Martijn J Koetsier

Peter A Jekel

Marco A Van Den Berg

Roel A L Bovenberg

Dick B Janssen
email: d.b.janssen@rug.nl.

Characterization of a phenylacetate-CoA ligase from Penicillium chrysogenum

Keywords: PCL, phenylacetate-CoA ligase, PAA, phenylacetic acid, POA, phenoxyacetic acid, MBP, maltose binding protein, PTS, peroxisomal targeting signal phenylacetate-coenzyme A ligase, CoA ligase, penicillin G, Penicillium chrysogenum

Enzymatic activation of phenylacetic acid (PAA) to phenylacetyl-CoA is an important step in the biosynthesis of the β-lactam antibiotic penicillin G by the fungus Penicillium chrysogenum. CoA esters of PAA and phenoxyacetic acid (POA) act as acyl donor in the exchange of the aminoadipyl side chain of isopenicillin N to produce penicillin G or penicillin V. The phl gene, encoding a phenylacetate-CoA ligase, was cloned in Escherichia coli as a maltose binding protein fusion and the biochemical properties of the enzyme were characterized. The recombinant fusion protein converted PAA to phenylacetyl-CoA in an ATP-and magnesium-dependent reaction.

PCL could also activate POA, but the catalytic efficiency of the enzyme was rather low with k cat /K m values of 0.23 ± 0.06 and 7.8 ± 1.2 mM -1 s -1 for PAA and POA, respectively. Surprisingly, PCL was very efficient in catalyzing the conversion of trans-cinnamic acids to the corresponding CoA thioesters (k cat /K m = (3.1 ± 0.4) • 10 2 mM -1 s -1 for trans-cinnamic acid). Of all the substrates screened, medium chain fatty acids, which also occur as the side chains of the natural penicillins F, DF, H and K, were the best substrates for PCL. The high preference for fatty acids could be explained by a homology model of PCL that was constructed on basis of sequence similarity with the Japanese firefly luciferase. The results suggest that PCL has evolved from a fatty acid activating ancestral enzyme that may have been involved in the β-oxidation of fatty acids.

INTRODUCTION

The final step in the biosynthesis of β-lactam antibiotics by the fungus Penicillium chrysogenum is the exchange of the L-α-aminoadipyl side chain for a more hydrophobic group, e.g. a phenylacetyl group in the case of penicillin G indicates that PCL is a member of the superfamily of adenylate-forming enzymes [START_REF] Babbitt | Ancestry of the 4-chlorobenzoate dehalogenase: analysis of amino acid sequence identities among families of acyl:adenyl ligases, enoyl-CoA hydratases/isomerases, and acyl-CoA thioesterases[END_REF]. CoA activation in these enzymes proceeds in a two-step reaction (Fig. 1). In the first half reaction, the substrate reacts with ATP to form an acyl-adenylate intermediate with the simultaneous formation of pyrophosphate. In the second half reaction, the adenylate group is replaced by CoA and AMP is released. The formation of an adenylate intermediate is characteristic for all members of the superfamily [START_REF] Babbitt | Ancestry of the 4-chlorobenzoate dehalogenase: analysis of amino acid sequence identities among families of acyl:adenyl ligases, enoyl-CoA hydratases/isomerases, and acyl-CoA thioesterases[END_REF].

Other family members include the adenylation domains of nonribosomal peptide synthetases, fatty-acid-CoA synthetases, luciferases and coumarate-CoA ligases 10.

Bacterial phenylacetate-CoA ligases involved in the catabolism of PAA in bacteria such as E. coli K12 and Azoarcus evansii [START_REF] Ferrandez | Catabolism of phenylacetic acid in Escherichia coli. Characterization of a new aerobic hybrid pathway[END_REF][START_REF] Mohamed | Biochemical and molecular characterization of phenylacetate-coenzyme A ligase, an enzyme catalyzing the first step in aerobic metabolism of phenylacetic acid in Azoarcus evansii[END_REF] and the extensively characterized enzyme from Pseudomonas putida U [START_REF] Martínez-Blanco | Purification and biochemical characterization of phenylacetyl-CoA ligase from Pseudomonas putida. A specific enzyme for the catabolism of phenylacetic acid[END_REF] are also members of the superfamily of adenylate forming enzymes, but the sequence identity between these enzymes and PCL from Penicillium is very low (approximately 15%). PCL contains a C-terminal peroxisomal SKI targeting signal sequence (PTS1) and probably localizes in the peroxisomal matrix [START_REF] Gidijala | Reprogramming Hansenula polymorpha for penicillin production: expression of the Penicillium chrysogenum pcl gene[END_REF]. Isopenicillin N acyltransferase was also shown to be a peroxisomal enzyme [START_REF] Müller | Localization of the pathway of the penicillin biosynthesis in Penicillium chrysogenum[END_REF], indicating that the last two steps of the penicillin biosynthesis pathway are localized in the peroxisome.

Although PCL is important for the biosynthesis of penicillin G, little information is available about its catalytic properties, which may be partly due to the instability of the enzyme [START_REF] Kogekar | Biosynthesis of penicillin in vitro: purification and properties of 6-aminopenicillanic acid-phenylacetyl-CoA synthetase from Penicillium chrysogenum[END_REF]. We have used a maltose binding protein (MBP) fusion to obtain a high level of expression in E. coli and to increase the stability of PCL.

Here we describe the catalytic properties and identify the substrate binding residues of this important enzyme of the penicillin G biosynthetic route.

of the pBAD vector. The QuikChange site-directed mutagenesis kit (Stratagene) was used to introduce alanine codons in the phl gene by replacing the target codon by GCT or GCC. All mutations were confirmed by sequencing at GATC Biotech (Konstanz, Germany).

Expression and purification of recombinant PCL

E. coli TOP10 cells (Invitrogen) containing pBAD derived phl constructs were precultured overnight in 5 ml LB. The preculture was used to inoculate 1 l of LB medium containing 0.001% (w/v) L-arabinose. Cells were harvested after 72 h of growth at 17 ºC, and resuspended in TANG buffer (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 0.002% (w/v) NaN 3 and 10% (w/v) glycerol). Subsequently, cells were sonicated and cell debris was removed by ultracentrifugation. The histidine-tagged enzyme was purified from cell-free extract using Ni affinity chromatography. The enzyme was bound to 3 ml of Ni-NTA resin (Qiagen) and eluted using 3 ml of TANG buffer containing 500 mM imidazole. The imidazole was removed using an Econo-Pac 10 DG chromatography column (Biorad). Enzyme concentrations were determined spectrophotometrically at 280 nm using a calculated extinction coefficient of 137,370 M -1 cm -1 .

To obtain MBP-free protein, the purified MBP-PCL-HIS was treated with Factor Xa (New England Biolabs). For this, CaCl 2 was added to a final concentration of 2 mM to 4 mg of purified enzyme in 1 ml TANG buffer. Subsequently, 10 µg of the protease was added and the sample was incubated for 4 days at 4 °C after which the fusion protein was completely cleaved. The cleaved PCL was purified from the mixture using the C-terminal histidine tag as described above. A c c e p t e d M a n u s c r i p t the cleaved protein was determined spectrophotometrically at 280 nm using a calculated extinction coefficient of 65,695 M -1 cm -1 .

CoA ligase activity assay

Phenylacetate-CoA ligase activity in cell-free extracts was determined using the hydroxamic acid assay [START_REF] Brunner | Phenacyl:coenzyme A ligase[END_REF]. Assay mixtures contained 50 mM Tris-HCl, pH 8.0, 10 mM PAA, 10 mM ATP, 1 mM CoA, and 10 mM MgCl 2 . One unit is defined as the catalytic activity resulting in the formation of one µmol of phenylacetylhydroxamate per minute.

Ligase activity with trans-cinnamic acid derivatives was determined by monitoring the conversion of the respective cinnamic acid to the corresponding CoA thioesters. The increase in absorbance during conversion was followed in a Perkin Elmer spectrophotometer at wavelengths of 309 nm for cinnamoyl-CoA, 340 nm for coumaroyl-CoA, 346 nm for feruloyl-CoA, 346 nm for caffeoyl-CoA acid, and 352 nm for sinapoyl-CoA [START_REF] Stöckigt | Chemical syntheses and properties of hydroxycinnamoyl-coenzyme A derivatives[END_REF]. Extinction coefficients for other cinnamoyl-CoA derivatives were estimated by measuring the final absorbance after complete conversion of 25 and 50 µM substrate. Standard reaction mixtures contained 50 mM Tris-HCl, 200 mM NaCl, 3 mM ATP, 1.5 mM CoA, 5 mM MgCl 2 , 10 µM to 20 mM of substrate, and 10 nM of purified recombinant enzyme in a total volume of 1 ml.

Reactions were carried out at 30ºC and at pH 8.5.

Ligase activity for nonchromogenic substrates was determined by following the substrate-dependent formation of AMP by reversed phase HPLC under the same reaction conditions as described above. Samples were diluted 50-fold in eluens to quench enzymatic conversion and subsequently AMP was measured using an Alltech Alltima HP C18 column in connection with Jasco PU-980 pumps and a Jasco MD-910 A c c e p t e d M a n u s c r i p t detector set at 259 nm. The nucleotides AMP and ATP were eluted isocratically using a solution containing 100 mM potassium phosphate, 20 mM acetic acid, 5 mM tetrabutylammonium bisulfate, and 10% acetonitrile, which was adjusted to pH 4.0. Typical retention times were 2 min for AMP and 8 min for ATP. Substrate independent ATP hydrolysis by PCL was not observed (k obs < 0.01 s -1 ).

Determination of kinetic parameters

Apparent kinetic constants K m and k cat for PCL substrates were determined by varying the concentration of one substrate at fixed concentration of the other substrates. For determining the apparent K m values with carboxylic acid substrates, fixed concentrations of 3 mM ATP, 1.5 mM CoA, and 5 mM Mg 2+ were used. The apparent kinetic parameters for trans-cinnamic acid derivatives were determined by fitting the initial rates of cinnamoyl-CoA formation with the Michaelis-Menten equation. Apparent K m and k cat values for nonchromogenic substrates were determined by fitting the initial rate of AMP formation. For substrates with low apparent K m values (fatty acids) the apparent K m values were determined by fitting the level of inhibition on the initial rate of conversion of the chromogenic substrate transcinnamic acid [START_REF] Alkema | The use of chromogenic reference substrates for the kinetic analysis of penicillin acylases[END_REF]. When two substrates are converted by a single enzyme at the same active site, the substrates behave as mutual competitive inhibitors and the K m value of the second (invisible) substrate (K mS2 ) is equal to the inhibition constant on the conversion rate of the visible substrate (v s1 ) (equation 1).

