Biochemical and Biophysical Research Communications 334 (2005)

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1 Biochemical and Biophysical Research Communications 334 (2005) BBRC Stimulus-induced phosphorylation of PKC h at the C-terminal hydrophobic-motif in human T lymphocytes q,qq Michael Freeley a, Yuri Volkov b,c, Dermot Kelleher b,c, Aideen Long a, * a Department of Biochemistry, Royal College of Surgeons in Ireland, 123 St. Stephens Green, Dublin 2, Ireland b Department of Clinical Medicine, Trinity College, St. JamesÕs Hospital, Dublin, Ireland c Dublin Molecular Medicine Centre, St. JamesÕs Hospital, Dublin 8, Ireland Received 13 June 2005 Available online 1 July 2005 Abstract Protein kinase C (PKC) is a family of serine/threonine kinases whose activity is controlled, in part, by phosphorylation on three conserved residues that are located on the catalytic domain of the enzyme, known as the activation-loop, the turn-motif, and the C-terminal hydrophobic-motif sites. Using a panel of phospho-specific antibodies, we have determined that PKC b I and d are constitutively phosphorylated on all three sites in unstimulated and activated T cells. Although PKC h is constitutively phosphorylated at the activation-loop and turn-motif sites in T cells, PMA or anti-cd3/cd28 stimulation results in an increase in phosphorylation at the hydrophobic-motif (Ser695), an event that coincides with translocation of the enzyme from the cytosol/cytoskeleton to the membrane. Studies on the stimulus-induced phosphorylation of PKC h demonstrate that an upstream kinase activity involving a conventional PKC isoform(s) and the PI3-kinase pathway, rather than autophosphorylation or the rapamycin-sensitive mtor pathway, regulates this site in T lymphocytes. However, hydrophobic-motif phosphorylation does not appear to control membrane translocation, suggesting that this site may control other aspects of PKC h signalling. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Protein kinase C h; Hydrophobic-motif; T lymphocyte Protein kinase C (PKC) is a family of phospholipiddependent serine/threonine kinases that play key roles in many of the signalling pathways that control cellular q This work was supported by the Royal College of Surgeons in Ireland Research Committee, The Health Research Board of Ireland and Enterprise Ireland. qq Abbreviations: apkc, atypical PKC; cpkc, conventional PKC; DAG, diacylglycerol; FBS, foetal bovine serum; IL-2, interleukin-2; ILK, integrin-linked kinase; LFA-1, lymphocyte function-associated antigen-1; MAPKAPK, mitogen-activated protein kinase-activated kinase-2; mtor, mammalian target of rapamycin; NF-jB, nuclear factor of jb; NP40, nonidet P40; npkc, novel PKC; PBS, phosphatebuffered saline; PDK-1, phosphoinositide-dependent protein kinase-1; PI3-kinase, phosphatidylinositide-3 0 -OH kinase; PKA, protein kinase A; PKB, protein kinase B; PKC, protein kinase C; PMA, phorbol 12- myristate 13-acetate; PS, phosphatidylserine. * Corresponding author. Fax: address: aclong@rcsi.ie (A. Long). growth, proliferation, differentiation, and cell death [1]. PKC isoforms are divided into three groups based on their N-terminal regulatory domain structure and requirements for lipid second messengers. Conventional PKCs (cpkcs) comprise the a, b I /b II,andc isoforms, and are regulated in a Ca 2+ and diacylglycerol (DAG)- dependent manner and also require the membrane-localised co-factor phosphatidylserine (PS) for optimal activity. The novel PKCs (npkc) comprise the d, e, g, h, andl isoforms, and function in a Ca 2+ -independent manner but require DAG and PS for activity. Finally, the atypical PKC family (apkc), comprising f and i/k, functions in a PS-dependent, but Ca 2+ and DAG-independent, manner. DAG and Ca 2+ ions accumulate in the cell following receptor-mediated activation of phospholipase C (PLC), which hydrolyses the inositol phospholipid PtdIns(4,5)P 2 into DAG (which activates X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi: /j.bbrc

2 620 M. Freeley et al. / Biochemical and Biophysical Research Communications 334 (2005) PKC) and Ins(1,4,5)P 3, the production of which leads to the release of Ca 2+ from intracellular stores. Stimulusinduced increases in the levels of DAG (and Ca 2+ for cpkcs) recruit these enzymes to membranes. Binding of DAG to the regulatory domain of PKC induces a conformational change in the enzyme, resulting in expulsion of the autoinhibitory pseudosubstrate from the catalytic cleft, thus permitting substrate binding and phosphorylation [1]. PKC activity is controlled by a complex set of processes that includes regulation by lipid second messengers (e.g., DAG), binding to anchoring proteins, and phosphorylation of the enzyme itself [2]. The cpkc and npkcs contain three conserved phosphorylatable serine/threonine residues located at the carboxyl-terminal catalytic domain that are essential for catalytic activity, intracellular distribution, and stability [3]. These sites are known as the activation-loop, the turn-motif, and the C-terminal hydrophobic-motif (the apkcs contain a phosphorylatable activation-loop and turn-motif residue but possess a negatively charged glutamic-acid at the hydrophobic-motif site, perhaps mimicking a constitutively phosphorylated state). These phosphorylation sites are also conserved on many other kinases termed ABC kinases (representing PKA, 1 PKB, PKC, and other kinase families such as p70 and p90 ribosomal S6 kinases). While PDK-1 phosphorylates the activationloop of many ABC kinases in vivo (including PKCs) [3], the mechanism of phosphorylation at the other two residues, in particular at the hydrophobic-motif site, is unclear. For many of these kinases, conflicting evidence in the literature suggests that these enzymes either autophosphorylate at the hydrophobic-motif [4] or that a heterologous kinase activity (putatively termed PDK- 2) phosphorylates this site (see Discussion). T lymphocytes predominantly express PKC family members a, b I, d, e, g, h, l, and f [5,8]. PKC h is the only member of the T cell-expressed PKCs that localises to the immunological synapse [6] and activates important transcription factors such as NF-jB [7]. Our group has demonstrated that the expression of PKC b I is crucial for integrin-mediated migration of T lymphocytes [9] and we (and others) have also shown that the expression of this enzyme is required for the secretion (but not production) of the cytokine interleukin-2 (IL-2) in response to T cell activation [10,11]. The phosphorylation status of endogenous PKC isoforms at these sites in response to T cell receptor activation stimuli has not been analysed in detail. In the present study, we have characterised the phosphorylation status of PKC isoforms b I, d, and h at these sites in resting and activated T cells. Unlike PKC b I and 1 PKA does not possess a hydrophobic-motif residue as the protein sequence terminates immediately prior to the hydrophobic-motif residue. d, PMA or anti-cd3/cd28 stimulation induces hydrophobic-motif phosphorylation (Ser695) of PKC h. We also provide evidence that an upstream kinase activity involving the PI3-kinase pathway and cpkc isoforms, rather than autophosphorylation or the rapamycin-sensitive mtor pathway, is responsible for the phosphorylation of this site on the enzyme. These findings suggest that cpkcs may act as PDK-2-like enzymes for PKC h and regulate its function in T lymphocytes. Although hydrophobic-motif phosphorylation of PKC h coincides with translocation of the enzyme to the membrane, phosphorylation of this site does not appear to be important for membrane localisation. Materials and methods Reagents and antibodies. The Jurkat leukaemic T cell line and the monoclonal anti-cd3 producing hybridoma cell line (OKT-3) were obtained from the American Type Culture Collection (Manassas, VA). RPMI-1640, foetal bovine serum (FBS), penicillin, streptomycin, L-glutamine, cell culture grade phosphate-buffered saline (PBS), and cell culture flasks were obtained from Gibco BRL (Life Technologies, Paisley, UK). Sterile six-well plates were obtained from Nunc (Roskilde, Denmark). Go6976, staurosporine, cyclosporine A, and rapamycin were obtained from Calbiochem (Nottingham, UK). LY was obtained from Alexis (Nottingham, UK). Protein G Sepharose beads were obtained from Amersham Biosciences (Bucks, UK). Polyvinylidene fluoride (PVDF) membrane was obtained from Pall Gelman Laboratories (Ann Arbor, MI). Nonidet P40 (NP40) and EDTA were obtained from B.D.H. chemical and laboratory supplies (Poole, UK). All other chemicals (including rottlerin and wortmannin) were from Sigma (St. Louis, MO). Rabbit anti-mouse IgG was obtained from Dako A/S (Denmark). Mitogenic antibody to human CD28 was obtained from Ancell (Bayport, MN). Monoclonal antibodies to PKC b were obtained from Seikagaku (Tokyo, Japan), BD Biosciences (Oxford, UK), and Zymed (San Francisco, CA) (the anti- PKC b antibody from Zymed was used for immunoprecipitation, whilst the anti-pkc b antibodies from Seikagaku and BD Biosciences were used for immunoblotting). Antibodies to total PKC d were obtained from BD Biosciences and Santa Cruz Biotechnology (Santa Cruz, CA) (the anti-pkc d antibody from BD Biosciences was used for immunoprecipitation, whilst the Santa Cruz antibody was used for immunoblotting). The anti-pkc h antibody was obtained from BD Biosciences and was used for both immunoprecipitation and immunoblotting. The anti-lfa-1 antibody was obtained from Serotec (Oxford, UK). The following antibodies were obtained from Cell Signalling Technology (Beverly, MA): phospho-pkc h (p-thr538) antibody (recognises the phosphorylated activation-loop), phospho- PKC d (p-ser643) antibody (recognises the phosphorylated turn-motif of PKC d and h), phospho-pkc a/b II (p-thr638/thr641) antibody (recognises the phosphorylated turn-motif of PKC a and b), and phospho-pan PKC (recognises the phosphorylated C-terminal hydrophobic-motif of all PKC isoforms). Goat anti-mouse IgG-HRP, goat anti-rabbit IgG-HRP, anti-erk 1/2 (total), and the Phototope HRP detection kit were also obtained from Cell Signalling Technology. The P500 antibody that recognises the phosphorylated activationloop threonine residue of all PKC isoforms was generated by Joanne Johnson in the Newton laboratory and was generously provided by Alexandra Newton (University of California, US). The specificity of this antibody has been characterised elsewhere and was used at a 1/ 1000 dilution [12]. Antisera generated against the phosphorylated forms of PKC h at Ser676 and Ser695 (phosphorylated turn-motif and hydrophobic-motif of PKC h, respectively) and their non-phosphory-

3 M. Freeley et al. / Biochemical and Biophysical Research Communications 334 (2005) lated blocking peptides were kindly donated by Dr. Stephen Shaw (NIH, US). To prevent these antisera from binding to non-phosphorylated PKC h, the antisera (at 1/1000 dilution) were pre-incubated for 1 h at room temperature with their respective non-phosphorylated peptides (also at 1/1000 dilution) prior to incubation with the PVDF membrane. The specificity of these phospho-pkc h antisera is characterised elsewhere [13]. Cell culture and cellular stimulation. The human Jurkat leukaemic T cell line (E6.1 clone) was cultured in RPMI-1640 containing 10% (v/v) heat-inactivated FBS, 50 U/ml penicillin, 50 lg/ml streptomycin, and 2 mm L-glutamine in a humidified chamber at 37 C containing 5% CO 2. Cell density was maintained between and cells/ml, and in all experiments the cells were seeded at cells/ml prior to stimulation. The PKC activator phorbol 12-myristate 13-acetate (PMA) was used at a final concentration of 10 ng/ml in all experiments. For anti-cd3/cd28 co-stimulation experiments, sterile six-well plates were coated with a 1/100 dilution of rabbit anti-mouse IgG in sterile PBS overnight at 4 C. Unbound rabbit anti-mouse IgG was carefully removed and the wells were gently washed in sterile warm PBS. The wells were then coated with a pre-mixed solution of anti- CD3 antibody (1/100 dilution of OKT-3 hybridoma supernatant in sterile PBS) and anti-cd28 (1/200 dilution in sterile PBS) and incubated for 3 h at 37 C. Unbound antibodies were removed and the wells were gently washed in sterile warm PBS. Jurkat cells (at a concentration of cells/ml in complete RPMI) were added to the wells and incubated at 37 C