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Originally published In Press as doi:10.1074/jbc.M602075200 on June 18, 2006

J. Biol. Chem., Vol. 281, Issue 32, 23218-23226, August 11, 2006
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Protein Kinase C {delta} ({delta}PKC)-Annexin V Interaction

A REQUIRED STEP IN {delta}PKC TRANSLOCATION AND FUNCTION*

Viktoria Kheifets, Rachel Bright, Koichi Inagaki, Deborah Schechtman, and Daria Mochly-Rosen1

From the Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94305

Received for publication, March 6, 2006 , and in revised form, May 17, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein kinase C (PKC) plays a critical role in diseases such as cancer, stroke, and cardiac ischemia, and participates in a variety of signal transduction pathways such as apoptosis, cell proliferation, and tumor suppression. Though much is known about PKC downstream signaling events, the mechanisms of regulation of PKC activation and subsequent translocation have not been elucidated. Protein-protein interactions regulate and determine the specificity of many cellular signaling events. Such a specific protein-protein interaction is described here between {delta}PKC and annexin V. We demonstrate, at physiologically relevant conditions, that a transient interaction between annexin V and {delta}PKC occurs in cells after {delta}PKC stimulation, but before {delta}PKC translocates to the particulate fraction. Evidence of {delta}PKC-annexin V binding is provided also by FRET and by in vitro binding studies. Dissociation of the {delta}PKC-annexin V complex requires ATP and microtubule integrity. Furthermore, depletion of endogenous annexin V, but not annexin IV, with siRNA inhibits {delta}PKC translocation following PKC stimulation. A rationally designed eight amino acid peptide, corresponding to the interaction site for {delta}PKC on annexin V, inhibits {delta}PKC translocation and {delta}PKC-mediated function as evidenced by its protective effect in a model of myocardial infarction. Our data indicate that translocation of {delta}PKC is not simply a diffusion-driven process, but is instead a multi-step event regulated by protein-protein interactions. We show that following cell activation, {delta}PKC-annexin V binding is a transient and an essential step in the function of {delta}PKC, thus identifying a new role for annexin V in PKC signaling and a new step in PKC activation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein-protein interactions determine the specificity of many cellular signaling events. One set of such interactions is mediated by RACKs2 (receptors for activated C-kinase) that localize different activated protein kinase C (PKC) isozymes to distinct subcellular sites and determine isozyme-specific roles by anchoring each active PKC next to its corresponding substrates (1). Other examples of anchoring proteins include AKAPs (2), and STICKs (3). The importance of anchoring for downstream signaling is well recognized (4, 5).

PKC isozymes are usually found in the cell cytosol when inactive. Following the generation of the second messenger diacylglycerol, active PKCs translocate to the cell particulate fraction (6). Although the molecular basis of PKC translocation has not been elucidated, the critical role of PKC-RACK interaction in mediating specific PKC functions both in vitro and in vivo has been demonstrated using peptides designed to inhibit or increase PKC-RACK binding (7-9).

We previously found that both of the isozyme-specific RACKs identified to date (betaIIRACK and {epsilon}RACK) share a short sequence of homology with their corresponding PKCs (10, 11); e.g. SVEIWD in betaIIPKC241-246 is homologous to SIKIWD in betaIIRACK-(255-260). The RACK homologous sequences in the corresponding PKCs were termed pseudoRACK sequences ({psi}RACKs) (8, 10). Peptides corresponding to the {psi}RACK site act as allosteric agonists by interfering with the auto-inhibitory intramolecular interaction between the {psi}RACK site and the RACK binding site within PKC, thus stabilizing a state in PKC in which the RACK binding site is available for protein-protein interaction (8, 12, 13).

{delta}PKC plays a critical role in diseases such as cancer (14), stroke, and cardiac ischemia (9, 15-17) and participates in a variety of signal transduction pathways such as apoptosis (18, 19), cell proliferation (20-22), and tumor suppression (23). Though much is known about its downstream signaling events, the process of {delta}PKC translocation and activation, and the proteins that regulate it have not been identified.

