 in yellow. (B) Ribbon representation of the pocket with sidechains of the residues discussed in the text. PDKl residues are red, blue or grey, whereas the RSK2 hydrophobic motif peptide is shown in green with the phosphogroup in yellow.

Figure 3 - Shows identification of Arg/Lys residues in PDKl required for interaction with the phosphorylated hydrophobic motif. (Aι_/2) COS7 cells were co-transfected with plasmids expressing wild-type or mutant myc-PDKl together with HA-RSK2 , GST-PRK2 or empty vector (Vec) . After 48 h and a final 3 h serum-starvation period, the cells were lysed subsequent to 35 min EGF-treatment of RSK-expressing cells. HA-RSK2 and GST-PRK2 were precipitated from the cell lysates using anti-HA Ab or glutathione beads, respectively. The precipitates were subjected to SDS-PAGE and immunoblotting with anti-myc Ab to detect co-precipitated myc-PDKl (upper panels) or to anti-HA immunostaining or protein staining to assess the amounts of HA-RSK2 and GST-PRK2 (lower panels) . Pre- precipitation lysates were subjected to immunoblotting for the myc-tag (middle panels). (Bχ_₃) Interaction of PDKl with the hydrophobic motif of RSK2 was analysed using surface plasmon resonance measurements in a BiaCore3000 system. Biotinylated peptides of the motif phosphorylated (pHM^RSK) or non-phosphorylated (HM^RSK) at Ser386 were used to coat Sensor Chips SA (10 response units) . (Bi) GST-PDK1 was injected at different concentrations (0.013-3.33 μM) onto pHM-coated chips. In the inset, the steady state binding is plotted against the various concentrations of PDKl. Kinetic constants were obtained by fitting the data to a hyperbola using Kaleidagraph software. (B₂) and (B₃) GST-PDK1 wild-type, -R131A or -R131M were injected at 400 nM onto chips coated with pHM^RSK and HM^RSK, respectively. (A and B) The experiments were performed three times with similar results .

Figure 4 - Shows that R131 is important for the ability of PDKl to phosphorylate S6K1. Wild-type or T412E His-S6Klι_₄₂₁ were incubated for 10 min with wild-type or mutant GST-PDK1 and Mg[γ-³²P]ATP. Thereafter, the kinase reactions were subjected to SDS-PAGE. Radioactivity incorporated into the S6K1 protein band was quantitated and expressed as per cent of the maximal value obtained. The results are representative of two independent experiments. Figure 5 - Identifies that a phosphate-binding pocket is conserved in major growth factor-activated AGC kinases. (A) Amino acid sequence alignment of selected AGC kinases in the region of the kinase domain that contains the hydrophobic pocket as well as the region forming the hydrophobic motif. Conserved residues predicted to interact with the phosphogroup, the first two aromatic residues and the last aromatic residue of the hydrophobic motif, are indicated by arrow, asterisk and circle, respectively. The ion pair formation between the Lys and Glu residues conserved in all kinases is indicated. (B and C) Model of the intramolecular interaction of the hydrophobic pocket of RSK2 and PKBα with their respective phosphorylated hydrophobic motifs,

FRGFpSFV (RSK2) and FPQFpSYS (PKBα) . (B) Electrostatic surface potential models of the pockets, with positive potential in blue and negative potential in red. The hydrophobic motif peptides are shown in white with the phosphogroup in yellow. (C) Ribbon representation of the pockets with sidechains of the residues discussed in the text. The hydrophobic motif peptides are shown in green with the phosphogroup in yellow.

Figure 6 - shows evidence for intramolecular activation of AGC kinases by the phosphorylated hydrophobic motif (A-E, upper panels) Wild-type or point- mutated kinase domains of RSK2, S6K1, MSK1, PKBα and

SGK1, all lacking their hydrophobic motif (except SGKl₆i_ ₄₁₅), were pre-phosphorylated or not by PDKl. Thereafter, PDKl was removed (except in the RSK2 assay) . The activity of each kinase was then determined in the absence or presence of phosphorylated (pHM) or non- phosphorylated (HM) hydrophobic motif peptide at 170 μM (A,B,E) or 360 μM (C,D) and expressed as per cent of maximal activity. Data are means +S.D. of (A) 4 to 10 observations from 2 to 4 independent experiments, (B-D) 3 independent experiments or (E) means of duplicates with less than 5% difference between duplicate samples from one experiment performed twice with similar results (A-D, lower panels) To control for equal protein amount and PDKl-induced phosphorylation of wild-type and mutant kinase, aliquots of the reactions were subjected to SDS- PAGE and protein staining/anti-HA blotting or to blotting with phospho-specific Ab to the PDKl site. However, in (A), phosphorylation of HA-RSK2ι_₃₇₃ was assessed by including [γ-³²P]ATP during preincubation with PDKl, followed by precipitation with anti-HA Ab, SDS-PAGE and autoradiography.