with 5% CO 2 for the indicated times. Cell lysis, immunoprecipitation, and Western blotting. Cellular stimulation was terminated by recovering the cells from the wells or flasks and centrifugation at 1400 rpm for 4 min. The cell pellets were quickly resuspended in ice-cold cell lysis buffer (1% NP40, 20 mm Tris HCl (ph 7.4), 150 mm NaCl, 5 mm EDTA, 5 mm EGTA, 5 mm sodium fluoride, 5 mm sodium pyrophosphate, 1 mm sodium orthovanadate, 1 mm PMSF, and 10 lg/ml leupeptin) for at least 30 min on ice with intermittent vortexing of the sample. Detergent-insoluble material, unlysed cells, and nuclei were removed by centrifugation for 5 min at 2500 rpm. Detergent-soluble lysates ( cells in 0.5 ml lysis buffer) were pre-cleared with washed protein G Sepharose beads for 2 h at 4 C with constant end-over-end mixing. The beads were removed by centrifugation and pre-cleared lysates were subjected to immunoprecipitation with antibodies to total PKC b I, PKC d, PKC h or an irrelevant IgG isotype control monoclonal antibody for 2 h on ice. The antibody antigen complexes were then collected by incubating the lysates with washed protein G Sepharose beads overnight at 4 C with end-over-end mixing. The immunoprecipitates were recovered by centrifugation and washed three times with ice-cold cell lysis buffer. The pellets were resuspended in SDS PAGE sample buffer, boiled for 4 min, the beads were removed by centrifugation, and the immunoprecipitate supernatants were resolved on 10% SDS PAGE gels. The separated proteins were electrophoretically transferred to PVDF membranes by the semi-dry transfer technique for 1 h. PVDF membranes were subsequently blocked in 5% non-fat dried milk in TBS 0.1% Tween (TBS-T) for 1 h at room temperature, washed three times in TBS-T, and incubated with primary antibodies (diluted according to the manufacturerõs instructions) overnight at 4 C with gentle rocking. After three washes in TBS-T, the membranes were incubated with the appropriate HRP-labelled secondary antibodies for 2 h at room temperature. The membranes were washed three times in TBS-T and immunoreactive bands were visualised with the Phototope HRP detection system and subsequent exposure to Kodak light-sensitive film (Cedex, France). In a number of experiments, the PVDF membrane was stripped of its antibodies by incubation in stripping buffer (62.5 mm Tris HCl (ph 6.8), 2% SDS, and 100 mm b-mercaptoethanol) for 30 min at 65 C, followed by washing, blocking, and reprobing with antibodies as outlined above. Subcellular fractionation. Jurkat T cells (typically cells) were harvested, resuspended in 2 ml of ice-cold subcellular fractionation buffer A (20 mm Tris HCl, ph 7.5, containing 0.25 M sucrose, 2 mm EDTA, 2 mm EGTA, 1 mm PMSF, 10 lg/ml leupeptin, and 1 mm sodium orthovanadate), and incubated on ice for at least 5 min. The sample was sonicated for 5 s and spun down at 600g to remove unlysed cells and nuclei. The supernatants were then centrifuged at 100,000g for 10 min at 4 C. The resulting supernatant was designated as the cytosolic-rich fraction. The pellet was resuspended in 0.5 ml of ice-cold subcellular fractionation buffer B (20 mm Tris HCl, ph 7.5, containing 1% NP40, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm PMSF, 10 lg/ml leupeptin, and 1 mm sodium orthovanadate) for 30 min on ice with intermittent vortexing, and centrifuged at 21,000g for 30 min at 4 C. The supernatant was designated as the detergent-soluble membrane-rich fraction. The pellet, representing the detergent-resistant cytoskeletal-rich fraction, was dissolved by boiling in subcellular fractionation buffer C (20 mm Tris HCl, ph 7.5, containing 1% w/v SDS, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm PMSF, 10 lg/ml leupeptin, and 1 mm sodium orthovanadate). Protein concentration was quantified by the Markwell (modified Lowry) assay, the samples were concentrated by acetone precipitation, resuspended in SDS PAGE loading buffer, and equal amounts of protein from each fraction were resolved on SDS PAGE gels and probed by Western blotting. The quality of the cytosolic and membrane fractions was routinely analysed by immunoblotting the fractionated cells for ERK (cytosolic distribution) and the membranelocalised b II integrin LFA-1. Detection of secreted IL-2. Secreted IL-2 was detected with an ELISA kit (R&D Systems Europe, Oxon, UK), according to the manufacturerõs instructions. Results Stimulus-induced phosphorylation of PKC h at the C-terminal hydrophobic-motif Immunoprecipitation of total PKC isoforms b I, d,and h from unstimulated or 30 min PMA-activated Jurkat T cells and immunoblotting with phospho-specific antibodies to the activation-loop, turn-motif, and hydrophobicmotif sites revealed basal levels of phosphorylation at all three sites for PKC b I and d (Fig. 1). Stimulation of the cells with PMA for 30 min did not alter the levels of phosphorylation at these sites on PKC b I or d to any great extent. Furthermore, culturing of the cells overnight in the absence of serum did not affect the basal or PMAstimulated levels of phosphorylation at these sites (data not shown). It should be noted however that PMA stimulation did induce a partial reduction (40% loss) in the amount of detergent-extractable PKC d, which may indicate translocation of the enzyme to a detergent-insoluble fraction or alternatively, degradation via the proteasome complex in response to cellular activation. In contrast to PKC b I and PKC d, the levels of phosphorylation of PKC h were inducible with PMA in a site-specific manner; although PMA did not alter the level of activation-loop phosphorylation of PKC h, a striking increase in the level of phosphorylation at the hydrophobic-motif site was observed whilst a slight increase in phosphorylation was observed at the turn-motif site following activation (Fig. 1). This PMA-induced increase in phosphorylation of PKC h at the hydropho-