] [S ) ]/ S [ (1 ] [S 1 2 mS 2 1 mS 1 1 catS + + ⋅ ⋅ - = K K k E v S1
Eq. 1 the apparent K m value. For the determination of the apparent kinetic parameters of the cosubstrates ATP and CoA, fixed concentrations of 1.5 mM cinnamic acid or 60 mM PAA were used. When ATP was varied, 1.5 mM of CoA was used. When CoA was varied the ATP concentration was 3 mM. All determinations of kinetic parameters were performed at least in duplicate.

Sequence comparison and modelling

The

RUSULTS

Expression and purification of PCL

CoA activation of PAA is an essential step in penicillin G biosynthesis.

However the biochemical properties of the enzyme catalyzing this reaction have never been extensively characterized. We have expressed PCL from P. chrysogenum in E.

coli in order to obtain large amounts of recombinant enzyme for characterization.

The expression of active PCL in E. coli initially proved to be difficult. An extensive investigation of the expression conditions was performed by varying the E. coli host strain, culture volume and aeration, growth temperature, arabinose concentrations, and time of induction, but PCL was not detected. SDS-PAGE analysis of whole cell lysates did not show the presence of the expected protein band at 62.6 kDa and CoA ligase activity was never detected in cell extracts, indicating that neither active nor inactive enzyme was produced. However, a high level of expression of the CoA ligase was observed for a construct in which the CoA ligase was fused to the C-terminus of MBP. SDS-PAGE showed that up to 20% of all protein in CFE of E. coli TOP 10 was MBP-PCL and the hydroxamic acid assay showed that the CFE contained up to 20 mU phenylacetate-CoA ligase activity per mg of protein.

Purification of MBP-PCL using amylose affinity chromatography was unsuccessful because the fusion protein did not bind to the amylose resin. Therefore further characterization of PCL was performed using a histidine-tagged MBP fusion enzyme. PCR was used to create a construct encoding a fusion protein that contained the N-terminal MBP that appeared essential for expression of the enzyme as well as a C-terminal hexahistidine tag for purification. The recombinant protein was purified to homogeneity using Ni-agarose, yielding a single protein band upon SDS-PAGE. From a culture of 500 ml, typically 20 mg of recombinant enzyme were obtained. The presence of 200 mM NaCl was essential for the stability of the enzyme and storage or conversions in buffer lacking NaCl resulted in rapid loss of activity. The presence of glycerol protected the enzyme when stored at -20 ºC. Purified protein could be stored in TANG buffer at -20ºC for several months without significant loss of activity.

Substrate specificity of PCL

Purified hexahistidine-tagged MBP-PCL was used to investigate the substrate specificity of PCL. The kinetic parameters for the physiological substrate PAA and for phenoxyacetic acid (POA) were determined using the AMP formation assay (Table 1). The enzyme had an apparent k cat of 1.4 ± 0. A c c e p t e d M a n u s c r i p t

6.1 ± 0.3 mM for PAA under the conditions used. POA appeared to be a much better substrate for the enzyme than PAA having both a 10-fold higher apparent k cat value and a 3-fold lower apparent K m . PCL also catalyzed the CoA activation of several substituted phenylacetic acids. Hydrophobic metaor para-substituted phenylacetic acids were substrates for PCL. The larger 3-phenylpropionic acid was also a reasonable substrate for PCL while the shorter benzoic acid was not converted. The amino acids phenylalanine, tyrosine, alanine and glycine were not substrates for PCL.

We also tested CoA ligase activity with trans-cinnamic acids, since these are the substrates for the homologous coumarate-CoA ligases from plants. The formation of cinnamoyl-CoA can easily be followed because of the strong absorbance of the product at around 309 nm. Both trans-cinnamic acid and trans-4-coumaric acid were converted very efficiently by PCL, providing a convenient spectrophotometric assay for the enzyme. The catalytic efficiency for cinnamic acid was more than a 1,000-fold higher than that for PAA. The difference is caused by the lower apparent k cat and a relatively high apparent K m value for the physiological substrate. The more substituted compounds ferulic acid, caffeic acid and sinapic acid, which are substrates for most 4coumarate CoA ligases [START_REF] Hamberger | The 4-coumaric acid:CoA ligase gene family in Arabidopsis thaliana comprises one rare, sinapic acid-activating and three commonly occurring isoenzymes[END_REF], were very poor substrates for PCL. Surprisingly, the very similar substrates 3-methoxy, 3-ethoxy and 3,4-methylenedioxycinnamic acid were very good substrates for PCL. To investigate whether the relatively low activity of para-hydroxy substituted compounds was caused by the strong electron-donating substituent of the substrate, we tested a set of para-substituted cinnamic acids with both electron withdrawing and donating groups (Table 2). The apparent K m values for these substituted trans-cinnamic acids were very similar to that for cinnamic acid and these substrates probably bind in a similar manner in the enzyme active site. The A c c e p t e d M a n u s c r i p t cinnamic acid phenyl ring was changed. A Hammett plot of log(k cat, x / k cat, H ) vs. σ p - [START_REF] Hansch | A Survey of Hammett substituent constants and resonance and field parameters[END_REF] shows a correlation between the observed apparent k cat and the substituent for both electron-donating and electron-withdrawing groups (Fig. 2). For substrates with electron-donating groups the slope is 2.5 (r = 0.69) and for substrates with electronwithdrawing groups the slope is -0.94 (r = 1.00). The Hammett plot is biphasic, indicating that both electron-donating and electron-withdrawing groups slow down the reaction. The observed correlation between the substituent and the apparent k cat suggests the formation or disappearance of a negative charge in the rate-limiting step.

The biphasic relationship suggests a change in the rate-determining step for substrates with electron-withdrawing substituents compared to substrates with electron-donating substituents [START_REF] Du | Acylation is rate-limiting in glucosylasparaginase-catalyzed hydrolysis of N4-(4'-substituted phenyl)-Lasparagines[END_REF].

The initial rate of cinnamic acid conversion was used to determine biochemical properties such as optimum pH and temperature and the kinetic parameters for the cosubstrates ATP and CoA and the cofactor magnesium. The pH optimum of the enzyme was 8.5 and the optimum temperature was between 35 and 40 ºC. The optimal Mg 2+ concentration was determined by varying the concentration of MgCl 2 at fixed concentrations of ATP (3 mM), CoA (1.5 mM) and cinnamic acid (1.5 mM) and was found to be 5 mM. At higher concentrations, inhibition was observed. When cinnamic acid was used as the substrate to determine the kinetic parameters of the cosubstrates, the apparent K m ATP was 0.48 ± 0.06 (apparent k cat was 42.9 ± 1.0 s -1 ) and the apparent K m CoA was 0.94 ± 0.10 mM (apparent k cat was 60.5 ± 3.2 s -1 ). When PAA was present as the carboxylic acid substrate and the AMP formation assay was used to determine the kinetic parameters, the apparent K m ATP was 0.65 ± 0.03 mM (apparent k cat was 1.7 ± 0.2 s -1 ), and the apparent K m CoA was 0.12 ± 0.01 mM (apparent k cat 1.5 ± 0.2 s -1 ).

Of the carboxylic acid substrates that we screened for, the highest activities were found for fatty acids (Table 1). With the exception of acetic acid, all short and medium chain fatty acids tested were converted by PCL. With increasing chain length the apparent K m value went down from more than 80 mM for propionic acid (C3) to less then 1 µM for myristic acid (C14). The apparent K m values for fatty acids with low K m values (C10 and larger) were determined by fitting the level of inhibition on the initial rate of conversion of the chromogenic substrate trans-cinnamic acid as described in Experimental. The apparent k cat values were in the same range for all fatty acids (Table 1), but a small decrease with increasing chain length was observed.

The apparent k cat value for long chain fatty acid myristic acid was approximately 10fold lower than for the short chain fatty acid valeric acid (C5). The cause of the observed decrease with increasing chain length is not immediately apparent but may be related to poor positioning of the larger substrate for nucleophilic attack on ATP, slow product release, or both.

During penicillin G producing fermentations of P. chrysogenum the formation of β-lactam side products has been reported [START_REF] Arnstein | The metabolism of the Penicillia in relation to penicillin biosynthesis[END_REF]. The main side products that are detected are the penicillins K, F and DF and these penicillins have caprylic acid, trans-3-hexenoic acid and caproic acid as side chains, respectively. PCL is able to activate all the side chains of these naturally occurring lactam side products (Table 3) and thus may be responsible for their formation during penicillin G production.

To determine whether the MBP fusion protein behaves similar to the native PCL protein, we prepared PCL without the N-terminal MBP-tag. For this, purified histidine tagged MBP-PCL was treated with Factor Xa protease. This yielded the two separate proteins MBP and the hexahistidine-tagged PCL. The PCL protein was purified from this mixture using the C-terminal histidine tag. The cleaved PCL-HIS

Homology modelling

The program BLAST was used to search the NCBI database with the amino acid sequence of PCL. This yielded strong hits with the extensively characterized 4-coumarate-CoA ligases from Arabidopsis thaliana, involved in the biosynthesis of plant phenylpropanoids, and a very similar bacterial cinnamate-CoA ligase from

Streptomyces coelicolor [START_REF] Hamberger | The 4-coumaric acid:CoA ligase gene family in Arabidopsis thaliana comprises one rare, sinapic acid-activating and three commonly occurring isoenzymes[END_REF][START_REF] Schneider | The substrate specificity-determining amino acid code of 4-coumarate:CoA ligase[END_REF][START_REF] Kaneko | Cinnamic acid:coenzyme A ligase from the filamentous bacterium Streptomyces coelicolor A3(2)[END_REF]. These enzymes are also able to catalyze the activation of trans-cinnamic acid derivatives to the corresponding CoA esters.

Many structures of members of the superfamily of adenylate forming enzymes were found in the PDB database, including two acetate-CoA ligases [START_REF] Gulick | The 1.75 A crystal structure of acetyl-CoA synthetase bound to adenosine-5'-propylphosphate and coenzyme A[END_REF][START_REF] Jogl | Crystal structure of yeast acetyl-coenzyme A synthetase in complex with AMP[END_REF] a chlorobenzoate-CoA ligase [START_REF] Gulick | Crystal structure of 4chlorobenzoate:CoA ligase/synthetase in the unliganded and aryl substrate-bound states[END_REF] and a fatty acid CoA ligase [START_REF] Hisanaga | Structural basis of the substrate-specific two-step catalysis of long chain fatty acyl-CoA synthetase dimer[END_REF]. These enzymes have less than 25 % sequence identity with PCL. Surprisingly, the most related sequences A c c e p t e d M a n u s c r i p t

to PCL are from two luciferases. The first luciferase structure that was solved is that of the North American firefly Photinus pyralis [START_REF] Conti | Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes[END_REF]. The structure was solved in the absence of substrate and the identification of the active site and the residues important for substrate binding remained unclear. More recently the structure of the Japanese firefly luciferase (Luciola cruciata) was solved [START_REF] Nakatsu | Structural basis for the spectral difference in luciferase bioluminescence[END_REF]. This structure was determined in complex with an adenylate intermediate analogue at a resolution of 1.3 Å, and was used as the template for homology modelling of PCL which was possible because the sequence identity was 29.%. Figure 3 shows a sequence alignment of PCL with the homologous CoA ligases and luciferases around the substrate binding pocket.