A potential insight into the {delta}PKC translocation process came from the observation that the small molecule JTV-519 (also known as K201) has been shown to protect hearts from ischemia and reperfusion damage (24) and to modulate the translocation of {delta}PKC (25). There is no evidence that JTV-519 binds to {delta}PKC directly, and therefore it is a possibility that JTV-519 modulates {delta}PKC translocation indirectly through a change in protein-protein interaction during the activation process of {delta}PKC. In a separate study, JTV-519 has been reported to modulate annexin V function (26, 27). Although we do not have access to JTV-519 for this study, based on the observations described above, we hypothesized that annexin V plays a role in the {delta}PKC translocation process.

Indirect evidence made this hypothesis plausible: members of the annexin family of proteins, like the PKC family, bind phospholipids, participate in a variety of similar cellular functions and co-localize with some PKC isozymes in cells (28-30). Members of the annexin family are also substrates of select PKC isozymes, and their cellular activity can be regulated by this PKC-mediated phosphorylation (31-33). However, no direct evidence for a role of annexins in PKC function has been suggested. Here, we determined that annexin V interacts with {delta}PKC and studied the consequences of this protein-protein interaction on {delta}PKC translocation and function. Our results have led us to propose that transient {delta}PKC-annexin V interaction is an essential step in {delta}PKC translocation and function.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Anti-MBP antibodies were obtained from New England Biolabs. Other antibodies, siRNA for annexins, and protein G-agarose beads were obtained from Santa Cruz Biotechnology. Secondary horseradish peroxidase-conjugated antibodies were purchased from Amersham Biosciences. pAnxV and pEAnxV peptides were synthesized and conjugated by a Cys S-S bond to TAT47-57 (34) by SynPep.

Sequence Alignments—Sequences of the human annexin family members (GenBankTM accession numbers: P04083 [GenBank] , NP_001002858, P12429 [GenBank] , P09525 [GenBank] , P08758 [GenBank] , P08133 [GenBank] , P20073 [GenBank] , P13928 [GenBank] , O76027 [GenBank] , NP_665876 [GenBank] , P50995 [GenBank] , P27216 [GenBank] ), and various annexin V species homologs (GenBankTM accession numbers: AAF25883 [GenBank] , BAA07708 [GenBank] , AAB60648 [GenBank] , BAA11012 [GenBank] , NP_001026709) were aligned using MULTALIN (35). {delta}PKC-annexin V fragment alignment (GenBankTM accession numbers: NP_579841 [GenBank] , NP_579841 [GenBank] ) was conducted with LALIGN (36).

Protein Purification—Annexin V, a kind gift from Joel Ernst, was purified from Escherichia coli as described (37). Briefly, transformed E. coli were grown to OD 0.8, then induced with 0.5 mM isopropyl-1-thio-beta-D-galactopyranoside for 4 h, pellets lysed in homogenization buffer (20 mM Tris, pH 7.4, 10 mM EGTA, 2 mM EDTA, 12 mM beta-mercaptoethanol, protease inhibitor mixture (Sigma)). The bacterial lysate was incubated with a liposome column (phospholipids were prepared as described (38), then conjugated to activated HZ beads (Bio-Rad) according to the manufacturer's instructions. After washing with 20 mM Tris, pH 7.4, annexin V was eluted in wash buffer containing 10 µM CaCl2. MBP-{delta}V1, MBP-{epsilon}V1, and GST-{delta}V5 lysates were induced as described for annexin V, and purified using amylose (New England Biolabs) and glutathione (Amersham Biosciences) beads, respectively. A partial PKC purification was conducted as described (38).

ELISA—0.1 µg of pure annexin V was bound to an ELISA plate in carbonate buffer at 4 °C overnight, and blocked with 1% bovine serum albumin. Bacterial lysate expressing {delta}PKC was incubated in 100 mM HEPES (pH 7.4) in the presence of 10 µM CaCl2 for 1 h 37 °C with 0.5 µM peptides where appropriate, and washed with 100 mM HEPES. Amount of {delta}PKC bound was then determined with anti-{delta}PKC antibodies, and followed with a secondary antibody conjugated to alkaline phosphatase, and developed for 3 h using PNPP (Pierce).

