Rapamycin-sensitive phosphorylation of PKC on a carboxy-terminal site by an atypical PKC complex

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Research Paper

Rapamycin-sensitive phosphorylation of PKC on a carboxyterminal site by an atypical PKC complex W.H. Ziegler*†, D.B. Parekh*, J.A. Le Good*, R.D.H. Whelan*, J.J. Kelly*, M. Frech‡, B.A. Hemmings‡ and P.J. Parker* Background: The protein kinase C (PKC) family has been implicated in the control of many cellular functions. Although PKC isotypes are characterized by their allosteric activation, phosphorylation also plays a key role in controlling activity. In classical PKC isotypes, one of the three critical sites is a carboxyterminal hydrophobic site also conserved in other AGC kinase subfamily members. Although this site is crucial to the control of this class of enzymes, the upstream kinase(s) has not been identified.

Addresses: *Protein Phosphorylation Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, UK. ‡Friedrich Miescher Institute, Maulbeerstrasse 66, PO Box 2543, CH4002 Basel, Switzerland.

Results: A membrane-associated kinase activity that phosphorylates the hydrophobic site in PKCα was detected. This activity was suppressed when cells were pretreated with the immunosuppresant drug rapamycin or the phosphoinositide (PI) 3-kinase inhibitor LY294002. These pretreatments also blocked specifically the serum-induced phosphorylation of the hydrophobic site in PKCδ in vivo. The most highly purified hydrophobic site kinase preparations (~10,000-fold) reacted with antibodies to PKCζ/ι. Consistent with this, rapamycin and LY294002 reduced the recovery of PKCζ from the membrane fraction of transfected cells. An activated mutant of PKCζ, but not wild-type PKCζ, induced phosphorylation of the PKCδ hydrophobic site in a rapamycinindependent manner, whereas a kinase-dead PKCζ mutant suppressed this serum-induced phosphorylation. The immunopurified, activated mutant of PKCζ could phosphorylate the PKCδ hydrophobic site in vitro, whereas wild-type PKCζ could not.

Correspondence: Peter J. Parker E-mail: [email protected]

Present address: †Technical University Braunschweig, Zoological Institut, Spielmannstrasse 7, D-38106 Braunschweig, Germany.

Received: 27 January 1999 Revised: 17 March 1999 Accepted: 9 April 1999 Published: 6 May 1999 Current Biology 1999, 9:522–529 http://biomednet.com/elecref/0960982200900522 © Elsevier Science Ltd ISSN 0960-9822

Conclusions: PKCζ is identified as a component of the upstream kinase responsible for the phosphorylation of the PKCδ hydrophobic site in vitro and in vivo. PKCζ can therefore control the phosphorylation of this PKCδ site, antagonizing a rapamycin-sensitive pathway.

Background Classical and novel protein kinase C (PKC) isotypes are direct targets for the second messenger diacylglycerol [1–3]. Allosteric activation by diacylglygcerol, together with the activation by the phorbol ester class of tumour promoters, has contributed to the view that PKC isotypes are involved in many signalling responses in cells. Recent studies have defined a further level of regulation involving phosphorylation. The cPKC isotypes have been shown to be phosphorylated on at least three sites in vivo including a conserved carboxy-terminal hydrophobic site Phe–X–X–Phe–Ser/Thr–Tyr/Phe (where X is any amino acid) [4–6]. In the PKCα protein this hydrophobic site (containing Ser657) has been shown to be rate-limiting for the accumulation of the active (latent), fully phosphorylated kinase [6]. A number of related members of the AGC subfamily of protein kinases (where AGC represents cyclic AMP-dependent protein kinase, cyclic GMPdependent protein kinase and protein kinase C family) [7] have been shown to be phosphorylated at a similar

hydrophobic site carboxy-terminal to their kinase domains. Notably, although certain related proteins including PKCζ and PKCι retain the hydrophobic motif, they have an acidic residue in place of the serine/threonine residue [8,9]. In addition to the carboxy-terminal hydrophobic site, a number of AGC kinases have phosphorylation sites in their activation loops that are also conserved in PKC isotypes. An activation loop kinase, 3′ phosphoinositide-dependent protein kinase (PDK1), has been identified for protein kinase B [10,11] and was recently shown to act also upon p70 S6 kinase (p70) [12,13] as well as PKCδ and PKCζ [14,15]. PDK1 does not phosphorylate the hydrophobic carboxy-terminal site, however, and to date the enzyme that is responsible for the phosphorylation of this motif has not been found. In p70, phosphorylation of this hydrophobic site is critical for the activity of p70 and it is also the site most sensitive to the potent immunosuppressant drug rapamycin [16]. Elucidation of the rapamycin-dependent signalling pathway that controls p70, and that operates on other AGC kinase