The model shows that PCL is composed of a large N-terminal domain and a small C-terminal domain similar to the other members of the adenylate forming superfamily. In the model 14 residues form the substrate binding pocket: H265, I266, Y267, V270, F307, F335, G337, A338, G361, T369, V370, and K557 (Fig. 4A).

Lysine 557 is located in the C-terminal domain and this invariant residue is involved in formation of the adenylate intermediate [START_REF] Branchini | The role of lysine 529, a conserved residue of the acyl-adenylate-forming enzyme superfamily, in firefly luciferase[END_REF][START_REF] Starai | Sir2-dependent activation of acetyl-CoA synthetase by deacetylation of active lysine[END_REF]. The other residues are located at the interface between the two domains and form the wall of a long tunnel for substrate binding. Close to the carboxylate binding site the tunnel is rather narrow because of the backbone of residues G337, A338 and G361 and the side chains of I266 and T369.

When PAA is modelled in the substrate binding channel, there seems be a steric conflict between the phenyl ring of PAA and the wall of the substrate binding pocket formed by the backbone of residues G337 and A338. This may explain the high apparent K m value observed for PAA. The additional oxygen atom in POA and carbon atom in trans-cinnamic acid may cause the phenyl ring of these substrates to become located in the tunnel; these substrates have low apparent K m values. In the model, the fatty acids are also able to rotate their aliphatic chain into the tunnel and do not have a steric conflict with the wall of the pocket (Fig. 4B).

Mutagenesis of substrate binding pocket residues

The role of the side chains of residues H265, I266, Y267, V270, F307, F335, T369 and V370 was investigated by mutating these residues to an alanine. All alanine mutations were made in the histidine-tagged MBP-PCL enzyme and mutant enzymes were expressed and purified as described for wild-type MBP-PCL. The kinetic parameters of the mutants for PAA and POA were determined. The effect of the mutations on the activity with the caproic acid and with trans-cinnamic and para-coumaric acid was also investigated (Table 4).

The affinity of wild-type PCL for PAA and POA was relatively low, with apparent K m values in the millimolar range. None of the alanine mutations had a significant effect on the affinity for PAA since the apparent K m of all the mutants was unaffected. However, the apparent k cat for PAA was strongly influenced by the mutations, indicating that the residues are important for CoA ligase activity. A c c e p t e d M a n u s c r i p t also observed for the substrates caproic acid, cinnamic acid, and coumaric acid but not for POA.

Activity with POA was affected in a similar way by the mutations as activity for PAA. The apparent k cat was affected strongly by all the mutations but the effect on the apparent K m was relatively small. Mutants Y267A and F307A and F335A did show a moderate (3 to 5-fold) increase in apparent K m for POA. In the model the phenyl ring of the substrate POA possibly interacts with the phenyl rings of Y267 and with residues F307 and F335 located further away in the substrate binding tunnel.

Mutation of one of these residues into an alanine removes this interaction causing a lower affinity for the substrate. This effect on apparent K m was also observed for the other aromatic substrates cinnamic acid and coumaric acid, but not for the aliphatic caproic acid, suggesting that these residues are important for the binding of aromatic substrates. This effect on affinity was not observed for PAA. The model suggest that the phenyl ring of the smaller substrate PAA is unable to rotate towards the substrate binding pocket, making it impossible for PAA to interact with these aromatic residues (Fig. 4B).

Mutations V270A and V370A had relatively small effects on CoA ligase activity for all five substrates. The apparent K m values for both mutants were similar to those of wild-type PCL. The effect on the apparent k cat was also relatively small with the exception that mutant V270A showed a 10-fold lower apparent k cat for POA and mutant V370A a 10-fold lower apparent k cat for PAA. These valine residues may help to form a hydrophobic substrate binding pocket and are probably not very important in determining the substrate specificity.

DISCUSSION

CoA activation of PAA is an essential step in the biosynthesis of penicillin G by P. chrysogenum. Previous work has indicated that multiple CoA ligases may play a role in the formation of phenylacetyl-CoA but a large part is formed by PCL [START_REF] Lamas-Maceiras | Amplification and disruption of the phenylacetyl-CoA ligase gene of Penicillium chrysogenum encoding an aryl-capping enzyme that supplies phenylacetic acid to the isopenicillin N-acyltransferase[END_REF].

Purification of PAA-CoA ligase activity from P. chrysogenum cultures has been reported but biochemical data on the enzyme responsible for this activity are scarce, which may be due to the reported instability of the enzyme [START_REF] Kogekar | Biosynthesis of penicillin in vitro: purification and properties of 6-aminopenicillanic acid-phenylacetyl-CoA synthetase from Penicillium chrysogenum[END_REF]. In this study we report the overexpression of phenylacetate-CoA ligase as an MBP fusion in E. coli to produce a stable fusion enzyme, and the characterization of this key enzyme for penicillin G biosynthesis.

MBP from E. coli is often used as an expression tag to increase the recombinant expression of proteins [START_REF] Di Guan | Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein[END_REF]. In the case of PCL the fusion not only increased the expression level of the protein, but also improved the stability of the protein. The MBP part of the MBP-PCL-HIS protein could be removed by digestion with Factor Xa protease, but the cleaved PCL-HIS could be purified and showed CoA ligase activity but was less stable.

Recombinant MBP-PCL-HIS was used to investigate the substrate specificity of the CoA ligase. For cinnamic acids the spectrophotometric assay was used to follow CoA ligase activity. The extinction coefficients of the aromatic enoyl-CoA products is relatively high (around 20 mM -1 cm -1 ), providing a practical and sensitive activity assay for PCL. Formation of as little as one micromolar of product is easily detected. To measure CoA ligase activity with other carboxylic acids, the AMP formation assay was preferred over the hydroxamate assay. The release of the coproduct AMP is independent of the carboxylic acid substrate used and is also sensitive because of the high UV absorbance of nucleotides at 259 nm. In the hydroxamate assay the extinction coefficient of the formed iron-hydroxamate complex depends on the acid used and is relatively low (e.g. ε max = 0.9 mM -1 cm -1 for phenylacetyl-hydroxamate), making the assay not very sensitive. For some substrates, such as the amino acid phenylglycine, the extinction coefficient was even ten times lower, making it almost impossible to detect CoA ligase activity with such a substrate.

To validate the use of AMP detection as a CoA ligase activity assay we followed the conversion of trans-cinnamic acid both with the spectrophotometric method as well as with AMP formation on HPLC, which yielded the same results.

Comparison of the biochemical properties indicates that PCL, encoded by phl, differs from the CoA ligase that was earlier purified from P. chrysogenum by Kogekar et al. [START_REF] Kogekar | Biosynthesis of penicillin in vitro: purification and properties of 6-aminopenicillanic acid-phenylacetyl-CoA synthetase from Penicillium chrysogenum[END_REF]. We found apparent K m values of 6.1 and 2.0 mM for PAA and POA, respectively, whereas Kogekar et al. reported an apparent K m of 2.9 µM for both substrates. Furthermore, unlike PCL, the latter enzyme was unable to use phenylpropionic acid or fatty acids as substrates. The previously constructed P. chrysogenum phl knockout was shown to be still able to produce some penicillin G [START_REF] Lamas-Maceiras | Amplification and disruption of the phenylacetyl-CoA ligase gene of Penicillium chrysogenum encoding an aryl-capping enzyme that supplies phenylacetic acid to the isopenicillin N-acyltransferase[END_REF] and consequently there must be at least one more enzyme with phenylacetyl-CoA ligase activity, which may be the enzyme purified by Kogekar et al. Recently, the cloning of a peroxisomal CoA ligase from P. chrysogenum was reported, but the biochemical properties were not investigated in detail [START_REF] Wang | Molecular cloning and functional identification of a novel phenylacetyl-CoA ligase gene from Penicillium chrysogenum[END_REF]. The recombinant enzyme was reported to have phenylacetyl-CoA ligase activity, and it was suggested that this enzyme may be involved in penicillin biosyntheses, but no quantitative activity data were given. A further candidate for phenylacetate activation that could be considered in P. chrysogenum is the previously purified acetate-CoA ligase [START_REF] Martínez-Blanco | Isolation and characterization of the acetyl-CoA synthetase from Penicillium chrysogenum. Involvement of this enzyme in the biosynthesis of penicillins[END_REF]. This protein was described to activate phenylacetic acid and may be responsible for the remaining phenylacetyl-CoA ligase activity that is used for penicillin G biosynthesis in a PCL knockout strain [START_REF] Lamas-Maceiras | Amplification and disruption of the phenylacetyl-CoA ligase gene of Penicillium chrysogenum encoding an aryl-capping enzyme that supplies phenylacetic acid to the isopenicillin N-acyltransferase[END_REF]. However, this acetate-CoA ligase does not contain a PTS1 or PTS2 sequence [START_REF] Martínez-Blanco | Characterisation of the gene encoding acetyl-CoA synthetase in Penicillium chrysogenum: conservation of intron position in plectomycetes[END_REF] and probably localizes in the cytosol and it is unclear whether phenylacetic acid activated in the cytosol can be used for the acyltransferase reaction which occurs in the peroxisome [START_REF] Müller | Localization of the pathway of the penicillin biosynthesis in Penicillium chrysogenum[END_REF].

The substrate range of PCL is rather broad. The enzyme showed CoA ligase activity with PAA and with POA, but the activity with these substrates was low compared to that with trans-cinnamic acids and fatty acids. Nonetheless, we maintain the name phenylacetate-CoA ligase because of its role in penicillin G biosynthesis.

The broad substrate range combined with relatively low activity for PAA makes PCL very different from phenylacetate-CoA ligases involved in PAA catabolism from bacteria such as A. evansii [START_REF] Mohamed | Biochemical and molecular characterization of phenylacetate-coenzyme A ligase, an enzyme catalyzing the first step in aerobic metabolism of phenylacetic acid in Azoarcus evansii[END_REF] and P. putida [START_REF] Martínez-Blanco | Purification and biochemical characterization of phenylacetyl-CoA ligase from Pseudomonas putida. A specific enzyme for the catabolism of phenylacetic acid[END_REF]. Indeed, sequence comparison shows that PCL from P. chrysogenum is more closely related to plant coumarate-CoA ligases and firefly luciferases than to the bacterial phenylacetate-CoA ligases. The bacterial CoA ligases are specifically expressed in the presence of PAA and the substrate profile shows that these enzymes are highly specific for PAA. Despite the strong preference for PAA, the P. putida CoA ligase was reported to accept a broad range of acid substrates in reactions coupled with isopenicillin N acyltransferase from P. chrysogenum. In these in vitro experiments, the CoA ligase was found to activate a wide range of side chains leading to the formation of many both natural and nonnatural penicillins [START_REF] Luengo | Enzymatic synthesis of hydrophobic penicillins[END_REF].