Figure 7 - Shows that interaction of RSK2 with its phosphorylated hydrophobic motif. The interaction of RSK2 with its own hydrophobic motif was analysed by surface plasmon resonance measurements. Biotinylated pHM^RSK peptide was used to coat Sensor Chips SA (500 response units) and tested for binding to GST-HA-RSK2 _λ _ ₃₇₃ or GST-HA-RSK2 _!_ ₃₇₃R119A. The experiments were performed several times with similar result. Figure 8 - Shows the role of the phosphate-binding arginines in activation and phosphorylation of AGC kinases in vivo. COS7 cells were transfected with plasmids expressing HA- or GST-tagged wild-type or mutant kinase. After 48 h and a final 4 h serum-starvation period, cells were exposed to 20 nM EGF for 20 min or 1 μM insulin for 8 min as indicated and then lysed. Thereafter, the kinases were precipitated from the cell lysates with antibody to the HA tag or with glutathione beads. (A) Kinase activity was determined and expressed as per cent of the wild-type stimulated value. Data are means ±S.D. of 3 (RSK2, S6K1, MSK1, SGK1) or 4 (PKBα) independent experiments performed in duplicate. (B) Precipitated kinase from (A) were subjected to SDS-PAGE. The gel was subjected to immunoblotting with the indicated anti-phosphopeptide Ab or stained for protein.

Materials and methods

Peptide sequences were: S6 peptide (RRLSSLRA) ,

CREBtide (EILSRRPSYRK) , Crosstide (GRPRTSSFAEG) , pHM^RSK (KKPPSANAHQLFRGFSFVAITSDDE of mRSK2), pHM^S6K (TLSESANQVFLGFTYVAPSC of rS6Kl) , pHM^SG (VKEAAEAFLGFSYAPPTP of hSGKl) and pHM^ROK

(VGNQLPFIGFTYFRENL of hROKα) , with underlined residues phosphorylated. Transfection and immunoprecipitation

Kinases were expressed and purified from COS7 cells as described in Frodin et al . , (2000) or as described herein.

In vivo AGC kinase assays

Beads with precipitated kinase were resuspended in 20μl 1.5x buffer A (30 mM Tris-HCl (pH 7.5), 10 mM MgCl₂ 1 mM dithiothreitol) . The kinase reaction was initiated by the addition of 10 μl (final concentrations) ATP (200 μM, 0.2 μCi [γ-³²P]ATP) and peptide substrate: 800 μM S6 peptide (RSK, S6K) , 166 μM Crosstide (PKB, SGK) or 200 μM CREBtide (MSK) . After 10 min at 28°C with vigorous shaking (the reaction was linear with time) , 20 μl of the supernatant was removed (leaving behind precipitated kinase) and spotted onto phosphocellulose paper (Whatman p81) . After washing with 150 mM orthophosphoric acid, [ P]phosphate incorporated into protein substrate was quantified on a Phospholmager (Molecular Dynamics inc.).

In vitro AGC kinase assays

In the RSK2 assay, 200 ng GST-HA-RSK2₁_ ₃₇₃ (wild- type or R119A) were incubated in 20 μl buffer A containing 60 μM MgATP with or without 100 ng His-PDKl for 1 h at 28C in vitro . At this time, phosphorylation of RSK by PDKl had reached saturation, as determined by time-course experiments. In the S6K1, MSK1 and PKBα assays, C0S7 cells were co-transfected with plasmids expressing HA-S6Kι_₃₆₄, HA-MSK1i_ ₃₇₀ , HA-PKBαι_₄₆₇ or point mutants hereoff together with mycPDKl or empty vector. S6K1, MSK1 or PKBα were then purified from the lysed cells using anti-HA Ab as described above and resuspended in 20 μl buffer A. In the SGK1 assay, GST- SGK1₆₁-₄₃₁ or GST-SGK1₆₁-₄₁₅ were incubated with or without His-PDKl and 100 μM MgATP for 1 h in vitro, whereafter His-PDKl was removed by precipitation with Ni- agarose beads. GST-SGKl₆ι- ₄₃₁ (40 ng) or GST-SGKl₆ι- ₄₁₅ (400 ng) were then resuspended in 20 μl buffer A. RSK, S6K, MSK, PKB or SGK activity was then determined in the absence or presence of the indicated hydrophobic motif peptide using kinase assay conditions as above. For RSK, a reaction blank (PDKl alone) was subtracted from all values. Phosphorylation of His-S6Klι_₄₂₁ by PDKl was performed as described (Biondi et al . , 2001).

Surface plasmon resonance measurements

BiaCore analysis was performed as described in herein or in Biondi et al . , (2000) and (2001).

Modelling procedures

Homology modelling and energy minimization of human

PDKl₇₅-₃₅₁, mouse RSK2₆₂-₃51 ^and human PKBα₁44-4₁7 sequences were performed using the Homology and Discover modules as implemented in INSIGHT II (97.0) (Biosym/MSI, San Diego) . The template for the modelling experiments was PKA (1YDR) (Engh et al., 1996), which has 39-45% sequence identity and 60-64% similarity to the targets. Energy minimization was performed using a steepest descents algorithm as implemented in Discover. The calculations were performed ignoring solvent interactions. The force field used was AMBER as implemented in INSIGHT II. Hydrophobic motif peptides were docked into the hydrophobic pockets using the program AUTODOCK (Ver. 3.0.3) from the Scripps Research Institute and Molecular Graphics Laboratory with default parameters and were subsequently refined manually. The hydrophobic motif peptides were considered rigid with no flexibility of the torsion angles. The program GRID (Ver. 20) (Goodford, 1984) was used to compute the hydrophobic surface contours, which were used to predict the extent of the hydrophobic pocket. The program PROCHECK (Laskowski et al . , 1993) was used to validate the geometry of the energy minimized structures.