4 622 M. Freeley et al. / Biochemical and Biophysical Research Communications 334 (2005) Fig. 1. Stimulus-induced phosphorylation of PKC h in Jurkat T lymphocytes. Jurkat T cells were activated with PMA for 30 min or left unstimulated. Total lysates were prepared and PKC isoforms were immunoprecipitated with antibodies to PKC b I, d, h or an irrelevant monoclonal control antibody. Immunoprecipitates were probed by Western blotting with phosphorylation-specific antibodies to PKC that recognise the activation-loop (P500 antibody), the turn-motif or the C-terminal hydrophobic-motif (both from Cell Signalling). As a relative loading control, all membranes were stripped and re-probed with antibodies that specifically recognise total PKC isoforms (not phosphorylation specific) and a representative blot is indicated here. Results shown are representatives of two similar experiments. A non-specific band in the control immunoprecipitate that migrates slightly faster than the PKC b I immunoprecipitate is evident in the phosphorylated activation-loop blot and is marked with *. bic-motif site in Jurkat leukaemic T cells was also observed in immunoprecipitates derived from peripheral blood lymphocytes (data not shown). We confirmed our findings on the stimulus-induced increase in phosphorylation of PKC h in resting and PMA-activated T cells by utilising an independent panel of phospho-antibodies specifically raised against human PKC h (phospho-thr538, phospho-ser676, and phospho-ser695 that recognise PKC h when phosphorylated at the activation-loop, turn-motif, and hydrophobic-motif sites, respectively) (Fig. 2). PKC h was immunoprecipitated from lysates derived from unstimulated or PMA-activated Jurkat T cells and immunoblotted with these phospho-antibodies. As shown in Fig. 2, PMA stimulation did not alter the levels of phosphorylation of PKC h at the activation-loop (Thr538). In contrast, the same stimulus induced a small, but detectable, increase in phosphorylation of PKC h at the turn-motif (Ser676) and hydrophobic-motif sites (Ser695). Jurkat T cells were also stimulated with plate-bound antibodies against the T cell receptor-associated complex (anti-cd3) in combination with mitogenic anti- CD28 antibodies. Cross-linking of the CD3 complex in combination with CD28 induces T cell signalling similar to that induced through the TCR, leading to PKC activation and IL-2 production. As shown in Fig. 3, stimulation of the cells for 5 60 min did not modulate the levels of phosphorylation of PKC h at the activationloop domain or turn-motif sites. In contrast, the levels of phosphorylation of PKC h were inducible at the Fig. 2. Confirmation of the phosphorylation status of PKC h in Jurkat T cells using site-specific antibodies. Jurkat T cells were activated with PMA for 30 min or left unstimulated. Lysates were immunoprecipitated with an anti-pkc h antibody and probed with site-specific antibodies to the phosphorylated forms of PKC h; (A) the activation-loop (phospho-thr538 PKC h; Cell Signalling); (B) turn-motif (phospho-ser676 PKC h; gift from Stephen Shaw), (C) hydrophobic-motif (phospho-ser695 PKC h; gift from Stephen Shaw) or (D) phospho-c-terminal hydrophobic-motif antibody (Cell Signalling). As a relative loading control, all blots were stripped and re-probed with an antibody to total PKC h.

5 M. Freeley et al. / Biochemical and Biophysical Research Communications 334 (2005) Fig. 3. Stimulus-induced phosphorylation of PKC h in anti-cd3/ CD28-stimulated Jurkat T cells. Jurkat T cells were stimulated with plate-bound antibodies to CD3 in combination with anti-cd28 for the indicated times. Total cell extracts were immunoblotted with an antibody to the phosphorylated activation-loop of PKC h (phospho- Thr538 PKC h; Cell Signalling) or total PKC h as a loading control (Transduction Labs). In addition, immunoprecipitates of PKC h (indicated with *) were probed with antibodies to the phosphorylated turn-motif or C-terminal hydrophobic-motif of PKC h (both from Cell Signalling). hydrophobic-motif, demonstrating that the stimulus-induced phosphorylation of this enzyme could be reproduced under more physiological conditions. Ligation of the T cell receptor (TCR) in combination with anti-cd28 induces PLC c-induced production of DAG, which leads to translocation and activation of PKCs [5,8]. Translocation and activation of PKC can also be achieved by directly treating cells with phorbol esters such as PMA, agents that mimic the effects of DAG. Changes in the subcellular localisation of PKCs are therefore often taken as markers of catalytic activation of these enzymes. To show that stimulation of the T cells led to activation of PKC b I, d, and h, unstimulated or PMA-activated Jurkat cells were fractionated into cytosol, membrane, and cytoskeletal-rich fractions and the presence of each PKC isoform in the fractions was determined by immunoblotting. As shown in Fig. 4A, the majority of PKC b I in unstimulated Jurkat cells resided in the cytosolic-rich fraction with minimal amounts present in the membrane and cytoskeletal-rich fractions. After the addition of PMA, however, the majority of PKC b I was present in the detergent-soluble membrane-rich fraction, with minimal amounts in the cytoskeletal-rich fraction. The increase in membranedistributed PKC b I correlated with a direct loss of the enzyme from the cytosolic fraction. In contrast to PKC b I, a higher proportion of PKC d was already pres- Fig. 4. Translocation of PKC h to the plasma membrane in PMA or anti-cd3/cd28-activated Jurkat T cells. (A) Unstimulated or 30-min PMAactivated Jurkat cells were processed for subcellular fractionation. Equal amounts of protein (50 lg) from each fraction were immunoblotted with an antibody to PKC b I, PKC d or PKC h. (B) Jurkat cells were left unstimulated or activated with anti-cd3/cd28 antibodies for the indicated times and processed for subcellular fractionation. Equal amounts of protein (30 lg) from each fraction were immunoblotted with antibodies to PKC b I, PKC d or PKC h. The detergent-insoluble cytoskeletal-rich fraction (S) was not analysed in (B). The quality of the fractions was assessed by immunoblotting parallel samples for ERK 1/2 (cytosol) or LFA-1 (membrane). C = cytosol-rich fraction, M = membrane-rich fraction, and S = cytoskeletal-rich fraction. The relative distribution of PKC in each fraction is expressed as a percentage value (e.g., C + M + S = 100%) and is indicated below each immunoblot.