The highest activities of PCL were observed for medium chain fatty acids. The formation of β-lactam side products containing these fatty acid side chains has been reported in penicillin G and V producing cultures of P. chrysogenum [START_REF] Arnstein | The metabolism of the Penicillia in relation to penicillin biosynthesis[END_REF].

The main β-lactam side products that are formed are the natural penicillin K, F and DF, which contain caprylic acid, trans-3-hexenoic acid and caproic acid as the side chain. The original strain from which current production strains are derived, Penicillium chrysogenum NRRL 1951, and early derivatives of this original isolate (e.g. strain Q176) were shown to produce a mixture of these penicillins when no (additional) PAA was supplied in the feed [START_REF] Macmorine | Some factors influencing the production of certain biosynthetic penicillins[END_REF]. It was shown that in a defined medium without any PAA, penicillin G is not produced, and the addition of medium or long chain fatty acids results in higher levels of penicillin K, F and DF [START_REF] Arnstein | The metabolism of the Penicillia in relation to penicillin biosynthesis[END_REF][START_REF] Thorn | Precursors for aliphatic penicillins[END_REF] .

The formation of these side products occurs because isopenicillin N acyltransferase is able to accept many CoA activated fatty acids as a donor in the acyltransferase reaction with isopenicillin N [START_REF] Alvarez | The isopenicillin-N acyltransferase of Penicillium chrysogenum has isopenicillin-N amidohydrolase, 6-aminopenicillanic acid acyltransferase and penicillin amidase activities, all of which are encoded by the single penDE gene[END_REF][START_REF] Luengo | Enzymatic synthesis of hydrophobic penicillins[END_REF][START_REF] Thorn | Precursors for aliphatic penicillins[END_REF]. The results presented here show that PCL also A c c e p t e d M a n u s c r i p t has a broad substrate range and is able to activate the side chains of all the natural β-lactam side products that are observed.

The observed selectivity could be explained by homology modelling. The model shows that PCL has a large narrow active site which is formed by hydrophobic residues. The large hydrophobic active site explains the observed broad substrate range for hydrophobic acids. The fact that the apparent K m value for the substrates PAA and POA is much higher than for fatty acid substrates indicates that PCL does not have an optimized binding site for PAA or POA. The alanine mutations do not cause a large increase in the apparent K m , also indicating that the substrate binding pocket is not optimized for these substrates. Modelling of acid substrates in the active site shows that α-unsubstituted and mono-β-substituted carboxylic acids are the best substrates for PCL. The narrow neck of the substrate binding pocket prevents the binding of α-substituted acids such as the amino acids and also the binding of the mono-α-substituted PAA is not optimal. The longer acids POA, trans-cinnamic acid and fatty acids do not have substituents on the α-carbon and the β-substituent and can rotate into the substrate binding tunnel. Therefore, these substrates have relatively low apparent K m values and high turnover numbers.

In summary, the homology model and the substrate specificity suggest that PCL is not optimized for the activation of PAA and POA but for medium chain fatty A c c e p t e d M a n u s c r i p t may have been recruited for the penicillin G biosynthesis. Interestingly, at the genetic level there is also evidence for the different evolutionary origin of PCL. The phl gene is not part of gene clusters that contain the other genes of the penicillin biosynthesis, penAB, penC and penD [START_REF] Diez | The cluster of penicillin biosynthetic genes. Identification and characterization of the pcbAB gene encoding the alpha-aminoadipyl-cysteinylvaline synthetase and linkage to the pcbC and penDE genes[END_REF]. The recruitment of a peroxisomal fatty acid CoA ligase has also been proposed for the evolution of firefly luciferases [START_REF] Oba | Cloning and characterization of the homologous genes of firefly luciferase in the mealworm beetle, Tenebrio molitor[END_REF]. The CoA ligase activity observed in some luciferases indicates the close relation between these two enzyme activities. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

k cat (s -1 ) K m (mM) k cat (s -1 ) K m (mM) k cat (s -1 ) K m (mM) k cat (s -1 ) K m (mM) k cat (s -1 ) K m (mM)

  Cleavage and purity of the resulting MBP-free enzyme were analyzed by SDS-PAGE. The concentration of 6 Biochemical Journal Immediate Publication. Published on 06 Oct 2008 as manuscript BJ20081257 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081257

  apparent k cat values varied between the 2 and 50 s -1 when the substituent on the 11 Biochemical Journal Immediate Publication. Published on 06 Oct 2008 as manuscript BJ20081257 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081257

  Biochemical Journal Immediate Publication. Published on 06 Oct 2008 as manuscript BJ20081257 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081257A c c e p t e d M a n u s c r i p t had the same steady state kinetic parameters for cinnamic acid as well as for PAA and POA as MBP-PCL-HIS (PAA: K m 5.2 ± 0.9 mM, k cat 1.9 ± 0.2 s -1 ; POA: K m 2.1 ± 0.1 mM, k cat 12 ± 2.1 s -1 and cinnamic acid: K m 0.19 ± 0.04 mM, k cat 41.2 ± 1.4 s -1 ). The cleaved enzyme was less stable than the MBP fusion enzyme and precipitation was observed during concentrating or freeze/thawing of the enzyme. Factor Xa cleavage of an MBP-PCL fusion that contained the hexahistidine tag to the N-terminus of the MBP instead of the C-terminus of PCL resulted in an even less stable PCL. The fusion protein HIS-MBP-PCL expressed normally in E. coli and could be purified using the histidine tag and showed a similar activity with trans-cinnamic acid, indicating that functional PCL was produced. However, Factor Xa cleavage of HIS-MBP-PCL to obtain untagged PCL resulted in almost immediate precipitation of the protein after the HIS-MBP part was separated from PCL. Both the N-terminal MBP and the C-terminal histidine tag appear to stabilize PCL.

Fatty

  acid activation by CoA ligases is also the first step in β-oxidation of fatty acids, which occurs in the peroxisomal matrix. PCL contains a C-terminal PTS1 sequence (SKI) for targeting and probably localizes in the peroxisomal matrix. Maximal CoA ligase activity was found at pH of 8.5, indicating that PCL is optimized for the 20 Biochemical Journal Immediate Publication. Published on 06 Oct 2008 as manuscript BJ20081257 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081257 A c c e p t e d M a n u s c r i p t peroxisome which has a slightly alkaline pH [40]. The β-oxidation of fatty acids has been investigated extensively in yeasts and it is believed that there are two routes for transport of fatty acyl-CoAs to the peroxisomal matrix. Fatty acids are either activated in the cytosol by a cytosolic ligase and then transported to the peroxisome as CoA thioesters, or CoA activation occurs during transport of the fatty acids across the peroxisomal membrane by a membrane-associated ligase [41]. CoA activation of fatty acids inside the peroxisome by a matrix protein has not been reported to play a role in β-oxidation in yeasts. It is unclear if fatty acid activation by PCL plays a physiological role in β-oxidation in Penicillium, but the formation of β-lactam side products containing fatty acyl side chains during fermentations is an indication that fatty acid activation occurs under penicillin producing conditions.

Biochemical

  Journal Immediate Publication. Published on 06 Oct 2008 as manuscript BJ20081257 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081257

  acids and PCL may have evolved from an enzyme that activated fatty acids, likely involved in the β-oxidation of fatty acids in the peroxisome. The broad substrate range of PCL for relatively large hydrophobic acids may have allowed the enzyme to become involved in the activation of penicillin side chains. During the evolution of β-lactam biosynthesis and perhaps even during the subsequent strain improvement towards higher penicillin G producing strains, this and other peroxisomal CoA ligases 22 Biochemical Journal Immediate Publication. Published on 06 Oct 2008 as manuscript BJ20081257 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081257

Figure 1 .

 1 Figure 1. The final step in the biosynthesis of penicillin G. The L-α-aminoadipyl side

Figure 2 .

 2 Figure 2. A Hammett plot of log(k cat, x / k cat, H ) vs. σ p -for the CoA ligase reaction with

Figure 3 .

 3 Figure 3. Sequence alignment of PCL, firefly luciferases and coumarate-CoA ligases.

Figure 4 .

 4 Figure 4. Active site residues in the structure of LcrLucif and the homology model of

Figure 1 37Figure 2 38Figure 3 39Figure 4 40

 1234 Figures

  Biochemical Journal Immediate Publication. Published on 06 Oct 2008 as manuscript BJ20081257 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081257

  The relatively high apparent K m of wild-type PCL for PAA, and the small effect that the mutations have on the affinity for PAA, indicate that the substrate binding pocket of PCL is not selectively optimized for PAA. The observed effect on apparent k cat might be caused by changes in orientation of the carboxylate group for reaction with ATP in the first half reaction. Interestingly, mutant T369A had a 3.7-fold increased apparent k cat for PAA. In the model T369 is located at the top of the tunnel close to the carboxylate binding site. Removal of the side chain of residue T369 may create some

more space at the neck of the substrate binding tunnel allowing the substrate to bind more favourably for catalysis. The increased apparent k cat value of mutant T369A was 16 Biochemical Journal Immediate Publication. Published on 06 Oct 2008 as manuscript BJ20081257 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081257

Table 1 .

 1 Steady state kinetic parameters of PCL. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

	Biochemical Journal Immediate Publication. Published on 06 Oct 2008 as manuscript BJ20081257
	Substrate	k cat	K m	k cat / K m
	aromatic acids	(s -1 )	(mM)	(mM -1 s -1 )
	phenylacetic acid phenoxyacetic acid 3-phenylpropionic acid trans-cinnamic acid trans-4-coumaric acid caffeic acid a ferulic acid a sinapic acid a 3-methoxycinnamic acid 3-ethoxycinnamic acid 3,4-methylenedioxy cinnamic acid A c c e p t e d M a n u s c r i p t OH O 1.4 ± 0.3 6.1 ± 0.3 OH O O 15.6 ± 1.6 2.0 ± 0.2 O OH 22.7 ± 1.9 2.1 ± 0.4 O OH 43.5 ± 2.7 0.14 ± 0.01 (3.1 ± 0.4) • 10 2 0.23 ± 0.06 7.8 ± 1.2 10.8 ± 2.3 O OH O H 3.4 ± 0.5 0.47 ± 0.08 7.2 ± 1.7 O OH O H OH >0.02 >1.0 0.02 ± 0.01 O OH O H OMe >0.07 >0.5 0.07 ± 0.01 O OH O H OMe MeO na b na na O OH OMe 11.6 ± 0.3 0.84 ± 0.09 14 ± 2 O OH OEt 12.1 ± 0.7 0.82 ± 0.02 15 ± 2 O OH O O 11.2 ± 1.9 0.54 ± 0.04 21 ± 5	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081257
	30

Table 2 .