Antibodies and chemicals

Anti-pS473, -pS386 and -pPRK2 Ab (a pan-PDKl phosphorylation site Ab used for pT229 S6K1 and pT256 SGK1) were from Upstate Biotechnology. Anti-pT389, - pT308 and -pS376 Abs were from Cell Signaling Technology. Anti-pS227 Ab is described (Merienne et al . , 2000). Anti-HA Ab for immunoprecipitation were from the 12CA5 mouse hybridoma cell line. Anti-HA or anti-myc Ab for s immunoblotting were rabbit polyclonal Abs from Santa Cruz Biotechnologies. Other chemicals were from Sigma. Plasmid constructs

PMT2-HA-RSK2 (mouse) (Zhao et al . , 1996) was kindly provided by Dr. Christian Bjørbsek (Beth Israel Hospital,

Boston, MA, USA) . pECE-PKBα-HA (human) and pRK5-GST-myc-

S6K1 (the rat 70 kDa splice variant) are described in (Kohn et al . , 1995) and (Pullen et al . , 1998), respectively. GST-HA-RSK2 _ ₃₇₃ was expressed from pGEX- 4T1 in E. coli BL21 and purified as described (Jensen et al . , 1999). pCMV5-myc-PDKl is described (Jensen et al . , 1999). GST-PDK1, His-PDKl, GST-PRK2 , GST-SGK1₆₁ - ₄₃₁ and GST-SGKlβi- ₄₁₅ , all human sequences, were expressed from pEBG-2T. For the in vitro experiments, expression was done in HEK 293 cells and proteins purified as described (Balendran et al . , 2000; Biondi et al . , 2001). pMT2-HA- MSK1 and pMT2-HA-MSKlι_₃₇₀ are described in Frδdin et al . (2000). HA-S6Klι_₃₆₄ in pMT2 was generated by PCR amplification of the desired rat p70 S6K1 sequence using a 5 ' end primer introducing a Xhol site and a 3 • end primer introducing a stop codon and a Kpnl site. RSK2 was removed from pMT2-HA-RSK2 by Xhol+Kpnl digestion and replaced with the PCR product. N-terminally HA-tagged PKBι_₄₅₇ in pMT2 was generated by PCR amplification of the desired human PKBα sequence using a 5 * end primer introducing a Xhol site and a 3 • end primer introducing a stop codon and a Sail site. RSK1 was removed from pMT2- HA-RSK1 (from Dr. Joseph Avruch, Massachusetts General Hospital, Boston, MA, USA) by Xhol+Sall digestion and replaced with the PCR product. SGKl₆i_₄i₅ was generated by introducing a stop-codon after residue 415 in pEBG-2T- SGK1₆₁-₄₃₁- All point-mutations described in this study were introduced using the QuickChange™ (Stratagene) utagenesis procedure.

COS7 cells were cultured and transfected using Lipofectamine reagent (Life Technologies inc.) as described (Jensen et al . , 1999). Cells were solubilized for 15 min in 500 μl lysis buffer (0.5% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 5% glycerol, 1 mM Na₃V0₄, 5 mM EDTA, 25 mM NaF, 10 nM calyculin A, 1 mM phenylmethylsulfonylfluoride, 10 μM leupeptin, 10 μM pepstatin and 200 kallikrein inhibitor units/ml aprotinin) on ice and manipulated cool thereafter.

Transfection, immunoprecipitation and protein staining

Cell extracts were clarified by centrifugation for 10 min at 14.000 g and the supernatant was incubated for 75 min with antibody with the addition of protein G agarose beads (Amersha Pharmacia Biotech) during the final 25 min or with glutathione-Sepharose beads (Amersham Pharmacia Biotech) all the time. The beads were then precipitated by centrifugation, washed 5 times with lysis buffer, drained and dissolved in SDS-PAGE sample buffer (2% sodium dodecyl sulfate, 62 mM Tris(pH

6.8), 10% glycerol, 5% 2-β-mercaptoethanol, 0.1% bromphenol blue) . For kinase assays, the final 2 washes were with buffer A (30 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM dithiothreitol) . Kinase expression levels were often evaluated by subjecting the precipitate to SDS-PAGE and staining of the gel with SimplyBlue™ protein staining (Invitrogen) .

Immunoblotting

Immunoblotting on precipitated kinases were performed as described (Jensen et al . , 1999) but with the following modification. PVDF membranes for immunoblots with phosphospecific Abs were blocked with Baileys liquor which proved superior in blocking background staining with this type of Ab as compared to skimmed milk, used for all other immunoblots.

Surface plasmon resonance measurements

BiaCore analysis was performed as described in Biondi et al . , (2000) and (2001). HM^RSK peptides were biotinylated with EZ-Link sulfo-NHS-LC-LC-Biotin (Pierce) and then subjected to SDS-PAGE. The gel was stained with coomasie brillant blue R250, the peptide band was cut out and peptide extracted by vigorous vortexing in 50 mM Tris pH 7.5.