Research Paper PKC phosphorylation by an atypical PKC complex Ziegler et al.

subfamily proteins, requires the identification of the proximal protein kinase activity. Here we show that phosphorylation of this hydrophobic site in PKCδ is a rapamycin-sensitive process and identify the atypical PKC isotype PKCζ as a component of the kinase responsible for this phosphorylation of PKCδ.

Results In PKC isotypes, the hydrophobic phosphorylation site is located in the carboxy-terminal variable 5 domain (V5 domain). To identify a V5 kinase activity in vitro, a V5 domain fragment of PKCα (as a glutathione-S-transferase fusion termed GST–V5α) was used as a substrate to screen fractionated cell extracts. The specificity of phosphorylation

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was monitored by use of antisera selective for the phosphorylated Ser657 residue (Ser657(P)) site, as well as use of mutant proteins containing serine/threonine to alanine mutations at either the Thr638 or Ser657 phosphorylation sites (T638A or S657A, respectively), or at both sites. It is evident that the kinase activity has some selectivity for the Ser657 site and that the Ser657-phosphorylated protein migrates more slowly than the substrate does (Figure 1a). A membrane-associated activity was identified in all cells types investigated (COS-7, U937, HeLa and Jurkat) as well as in rat brain tissue. In each case the activities displayed similar elution properties on anion exchange chromatography (for example, Figure 1b). On the basis of 32P-orthophosphate incorporation into the slower migrating GST–V5α

Figure 1 (a)

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Identification of a Ser657 V5 kinase activity. (a) Phosphorylation-site specificities of V5α kinase fractions were tested in vitro using GST–V5α constructs. GST–V5α contains two of the characterized in vivo PKCα phosphorylation sites, Thr638 and Ser657. The corresponding alanine substitution-mutants of GST–V5α, GST–V5α(T638A) and GST–V5α(S657A), confirm the site specificity of kinase fractions. GST–V5α(T638A) is phosphorylated like the wildtype protein (WT), while GST–V5α(S657A) and the double mutant GST–V5α(T638A,S657A), represented by AA in the figure, are not significantly phosphorylated. The Ser657(P)-specific bands on the immunoblot (black arrow, upper panel) correspond to 32P signals on the autoradiograph (black arrow, lower panel). The background immunoreactivity (arrowhead) does not alter in any of the mutants; this corresponds to the bulk of the unphosphorylated substrate, coinciding with the position of the Coomassie-blue-stained protein (data not shown). (b) MonoQ elution profile of a COS-7 cell membrane extract

(the first chromatographic step of kinase purification). The eluted V5 kinase activity is shown; no activity is detected in the unbound fraction (data not shown). The autoradiograph (inset) shows the 32P incorporation into GST–V5α by the eluted fractions, the arrow indicating the Ser657-phosphorylated GST–V5α and the arrowhead indicating the Ser657-nonphosphorylated protein. The asterisk indicates a non-specific phosphoprotein that is present at this first step of purification. (c) Bacterially expressed, full-length PKCδ is phosphorylated by the V5α kinase preparation on the Ser662 site as shown by the phosphorylation-site-specific antiserum (the specificity of this antiserum is illustrated in Figure 3). This bacterially expressed GST–PKCδ is autophosphorylated on Ser643 as isolated. On incubation in vitro, it will further autophosphorylate on an undefined site but not on Ser662. The V5 kinase phosphorylates Ser662. Phorbol myristate acetate (PMA) is an allosteric activator of PKCδ.

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Figure 2

equivalent sites on both PKCα (V5 domain) and PKCδ (intact protein).