6 624 M. Freeley et al. / Biochemical and Biophysical Research Communications 334 (2005) ent in the membrane-rich fraction of unstimulated Jurkat T cells. After the addition of PMA to the cells, translocation of additional PKC d molecules to the membrane-rich fraction was apparent. Minimal amounts of PKC d were recovered in the cytoskeletalrich fraction both before and after PMA stimulation. It should be noted that the characteristic 40% loss of detergent-extractable PKC d observed in immunoprecipitate samples after PMA stimulation (Fig. 1) was not observed when subcellular fractions from the same cells were analysed (Fig. 4A) (combined densitometry values for cytosol, membrane, and cytoskeleton in resting cells = 251,702 and PMA treated cells = 293,975). The reasons for this discrepancy are unclear but could be related to the inability of the PKC d antibody that was used for immunoprecipitation (Transduction Labs) to recognise certain forms of the enzyme following PMA activation, a finding that has been documented elsewhere [14]. It is unlikely therefore that PKC d undergoes PMA-induced degradation but is rather an inability of the immunoprecipitation antibody to recognise certain species of the activated enzyme. In unstimulated cells, PKC h was found in association with the membrane-rich fraction with lesser amounts present in the cytosol and cytoskeletal-rich fractions, which is consistent with a previous report of high levels of plasma membrane-associated PKC h in resting Jurkat cells [15]. PMA stimulation resulted in a striking loss of PKC h from the cytosol and cytoskeletal fractions and an increased association with the membrane fraction, such that virtually all of the extracted PKC h was present in the membrane-rich fraction. Translocation of PKC b I, d, and h to the plasma membrane following PMA stimulation was confirmed by immunofluorescence microscopy (data not shown). Translocation of PKCs from the cytosol to the membrane fraction was also observed following anti-cd3/ CD28 stimulation of Jurkat T cells (Fig. 4B). A small proportion of PKC b I translocated to the membrane fraction after 30 min of stimulation. This association with the membrane fraction was transient, because after 60 min of stimulation a smaller amount of enzyme was detected in this fraction. Although PKC d and PKC h both translocated to the membrane fraction following anti-cd3/ CD28 stimulation, the kinetics of translocation for these enzymes were notably different to PKC b I such that the amount of PKC d that associated with the membrane fraction increased steadily over the stimulation period. While significant amounts of PKC h were found in the membrane fraction of unstimulated cells, an increase in membrane-associated enzyme was detected after 30 min of stimulation and this association with the membrane fraction was also evident after 60 min of stimulation. These experiments demonstrate therefore that phosphorylation of PKC h at the hydrophobic-motif coincides with translocation and activation of the enzyme to the membrane fraction of stimulated cells. In contrast, PKC b I and d also undergo translocation and activation following PMA or anti-cd3/cd28 stimulation, but activation of these enzymes is not accompanied by changes in their phosphorylation state at the sites that we have analysed. The stimulus-induced C-terminal hydrophobic-motif phosphorylation of PKC h is insensitive to rapamycin It has been demonstrated that the mammalian target of rapamycin (mtor) complex controls the hydrophobicmotif phosphorylation of many protein kinases, including p70 ribosomal S6 kinases and some PKC isoforms [3]. mtor is an intracellular serine/threonine kinase related to the PI3-kinase family which plays key roles in cell signalling events, including proliferation, differentiation, cell cycle progression, and cell death [16]. Rapamycin is a commercially available inhibitor of certain forms of mtor (see Discussion) and has been approved for use as an immunosuppressant in organ transplantation and autoimmune diseases. We investigated whether mtor controlled the C-terminal hydrophobic-motif phosphorylation of PKC h in T cells and whether such control impacted on the immunosuppressive properties of rapamycin. Pretreatment with 1 lm rapamycin did not inhibit the stimulus-induced phosphorylation of PKC h at the hydrophobic-motif either in Jurkats (Fig. 5A) or in peripheral blood lymphocytes (data not shown). Furthermore, rapamycin did not inhibit the PMA or anti-cd3/ CD28-mediated translocation of PKC h to the plasma membrane, as analysed by immunofluorescence microscopy (data not shown). Nonetheless, rapamycin inhibited anti-cd3/cd28-mediated IL-2 secretion from Jurkat T cells almost as effectively as cyclosporine A (Fig. 5B). Unlike cyclosporine A, however (which blocks transcription of IL-2 by inhibiting nuclear translocation of NF-AT transcription factors), rapamycin-mediated inhibition of IL-2 is most likely a result of destabilisation of intracellular mrna levels of this cytokine [17]. Evidence for a cpkc-like kinase activity, but autophosphorylation-independent mechanism, in phosphorylating the C-terminal hydrophobic-motif of PKC h in T cells A pharmacological approach was next undertaken to investigate the pathways responsible for the C-terminal hydrophobic-motif phosphorylation of PKC h in T cells. Autophosphorylation of PKC h should be prevented by pretreatment of the cells with a catalytic inhibitor of this enzyme such as rottlerin [18,19]. Hence, Jurkat T cells were pretreated with catalytic inhibitors of PKC subfamily members (the cpkc inhibitor Go6976 or the npkc inhibitor rottlerin), inhibitors of PI3-kinase (the unrelated compounds LY and wortmannin) or the broad-spectrum ki-