Results

Phosphoserine/phosphothreonine recognition by the hydrophobic pocket of PDKl

Previous studies provided a partial characterization of the hydrophobic pocket of PDKl by showing that residues Lysll5, Ilell9, Glnl50 and Leul55 in the small lobe of the kinase domain form a hydrophobic pocket that likely binds the first two phenylalanines of the hydrophobic motif of PDKl target kinases (Biondi et al . , 2000 and Biondi et al . , 2001). The present inventors wished to identify amino acids near the pocket that could account for recognition of phosphoserine/threonine in the hydrophobic motif. The programme Swiss-Pdb Viewer (Guex and Peitsch, 1997) was used to model the sequence of PDKl over the crystal structure coordinates of PKA (Engh et al . , 1996), the only AGC kinase whose structure had been solved. The model of PDKl visualized that the above- mentioned pocket residues cluster in one end of a larger groove (data not shown) . Further into the groove and topografically close to this cluster, the present inventors noticed an Arg residue (131) located in the so- called αC-helix of the conserved kinase fold. Moreover, the inventors noticed four consecutive basic residues, ⁷⁵Arg-Lys-Lys-Arg⁷⁸ , N-terminal to β-strand 1. Although the basic cluster is outside the conserved kinase fold, and its position therefore less predictable, the start of β-strand 1 is topographically close to the hydrophobic pocket. It was decided to investigate whether these positively charged amino acids could be involved in phosphate-binding by performing more advanced molecular modelling and biochemical characterization.

Residues 82-351 of PDKl, encoding the kinase domain, were used to model PDKl based on the coordinates of a PKA structure in the closed, active conformation with phosphorylated activation loop (Engh et al . , 1996). The sequence of the hydrophobic motif of RSK2 phosphorylated at the regulatory site (Ser386) was then modelled into the pocket by first fixing the first two phenylalanines of the motif in the position held by the corresponding phenylalanines in the PKA crystal structure. The remaining residues of the motif were then modelled without template (ab initio modelling) into the pocket. Finally, residues 75-81 of PDKl (encompassing the basic cluster) were ab initio modelled onto the PDKl kinase domain. The resulting model of the docking interaction showed juxtaposition of the phosphogroup of Ser386 and Argl31 of PDKl with possibility of hydrogen bond formation between the two (Figures 2A and B) . Intriguingly, Argl31 is next to a Glu residue (130 in PDKl) that is one of very few residues conserved in all kinases and that has a regulatory role. Modelling also suggested binding of the phosphogroup of Ser386 to Arg75, Lys76 and Lys77 of the basic cluster in PDKl, but suggested that only one of these residues may bind the phosphate at a time. Moreover, the sidechains of Phe 82, Phel47 and Phel49 of PDKl appear to form a hydrophobic site that accommodates the last aromatic residue of the hydrophobic motif. Finally, the Val after the motif may form hydrophobic interactions with Leul45 of PDKl. In conclusion, the model suggests that the hydrophic motif completely occupies the groove in the small lobe of the PDKl kinase domain and that the phosphate in the motif is grabbed by two oppositely positioned basic residues.

The inventors next assessed the effect of mutating the predicted phosphate-binding residues in PDKl on its ability to bind RSK2 in vivo using co-immunoprecipitation experiments. Prior to lysis, the cells were exposed to epidermal growth factor (EGF) in order to induce phosphorylation of the RSK2 hydrophobic motif and recruitment of PDKl to RSK2 (Frδdin et al . 2000). RSK2 co-precipitated wild-type PDKl, but much lower amounts of PDK1-R131A mutant (Figure 3Aι, upper panel) or R131D, R131L and R131H mutants (data not shown) . Furthermore, Ala mutation of either Arg75, Lys76 or Lys77 in PDKl, all resulted in much reduced precipitation of PDKl by RSK2. PRK2 , that contains Asp in the position of phosphoserine/threonine in the hydrophobic motif, co- precipitated wild-type PDKl, but not PDK1-R131A or PDK1- R131D (Figure 3A₂, upper panel), or R131L and R131H (data not shown) . All PDKl constructs expressed protein at a similar level (Figure 3A, middle panels) .

Surface plasmon resonance measurements showed that wild-type PDKl bound with high affinity (K_d ≡400 ± 20 nM) to a synthetic peptide of the RSK2 hydrophobic motif phosphorylated at Ser386 (pHM^RS ), whereas PDK1-R131M or PDK1-R131A had no detectable affinity for pHM^RS (Figures 3Bι and B₂) • Unphosphorylated RSK2 hydrophobic motif peptide (HM^RSK) showed no binding to wild-type PDKl or PDK1-R131A (Figure 3B₃).