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Loss of membrane-associated V5 kinase activity in response to rapamycin and LY294002. U937 cells were pretreated for 20 min with rapamycin (20 nM) or LY294002 (10 µM), or were left untreated (control). V5 kinase activity was purified and assayed with GST–V5α as substrate (see Materials and methods section). A representative western blot of GST–V5α phosphorylation is shown in the upper panel. The mean activities (± standard deviations) for three independent experiments are indicated in the lower panel.

domain and the immunodetection with the Ser657(P)-specific antibody, this membrane-derived activity was further purified by sequential chromatography on MonoQ, MonoS and MiniQ columns (see Materials and methods section). Although the purification yielded an overall enrichment of around 10,000-fold relative to the crude extract, the protein was not pure as judged by Coomassie blue staining following polyacrylamide gel electrophoresis (this estimation of the enrichment is a minimum estimate because there are likely to be contaminating kinase activities at the earlier steps in the purification). To investigate the specificity of this highly purified activity, PKCδ was tested as a substrate. Unphosphorylated PKCδ was purified as a GST-fusion protein from bacteria. This recombinant protein has a low basal activity (~20 units/mg), is phosphorylated at Ser643 [17] (corresponding to the Thr638 site in PKCα) and autophosphorylates in vitro, but not on either of the sites Thr505 [14] or Thr662 (Figure 1c). Only in the presence of the V5 kinase does GST–PKCδ become phosphorylated at the carboxy-terminal hydrophobic site Ser662, as evidenced by immunoreaction with the site-specific antibody (Figure 1c). These results indicate that this V5 kinase preparation appears to have non-exclusive substrate specificity in vitro, because it phosphorylates the

Phosphorylation of the homologous hydrophobic site (Thr389) of p70 is sensitive to treatment of cells with rapamycin and phosphoinositide (PI) 3-kinase inhibitors, such as LY294002 ([18] and references therein). This precedent suggested that the V5 kinase activity itself might be sensitive to these inhibitors. Isolation of membranes from U937 cells pretreated with such inhibitors revealed that V5 kinase activity was substantially reduced compared with control cells that had not been pretreated with inhibitors (Figure 2). An assessment of PKC phosphorylation sensitivity to rapamycin has not been carried out previously. To determine the sensitivity of PKCδ phosphorylation at the Ser662 site in vivo, 293 cells were transfected with a plasmid containing PKCδ and were serum-starved. Phosphorylation of Ser662 was monitored with a site-specific antiserum that showed selective recognition of the phosphorylated Ser662 site and specific competition by the phosphopeptide antigen (Figure 3a,b). Under these serumstarved conditions a decrease in Ser662 PKCδ phosphorylation was observed (Figure 3b). Stimulation of starved cells with 10% serum induced the phosphorylation of Ser662 (Figure 3c). In the presence of rapamycin or LY294002, however, there was an inhibition of serum-induced Ser662 phosphorylation (Figure 3c,d). PKCδ, like p70 [16], therefore displays a rapamycin sensitivity in vivo, suggesting that aspects of the responses of cells to rapamycin treatment might be mediated through alteration in PKCδ function. To identify the kinase responsible and reconstitute the phosphorylation, direct protein sequence analysis of the V5 kinase preparation was performed. Mass spectrometry of tryptic fragments from the highly purified protein revealed that the major protein staining bands were heterogeneous and no known kinase or kinase-related sequence was identified. Because the kinase was below the levels of detection by sequence, we screened a number of kinase candidates by immunological methods. A commercial antibody (Santa Cruz) recognises both atypical PKC (aPKC) isotypes (ζ and ι) because it is specific for a peptide corresponding to residues 573–592 of PKCζ, which is almost entirely conserved in PKCι. This antibody identified an 80 kDa protein and a predicted PKCζ/ι kinase domain fragment in the most highly purified preparations. These immunoreactive bands were not coincident with the major staining proteins (Figure 4a), consistent with the fact that aPKC tryptic sequences were not identified. The related PKCα was not present in these preparations (data not shown). Consistent with the notion that PKCζ might contribute to this activity, we analysed the membrane-associated PKCζ in control untreated cells and in cells treated with LY294002 or rapamycin. As observed for the V5 kinase activity there was an inhibitor-dependent reduction in the recovery of PKCζ (Figure 4b).

Research Paper PKC phosphorylation by an atypical PKC complex Ziegler et al.