7 M. Freeley et al. / Biochemical and Biophysical Research Communications 334 (2005) Fig. 5. Rapamycin does not inhibit the PMA or CD3/CD28- mediated C-terminal hydrophobic-motif phosphorylation of PKC h. (A) Jurkat T cells were pre-treated with 1 lm rapamycin or methanol (MeOH) drug solvent control for 60 min prior to stimulation with plate-bound anti-cd3/cd28 antibodies or PMA for the indicated times. Lysates were immunoprecipitated with an antibody to PKC h (Transduction Labs). Immunoprecipitates were probed by Western blotting with the C-terminal hydrophobic-motif antibody (Cell Signalling). As a relative loading control, the blot was stripped and re-probed with an antibody to PKC h. (B) Jurkat T cells were pretreated with 1 lm rapamycin, 1 lm cyclosporine A or methanol control (MeOH) for 30 min prior to stimulation with anti-cd3/cd28 for 24 h. Cell culture supernatants were analysed for secreted IL-2 by ELISA. nase inhibitor staurosporine prior to stimulation with antibodies to CD3 plus CD28. As shown in Fig. 6, anti-cd3/cd28 stimulation of Jurkat T cells induced the characteristic phosphorylation of PKC h at Ser695. Pretreatment with rottlerin, an inhibitor of PKC d [20] and PKC h [21 23], did not inhibit the phosphorylation of this site on PKC h. However, the stimulus-induced phosphorylation of PKC h at this site was inhibited by the broad-spectrum kinase inhibitor staurosporine (which also inhibits PKC isoforms), inhibitors of PI3-kinase, and by the cpkc inhibitor Go6976. Importantly, the catalytic activity of PKC h has been reported to be unaffected by Go6976 [21,22], suggesting that Go6976-mediated inhibition of PKC h hydrophobic-motif phosphorylation is not via direct inhibition of PKC h. Instead, a cpkc isoform such as PKC a/b I may operate upstream of PKC h in T cells and phosphorylate (either directly or indirectly) the C- terminal hydrophobic-motif site, leading to full activation and/or modulation of this enzyme. Inhibiting hydrophobic-motif phosphorylation of PKC h does not prevent its translocation to the membrane-rich fraction Inhibitors of PI3-kinase have been shown to block the anti-cd3/cd28-mediated translocation of PKC h to the membrane fraction in T cells [24]. Because we determined that: (a) hydrophobic-motif phosphorylation of PKC h coincides with activation and translocation to the membrane fraction and (b) that inhibitors of PI3-kinase block hydrophobic-motif phosphorylation, we investigated whether inhibitors of hydrophobic-motif phosphorylation of PKC h such as Go6976 and staurosporine blocked membrane translocation of the kinase. As shown in Fig. 7, anti-cd3/cd28 stimulation induced membrane translocation of PKC h. However, the proportion of PKC h that was present in the membrane fraction of stimulated cells was not modulated to any great extent by any of the inhibitors tested. We conclude therefore that the hydrophobic-motif of PKC h does not control membrane translocation in response to anti-cd3/cd28 stimulation. Discussion Mutation of the activation-loop, turn-motif, and hydrophobic-motif sites on different PKC family members to non-phosphorylatable residues has provided important information on the role of these sites in controlling the catalytic activity, intracellular distribution, and stability of this family of kinases [3]. For cpkcs (such as PKC b II ), such studies have demonstrated that

8 626 M. Freeley et al. / Biochemical and Biophysical Research Communications 334 (2005) Fig. 6. Inhibition of anti-cd3/cd28-induced phosphorylation of PKC h in Jurkat T cells by PKC inhibitors Go6976, staurosporine, and PI3-kinase inhibitors LY and wortmannin. (A) Jurkat T cells were pre-treated with the following inhibitors (or DMSO vehicle control) for 30 min prior to stimulation with anti-cd3/cd28 for an additional 60 min: Go6976 (conventional PKC inhibitor, 2 lm); rottlerin (novel PKC inhibitor, 10 lm); staurosporine (broad-spectrum kinase inhibitor, 0.1 lm); LY (PI3-kinase inhibitor, 50 lm), and wortmannin (PI3-kinase inhibitor, 0.2 lm). Total cell lysates were immunoprecipitated with an antibody to PKC h and immunoprecipitates were probed by Western blotting with an antibody to the C-terminal hydrophobic-motif of PKC (Cell Signalling). As a relative loading control, the blot was stripped and re-probed with an antibody to PKC h. (B) Densitometry values for the blot in (A) were determined by calculating the ratio of phosphorylated PKC h to total PKC h for each treatment. The ratio of phospho/total PKC h for untreated cells was taken as unity and all other values are expressed relative to untreated cells. activation-loop and turn-motif phosphorylation are necessary for catalytic activity, whilst hydrophobic-motif phosphorylation controls intracellular distribution and/or enzyme stability [3]. Phosphorylation at these sites on cpkcs appears to be a post-translational maturation process that occurs soon after synthesis and is constitutive. However, this concept of constitutively phosphorylated PKCs has recently been challenged with reports of inducible phosphorylation of these sites on PKC b [25] and other family members, in certain cell types following stimulation. The regulation of phosphorylation status of endogenous PKCs at these three sites has not been characterised in detail in T lymphocytes. In this study, we found that PKC b I and d are constitutively phosphorylated, whilst PKC h phosphorylation at the hydrophobic-motif (Ser695) is inducible with cellular stimulation. Stimulus-induced phosphorylation of PKC h correlates with translocation to the plasma membrane, a classical activation marker for this enzyme in T cells that also induces kinase activation in parallel [22]. PKCb I and d translocation to the plasma membrane is not accompanied by changes in phosphorylation at any of the sites that we have analysed. Hence, this study highlights site-specific phosphorylation of PKC h in response to T cell activation in vitro. Inducible hydrophobic-motif phosphorylation and membrane translocation of PKC h in anti-cd3/cd28- stimulated Jurkat T cells has been already demonstrated [24] and is in agreement with our findings presented here. Importantly, however, the pathways that control this stimulus-induced phosphorylation of PKC h were not deduced in [24], other than demonstrating that a pharmacological inhibitor of PLC, U73122, failed to block the anti- CD3/CD28-mediated membrane translocation and hydrophobic-motif phosphorylation of PKC h. Furthermore, Altman and co-workers [24] did not investigate the phosphorylation status of PKC h at the activation-loop and turn-motif sites (or the phospho-status of other PKC isoforms) in response to T cell activation. In primary murine T lymphocytes, TCR stimulation induces a serine/threonine phosphorylation-dependent mobility