 2 Kinetic parameters and σ p -values for para substituted trans-cinnamic acids

	k cat	K m	k cat / K m	σ p -a
	(s -1 )	(mM)	(mM -1 s -1 )

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Table 3 .

 3 CoA ligase activity of PCL with side chains precursors of natural penicillins. Biochemical Journal Immediate Publication. Published on 06 Oct 2008 as manuscript BJ20081257 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081257 A c c e p t e d M a n u s c r i p tLicenced copy. Copying is not permitted, except with prior permission and as allowed by law.

	Product	Side-chain precursor					k cat	K m	k cat / K m
							(s -1 )	(mM)	(mM -1 s -1 )
	penicillin G	phenylacetic acid			O		1.4 ± 0.3	6.1 ± 0.3	(2.3 ± 0.6) 10 -1
						OH
	penicillin V	phenoxyacetic acid		O	O	OH	15.6 ± 1.6	2.0 ± 0.2	7.8 ± 1.2
	penicillin F	trans-3-hexenoic acid			O		14.7 ± 1.2	0.12 ± 0.01	(1.2 ± 0.2) • 10 2
						OH
	penicillin DF caproic acid			O		15.8 ± 0.5	0.24 ± 0.02	(6.6 ± 0.7) • 10 1
						OH
	penicillin H	enanthic acid			O		12.3 ± 3.9	0.05 ± 0.03	(2.5 ± 1.8) • 10 1
						OH
	penicillin K	caprylic acid			O		11.2 ± 0.7	0.06 ± 0.01	(1.9 ± 0.4) • 10 1
						OH
	penicillin X	hydroxyphenylacetic acid	H	O	O		0.08 ± 0.02	5.7 ± 0.4	(1.4 ± 0.4) • 10 -2
						OH
							33
	© 2008 The Authors Journal compilation © 2008 Biochemical Society

Table 4 .

 4 Kinetic parameters of PCL wild type and mutants with substrates PAA, POA, caproic acid, trans-cinnamic acid and para-

	coumaric acid.
	Wild type	1.4 ± 0.3	6.1 ± 0.3	15.6 ± 1.6	2.0 ± 0.2	15.8 ± 0.5	0.24 ± 0.02	43.5 ± 2.7	0.14 ± 0.01	3.4 ± 0.5	0.47 ± 0.08
	H265A	0.009 ± 0.001	8.5 ± 0.8	0.41 ± 0.09	4.8 ± 0.7	4.4 ± 0.3	1.6 ± 0.2	1.2 ± 0.2	4.0 ± 0.2	0.08 ± 0.03	2.8 ± 0.6
	I266A	0.49 ± 0.05	5.5 ± 1.2	1.1 ± 0.1	3.3 ± 0.4	41.8 ± 1.6	0.30 ± 0.01	10.4 ± 1.1	0.8 ± 0.3	0.6 ± 0.1	1.8 ± 0.3
	Y267A	0.025 ± 0.002	6.3 ± 1.1	0.13 ± 0.01	10.7 ± 1.5	0.13 ± 0.02	0.13 ± 0.01	0.4 ± 0.2	3.0 ± 1.0	0.011 ± 0.001	0.57 ± 0.03
	V270A	0.68 ± 0.04	3.9 ± 0.3	1.5 ± 0.1	2.4 ± 0.2	10.0 ± 1.5	0.13 ± 0.01	11.5 ± 2.8	0.54 ± 0.04	0.30 ± 0.01	0.41 ± 0.03
	F307A	0.017 ± 0.002	6.8 ± 0.3	0.53 ± 0.08	5.3 ± 0.7	5.3 ± 0.7	0.24 ± 0.08	4.5 ± 0.3	1.0 ± 0.3	0.11 ± 0.02	1.1 ± 0.1
	F335A	0.04 ± 0.01	8.0 ± 0.5	0.91 ± 0.09	10.3 ± 0.9	11.3 ± 0.2	0.8 ± 0.2	12.1 ± 1.4	2.5 ± 0.4	0.8 ± 0.2	0.17 ±0.02
	T369A	5.2 ± 0.6	4.7± 0.3	2.2 ± 0.2	1.3 ± 0.2	47.6 ± 1.3	0.11 ± 0.01	56 ± 11	0.5 ± 0.2	3.0 ± 0.2	0.47 ± 0.01
	V370A	0.14 ± 0.01	7.6 ± 1.0	4.2 ± 0.3	4.0 ± 0.5	13.9 ± 1.2	0.15 ± 0.04	24.6 ± 2.9	0.25 ± 0.03	1.3 ± 0.1	0.14 ± 0.02

34 Biochemical Journal Immediate Publication. Published on 06 Oct 2008 as manuscript BJ20081257 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081257

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society

ACKNOWLEDGEMENTS

This work was part of the Dutch EET Program (Project No. EETK20002) supported by the Dutch Ministry of Economic Affairs and of the IBOS (Integration of Biosynthesis and Organic Synthesis) Program of Advanced Chemical Technologies for Sustainability (ACTS), supported by the Dutch Ministry of Economic Affairs and The Netherlands Organization for Scientific Research (NWO).


Katja Lehmann

Jörg P Müller

Bernhard Schlott

Philipp Skroblin

Dagmar Barz

Johannes Norgauer

Reinhard Wetzker

PI3Kγ controls oxidative burst in neutrophils

Keywords: PI3Kγ, PKCα, interaction, protein kinase, reactive oxygen species, neutrophils ROS, reactive oxygen species, PI3Kγ, phosphoinositide 3-kinase γ, PKCα, protein kinase C α, fMLP, N-formyl-Met-Leu-Phe, BIM, bisindolylmaleimide, PVDF, poly vinylidene

INTRODUCTION

Neutrophils are key mediators of innate immunity and host defense. After exposure to chemotaxins, freely circulating neutrophils adhere to endothelial cells and migrate into infected tissue. Once they have reached the inflamed area, the neutrophils begin to phagocytize the pathogens. Secretion of reactive oxygen species (ROS) and lysosomal enzymes contributes to the killing of bacteria. Neutrophil defense reactions are tightly controlled by inflammatory mediators, including chemokines, bacterial peptides, and complement fragments.

Bacterial cell wall-derived chemotactic peptides, such as N-formyl-Met-Leu-Phe (fMLP) or complement fragment C5a, are prototypical peptides that affect the cell responses of neutrophils by binding to specific G protein-coupled receptors [START_REF] Wymann | Structure and function of phosphoinositide 3kinases[END_REF]. The interactions of these chemotaxins with their receptors trigger multiple intracellular signalling pathways, including rapid stimulation of phosphoinositide 3-kinase γ (PI3Kγ) [START_REF] Stephens | The G beta gamma sensitivity of a PI3K is dependent upon a tightly associated adaptor, p101[END_REF][START_REF] Kular | Co-operation of phosphatidylinositol transfer protein with phosphoinositide 3-kinase gamma in the formylmethionyl-leucylphenylalaninedependent production of phosphatidylinositol 3,4,5-trisphosphate in human neutrophils[END_REF]. This signalling protein is critically involved in the control of neutrophil migration and ROS secretion induced by fMLP and chemokines [START_REF] Hirsch | Central role for G proteincoupled phosphoinositide 3-kinase gamma in inflammation[END_REF][START_REF] Sasaki | Function of PI3Kgamma in thymocyte development, T cell activation, and neutrophil migration Science[END_REF][START_REF] Li | Roles of PLCbeta2 and -beta3 and PI3Kgamma in chemoattractant-mediated signal transduction[END_REF]. In neutrophils and other cell types, PI3Kγ exists in a heterodimeric form associated with the adapter protein p101. p101 strongly enhances G protein-dependent stimulation of the lipid kinase activity of PI3Kγ in vitro [START_REF] Suire | p84, a new Gbetagamma-activated regulatory subunit of the type IB phosphoinositide 3-kinase p110gamma[END_REF]. Comparative investigations have revealed a substantial decrease in PI3Kγ lipid kinase activity and phosphatidylinositol-3,4,5triphosphate production provoked by chemoattractants in p101 -/-neutrophils in comparison with wild-type littermates. In addition, simultaneous loss of chemoattractant-dependent migration has been observed in p101-deficient neutrophils. In compelling contrast with these findings, p101 -/-neutrophils exhibit an unaltered oxidative burst induced by fMLP or C5a [START_REF] Leopoldt | Gbetagamma stimulates phosphoinositide 3-kinase-gamma by direct interaction with two domains of the catalytic p110 subunit[END_REF].

These intriguing data point to differential functions for p101 in migration and chemoattractantinduced ROS production of neutrophils. Whereas migration of neutrophils requires interactions between p101 and PI3Kγ, ROS production induced by chemoattractants appears to be independent of p101. Consequently, the distinct regulatory effects of PI3Kγ on both inflammatory reactions of neutrophils become apparent.

MATERIALS AND METHODS

Materials

Chemoattractant N-formyl-Met-Leu-Phe (fMLP), PKC inhibitor bisindolylmaleimide I (BIM), bis-N-methylacridinium nitrate (lucigenin), histone type H1 calf thymus (SIGMA, H4524), histopaque 1077, and histopaque 1119 were from Sigma-Aldrich (Taufkirchen, Germany).

PI3Kγ inhibitor AS605240 was from Alexis; monoclonal mouse PKCα antibody for immunoprecipitation was from BD Biosciences (Heidelberg, Germany); polyclonal rabbit PKCα for immunodetection and polyclonal rabbit p47phox antibody were from Santa Cruz Biotechnology (Santa Cruz, CA); [γ-32 P]-ATP was from Hartmann Analytik GmbH (Braunschweig, Germany); and monoclonal mouse PI3Kγ antibody H1 [START_REF] Leopoldt | Gbetagamma stimulates phosphoinositide 3-kinase-gamma by direct interaction with two domains of the catalytic p110 subunit[END_REF] used for immunodetection was from Jena Bioscience (Jena, Germany). Sequencing-grade modified trypsine was purchased from Promega (Mannheim, Germany). The HEK293 cell line stably expressing the catalytic subunit p110γ was from our laboratory collection.