S6K1₁-₄₂₁ becomes a much better substrate of PDKl when Thr412, i.e. the phosphorylation site of the hydrophobic motif, is mutated to glutamic acid (Alessi et al . , 1998; Dennis et al . , 1998), which is due to enhanced docking of PDKl to S6K1 via the hydrophobic pocket (Biondi et al . , 2001). S6K1₁-₄₂₁ has a deletion of the C-terminal inhibitory domain, which blocks PDKl interaction with S6K1. The inventors used this assay to evaluate the importance of Argl31 in PDKl for the ability of PDKl to phosphorylate S6K1. As shown in Figure 4, PDKl, PDK1-R131A and PDK1-R131M all phosphorylated S6Klι_ ₄₂i poorly. PDKl phosphorylated S6Klι_ ₂ ιT412E with high efficiency. In contrast, PDK1-R131A and PDK1-R131M showed reduced ability to phosphorylate S6Klι_ ₄₂ ιT412E, suggesting that these mutants are impaired in recognizing the negative charge of E412. The reduced ability of PDK1-R131A or -R131M to phosphorylate S6KI₁ _ ₄₂ ιT412E was not due to impairment of PDKl catalytic activity by the mutations. In fact, these and other mutations in the hydrophobic pocket of PDKl increased 2-3 fold the kinase activity towards the PDKl peptide substrate T308tide (data not shown) . This suggests that the unoccupied pocket can suppress PDKl activity, as previously proposed (Biondi et al . , 2000).

In contrast to Ala, Asp, Leu, His or Met (data above) , the basic amino acid Lys could partially substitute for Arg at position 131 in PDKl. Against T308tide, PDK1-R131K showed basal activity similar to PDKl, but the dose-response curve for activation by pHM^s6K and HM^PRK2 was shifted to about 10- and 100-fold higher concentrations, respectively (data not shown) .

In conclusion, the present data strongly suggest that PDKl utilizes Argl31 in C-helix and the N-terminal basic residues Arg75, ys76 and Lys77 in order to recognize phosphoserine/phosphothreonine (or mimicking acidic residues) in the hydrophobic motif of its target kinases.

A phosphate-binding pocket is conserved in the growth factor-activated AGC kinases

PDKl itself belongs to the AGC kinase family, but is atypical since it does not contain a hydrophobic motif. Interestingly, sequence alignment suggests that a phosphate-binding pocket is present also in RSK2, S6K1, PKBα, MSK1, SGK1 and PKCα (Figure 5A) . First, an Arg or Lys residue equivalent to Argl31 in C-helix of PDKl is conserved. Second, a basic residue equivalent to Lys76 of PDKl is conserved. Finally, the residues for binding the three aromatic residues of the hydrophobic motif are conserved. This raised the possibility that the phosphorylated hydrophobic motif of these kinases may interact with a phosphate-binding pocket within the kinase domain, which could serve to stimulate their catalytic activity by an intramolecular mechanism. To address this question, the inventors first modelled the sequence encompassing the kinase domains of RSK2 (the N- terminal kinase domain) or PKBα over the structure of PKA and then modelled their respective phosphorylated hydrophobic motif into the presumed pocket. As shown in Figures 5B and C, the hydrophobic motif fitted perfectly into the hydrophobic pocket of RSK2 and PKBα, respectively. Moreover, in the two kinases, the phosphogroup of the hydrophobic motif was juxtaposed to the conserved Arg in C-helix with possibility of hydrogen bond formation between the two. Both models also allowed binding of the phosphate to the positively charged residue N-terminal to the kinase domain, i . e . K62 in RSK2 and R144 in PKBα. Very similar results were obtained through modelling of S6K1, MSK1 and SGK1 (data not shown) .

Evidence for intramolecular activation of AGC kinases by the hydrophobic motif via the phosphate-binding pocket

To test for an intramolecular function of the hydrophobic motif, a reconstitution assay was designed that could assess the contributions of hydrophobic motif phosphorylation, activation loop phosphorylation and their interaction in catalytic stimulation of RSK2, S6K1,

MSK1, PKBα and SGK1. The assay was based on isolated kinase domains with deletion of the hydrophobic motif and other C-terminal domains, i.e. the C-terminal kinase domain of RSK2 and MSK1 and the inhibitory domain in S6K1. In this assay, hydrophobic motif phosphorylation was conferred by the addition of synthetic peptides of the motif phosphorylated at the regulatory site (pHM) . Activation loop phosphorylation was conferred by pre- exposure to PDKl, which was then removed, except in the RSK2 assay. Although not a physiological substrate, MSK1 can be phosphorylated and activated by PDKl (Frodin et al., 2000), which was exploited here. The kinase domain of RSK2 (RSK2ι_₃₇₃) was completely inactive when incubated alone (Figure 6A) . No activation was achieved by the addition of pHM^RSK peptide, whereas phosphorylation of the activation loop by preincubation with PDKl resulted in stimulation of kinase activity. However, the combination of pHM^RSK and activation loop phosphorylation resulted in synergistic stimulation of RSK2₁-₃₇₃ catalytic activity. Half- maximal activation was observed at 20-30 μM pHM^RSK (data not shown) . In contrast, the non-phosphorylated hydrophobic motif peptide, HM^RSK, showed no ability to stimulate RSK2ι_₃₇₃ up to 350 μM peptide, the highest concentration possible here. To assess the role of the predicted phosphoserine-binding Argll9 in C-helix, similar experiments were performed with RSK2ι_₃₇₃ in which Argll9 had been mutated to Ala. RSK2ι_ ₃₇₃R119A showed normal stimulation upon activation loop phosphorylation. However, pHM^RSK failed to stimulate

RSK2i_₃₇₃R119A catalytic activity in synergy with activation loop phosphorylation. This suggests that activation of RSK2ι_₃₇₃ by pHM requires binding of pHM to Argll9, and moreover, that the R119A mutation does not compromise tertiary structure, since RSK2ι_₃₇₃RII9A was normally activated by PDKl.