Overlay—All bacterial lysates were prepared as described above. Binding of {delta} and {epsilon}PKC fragments and full-length enzyme to annexin V was determined as described (39). Briefly, annexin V bacterial lysate was chromatographed on 12% SDS-PAGE, transferred to nitrocellulose, and incubated with recombinant PKC isoforms or fragments in the presence of 10 µM Ca2+ and phospholipids where necessary. After washing as described (39), the strips were probed with anti-MBP, anti-GST, or anti-PKC antibodies and visualized by ECL.

Co-immunoprecipitation of the {delta}PKC and Annexin V Complex—CHO-K1 cells (ATCC) were grown in F-12 (HAM) nutrient mixture supplemented with Glutamax, 10% fetal bovine serum, and antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin sulfate, all from Invitrogen). Cells were cultured at 37 °C 5% CO2. 48 h prior to experiments, cells were serum-starved. They were stimulated with 10 nM phorbol 12-myristate 13-acetate (PMA) (LC Laboratories) or with 5 mM H2O2 (Sigma) for the indicated duration, as described by Konishi et al. (40). Cells were washed with cold phosphate-buffered saline, and homogenized on ice with trituration in homogenization buffer (20 mM Tris, pH 7.4, 2 mM EDTA, 10 mM EGTA, 0.25 M sucrose, 12 mM beta-mercaptoethanol, 0.1% Triton X-100, protease inhibitor mixture (Sigma), and 1% formaldehyde). We found that 0.1% Triton extracts all the translocatable PKC from the cell. After 30 min at 4 °C, the lysates were quenched with 0.14 M glycine for 20 min at 4 °C, and samples spun at 14,000 rpm at 4 °C. The supernatant was incubated with 1 µg of anti-{delta}PKC antibody (Santa Cruz Biotechnology) for 1 h, followed by protein G beads (Invitrogen) for 3 h at 4 °C. The beads were then washed with wash buffer (20 mM Tris, pH 7.5, 2 mM EDTA, 100 mM NaCl, 12 mM beta-mercaptoethanol, 0.1% Triton), and separated on 7.5% SDS-PAGE, transferred to nitrocellulose, and probed with anti-annexin V (Santa Cruz Biotechnology), followed by visualization with ECL.

Translocation of {delta}PKC—Cells were fractionated as described in Ref. 41. Briefly, after stimulation, cells were washed with cold phosphate-buffered saline, scraped in homogenization buffer as described above (but without cross-linker), and spun at 100,000 x g for 30 min at 4 °C, resulting in the soluble fraction. The pellet was then resuspended in homogenization buffer with 1% Triton X-100, and spun under the same conditions. Where applicable, the cells were preincubated with 1 µM peptide for 15 min prior to stimulation. The samples were then analyzed by Western blot, and loading corrected based on protein concentration, using an internal control such as actin or by Bradford.

siRNA Knockdown of Annexin Protein Level—40% confluent CHO (as described above) or HeLa cells (ATCC) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, and antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin sulfate). Cells were transfected using GeneSilencer (Gene Therapy Systems, Inc) according to the manufacturer's instructions with 20 nM siRNA for annexin family members (Santa Cruz Biotechnology). 24-h post-transfection, cells were serum-starved for an additional 24 h. CHO cells were then stimulated with 10 nM PMA and HeLa cells with 100 µM UDP (Sigma) for the indicated times and fractionated as described above.