Recombinant proteins

For recombinant protein production, the PKCα-encoding gene was amplified by PCR using primers PKC-FW, containing an EcoRI restriction site (5′-GCGAATTCGGCATGGCTGACGTTTTCCCGGGC-3′), and PKCα-REV, containing an XbaI restriction site (5′-TGCTCTAGAATCATACTGCACTCTGTAAGATGGGG-3′). The amplifed gene was subsequently subcloned into plasmid pcDNA [START_REF] Adomeit | Bradykinin B(2) receptor-mediated mitogen-activated protein kinase activation in COS-7 cells requires dual signalling via both protein kinase C pathway and epidermal growth factor receptor transactivation[END_REF]. In parallel, a DNA region encoding the GST epitope was amplified using primer GST-FW, containing a BamHI restriction site (5′-CGGGATCCGGCATGTCCCCTATACTAGG-3′), and GST-REV, containing an EcoRI restriction site (5′-GAGAATTCAGAACGCGGAACCAGATCCGA-3′). To produce GST-PKCα, the subcloned DNA fragments were fused and inserted into pFastBac1 (Invitrogen, Karlsruhe, Germany). The resulting plasmid pFastBac1-GST-PKCa was chemically transformed into E. coli DH10BacTM cells and integrated by site-specific recombination into bacmids containing the host DNA. GST-PKCα-Bacmid was transfected into SF9 cells, and baculoviruses were produced in accordance with the manufacturer's instructions. The baculovirus SF9 system was used to produce the recombinant proteins PKCα, GST-PKCα, and His-tagged wild-type PI3Kγ or catalytically inactive PI3Kγ-KR [START_REF] Stoyanova | Lipid kinase and protein kinase activities of G-protein-coupled phosphoinositide 3-kinase gamma: structure-activity analysis and interactions with wortmannin[END_REF], as described previously [START_REF] Rubio | Farnesylation of Ras is important for the interaction with phosphoinositide 3-kinase gamma[END_REF]. Recombinant p47phox and p67phox proteins were produced in E. coli as described previously [START_REF] Abo | Reconstitution of neutrophil NADPH oxidase activity in the cell-free system by four components: p67phox, p47-phox, p21rac1, and cytochrome b-245[END_REF]. GST-tagged proteins were purified by affinity chromatography on glutathione-agarose. Subsequently, the GST epitope was cleaved using thrombin.

Affinity precipitation of recombinant proteins

Purified recombinant proteins (1 μg each) were incubated in kinase buffer for 30 min at 30 °C. Subsequently, epitope-labeled proteins were purified using affinity purification. His-PI3Kγ was purified using Ni-NTA-agarose and GST-PKCα was purified using glutathione beads.

Affinity chromatography material (50 μl slurry) was incubated for 1 h with ongoing agitation.

Beads were washed extensively at least four times. For purification of His-PI3Kγ, imidazole (20 mM) was added to the buffer to reduce nonspecific binding. Affinity-purified material was boiled in protein loading buffer and directly subjected to SDS-PAGE.

Kinase assays

Protein kinase assays using purified His-PI3Kγ proteins and GST-PKCα protein were carried out as described in Stoyanova et al. at 30 °C [START_REF] Stoyanova | Lipid kinase and protein kinase activities of G-protein-coupled phosphoinositide 3-kinase gamma: structure-activity analysis and interactions with wortmannin[END_REF]. As indicated, TPA (300 nM), BIM (100 nM), wortmannin (100 nM), or liposomes were added to the reaction mixture. Lipids (100 mM total lipid) were composed of diacylglycerol:phosphatidylinositol-3,4,5triphosphate:phosphatidylserine:phosphatidylcholine (5:10:40:45 molecular ratio). Specific PKCα activity was determined using calf thymus histone H1 or p47phox as a substrate (total amount, 0.1 μg/ml) in presence of 5 μCi [γ-32 P]-ATP and incubating at 30 °C. In vivo activity of PKCα was determined from freshly prepared neutrophiles. PKCα was immunoprecipitated from cell lysates of 3 x 10 7 cells and subsequently used for kinase assays. Reaction mixtures (25 μl) were subjected to SDS-PAGE on 12.5 % PAA gels. After electrophoresis, the gels A c c e p t e d M a n u s c r i p t were washed three times to remove unincorporated ATP and subsequently exposed to Pimage analysis. The amount of incorporated 32 P was quantified using a phospho-imaging system (FLA500, Fuji, Raytest) and AIDA image analysis software. Differences were considered significant when P < 0.05.

Mass spectrometric analysis

Protein gels were stained with Coomassie brillant blue R250 (Sigma) and appropriate gel sections were subjected to in-gel digestion with trypsin as described by Shevchenko et al. [START_REF] Shevchenko | In-gel digestion for mass spectrometric characterization of proteins and proteomes[END_REF] with one modification: reduction and oxidation of thiol groups was performed with a mixture of 0.1 mM tributylphosphine (Sigma-Aldrich, Taufkirchen, Germany) and 0.82 mM 4vinylpyridine (Aldrich). The trypsin in-gel protein digests were analyzed with LC-ESI-MS equipment consisting of an Ettan micro LC-HPLC (GE Healthcare, Munich, Germany) online coupled to a Finnigan LTQ mass spectrometer (Thermo Electron Corporation, Erlangen, Germany). The HPLC was equipped with a Zorbax 300SB 5 µM, 5 × 0.3 mm trapping column and a Zorbax 300SB 5 µM, 150 × 0.075 mm separation column (Agilent, Böblingen, Germany). The separation of peptides on the HPLC occurred by applying a linear gradient running from 0% to 47% acetonitrile, followd by a stepwise elution with 84% acetontrile in 0.1% formic acid, each under control of Unicorn software (GE Healthcare). The LTQ was operated under control of Xcalibur 1.4 software (Thermo Electron Corporation, Erlangen, Germany). For processing of the CID spectra (MS/MS-spectra) and final protein identification, BioWorks 3.2 software (Thermo Electron Corp., Erlangen, Germany) and the NCBI human protein database were used. Site-specific phosphorylation analysis was performed by scanning pSer and pThr for loss of phosphoric acid (neutral loss of 98) in the respective MS/MS-spectra.

Preparation of neutrophils

Human neutrophils were isolated from peripheral blood from healthy volunteers. The erythrocytes were removed by dextran sedimentation, and the samples were then loaded on a Histopaque gradient to exclude lymphocytes, monocytes, and platelets. Any remaining erythrocytes were removed by hypotonic lysis in distilled water. Finally, the cells were washed twice with PBS and resuspended in the appropriate buffers. Trypan blue staining revealed that at least 95% of the cells were neutrophils with greater then 95% viability.

Neutrophil stimulation and treatment with inhibitors

Freshly prepared neutrophils were incubated at 37 °C in resuspension buffer (30 mM Hepes, 110 mM NaCl, 10 mM KCl, 1 mM MgCl 2 , 1.5 mM CaCl 2 , 10% glycerol, and 0.5 mg/ml BSA).

If indicated, inhibitors (10 µM AS605240 and 1 µM BIM) were added 30 min before stimulation. Cells were stimulated with 1 µM fMLP for the indicated time periods at 37 °C and stopped by rapid, short centrifugation at 4 °C.

Preparation of membrane and cytosolic fractions

Human neutrophils were isolated as described, aliqoted (3 x 10 7 cells) in resuspension buffer and stimulated. Cells were then rebuffered in fractionation buffer (25 mM Hepes, 25 mM βglycerophosphat, 2 mM EDTA, 0.5 mg/ml BSA, 1 mM Na3VO4, 1 mM PMSF, 21 µg/ml Aprotinin, 5 µg/ml Leupeptin, 5 µg/ml Pepstatin A, 5 µg/ml Benzamidine, 12 µg/ml AEBSF).

Cells were broken up with an ice cold Dounce homogenicer. After mild centrifugation (5 min, 500 g) to remove unbroken cells and the nuclei the supernatant was ultracentrifuged at 100000 g for 45 minutes at 4°C. The pellet representing the membrane fraction and the supernatant representing the cytosolic fraction were resuspended in protein sample buffer.

Immunoprecipitation

Neutrophils were lysed in ice cold lysis buffer (20 mM Tris-HCl pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 0.5% NP40, 10% (v/v) glycerol, 0.5 mg/ml BSA, 1 mM Na 3 VO 4 , 1 mM PMSF, 21 µg/ml aprotinin, 12 µg/ml AEBSF, 5 µg/ml leupeptin, 5 µg/ml pepstatin A, and 5 µg/ml benzamidine). A cleared cell lysate was received by rapid centrifugation for 15 min at 4 °C. For immunoprecipitation, the lysates was preincubated with the appropriate antibodies for 2 h, before protein A sepharose beads were added for another 2 h. Finally, the beads were washed three times in ice-cold lysis buffer.

Immunoblotting

Equal amounts of proteins were separated by SDS-PAGE via 10% polyacrylamide gels and subsequently transferred to PVDF (polyvinylidene fluoride) membranes. Blots were incubated in NET-G buffer (Tris-HCl pH 8.0, 10 mM NaCl, 150 mM EDTA, 5 mM Tween-20, 0.05% (v/v) ; and 0.05 % (g/v) gelatine for 1 h. Immunoblotting was performed using the indicated primary antibodies in NET-G buffer. After subsequent incubation with horseradish peroxidase-conjugated secondary antibody, the blots were quantitatively assayed using Western Lightning chemiluminescence detection (Perkin-Elmer Life Sciences, Boston, USA) and a LAS3000 CCD camera (Fujifilm, Düsseldorf, Germany). Densitometric analysis of specific bands was used for the statistical analysis.

ROS production in neutrophils

Production of reactive oxygen species was monitored using lucigenin-dependent chemiluminescence. Neutrophils were resuspended to a density of 1 x 10 6 cells/ml, containing 200 μM lucigenin. Measurements were performed in triplicate at 37 °C [START_REF] Dichmann | Adenosine triphosphate-induced oxygen radical production and CD11b up-regulation: Ca(++) mobilization and actin reorganization in human eosinophils[END_REF].

RESULTS

PI3Kγ associates with PKCα

Using a monoclonal antibody specific to the p110 catalytic subunit of PI3Kγ [START_REF] Leopoldt | Gbetagamma stimulates phosphoinositide 3-kinase-gamma by direct interaction with two domains of the catalytic p110 subunit[END_REF], we initially produced immunoprecipitates from different cell types. Analyses of these precipitates by mass spectroscopy revealed numerous p110γ cell-specific interaction partners. The PI3Kγ immunoprecipitates obtained from human neutrophils were separated via SDS-PAGE.

Copurified proteins were subsequently trypsin digested and identified via mass spectrometry.

In the 75 kDa molecular weight range, we identified peptides that originated from PKCα. In addition, we found indication for association of matrix metalloproteinase MMP 9, HSP70, and alpha subunit of heterotrimeric Gi proteins to the immunoprecipitated complex. To validate the interaction of PI3Kγ with PKCα, we analyzed immunoprecipitates of PI3Kγ by western blotting for the presence of PKCα (Fig. 1A). These data revealed that PKCα was a specific interaction partner of PI3Kγ in human neutrophils.