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Phosphorylation of PKCδ Ser662 is dependent on growth conditions and is inhibited by LY294002 (10 µM) and rapamycin (20 nM). (a) The specificity of the ‘FSY’-site-specific antiserum on Ser662 phosphorylation of PKCδ expressed in 293 cells cultured in the presence or absence of 10% foetal calf serum (FCS) is illustrated. Immunoreactivity is specifically eliminated by competition of antiserum with the phosphopeptide antigen (0.5 µg/ml); the dephosphopeptide (1 µg/ml) used in (b–d) suppresses non-specific immunoreaction with the dephosphorylated protein. (b) Ser662 phosphorylation (upper panel) of bacteriallyexpressed GST–PKCδ (negative control) and of PKCδ from serum-maintained 293 cells (10% FCS, + FCS) and after 24 h serum starvation (– FCS). The protein loading was assessed using a PKCδ carboxyl-terminusspecific antiserum (lower panel). (c) Ser662 phosphorylation of PKCδ (left panel) is induced by FCS treatment of serum-starved 293 cells and is optimal by 30 min after serum stimulation (no further increase is seen at 60 min; data not shown). The serum-induced phosphorylation is markedly reduced in cells pretreated for 30 min with LY294002 or

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in control and rapamycin- or LY294002pretreated cells and quantified as a function of PKCδ protein. The results are from four independent experiments (mean ± SD).

of its activated form removes the rapamycin sensitivity of both the basal and serum-induced responses. To further assess whether PKCζ could be responsible for PKCδ phosphorylation, immunoprecipitated PKCζ(T410E), was incubated in vitro with bacterially expressed PKCδ. Under these conditions the PKCδ Ser662 site was phosphorylated by the PKCζ(T410E) immunocomplex (Figure 5c). When immunoprecipitated wild-type PKCζ was assessed in the same assay it was unable to induce PKCδ phosphorylation at the Ser662 site, indicative of a requirement for activation of PKCζ (data not shown).

These observations indicated that aPKC might be a component of the PKC kinase, phosphorylating hydrophobic carboxy-terminal sites. To test this in intact cells we used PKCδ as a readout and examined the effects of an activated mutant of PKCζ that has threonine to glutamic acid mutations at the activation loop phosphorylation site (Thr410) and at the predicted autophosphorylation site (Thr560) [14]. When PKCδ is coexpressed with this PKCζ T410E/T560E mutant, PKCδ remained partially phosphorylated at the Ser662 hydrophobic site despite serum deprivation (Figure 5a). This effect was site specific as no equivalent phosphorylation was seen for the PKCδ activation loop site (Thr505). Upon stimulation with serum there was an increase in PKCδ Ser662 phosphorylation and this response is rapamycin insensitive (Figure 5a). Consistent with this insensitivity, in the presence of PKCζ T410E/T560E, the constitutive Ser662 phosphorylation was also not suppressed in the presence of rapamycin or LY294002 (data not shown). Despite the PKCζ T410E/T560E-dependent change in rapamycin sensitivity of the PKCδ Ser662 phosphorylation, serum-induced Thr505 phosphorylation remained sensitive to LY294002 (Figure 5a). To assess whether PKCζ might act on the serum-induced pathway, we determined the effect of a dominant-negative PKCζ mutant [19]. Coexpression of PKCδ with PKCζ (T410A) blocked the seruminduced phosphorylation of PKCδ Ser662 (Figure 5b).

These results demonstrate that an aPKC isotype is at least a component of a PKC V5 kinase, phosphorylating the Phe–X–X–Phe–Ser–Tyr motif. The initial evidence for this came from the isolation of a PKCα V5 kinase activity, which was shown to contain an aPKC antigen in the most highly purified preparations (~10,000-fold purification). The demonstration that immunopurified activated PKCζ phosphorylated PKCδ in vitro and promotes this phosphorylation upon coexpression in vivo confirms this assignment. It is also shown that the phosphorylation of this hydrophobic V5 domain site in PKCδ is controlled through a rapamycin-sensitive pathway, a property that is overcome by expression of the activated PKCζ mutant.