Microscopy and Analysis—CHO cells were grown on chambered no. 1 borosilicate coverglass (Lab-Tek), transfected at 50% confluency using FuGENE 6 (Roche) according to the manufacturer's instructions with CFP-annexin V and YFP-{delta}PKC cloned into the pECFP-C1 and pEYFP-C1 vectors, respectively (Clontech), and serum-starved for 24 h. Real-time confocal imaging was conducted on a spinning disk Nipkow confocal microscope. Cells were viewed using an inverted Olympus IX70 microscope with a x40 oil immersion Olympus objective (1.35 NA), and images were acquired with a CCD camera (Hamamatsu) and 2x2 pixel binning. CFP was excited with the 442 nm laser line of a helium-cadmium laser (Kimmon), whereas YFP was imaged with the 514 nm line of an argon ion laser (Melles-Griot). Images were acquired at 27 °C every 5-10 s for 10 min. 100 nM PMA was added to the cell chamber after the 10th image in each time series. Where indicated, the cells were pretreated with nocodazole (10 µM, 30 min). Exposure time was adjusted for photobleaching levels lower than 10%. Fluorescence intensity was measured using Metamorph[regs] data analysis software (Universal Imaging). To monitor the translocation of PKC and increases in FRET signal, a small region of interest was selected from each cell and fluorescence intensity values graphed against time and normalized to the initial fluorescence. FRET was calculated using the formula that corrects for bleed-through and donor concentration as previously described (42) in Equation 1.

Formula(Eq.1)

{alpha} and beta coefficients were calculated from singly transfected cells, where {alpha} = I(442-530)/I(442-480) = 47.6% of CFP bleed-through into the YFP channel, and beta = I(442-530)/I(515-530) = 14.4% of YFP excitation from the CFP channel.

Ex Vivo Model of Ischemia and Reperfusion—Wistar rats (300-350 g) were heparinized (2000 units/kg IP) and then anesthetized with sodium pentobarbital (100 mg/kg IP). The hearts were rapidly excised and then perfused with an oxygenated Krebs-Henseleit solution containing (in mmol/liter) NaCl 120, KCl 5.8, NaHCO3 25, NaH2PO4 1.2, MgSO4 1.2, CaCl2 1.0, and dextrose 10, pH 7.4, at 37 °C in a Langendorff coronary perfusion system (43). The coronary flow rate was kept constant during the experiment at 10 ml/min. Hearts were submerged into a heat-jacketed organ bath set at 37 °C. Coronary effluent was collected to determine creatine phosphokinase (CPK) release from necrotic cells. After 10 min of equilibration, the hearts were subjected to a 30-min global ischemia and a 60-min reperfusion to measure ischemic damage and CPK release during reperfusion. The hearts were perfused with 1 µM TAT47-57 carrier peptide conjugated to pAnxV, TAT47-57 peptide conjugated to EpAnxV, TAT47-57 peptide alone, or vehicle for either 10 min prior to ischemia and/or during the first 10 min of reperfusion.

Infarct Size Measurement—At the end of the reperfusion period, hearts were sliced into 1-mm thick transverse sections and incubated in triphenyltetrazolium chloride solution (1% in phosphate buffer, pH 7.4) at 37 °C for 15 min, as reported previously (44). Infarct size was expressed as a percentage of the total LV muscle mass.

Statistical Analysis—For quantitative analysis, autoradiographs were scanned and quantified using NIH Image software. Statistical significance for all analyses was calculated using 2-tail type 2 Student's t test (Microsoft Excel).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
A Short Homologous Sequence Indicative of Protein-Protein Interaction Is Found in Annexin V and {delta}PKC—As mentioned above, PKCs and their corresponding PKC-binding proteins, RACKs, share a short sequence of homology (6-8 amino acids long, Ref. 8). The RACK-like sequence on the PKC was termed {psi}RACK, as it participates in an inhibitory intramolecular interaction with the RACK binding site on the PKC. This intramolecular interaction is broken upon PKC activation, allowing PKC binding to its RACK. We reasoned that if annexin V binds selectively to {delta}PKC during its activation process, annexin V might also have a short {delta}PKC homologous sequence. Using LALIGN (36), we searched for a short homology between annexin V and {delta}PKC. We focused on the V1/C2 domain of {delta}PKC (amino acids 1-123), because that domain is critical for {delta}PKC anchoring and mediation of many critical intracellular processes (45-47). A portion of the highest ranked region of homology from the LALIGN output features the characteristic charge difference (glutamate, Glu78 in {delta}PKC to arginine, Arg161 in annexin V) (red in Fig. 1A), which we have previously found to be indicative of a potential protein-protein interaction site for PKC (8, 10). Importantly, as expected for a selective protein-protein interaction, the {delta}PKC homologous sequence in annexin V is unique and not found in other members of the annexin family (Fig. 1B), and is conserved among different species (Fig. 1C).