9 M. Freeley et al. / Biochemical and Biophysical Research Communications 334 (2005) Fig. 7. Inhibiting hydrophobic-motif phosphorylation of PKC h in T cells does not abolish membrane translocation in response to anti-cd3/cd28 stimulation. Jurkat T cells were pre-treated with the following inhibitors (or DMSO vehicle control) for 30 min prior to stimulation with anti-cd3/ CD28 for an additional 60 min: Go6976 (conventional PKC inhibitor, 2 lm); rottlerin (novel PKC inhibitor, 10 lm); staurosporine (broad-spectrum kinase inhibitor, 0.1 lm), LY (PI3-kinase inhibitor, 50 lm), and wortmannin (PI3-kinase inhibitor, 0.2 lm). The cells were processed for subcellular fractionation. Equal amounts of protein (30 lg) from each fraction were immunoblotted with an antibody to PKC h. The detergentinsoluble cytoskeletal-rich fraction (S) was not analysed in this experiment. The quality of the fractions was assessed by immunoblotting parallel samples for ERK 1/2 (cytosol) or LFA-1 (membrane). C = cytosol-rich fraction and M = membrane-rich fraction. shift on PKC h that coincides with translocation of the kinase to the cell membrane [19]. Phosphorylation of Ser695 of PKC h was identified as one site that contributed to the mobility shift on the enzyme. In addition, phosphorylation of Ser695 was required to trigger further phosphorylation events that also contributed to the anti-cd3/cd28-induced mobility shift (notably these additional phosphorylation sites on murine PKC h are not conserved with human PKC h which may explain why we did not see any changes in the electrophoretic mobility of PKC h in stimulated human T cells). Hydrophobic-motif phosphorylation of PKC h in stimulated murine and human T lymphocytes, therefore, indicates that it is a physiologically relevant event. Conflicting evidence centres on whether ABC kinases (including PKC, PKB, and p70 S6 kinase families) phosphorylate themselves at the hydrophobic-motif or whether an upstream kinase(s) (putatively termed PDK-2) is responsible. For PKB, both autophosphorylation [4] and heterologous kinases have been implicated as mechanisms of hydrophobic-motif phosphorylation. A bewildering number of putative PDK-2-like enzymes have been described as hydrophobic-motif kinases of PKB. For example, the elusive PDK-2 has been identified as a ÔmodifiedÕ form of PDK-1 [26], MAPKAPK-2 kinase [27,28], integrin-linked kinase (ILK) [29,30], a PKC f-associating activity [31], cpkc isoforms [32 34], mtor [35], or DNA-dependent protein kinase [36]. Why have so many different hydrophobic-motif kinases been identified for PKB? It appears that phosphorylation of this site may be stimulus and cell-type specific [32 34]. For PKC b II and PKC e, biochemical studies have shown that these enzymes autophosphorylate at the hydrophobic-motif [37,38]. However, other reports suggest that the hydrophobic-motif of PKC d and e is phosphorylated in a rapamycin- and PI3-kinase-sensitive manner, demonstrating that the rapamycin-sensitive mtor pathway controls phosphorylation of this site [18]. Furthermore, PKC f has also been identified as a component of the rapamycin-sensitive complex that phosphorylates the hydrophobic-motif of PKC d and e [39]. In this study, we analysed the mechanism of stimulus-induced phosphorylation of PKC h at the hydrophobic-motif in T lymphocytes. We demonstrate that the rapamycin-sensitive mtor pathway does not control hydrophobic-motif phosphorylation of PKC h in response to PMA or anti-cd3/cd28 stimulation. This is in contrast to reported studies of interferon stimulation of T lymphocytes in which rapamycin-sensitive phosphorylation of PKC h on the activation-loop is induced [40]. It has recently been uncovered that mtor exists in at least two distinct complexes within cells; one that binds to a protein called raptor (raptor mtor) that is sensitive to the effects of rapamycin and another form that instead binds to rictor and is largely rapamycin-insensitive (rictor mtor) discussed in [35]. Recent studies have deduced that the hydrophobic-motif of PKB, which is insensitive to rapamycin treatment, is phos-

10 628 M. Freeley et al. / Biochemical and Biophysical Research Communications 334 (2005) phorylated by the rapamycin-insensitive rictor mtor complex [35]. Although we ruled out an effect of the rapamycin-sensitive raptor mtor complex in phosphorylating the hydrophobic-motif of PKC h, we cannot exclude the possibility that the rapamycin-insensitive rictor mtor complex controls PKC h hydrophobicmotif phosphorylation. Our data imply that the hydrophobic-motif of PKC h is not a site of autophosphorylation in T cells, due to the fact that rottlerin, a pharmacological inhibitor of PKC h [21 23], did not inhibit the anti-cd3/cd28- mediated phosphorylation of PKC h at the hydrophobic-motif. Furthermore, in a recent study in murine T cells [19], a pan-pkc inhibitor, Go6983, did not block the anti-cd3/cd28-induced hydrophobic-motif phosphorylation of PKC h, thus arguing against autophosphorylation of this site. Instead, this stimulus-induced