Remarkably similar results were obtained in experiments performed with the isolated kinase domains of

S6K1, MSKl, and PKBα (S6Klι_₃₆₄, MSKlι_₃₇₀ and PKBαι_

₄₆₇ in Figures 6B-D, respectively). The experiments shown with MSKl and PKB were performed with pHM peptides derived from S6K1, since pHM^MSK peptide was not available and pHM^PKB peptide induced only a 2 to 3-fold activation of PKBαι_₄₆₇ (data not shown). In contrast to the other

kinases, pHM activated PKBαι_ ₆₇ although slightly, in the absence of PDKl co-expression, most likely because PKBαι_₄₆₇ was partially phosphorylated by endogenous

PDKl (Figure 6D, lower panel). In S6Klι_₃₆₄ and PKBαι_ ₄₆₇ the inventors also mutated the N-terminal phosphate- binding residue, Lys62 and Argl44, respectively. This resulted in 50% reduced activation of S6Klι_₃₆₄ by pHM^s6K (S.D.=12%, n=3, data not shown) and 40% reduced activation of PKBαι__4δ7 by pHM^{s 6 κ} (Figure 6D) . EC₅₀ experiments suggested that these mutants had 3- to 4-fold lower affinity towards pHM compared to the wild-type kinase (data not shown) . Control experiments showed that all mutants of the phosphate-binding basic residues expressed normally and were phosphorylated in the activation loop by PDKl similar to the wild-type enzymes (Figures 6A-D, lower panels) . Phosphorylation of MSKlι_ ₃₇₀ was evaluated by mobility shift in prolonged electrophoresis, since no phosphospecific Ab to the activation loop site has been reported. The maximal specific activities of RSK2ι_₃₇₃, S6Klι_₃₆₄, MSKlι_₃₇₀ and PKBαι_ ₆₇ obtained by combined activation loop phosphorylation and pHM were similar or several-fold higher than that of the full-length, recombinant proteins activated by EGF or insulin in vivo (data not shown) . This may reflect that the in vitro experiments were performed under conditions with a higher degree of phosphorylation of the hydrophobic motif and the activation loop than obtained in vivo .

Also SGK1 was activated by its phosphorylated, but not unphosphorylated, hydrophobic motif (Figure 6E) . This was observed both with SGKI₆₁-₄₁₅, in which the hydrophobic motif was deleted, and with SGKlei-₄₃₁ which retained its hydrophobic motif. Both SGK1 constructs lack residues 1-60, as the full-length protein does not express at sufficient levels for protein purification (Kobayashi and Cohen, 1999) . SGK1 could be even further activated by pHM peptide from Rho-kinase-α (also an AGC kinase), but not by HM^ROK. In contrast, pHM from RSK2 , S6K1, PKBα or myotonic dystrophy kinase (DMK) , another

AGC kinase, or HM from PRK2 or PKCζ showed little ability to activate SGK1 (data not shown) . Thus, in some instances an AGC kinase can be activated by pHM from another AGC kinase. However, since the EC₅₀ values for heterologous activation were always >10 μM, such activation may not be physiological. o

Surface plasmon resonance analysis showed that pHM^RSK is able to bind RSK2ι_₃₇₃, but not RSK2ι_ ₃₇₃R119A (Figure 7). The affinity, however, was too low for quantitative estimates of the dissociation constant in a BiaCore instrument, in agreement with the EC₅₀ value of 20-30 μM pHM^RSK in catalytic activation of RSK2ι_₃₇₃. HM^RSK showed no binding to RSK2ι_₃₇₃ or RSK2₁_₃₇₃R119A (data not shown) . In conclusion, these experiments demonstrate that the phosphorylated hydrophobic motif stimulates the catalytic activity of the growth factor-activated AGC kinases in synergy with the phosphorylation event in the activation loop. Moreover, the data suggest that binding and activation by pHM requires the conserved Arg in C- helix, and to a lesser degree, the conserved basic residue N-terminal to the kinase domain.