Surprisingly, the annexin V-like homology sequence in {delta}PKC is located within the previously identified {psi}{delta}RACK sequence ({delta}PKC-(74-81); Ref. 7), further suggesting that that annexin V might be a {delta}PKC-binding protein. In addition, the {psi}{delta}RACK-like sequence in annexin V is in close proximity (3.2 Å, Fig. 1E) to the binding site on annexin V for JTV-519 (27), a compound that regulates {delta}PKC function (25), indicating the potential importance of the {psi}{delta}RACK-like sequence on annexin V for {delta}PKC binding. We therefore hypothesized that there is a specific protein-protein interaction between {delta}PKC and annexin V that is mediated, at least in part, via the region of annexin V that is homologous between the two proteins.

{delta}PKC Binds Annexin V in Vitro—We first set out to determine whether {delta}PKC binds annexin V in vitro, using immobilized annexin V and recombinant full-length {delta}PKC as well as the {delta}V1 (the C2 domain of {delta}PKC), {delta}V5, and {epsilon}V1 fragments. All these domains have been previously shown to participate in important protein-protein interactions (45, 46, 48). Domains V1/C2 and V5 of {delta}PKC (Fig. 1F, red), as well as full-length {delta}PKC bound to annexin V (Fig. 1G). In contrast, the V1 region of another member of the novel PKC isozymes, {epsilon}PKC, did not bind to annexin V (Fig. 1G), indicating selectivity of this interaction. Phosphatidylserine and diacylglycerol micelles, to which both annexin V and {delta}PKC can bind independently, did not affect {delta}PKC-annexin V association in vitro (data not shown), suggesting that annexin V binding to {delta}PKC does not depend on a lipid bridge between these two lipid-binding proteins (49).

Association of {delta}PKC-Annexin V Precedes {delta}PKC Translocation in Cells—We next determined whether, in the absence of overexpression of any of the proteins in question, {delta}PKC and annexin V interact in cells. The complexes formed with {delta}PKC were then immunoprecipitated with anti-{delta}PKC antibodies and probed for the presence of annexin V at the combined molecular mass of ~110 kDa. Stimulation of CHO cells with PMA, 10 nM caused about a 4-fold increase in the amount of annexin V-{delta}PKC complex within 0.5 min of treatment (Fig. 2A, left). The interaction was short-lived, and after 1 min of stimulation the amount of annexin V-{delta}PKC complex was reduced often below the levels obtained prior to PMA treatment. Transient association between annexin V and {delta}PKC was also observed following activation of {delta}PKC with H2O2 (5 mM, 0.5-1 min of treatment, Fig. 2A, right). Therefore, annexin V-{delta}PKC interaction occurs early after cell stimulation, regardless of the means used to stimulate {delta}PKC.


Figure 1
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  		FIGURE 1.
Selective binding of {delta}PKC to annexin V in vitro. A, sequence similarity between annexin V and {delta}PKC sequences, found within the {psi}{delta}RACK sequence (Ref. 7, blue box), with an amino acid charge difference characteristic of a PKC-RACK relationship (highlighted in red). B, {psi}{delta}RACK-like sequence in annexin V is not found in other members of the annexin family. In the consensus bottom line, + indicates no homology, capital letters indicate >90% identity, lowercase letters indicate >50% identity. C, alignment of the {psi}{delta}RACK-like region in rat annexin V with annexin V from other species was obtained as in B. In the consensus bottom line, + indicates no homology, capital letters indicate >90% identity, lowercase letters indicate >50% identity. D, structure of annexin V with the {psi}{delta}RACK-like site highlighted in light blue and JTV-519 in yellow, shown in stick representation (27). E, closer view of the boxed region in D (rotated slightly, relative to the image in D). JTV-519 binds adjacent to the {psi}{delta}RACK-like site on annexin V. F, schematic of {delta}PKC domains structure. {delta}PKC is a member of the calcium-independent novel family of PKC isozymes. The V1 and the V5 domains (in red) have been reported to participate in protein-protein interactions. G, recombinant {delta}PKC domains, MBP-{delta}V1 and GST-{delta}V5, bind to annexin V; partially purified rat brain {delta}PKC full-length, but not {epsilon}PKC, binds to annexin V. Western blots were probed for the respective PKC isozymes. A representative figure of four independent experiments.