These results suggest that PKCζ can specifically effect PKCδ Ser662 phosphorylation and that the overexpression

The ability of coexpressed PKCζ(T410E,T560E) to increase the phosphorylation of the PKCδ Ser662 site in

Discussion

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Figure 4 (a)

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PKCδ The aPKC correlates with V5 kinase activity. (a) The most highly purified sample of the V5 kinase preparation was subjected to polyacrylamide gel electrophoresis and was stained with Coomassie blue to detect protein (left panel). The same sample was immunoblotted (right panel) with a PKCζ/ι antiserum (Santa Cruz) in the absence or presence of competing antigen (competition). The major immunoreactive bands are not coincident with the stained proteins and migrate at 80 kDa (PKCζ/ι) and 45 kDa (PKCζ/ι). Immunoreactivity is completely competed by the antigen. (b) A plasmid encoding PKCζ was transfected into 293 cells. Membranes were prepared from these transfected cells that had been pretreated with rapamycin (20 nM) or LY294002 (10 µM) or left untreated (control) as indicated. Protein was extracted and separated by SDS–PAGE and immunoblotted for aPKC, here transfected PKCζ. The recovery of PKCζ was quantified by scanning densitometry yielding recoveries of 62% (rapamycin) and 48% (LY294002) as shown in the graph. The immunoblot shown is one of two similar independent experiments.

vivo is site specific because the PKCδ activation loop Thr505 site is not affected by PKCζ(T410E,T560E) expression. The activated PKCζ mutant is able to bypass rapamycin sensitivity of PKCδ Ser662 phosphorylation, indicating that expression does not simply induce an autocrine serum-like response; PKCζ-transfected cells do not condition their media with factors that stimulate Ser662 phosphorylation (D.B.P., unpublished observations). Although it can be concluded that aPKC appears to be a component of a V5 kinase, it should be noted that the membrane-associated aPKC showing V5 kinase activity appears to be part of a complex because the native protein is not immunoprecipitated by an antibody that will deplete uncomplexed native recombinant PKCζ (unpublished observations). Thus, the V5 kinase activity is associated

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PKCδ Ser662 phosphorylation is controlled by PKCζ. (a) Plasmids encoding PKCδ and PKCζ(T410E/T560E) were transfected into 293 cells as shown. Cells were serum-starved and then restimulated with 10% FCS for 20 min after pretreatment with rapamycin (20 nM) or LY294002 (10 µM) as indicated. Extracts were prepared and immunoblotted for PKCδ phosphorylation (Ser662 and Thr505 sites) and PKC expression as indicated. Expression of the PKCζ mutant bypasses the sensitivity of Ser662 phosphorylation to the two inhibitors; no significant effect is seen for the Thr505 site. (b) Plasmids encoding PKCδ and PKCζ(T410A) were transfected into 293 cells as shown. Starved cells were then stimulated with 10% FCS for 20 min after pretreatment with rapamycin or LY294002 and extracts analysed for PKCδ expression and Ser662 phosphorylation. The mutant PKCζ is seen to inhibit the serum-induced PKCδ phosphorylation. (c) Immunopurified PKCζ(T410E) was incubated with purified GST–PKCδ and the phosphorylation of the PKCδ Ser662 site monitored by immunoblotting. Only the immunocomplex containing PKCζ(T410E) catalysed phosphorylation of PKCδ. In this figure Rap denotes rapamycin and LY denotes LY294002.

with a particular form of PKCζ that is complexed and/or modified. The importance of some activating step for PKCζ is directly evidenced here by the differential behaviour of wild-type PKCζ and the activated PKCζ mutant. Although the former has no effect on PKCδ phosphorylation in vivo or in vitro, the latter supports phosphorylation in both situations. The sensitivity of PKC phosphorylation to rapamycin has not been addressed previously. Interestingly, it has been reported that rapamycin can inhibit PKC-dependent stimulation of sodium transport [20] and this was thought to be due to the direct inhibition of PKC activity by rapamycin.

Research Paper PKC phosphorylation by an atypical PKC complex Ziegler et al.