The inventors next analysed the role of the phosphate-binding residues for in vivo activation (Figure 8A) and phosphorylation state (Figure 8B) of full-length AGC kinases in response to physiological stimuli. In RSK2 , EGF-induced activation was abolished by mutation of Argll9. RSK2-R119A showed normal expression and phosphorylation of the activation loop and the hydrophobic motif, as demonstrated by immunoblotting. Mutation of the N-terminal basic residue, Lys62, reduced kinase activity by 21% (S.D. =7.0%, n=3 , data not shown). In S6K1, basal and EGF-induced activity was largely abolished by mutation of Argl21 in C-helix. S6K1-R121A showed normal expression and phosphorylation of the activation loop, but interestingly, phosphorylation of the hydrophobic motif was largely absent. The loss of phosphorylation occurred in vivo and not during immunoprecipitation, since S6K1-R121A from cells lysed with Laemmli buffer after EGF-treatment showed a similar lack of phosphorylation in the hydrophobic motif (data not shown) . This suggests that in S6K1, the hydrophobic motif phosphorylation site is subject to rapid dephosphorylation when not bound to the hydrophobic pocket and thereby protected from phosphatase action. To evaluate the role of Argl21 for S6K1 activity in vivo, the inventors therefore generated a S6K1-T389E mutant, in which the phosphorylation site in the hydrophobic motif was mutated to glutamic acid. The residue Thr389 is the same hydrophobic motif residue as Thr412 in Figure 4, but the numbering differs according to the long and short S6K1 splice variants used in Figures 4 and 8, respectively. S6K1-T389E had high basal activity that was not increased much by EGF under the conditions used (data not shown) . Introduction of the R121A mutation in S6K1-T389E, abolished kinase activity without affecting expression level or phosphorylation of the activation loop. In MSKl, mutation of Argl02 in C-helix resulted in complete loss of kinase activity as well as phosphorylation of the hydrophobic motif, whereas the expression level was unaffected. Mutation of the N- terminal basic residue, K43, had no effect on kinase activity (data not shown) . In PKBα, mutation of the N- terminal Argl44 and C-helix Arg200 resulted in partial and complete loss of kinase activity, respectively. Both mutations resulted in somewhat reduced phosphorylation of the activation loop and the hydrophobic motif, without affecting the expression level. Finally, since SGK1 was not activated consistently by insulin or EGF under the conditions used, the inventors generated a mutant which contains Asp at the phosphorylation site of the hydrophobic motif. SGK1-S422D showed basal activity 3-4 times that of stimulated wild-type SGK1 (data not shown) . Mutation of the C-helix Argl47 in SGK1-S422D significantly reduced kinase activity without affecting expression level or phosphorylation of the activation loop.

These findings show that the phosphate-binding basic residues of the hydrophobic pocket are important for the in vivo activity of the growth factor-activated AGC kinases. Moreover, the results suggest that the phosphate-binding basic residues may have a dual role by promoting catalytic activation as well as protecting the hydrophobic motif from dephosphorylation by intracellular phosphatases.

Discussion

Structural modelling and biochemical analysis performed here, suggest that the recognition of phosphoserine/threonine by the phosphate-binding site is similar in AGC kinases. When the hydrophobic motif is docked in the pocket, the phosphogroup is grabbed by two oppositely positioned Arg or Lys residues located N- terminal to the kinase domain and in C-helix, respectively. The two phosphate-binding residues do not appear equally important, since mutation of the N- terminal Arg/Lys and the C-helix Arg typically resulted in partial and complete loss of kinase activation, respectively. The phosphate-binding pocket of PDKl may be different from that of the other kinases, since PDKl contains four N-terminal basic residues, of which three may be involved in docking to the hydrophobic motif as judged from the mutational analysis. The cluster of positive charge near to the pocket may function to attract the phosphate in the motif of the target kinases followed by docking of the motif in the pocket. In agreement with this possibility, PDKl has much higher affinity for the phosphorylated hydrophobic motif of its target kinases than the kinases have for their own motif, as evidenced by BiaCore and EC₅o measurements. Presumably, the affinity in the intramolecular interaction can be low because the effective concentration of pHM is high. Moreover, it may be important to have a low affinity in the intramolecular binding reaction in order to allow exposure of the phosphorylated motif for dephosphorylation and inactivation of the kinase.

The present study provides direct evidence that the phosphorylated hydrophobic motif can stimulate catalytic activity of AGC kinases, such as, the growth factor- activated AGC kinases by an intramolecular mechanism. This was demonstrated using an in vitro reconstitution assay with isolated RSK2 , S6K1, MSKl, PKBα and SGK1 kinase domains and addition of hydrophobic motif peptides. The results demonstrate that the phosphorylated hydrophobic motif cannot induce activation alone, whereas phosphorylation of the activation loop turns on activity many-fold. However, the combined effect of the phosphorylated hydrophobic motif and phosphorylated activation loop results in synergistic stimulation of catalytic activity.

In vitro, pHM^PKB and HM^PRK2 activated 4- and 15- fold, respectively, PKB, but not PKB-R200D, pre- phosphorylated by PDKl. Finally, calorimetric measurements suggested that HM^PRK2 induced order in PKB, interpreted as stabilization of C-helix and the active kinase conformation in general.

The present study was performed with kinases that are members of 5 different AGC kinase subfamilies. It is therefore reasonable to assume that the mechanism described here is a general activation mechanism among AGC kinases that contain a phophorylatable hydrophobic motif. Sequence alignment shows that the hydrophobic motif phosphorylation site and the Arg/Lys residue in C- helix are strictly co-conserved in AGC kinase subfamilies not studied here, such as PKCs, Rho-kinases and Ndr- related kinases. Only in rare cases, do the negative charge in the hydrophobic motif not appear to be required for kinase activity. An example is the Glu residue in the PKCξ hydrophobic motif, but the residue increases thermal stability suggesting that binding to the pocket does occur and have a functional role (Parekh et al . , 2000; Balendran et al . , 2000). The N-terminal basic residue is conserved in about 80% of AGC kinases with a phosphorylatable hydrophobic motif. In other kinase families, the two phosphate-binding basic residues are absent, suggesting that the basic residues do not have a structural function in the kinase fold, but only serve to bind phosphate. In conclusion, the intramolecular activation mechanism based on phosphorylation-dependent interaction between the hydrophobic motif and the hydrophobic pocket may be general within AGC kinases. If so, the mechanism would represent the most general activation mechanism described so far in protein kinases, next to activation loop phosphorylation.