 
Activation of PKC results in its translocation from the cell soluble to the cell particulate fraction (6). We therefore determined the timing of annexin V-{delta}PKC complex formation relative to the timing of {delta}PKC translocation to the particulate fraction. Whereas maximum annexin V-{delta}PKC complex accumulation was found between 0.5 and 1 min (Fig. 2, A and C), translocation of {delta}PKC from the soluble to the cell particulate fraction occurred later; it began only after 1 min of PMA stimulation and reached a maximum after 5 min of stimulation (Fig. 2, B and C). Based on the above observations, we concluded that formation of the {delta}PKC-annexin V complex preceded {delta}PKC translocation from the cytosol to the cell particulate fraction.

ATP and Microtubules Are Important for {delta}PKC-Annexin V Complex Disassociation—Because of the fast and transient PMA-induced association/dissociation of {delta}PKC with annexin V, we next determined whether {delta}PKC-annexin V complex formation requires energy. Whereas under native conditions (37°C), the disassociation of the complex was completed by 1 min of treatment with 10 nM PMA (Fig. 2D, left top panel), substantial amounts of {delta}PKC in complex with annexin V were found even after 5 min of PMA stimulation, when the cells were treated with 2-deoxyglucose to deplete cellular ATP (50) (Fig. 2D, bottom left). ATP might be required for enzymatically mediated disassociation of the complex or the actual movement of the complex to its site of disassociation, but determination of the exact role of ATP was beyond the scope of this study. As annexins are well known to bind to actin cytoskeleton and to affect the dynamics of actin fibers (51), we tested different cytoskeleton perturbation agents. Whereas disrupting actin elements with cytochalasin D did not affect kinetics of the {delta}PKC-annexin V complex (Fig. 2D, top right), pretreatment with the microtubule-destabilizing agent, nocodazole, greatly delayed complex dissociation (Fig. 2D, bottom right). Based on this observation, we tested the role of microtubles in {delta}PKC translocation, and found that nocodazole blocks PMA-induced translocation of {delta}PKC (Fig. 2E), thus confirming the importance of microtubules in {delta}PKC translocation process. Together, our data suggest that an initial step of {delta}PKC translocation to the cell particulate fraction involves its association with annexin V, and that dissociation of the complex is an energy-dependent process that requires intact microtubule filaments, but is independent of actin polymerization.

{delta}PKC-Annexin V Interaction as Observed by FRET—Both {delta}PKC and annexin V are cytosolic proteins in inactive cells, and co-localization studies by immunohistochemistry can be misleading, because they may be interpreted to show that the two proteins interact in unstimulated cells. Therefore, real-time fluorescent imaging was conducted with CFP-annexin V and YFP-{delta}PKC, and FRET was measured upon stimulation of CHO cells with PMA. Whereas annexin V remains cytosolic throughout the experiment (Fig. 3A, top panel), {delta}PKC translocation from the cytosol to the membrane can be observed starting from 2 min of stimulation and reaches its maximal translocation by 9 min (Fig. 3A, middle panel). FRET signal increased predominantly in the cytosolic region of the stimulated cells (Fig. 3A, bottom panel, quantitated in Fig. 3B). The basal FRET level at the cell membrane was higher than found in the cytosol, but did not increase after cell stimulation. Interestingly, the time course of FRET increase followed the time course of {delta}PKC translocation (Fig. 3C). Importantly, the fold increase of the FRET signal was comparable to the fold increase of complex association as seen in the co-immunoprecipitation study (Fig. 2B). It is important to note that while physiological expression of annexin V and {delta}PKC resulted in transient interaction upon stimulation (Fig. 2), the overexpressed proteins interacted throughout the presence of stimulus, suggesting impairment of the proper translocation machinery. In fact, pretreatment of cells overexpressing {delta}PKC with nocodazol did not inhibit {delta}PKC translocation, unlike that seen in the non-overexpressing cells (Fig. 2E versus Fig. 3C). These results suggest that whereas {delta}PKC and annexin V interact in the overexpressing conditions upon {delta}PKC stimulation, the cellular machinery involved in translocation and anchoring may be limited in its amounts and only functions properly when the proteins are present at endogenous levels.