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The results presented here, however, indicate that the inhibition might occur upstream at the level of a rapamycinsensitive phosphorylation. Dephosphorylation of PKCδ by serum starvation and rapamycin treatment dramatically reduces its activity in in vitro kinase assays (D.B.P. and P.J.P., unpublished observations). Thus, rapamycin influences the catalytic activity of PKCδ through the control of Ser662 phosphorylation. A clear precedent exists in the form of p70 which is well established as a downstream target of rapamycin (and PI 3-kinase inhibitors) [21]. Like PKC, p70 has a Phe–X–X–Phe–Ser/Thr–Tyr/Phe phosphorylation motif carboxy-terminal to the kinase domain, the phosphorylation of which is known to be sensitive to rapamycin [16]. The additional rapamycin-induced loss of phosphorylation of p70 in the activation loop site [22] might mirror the situation in PKCα in which we have established that alanine mutations in either of the carboxy-terminal sites (the autophosphorylation site Thr638 and the hydrophobic site Ser657) sensitises the protein to dephosphorylation at the activation loop site [6,23]. It would seem that these various AGC family kinases retain some similar structural requirements for activity.

component of an upstream kinase for PKC is attractive, given that aPKCs themselves escape an equivalent regulatory input to their carboxy-terminal hydrophobic region, having a glutamic acid residue in place of serine/threonine in the hydrophobic motif (Phe–X–X–Phe–Glu–Tyr). This negatively charged residue can partially mimic phosphorylation at this position, as evidenced by an acidic mutation in PKCα [6]. The aPKC isotypes, in particular PKCζ, have been implicated in many cellular functions but whether this is effected through their action as PKC family (or even AGC superfamily) kinases remains to be determined.

Previous studies in yeast and mammals have identified members of the target of rapamycin (TOR) family as targets of rapamycin ([24] and reviewed in [21]). Mammalian TOR (mTOR) is itself a member of the PI 3-kinase superfamily and is also sensitive to PI 3-kinase inhibitors such as LY294002 [25]. The Ser657-specific V5 kinase/PKCζ is not inhibited directly by LY294002 (data not shown) and PI 3-kinases are not sensitive to rapamycin, hence our current understanding would suggest that mTOR can control V5 domain phosphorylation on PKC. Whether or not this effect is principally through control of aPKC or through control of a phosphatase activity is unresolved. The data here indicate a reduced PKCζ/ι recovery in membrane preparations from cells pretreated with rapamycin or LY294002, as observed for the V5 kinase activity (see Results section). It seems possible, however, that the inhibitory effect might be channelled additionally through a protein phosphatase. For the related phosphorylation of p70 and the rapamycin -sensitive phosphorylation of eukaryote initiation factor 4E binding protein 1 (eIF-4E-BP1) there is evidence that mTOR will phosphorylate relevant sites directly [26]. There is also evidence, however, that the rapamycin-sensitive control of p70 is effected through a phosphatase because an amino-terminal deletion in p70 converts the kinase to a rapamycin-resistant form but does not affect its phosphorylation in response to serum [27–29]. Whether the kinases or phosphatases are dominant in controlling these rapamycin-sensitive phosphorylations and those of PKC awaits further analysis.

Materials and methods

There are very few ‘in vivo’ substrates for aPKC isotypes that are firmly established. The assignment of aPKCs as a

Conclusions The evidence presented here demonstrates that PKCδ phosphorylation at its conserved hydrophobic carboxy-terminal site, Ser662, is under the control of a rapamycin-sensitive pathway. The in vitro and in vivo studies presented indicate that a component of the kinase activity responsible for this phosphorylation is an aPKC. Thus, aPKC appears to act alongside PDK1 to control the phosphorylation of other PKC isotypes.

Expression and purification of recombinant proteins. PKCα–V5 domain (D615–V672), wild-type and the alanine-substitution mutants T638A, S657A, AA (T638A,S657A), as well as PKCδ (full-length) were cloned with a linker in pGex-2T (Pharmacia Biotech) and expressed in Escherichia coli BL21 (DE3) cells (Novagen). GST-fusion proteins were purified and eluted according to the ‘GST Gene Fusion System’ manual (Pharmacia Biotech). The dominant-negative PKCζ mutant (T410A) has been published [19] and the activated mutants were obtained by site-directed mutagenesis of the Thr410 and Ser560 sites using conventional procedures (J.A.L.G., unpublished observations). All mutant cDNAs were sequenced to confirm integrity. Myc-tagged PKCζ (T410E) or wild-type Myc-tagged PKCζ were expressed in 293 cells. Protein was immunopurified from extracts made in 1% Triton X-100, 0.1 M NaCl, 50 mM Tris-HCl pH 7.5, 2 mM EDTA, 2 mM DTT, 1 µM microcystin, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 0.5 mM phenylmethylsulphonylfluoride (PMSF), using affinity-purified anti-Myc antibody and protein-G–agarose beads. Immunocomplexes were washed twice with 1% Triton X-100, 0.2 M NaCl, 20 mM Tris-HCl pH 7.5, 2 mM EDTA, 2 mM DTT, 1 µM microcystin, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 0.5 mM PMSF and then once with an equivalent buffer containing only 0.1 M NaCl and 0.05% Triton X-100. Immunocomplexes were employed directly in kinase assays, with the equivalent of one sixth of a 15 cm plate of transfected cells employed in each phosphorylation reaction.