phosphorylation was dependent upon Src and PI3-kinase activities [19]. Our results in human cells are in partial agreement with the studies from murine cells, because we also found that hydrophobic-motif phosphorylation of PKC h is not due to autophosphorylation but instead requires PI3-kinase activity and cpkc activity, suggesting that PKC a/b I may signal upstream of PKC h in human T cells. Our finding that the hydrophobic-motif site of PKC h is sensitive to pharmacological inhibitors of cpkc isoforms is consistent with a report that PKC a functions upstream of PKC h to activate the NF-jB pathway following anti-cd3/cd28 stimulation [41]. An intriguing possibility is that similar to cpkcs controlling hydrophobic-motif phosphorylation of PKB in certain cell types [32 34], this PKC subfamily may also regulate hydrophobic-motif phosphorylation of PKC h in T lymphocytes. Although the phosphorylation of PKC h at the hydrophobic-motif in T lymphocytes is inducible with cellular stimulation, the impact on enzyme function is unclear. Data from in vitro kinase assays have demonstrated that mutation of Ser695 to alanine (Ser695A) substantially impairs the catalytic activity of the protein [13], unlike what was been described for this site on PKC b II [3]. However, expression of a Ser695A PKC h mutant in T cells does not affect the ability of the enzyme to activate NF-jB in response to anti-cd3/ CD28 stimulation in vivo [13]. This suggests that the hydrophobic-motif site of PKC h may play additional roles beyond regulation of kinase activity, such as intracellular distribution, binding to or disassociation from regulatory proteins (e.g., kinases such as PDK-1 or phosphatases), promoting the phosphorylation of other sites, and stabilisation of the active conformation (increased DAG binding, resistance to intracellular phosphatases, thermal inactivation, oxidation, and proteolysis). The hydrophobic-motif of PKC has been shown to act as a docking site onto which PDK-1, the activation-loop kinase, can attach and catalyse activationloop phosphorylation [3]. In our experiments, we were unable to demonstrate co-immunoprecipitation of endogenous PDK-1 and endogenous PKC h in unstimulated or activated Jurkat T lymphocytes (data not shown) and second, as already described in Figs. 1 3, we did not observe any increase in activation-loop phosphorylation following stimulation. It seems unlikely therefore that the physiological role of Ser695 phosphorylation of PKC h is to act as a docking site for PDK-1 in stimulated T cells. Because Ser695 phosphorylation of PKC h coincides with translocation and activation, we reasoned that phosphorylation of this site could control membrane translocation in response to anti-cd3/cd28 stimulation. However, as demonstrated in Fig. 7, pharmacological agents that inhibited hydrophobic-motif phosphorylation of PKC h, such as Go6976 or staurosporine, did not inhibit the anti-cd3/cd28 membrane translocation. This demonstrates therefore that membrane translocation of PKC h does not absolutely require Ser695 phosphorylation. Unlike anti-cd3/cd28 stimulation of T cells, which induces membrane translocation of PKC h (and lipid raft localisation), antigen stimulation of T lymphocytes induces selective translocation of PKC h (but not other PKCs) to the immunological synapse, a subdomain of the plasma membrane on the T cell that contacts the antigen-presenting cell [6]. The possibility that Ser695 phosphorylation is required for localisation of PKC h to the immunological synapse in antigen-stimulated T lymphocytes (rather than the plasma membrane) therefore warrants further investigation. It was reported that translocation of PKC h to the membrane-rich fraction in response to anti-cd3/cd28 stimulation in Jurkat T cells is sensitive to inhibitors of PI3-kinase [24], an effect that we have not observed in our study. The reasons for these differences are unclear at present. Importantly, inhibitors of PI3-kinase do not block the translocation of PKC h to the immunological synapse in antigen-stimulated T cells [42]. The crystal structure of the catalytic domain of human PKC h has recently been elucidated [43]. Similar to the solved crystal structure of PKB, it has been proposed that the function of a phosphorylated Ser695 residue on PKC h is to tighten intramolecular association between the hydrophobic-motif and the N-terminal lobe of the kinase. The consequence of this intramolecular association is predicted to align the kinase domain favourably for catalysis and/or to increase protein stability. A role for phosphorylated Ser695 in controlling PKC h activity and protein stability therefore seems likely. However, signalling downstream of PKC h (i.e., NF-jB activation) is not impaired in the Ser695A mutant [13] and as such implies that the predominant role of this site, like what has been described for other PKCs, could be to control protein stability [44 46].

11 M. Freeley et al. / Biochemical and Biophysical Research Communications 334 (2005) In summary, we have shown that T cell activation induces hydrophobic-motif phosphorylation and membrane translocation of PKC h. This stimulus-induced phosphorylation of PKC h at this site is independent of the rapamycin-sensitive mtor pathway but is sensitive to inhibitors of PI3-kinase and a conventional PKC inhibitor. This suggests that the PI3-kinase pathway and cpkcs (such as PKC a) control PKC h phosphorylation at this site and regulate its enzymatic activity and intracellular function in T cells. It appears therefore that cpkcs function as PDK-2-like enzymes for this critical mediator of T cell signalling. Hydrophobic-motif phosphorylation is not critical for membrane translocation however, which suggests that this site controls other important aspects of kinase function. 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