The results presented herein were not evident from the inventors previous work since they had found that for the peptide PIFtide, the mutaton of Asp to Ala provided a ten fold decrease in the activation ability, which correlated well with a 10 fold less binding affinity for PDKl, suggesting that the presence of a negatively charged residue only participated in increasing the binding to PDKl and not in the activation process. The inventors have now shown that the phosphate binding site for a protein kinase that does not have a hydrophobic motif C-terminal to the catalytic core (PDKl) provides binding to a subset of substrates of PDKl where a previously characterised phosphate dependent docking interaction had been suggested. In another subset of protein kinases, the phosphate binding site provides specific interaction with the hydrophobic motif present at the C-terminal extension of the kinase, directly participating in the activation of the kinase. Furthermore, the inventors have provided evidence that specific polypeptides different from the C-terminal hydrophobic motif of the given protein kinase can modulate the protein kinase activity of the protein kinase containing the hydrophobic motif, when the polypeptides are phosphorylated, but not when they are not phosphorylated. In fact the inventors found a 13 fold activation of SGK by the phosphorylated hydrophobic motif in ROK, and an inhibition of SGK activity by the dephosphopeptide. This finding provides evidence that compounds able to interact with AGC kinases and mimmic the binding of the phosphate to the phosphate binding site could be used as activators of AGC kinase activities when used in "trans", and compounds that bind without mimmicking the effects of the phosphate could be developed as specific non ATP inhibitors of AGC kinase activities.

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Claims

1. A method of identifying agents which modulate activity of an AGC protein kinase, comprising the steps of: a) providing a polypeptide comprising a kinase domain of an AGC protein kinase, and associated activation loop which may be phosphorylated or unphosphorylated; b) optionally providing a hydrophobic motif polypeptide from a or said AGC protein kinase, comprising a sequence F/Y-X-X-F/Y wherein said hydrophobic motif polypeptide may be phosphorylated or unphosphorylated; c) bring a test agent and said polypeptide(s) into contact under conditions conducive to allow kinase activity to be detected in the absence of said test agent; and d) detecting an effect said test agent has on kinase activity.

2. The method according to claim 1 wherein the AGC kirfese is PDKl, RSK, SCK, PKB, MSK or SGK.

3. The method according to claim 1 wherein the AGC kinase is an AGC kinase which has a phosphate-binding pocket as an essential part of its activation mechanism.

4. The method according to any preceding claim wherein the AGC kinase comprises a kinase domain with an activation loop phosphorylation site and a hydrophobic motif or partial hydrophobic motif C-terminal to the kinase domain, comprising the sequence F/Y-X-X-F/Y.

5. The method according to any preceding claim for identifying an activator of AGC protein kinase activity.

6. The method according to any preceding claim for identifying an inhibitor of AGC protein kinase activity.

7. The method according to any preceding claim wherein the polypeptide comprising the kinase domain and phosphorylatable activation loop comprises at least 70 - 90 amino acids.

8. The method according to any preceding claim wherein the polypeptide does not include the full length sequence of the AGC kinase.

9. The method according to any preceding claim wherein the polypeptide is a mutant form of a native AGC kinase polypeptide.

10. The method according to claim 9 wherein the mutant form comprises a mutation in the kinase and/or activation domain.

11. The method according to any preceding claim wherein a wild-type kinase polypeptide and a mutant polypeptide are used in order to identify an agent which effects one or both of said polypeptides.

12. The method according to any preceding claim wherein a single polypeptide corresponding to the polypeptides identified in steps a) and b) , is utilised.

13. The method according to any preceding claim wherein a test agent is tested for its effect on a full length kinase and a truncated kinase which lacks a hydrophobic motif.

14. The method according to any preceding claim wherein the hydrophobic motif comprises the sequence F/Y- X-X-F/Y-S/T-F/Y.

15. The method according to any one of claims 1 - 13 wherein the hydrophobic motif comprises the sequence F-X-X-F-COOH.

16. A truncated AGC kinase polypeptide for use in a method according to any preceding claim.

17. Use of a truncated AGC kinase polypeptide in a method according to any one of claims 1 - 15.

18. A mutated AGC kinase comprising a mutation in a kinase domain and/or activation loop in order to reduce binding ability to the hydrophobic motif for use in a method according to any one of claims 1 - 15.

19. Use of a mutated AGC kinase comprising a mutation in a kinase domain and/or activation loop in order to reduce binding ability to the hydrophobic motif in a method according to any one of claims 1 - 15.

20. A compound/agent identified by a method according to any one of claims 1- 15 for use in therapy.

21. Use of an AGC kinase modulation agent identified by a method according to any one of claims 1 - 15 for the manufacture of a medicament for use in therapy.

22. A method of treating a subject by administering to a subject in need of modulation of an AGC kinase, an effective amount of any agent, identified by a method according to any one of claims 1 - 15.

23. A phosphorylated peptide comprising the sequence F/Y-X-X-F/Y-S/T-F in which the S/T residue is phosphorylated.

24. A peptide comprising the sequence F/Y-X-X-F/Y- E/D-F.