Purification of V5 kinase activity Cells or rat brains were disrupted on ice in harvest buffer (20 mM Tris pH 7.5, 10 mM EGTA, 5 mM EDTA, 1 mM DTT, 10 µg/ml leupeptin/ aprotinin, 10 mM benzamidine, 1 mM PMSF, 1µM microcystin) and spun at low speed to clear extracts from debris and nuclei. Supernatants were spun for 1 h at 100,000 × g. Pellets were resuspended in harvest buffer, with 0.5 M NaCl added, and spun again for 1 h at 100,000 × g. Triton X-100 (1% (v/v) final) was added to supernatants, which were then diluted 1:4 in buffer A (20 mM Tris (pH 7.5), 2 mM EDTA, 10 mM benzamidine, 1 mM DTT, 0.02% Triton X-100) and loaded onto a HiTrapQ column (5 ml) (FPLC-System, Pharmacia Biotech). The protein was eluted in buffer A by application of a salt

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gradient (0–1 M NaCl). The V5 kinase activity was tested as described below. The activity eluted in a single peak at 350–400 mM NaCl. The active fractions were pooled and diluted 1:4 in buffer B (20 mM PIPES (pH 6.5), with further components as buffer A). The protein fraction was loaded onto a MonoS PC 1.6/5 column and the protein eluted in buffer B using a salt gradient (0–1 M NaCl). The V5 kinase activity eluted at 750–800 mM NaCl. The fractions were adjusted to pH 7.5, diluted 1:5 in buffer A and loaded onto a MiniQ column (SmartSystem, Pharmacia Biotech). The V5 kinase activity was eluted at 350–400 mM salt (50 µl fractions). The V5 kinase activity was assayed on GST–V5α protein substrate (5 µg/reaction) bound to glutathione beads. Between 2.5 µl and 5.0 µl of kinase fraction were added to the reaction mix (20 mM Tris pH 7.5, 10 mM MgCl2, 20µM ATP, 5 µCi [γ-32P]ATP in a final volume of 50 µl. The samples were shaken for 20 min at 30°C. The beads were spun down, the supernatant removed and the pellet washed twice in PBS (with Triton X-100 (1%), 10 µg/ml aprotinin/leupeptin). The GST–V5α substrate protein was eluted into SDS sample buffer and an aliquot separated by SDS–PAGE. The protein was transferred onto nitrocellulose and autoradiographed. The slow migrating, 32P-labelled GST–V5α protein at 38 kDa, lines up exactly with the Ser657(P)-specific antibody staining in the western analysis (see below) and was used to follow the purification. The specific activity of the V5 kinase preparations was calculated on basis of the specific 32P incorporation into the substrate. The protein concentrations of the fractions were determined using the Biorad protein assay (Biorad).

Western analysis PKC isotypes were immunoblotted essentially as described previously [30]. In addition, where stated, PKCζ/ι were detected with a commercial antiserum to PKCζ (Santa Cruz) diluted 1:2,000 in the presence or absence of peptide antigen (0.4 µg/ml) as indicated. The Ser657(P) site specific polyclonal antiserum was raised against a phospho-peptide ‘FGFS[P]YVNP’, taken from the region flanking the FSY-motif in PKCα, and tested against fully phosphorylated versus dephosphorylated PKCα (D.B.P. and P.J.P., unpublished observations). The Thr505 antiserum was as described previously [14]. Given that the Ser657(P) site specific serum shows some cross-reactivity with the unphosphorylated GST–V5α substrate protein, western analyses were always performed in the presence of the cognate dephosphorylated peptide (dephosphopeptide) at 1 µg/ml. As shown in Figure 1a, the antiserum (dilution 1:1,000–1:4,000) clearly detects a slower migrating, phosphorylated GST–V5α substrate band in the presence of dephosphopeptide. This band matches exactly the 32P signal on the corresponding autoradiograph and migrates slower than the Coomassie-blue-stainable GST–V5α substrate bands (data not shown). Western blots were developed using horseradish peroxidase (HRP)-coupled donkey anti-rabbit IgG secondary antiserum (Amersham; 1:5000) and ECL (Amersham).