In order to validate the interaction of PI3Kγ and PKCα in vitro, identical amounts of His epitope-labeled recombinant PI3Kγ were incubated with GST epitope-labeled PKCα.

Purification of the tagged proteins was carried out using Ni-NTA agarose beads for His-PI3Kγ and glutathione sepharose beads for GST-PKCα. As a control for nonspecific binding to the column material, identical reaction mixtures were prepared excluding the corresponding bait proteins. While in the His-PI3Kγ pull-down experiment PKCα was efficiently copurified, in the absence of His-PI3Kγ, only traces of GST-PKCα bound to the Ni-NTA agarose column. On the other hand, glutathione beads copurified His-PI3Kγ via GST-PKCα. Only traces of His-PI3Kγ could be detected if PKCα was omitted (Fig. 1B), or if the GST epitope alone was used as a bait protein (data not shown). In another experiment, His-PI3Kγ and GST-PKCα were coexpressed in Sf9 insect cells. After 72 h, the cells were lysed and the cleared lysates were applied to GST-or His-specific pull-down assays. Essentially, the interaction profiles obtained were identical to those resulting from the biochemical studies described above

PI3Kγ phosphorylates and stimulates PKCα in vitro

The direct binding of PI3Kγ to PKCα suggests that trans-phosphorylation reactions may occur between these signalling proteins. Both PKCα and PI3Kγ have been shown to exhibit protein kinase activities. Incubation of the recombinant proteins in the presence of [γ-32 P]-ATP did not reveal PKCα-mediated phosphorylation of PI3Kγ (data not shown). In contrast, pronounced phosphorylation of PKCα by PI3Kγ could be demonstrated (Fig. 2A). To exclude auto-phoshorylation of the enzyme, PKCα activity was suppressed using BIM as an inhibitor of classical PKC species. Unaltered incorporation of 32 P confirmed PI3Kγ-mediated phosphorylation of the protein. Impaired phosphorylation of PKCα in the presence of the enzymatically inactive variant PI3Kγ-KR or the PI3Kγ inhibitor wortmannin verified PI3Kγmediated trans-phosphorylation of PKCα (Fig. 2A).

Activation of PKCα by other protein kinases, such as PDK1, has been described as involving the successive phosphorylation of amino acid residues Thr497, Thr638, and Ser657 in the kinase domain [16,[START_REF] Parekh | Multiple pathways control protein kinase C phosphorylation[END_REF]. These target sites were analyzed after PI3Kγ-mediated phosphorylation. Both recombinant proteins were incubated in the presence of nonradioactive ATP, and PKCα was repurified using SDS-PAGE, extracted from the PAA gel, trypsinized, and subjected to mass spectrometry. While the kinase reaction in the absence of PI3Kγ or ATP did not result in phosphorylated amino acid residues, coincubation with PI3Kγ revealed peptides with specific mass shifts, suggesting the specific transphosphorylation of PKCα. Phosphorylation of Ser or Thr could be identified at 3 different regions of the protein (Suppl. Fig. 2). The most pronounced phosphorylation (residues Thr48, Ser51, and/or Thr54) according to number of identified peptides and the score of these 2). These data confirm the serine-threonine kinase activities of PI3Kγ, and explore, for the first time to our knowledge, a specific site that is trans-phosphorylated by the enzyme.

The putative PI3Kγ-specific phosphorylation site of PKCα has some similarities to the autophosphorylation site of PI3Kγ, which was recently identifyed by Czupalla et al. [START_REF] Czupalla | Identification and Characterization of the Autophosphorylation Sites of Phosphoinositide 3-Kinase Isoforms β and γ[END_REF]. As shown in Fig. 2B there is distinct homology between amino acids 36-61 of PKCα and the autophosphorylation site located at the C terminus of PI3Kγ. The resemblance between these two target sites for PI3Kγ protein kinase activity prompted us to search for further homology patterns in proteins that have been shown to act as substrates for PI3Kγ protein kinase. In recent reports, PI3Kγ-specific phosphorylations of MEK [START_REF] Bondev | Differential regulation of lipid and protein kinase activities of phosphoinositide 3-kinase gamma in vitro[END_REF][START_REF] Bondeva | Bifurcation of lipid and protein kinase signals of PI3Kgamma to the protein kinases PKB and MAPK[END_REF][START_REF] Bondev | Regulation of phosphoinositide 3-kinase γ protein kinase activity in vitro and in COS-7 cells[END_REF], p101 [START_REF] Bondev | Regulation of phosphoinositide 3-kinase γ protein kinase activity in vitro and in COS-7 cells[END_REF], Ras [START_REF] Foukas | eIF4E binding protein 1 and H-Ras are novel substrates for the protein kinase activity of class-I phosphoinositide 3-kinase[END_REF], 4EBP1 [START_REF] Foukas | eIF4E binding protein 1 and H-Ras are novel substrates for the protein kinase activity of class-I phosphoinositide 3-kinase[END_REF], and tropomyosin [START_REF] Prasad | Protein kinase activity of phosphoinositide 3-kinase regulates β-adrenergic receptor endocytosis[END_REF] were detected using recombinant proteins and cellular systems. Indeed, all of these PI3Kγ protein kinase substrates exhibit peptide sequences with different degrees of similarity to the PI3Kγ auto-phosphorylation site [START_REF] Czupalla | Identification and Characterization of the Autophosphorylation Sites of Phosphoinositide 3-Kinase Isoforms β and γ[END_REF] and the transphosphorylation site in PKCα that was identified in the present report (Fig. 2B).

After elucidating the binding and trans-phosphorylation of PKCα by PI3Kγ, we investigated the effects of the interaction of both signalling proteins on the specific activity of PKCα in vitro. The assay was carried out using histone H1 as a substrate, and PKCα kinase activity was monitored by incorporation of [γ-32 P]-ATP-derived phosphate. While PI3Kγ alone did not phosphorylate histone H1, 32 P was efficiently incorporated in the presence of PKCα. This reaction could be specifically inhibited by addition of the PKC inhibitor BIM (Fig. 2C). We next explored the effects of PI3Kγ on PKCα activity. As shown in Fig. 2D, a pronounced stimulation of substrate phosphorylation was observed in the presence of PI3Kγ in a time range up to 20 min.

We further explored the NADPH oxidase components p47phox and p67phox as substrates in PKCα kinase assays. While p47phox was phosphorylated with similar efficiency to histone H1 (Fig. 2E), phosphorylation of p67phox was only marginal (data not shown). Preincubation of PKCα with PI3Kγ induced only weak stimulation of p47phox phosphorylation compared with the kinase reaction in the absence of PI3Kγ.

fMLP induces PI3Kγ and PKCα complex formation in human neutrophils

To gain insight into the physiological role of the interactions between PI3Kγ and PKCα, we studied the complex formation of these proteins following stimulation of neutrophils with fMLP. PI3Kγ was immunoprecipitated at the indicated time points, and coprecipitated PKCα was assayed by western blotting (Fig. 3). The addition of fMLP markedly increased PKCα association with PI3Kγ in a transient manner (Fig. 3A). In unstimulated neutrophils we did not observe any alteration in PKCα binding to the immunoprecipitate in the given period (Fig. 3B). Remarkably, the p47phox subunit of NADH oxidase associated with identical kinetics to this complex (Fig. 3A).

It has been demonstrated in different cellular systems that activation of both PI3Kγ and PKCα causes the association of these proteins with the plasma membrane [START_REF] Brock | Roles of G beta gamma in membrane recruitment and activation of p110 gamma/p101 phosphoinositide 3-kinase gamma[END_REF]25]. To correlate this process with the fMLP-induced interaction of the signalling proteins, we analyzed the kinetics of membrane association after fMLP stimulation. The data shown in Fig. 3C demonstrate that the distribution of both proteins was altered in response to fMLP stimulation. While PI3Kγ segregated to the membrane fraction, peaking after 120 s of stimulation, most PKCα molecules associated with membranes 60 s after fMLP treatment. These data reveal that the formation of fMLP-mediated PI3Kγ-PKCα complexes is a process paralleling the membrane association of these proteins. To test the involvement of PI3Kγ enzymatic activity in the fMLP-dependent stimulation of PKCα, PKCα activity was analyzed in cells treated with the specific PI3Kγ inhibitor AS605240 [START_REF] Camps | Blockade of PI3Kgamma suppresses joint inflammation and damage in mouse models of rheumatoid arthritis[END_REF]. While the untreated cells corroborated the activity profile shown in Fig 4A ,   PKCα immunoprecipitated from neutrophils after treatment with AS605240 exhibited notably impaired PKC activity (Fig. 4B). This observation points to a strict dependence of PKCα activity on functionally active PI3Kγ. To exclude nonspecific effects of AS605240 on PKCα kinase activity, recombinant PKCα was preincubated with PI3Kγ inhibitors and subsequently assayed for its protein kinase activity in vitro. While treatment with the PKC inhibitor BIM completely abolished PKCα activity, neither wortmannin nor AS605240 significantly affected the enzymatic activity of PKCα (Fig. 4C).

Enzymatic activities of PI3Kγ and PKCα control fMLP-dependent ROS production in neutrophils

A role for PI3Kγ in the production of ROS was elucidated in detail using specific inhibitors and PI3Kγ-deficient mice [START_REF] Condliffe | Sequential activation of class IB and class IA PI3K is important for the primed respiratory burst of human but not murine neutrophils[END_REF]. To study the involvement of PKCα in the control of oxidative

DISCUSSION

There is an increasing amount of evidence indicating that the differential interactions of PI3Kγ with other signalling proteins depend upon the cell type [START_REF] Stephens | The G beta gamma sensitivity of a PI3K is dependent upon a tightly associated adaptor, p101[END_REF]25,[START_REF] Patrucco | PI3Kgamma modulates the cardiac response to chronic pressure overload by distinct kinase-dependent andindependent effects[END_REF]. Building upon these initial findings, we searched for PI3Kγ binding partners in different human cell types. Mass spectroscopy of PI3Kγ immunoprecipitates obtained from human neutrophils revealed PKCα as a novel interaction partner of PI3Kγ. Analysis of PI3Kγ binding to PKCα in vitro clearly indicated the direct and specific association of these two signalling proteins. In addition, we demonstrated PKCα phosphorylation by PI3Kγ protein kinase activity. These results expand the list of substrates known to be phosphorylated by PI3Kγ protein kinase activity under in vitro conditions. In addition to substantial auto-phosphorylation activity, the current list includes trans-phosphorylation of MEK, p101, H-Ras, and tropomyosin [10;19-23]. In spite of preliminary data hinting at a regulatory function for the PI3Kγ protein kinase in cellular systems [START_REF] Bondeva | Bifurcation of lipid and protein kinase signals of PI3Kgamma to the protein kinases PKB and MAPK[END_REF], compelling evidence of a physiological role for this enzymatic activity is still missing.