Phosphorylation of PKCδ in vitro

For assays with the highly purified V5 kinase, eluted GST–PKCδ (1 µg per reaction) was incubated for 2 h at 30°C with the kinase fraction (2 µl) and PMA (0.5 µM) as indicated, in a reaction buffer (see kinase assay above) containing 1 µM microcystin, 100 µM phosphatidylcholine, 100 µM phosphatidylserine. An aliquot of each reaction was analysed using the PKCα FSY site specific antiserum (1:2,000). For phosphorylation with the immunopurifed PKCζ(T410E) mutant or wild-type PKCζ, eluted GST–PKCδ was incubated for 60 min at 30°C in a reaction buffer containing: 20 mM Tris-HCl pH 7.5, 10 mM MgCl2, 20 µM ATP, 100 nM TPA, 100 µM phosphatidylserine, 100 µM phosphatidylcholine, 1 µM microcystin. These experiments were carried out additionally in the presence of the PKC inhibitor bisindolylmaleimide I, which inhibits PKCδ but not aPKC isotypes except at considerably higher concentrations (IC50, 5.8 µM [31]). Reactions were stopped by addition of 10 mM (final) EDTA and Laemmli sample buffer. An aliquot was subjected to immunoblotting to monitor PKCδ Ser662 phosphorylation as described above.

Inhibition of V5 kinase activity U937 cells were grown in RPMI 1640 medium (10% FCS) to a density of 4–5 × 105 cells/ml and cells were treated where indicated with LY294002 (10 µM) or rapamycin (20 nM) for 20 min prior to protein extraction. The V5 kinase preparation was performed as described in the purification protocol. The salt extracts of the membrane pellets were applied to analyze the V5 kinase activity (as described above). The extent of specific GST–V5α substrate phosphorylation was determined based on the immunoreactivity signals (each experiment run in duplicate) and normalized as a function of background immunoreactivity. The data are derived from three independent protein preparations.

Membrane-associated PKCζ in 293 cells PKCζ in pEFlink was transfected into 293 cells [14]. Cells were treated as indicated with rapamycin (20 nM) or LY294002 (10 µM) for 20 min prior to membrane preparation and protein extraction as described in the purification of V5 kinase activity section. Equal amounts of total protein were separated on SDS–PAGE and PKCζ protein recovery analysed using a commercial antiserum to PKCζ (Santa Cruz).

PKCδ S662 phosphorylation in 293 cells PKCδ was transfected into 293 cells [32] in pEFLink with or without PKCζ (T410E,T560E or T410A) as indicated and starved for 24 h before stimulation with 10% FCS for the indicated period of time. Where present, LY294002 (10 µM) and rapamycin (20 nM) were added to cells 30 min prior to stimulation. Whole cells were lysed in SDS sample buffer and protein samples were separated by SDS–PAGE. Protein was blotted and analysed for PKCδ Ser662 phosphorylation using the PKCα FSY-site specific antiserum (1:4000, with dephosphopeptide 1 µg/ml), parallel samples were probed with a PKCδ carboxy-terminus-specific antiserum [32]. In some instances, the Thr505(P)-specific antiserum was employed to monitor phosphorylation at the PKCδ Thr505 site. The extent of Ser662 phosphorylation was determined based on the immunoreactivity signals and normalized as a function of total PKCδ protein. The data are derived from four independent experiments.

Acknowledgements We are grateful to the DFG (W.H.Z.) and the E.C. BIOMED 2 BMH4–CT98–3328 (B.A.H., P.J.P.) for support of the work. We also thank Ben Webb for the GST–PKCδ protein and members of the Parker laboratory for helpful discussions.

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