Serine and threonine residues phosphorylated by PI3Kγ have been identified in three regions of PKCα. Interestingly, the most pronounced phosphorylation was found in a PKCα peptide localized the second C1 domain of the DAG-binding domain of the protein. It shows significant homology to the C-terminal end of PI3Kγ, which has been identified as the autophosphorylation site [START_REF] Czupalla | Identification and Characterization of the Autophosphorylation Sites of Phosphoinositide 3-Kinase Isoforms β and γ[END_REF]. Further similarities of this putative trans-phosphorylation site in PKCα have been identified to potential phosphorylation sites in other proteins in MEK, p101, Ras, 4EBP1, and tropomyosin. All of these intracellular proteins have been found to act as substrates for PI3Kγ protein kinase activity [START_REF] Bondev | Differential regulation of lipid and protein kinase activities of phosphoinositide 3-kinase gamma in vitro[END_REF][START_REF] Bondeva | Bifurcation of lipid and protein kinase signals of PI3Kgamma to the protein kinases PKB and MAPK[END_REF][START_REF] Bondev | Regulation of phosphoinositide 3-kinase γ protein kinase activity in vitro and in COS-7 cells[END_REF][START_REF] Foukas | eIF4E binding protein 1 and H-Ras are novel substrates for the protein kinase activity of class-I phosphoinositide 3-kinase[END_REF][START_REF] Prasad | Protein kinase activity of phosphoinositide 3-kinase regulates β-adrenergic receptor endocytosis[END_REF] This conclusion may be in accordance with recent findings by Suire et al. [START_REF] Suire | p84, a new Gbetagamma-activated regulatory subunit of the type IB phosphoinositide 3-kinase p110gamma[END_REF]. These authors initially investigated the fMLP-dependent synthesis of PIP 3 (the product of PI3Kγ lipid kinase activity) in p101-deficient mouse neutrophils. Compared with wild-type neutrophils, they observed a massive decrease in fMLP-sensitive PIP 3 synthesis, indicating an essential role for the p101 adapter protein in the control of PI3Kγ lipid kinase activity by G protein-coupled receptors. As a second parameter, Suire et al. also performed comparative analysis of fMLPdependent oxidative burst in wild-type and p101-deficient neutrophils [START_REF] Suire | p84, a new Gbetagamma-activated regulatory subunit of the type IB phosphoinositide 3-kinase p110gamma[END_REF]. Surprisingly, they did not observe any significant differences in ROS production indicating a marginal role for p101 in fMLP/PI3Kγ-dependent ROS production. Our investigations suggest the involvement of PI3Kγ protein kinase activity in the control of NADH oxidase activity. They also provide evidence for a consecutive signalling path: fMLP→fMLP receptor→PI3Kγ protein kinase→PKCα→p47phox→NADH-oxidase, which does not involve p101/p110γ-dependent PIP 3 production.

In fMLP-induced signalling pathways, PI3Kγ may act as a bifurcation point. Whereas PIP 3 produced by PI3Kγ lipid kinase activity controls the migration induced by chemoattractants, PI3Kγ protein kinase activity may affect the oxidative burst via phosphorylation and concomitant stimulation of PKCα. Hence, these findings provide additional evidence for the differential cell-specific regulatory functions of PI3Kγ. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

FIGURES FIGURE 1: PI3Kγ binds PKCα

(A) PI3Kγ and PKCα associate in human neutrophils. Freshly isolated human neutrophils were aliquoted (3-4 x 10 7 cells) and treated with 1 µM fMLP. A whole-cell lysate was prepared and equal amounts of protein were immunoprecipitated with PI3Kγ antibody and immunoblotted with an antibody for PKCα. Antibodies against the above-mentioned proteins were precipitated with Protein A agarose beads, extensively washed, subjected to SDS-PAGE, and blotted. Bait and cobound proteins were analyzed immunologically using specific antibodies (lane 1). To show nonspecific binding, primary antibodies were replaced by nonspecific IgG in a parallel sample (lane 2).

(B) PI3Kγ and PKCα associate in vitro. Purified recombinant His-PI3Kγ and GST-PKCα protein (1μg each) were incubated in 50 μl of protein kinase buffer for 30 min at room temperature. Complex formation was analyzed using 50 μl of glutathione beads to purify GST-PKCα (PKCα pull down) or 50 μl of Ni-NTA beads to purify His-PI3Kγ (PI3Kγ pull down). After further incubation for 30 min, extensively washed beads were subjected to SDS-PAGE and blotted. Bait and cobound proteins were analyzed immunologically using specific antibodies. To show nonspecific binding, bait proteins were omitted from a parallel sample as indicated. Representative data are shown of experiments that were repeated several times. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

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  Biochemical Journal Immediate Publication. Published on 05 Nov 2008 as manuscript BJ20081268 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081268 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

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  Biochemical Journal Immediate Publication. Published on 05 Nov 2008 as manuscript BJ20081268 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081268 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

10 Biochemical

 10 Journal Immediate Publication. Published on 05 Nov 2008 as manuscript BJ20081268 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081268 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society (Suppl. Fig. 1). These results indicate that the association of PI3Kγ with PKCα is direct and specific.

  peptides was identified in peptide QPTFCSHCTDFIWGFGK (amino acid residues 46-62), 11 Biochemical Journal Immediate Publication. Published on 05 Nov 2008 as manuscript BJ20081268 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081268 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society which is localized in the second C1 domain of the DAG-binding domain of PKCα. Two less abundant phosphorylation clusters have been found in the catalytic domain of the protein (Suppl. Fig.

  fMLP induces PI3Kγ-mediated activation of PKCα 13 Biochemical Journal Immediate Publication. Published on 05 Nov 2008 as manuscript BJ20081268 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081268 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society We next explored the functional relevance of the fMLP-induced association of PI3Kγ, PKCα, and p47phox. Classical PKC species have been shown to stimulate NADH oxidase activity via phosphorylation of p47phox [26]. However, the signalling reactions affecting PKC activity in neutrophils are poorly understood. Here, we first analyzed specific PKCα activity in response to fMLP stimulation. Neutrophils were stimulated for the indicated time points, aliquots were withdrawn and lysed, and PKCα was immunoprecipitated. The protein kinase activity of PKCα was analyzed by protein kinase assay using histone H1 as a substrate for 32 P incorporation. As shown in Fig. 4A, specific PKCα kinase activity was markedly enhanced in response to neutrophil stimulation by fMLP. Maximum activity was reached 30 sec after fMLP addition, which correlates with the onset of PI3Kγ/PKCα/p47phox complex formation.

14 Biochemical

 14 Journal Immediate Publication. Published on 05 Nov 2008 as manuscript BJ20081268 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081268A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  . The homology of the PKCα phosphorylation site to other known substrates suggests a widespread functional relevance of trans-phosphorylation reactions catalyzed by PI3Kγ protein kinase activity. In our hands, the in vitro interactions of PKCα with PI3Kγ induced a marked increase in PKCα activity. Part of the stimulatory effect was observed after addition of a kinase-negative mutant of PI3Kγ, supporting the hypothesis that association with PI3Kγ directly augments 16 Biochemical Journal Immediate Publication. Published on 05 Nov 2008 as manuscript BJ20081268 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081268 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society PKCα activity. Together with our in vitro investigations, these data provide evidence of the direct and specific binding of PI3Kγ to PKCα, and identify PKCα as a potential substrate, whose enzymatic activity can be stimulated by phosphorylation triggered by PI3Kγ protein kinase activity. It can not be excluded that immunoprecipitated PKCα contained additional proteins affecting total kinase activity, but since 32 P incorporation in the PKCα substrate histone H1 was drastically abrogated in presence of PI3Kγ selective inhibitor AS605240, a specific PI3Kγ mediated stimulation of PKCα activity can be concluded. To explore the physiological function of PI3Kγ and PKCα interactions in human neutrophils, we stimulated the cells with the bacterial peptide fMLP. fMLP has been shown to affect both directed neutrophil migration and ROS production. Comparative investigations of neutrophils from PI3Kγ-deficient mice have demonstrated that both fMLP-dependent processes are mediated by PI3Kγ [4-6]. PKCα has been described as a mediator of fMLP-induced oxidative burst, and the regulatory subunit of NADH oxidase p47phox was identified as an important target of PKCα activity [26]. The signalling pathway linking fMLP, the fMLP receptor, and PKCα is poorly understood. Thus, our experimental data regarding the fMLP-dependent association of PKCα with PI3Kγ provide a first clue for signalling reactions that induce stimulation of PKCα activity by fMLP. The fMLP-induced interaction between PI3Kγ and PKCα and the translocation of both proteins to the plasma membrane may contribute to the stimulation of PKCα activity observed after agonist addition. The PKCα substrate p47phox joins the complex with similar kinetics, possibly facilitating phosphorylation of this subunit and subsequent stimulation of NADH oxidase. As shown by our inhibition experiments, PI3Kγ enzymatic activity is an essential mediator of fMLP-dependent stimulation of PKCα activity in neutrophils. Due to the lack of specific inhibitors, these results do not allow for a definitive correlation of the blocking effect of AS605240 on PI3Kγ lipid or protein kinase activities. However, our findings showing PKCα phosphorylation by PI3Kγ protein kinase in vitro, and the concomitant increase of PKCα activity, provide evidence for a lipid kinase-independent signalling reaction. Biochemical Journal Immediate Publication. Published on 05 Nov 2008 as manuscript BJ20081268 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081268 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Biochemical

  Journal Immediate Publication. Published on 05 Nov 2008 as manuscript BJ20081268 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081268

FIGURE 2 :

 2 FIGURE 2: PI3Kγ phosphorylates PKCα and stimulates its activity

FIGURE 3 :

 3 FIGURE 3: fMLP-dependent interaction of PI3Kγ and PKCα in human neutrophils

FIGURE 4 :

 4 FIGURE 4: Stimulation of human neutrophils induces PI3Kγ-mediated activation of

FIGURE 5 :Figure 1 Biochemical

 51 FIGURE 5: PI3Kγ-and PKCα-dependent ROS production in human neutrophilsFreshly isolated human neutrophils were isolated and diluted to 1 x 10 6 cells in Hanks

Figure 2

 2 Figure 2

  Figure 2

  Figure 2

  Figure 2

+C 4 BC

 4 Figure 3

  Figure 5

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  Biochemical Journal Immediate Publication. Published on 05 Nov 2008 as manuscript BJ20081268 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081268

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society

ACKNOWLEDGEMENTS

This work was supported by the Deutsche Forschungsgemeinschaft SFB 604. KL and JPM contributed equally to this work.