Figure 2
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  		FIGURE 2.
Binding of {delta}PKC and annexin V in cells. A, representative blot of co-immunoprecipitation of {delta}PKC-annexin V complex with anti-{delta}PKC antibodies from cross-linked CHO cell lysates, probed with anti-annexin V and then with anti-{delta}PKC. Molecular mass of the complex corresponds to ~110 kDa, the sum of 73 kDa {delta}PKC and 35 kDa annexin V. Comparable amounts of {delta}PKC are immunoprecipitated in each condition. Maximum complex formation is observed at 0.5-1 min of 10 nM PMA stimulation (left panel). Stimulation with 5 mM H2O2 resulted in a similar time course of complex association (right panel). B, {delta}PKC translocation of PMA stimulated CHO cells: time course. {delta}PKC translocation to the membrane begins only after 1 min of PMA stimulation and is completed only after 5 min. This is a representative Western blot. C, dPKC-annexin V complex formation precedes {delta}PKC translocation. Quantitation of four independent co-immunoprecipitation experiments as described in A (*, p < 0.02 relative to basal level of complex prior to stimulation) and quantitation of four independent translocation experiments as described in B. (*, p < 0.02 relative to the basal level prior to stimulation.) D, disassociation of the {delta}PKC-annexin V complex. Whereas during PMA stimulation, the complex disassociates to the basal level after 1 min of stimulation, ATP depletion using 2-deoxyglucose (20 mM, 4-h pretreatment in glucose-free buffer) slows down the disassociation. Pretreatment of cells with nocodazole (10 µM, 30-min pre-treatment) blocked complex disassociation, whereas cytochalasin D (2 µM, 1-h pretreatment) had no effect. This is a representative blot of three independent experiments. E, nocodazole prevents {delta}PKC translocation. {delta}PKC translocates from the cytosolic fraction upon PMA stimulation (10 nM for indicated times). Pretreatment of cells for 30 min with 10 µM nocodazole prevents the disappearance of {delta}PKC from the cytosolic fraction regardless of the duration of PMA stimulation. Actin loading control confirms equal loading. This is a representative blot of four independent experiments.

 


Figure 3
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  		FIGURE 3.
Interaction of {delta}PKC with annexin V as determined by FRET. A, snapshots of real-time imaging after 100 nM PMA stimulation. Whereas annexin V (top panel) remains cytosolic, {delta}PKC (middle panel) translocates from cytosol to the cell membrane. FRET (bottom panel) in pseudocolor indicates protein-protein interaction. Regions chosen for quantitation are shown in the first panel of FRET. The calibration bar (bottom left) shows fold increase of the FRET signal. A representative set of frames from ten independent experiments. B, quantitation of FRET. Pixel intensity was monitored in the selected regions as a function of time after 100 nM PMA stimulation. FRET signal indicates the interaction of {delta}PKC and annexin V, and is seen to increase upon PMA stimulation in a time-dependent manner. Representative of 30 cells from 10 independent experiments. C, quantitation of {delta}PKC translocation. Drawing a region in the cytosolic region of the cell and monitoring decline in pixel intensity allows the measurement of {delta}PKC translocation. {delta}PKC begins translocation from the cytosol after 1 min of PMA stimulation. 10 µM nocodazole pre-treatment for 30 min did not alter the translocation profile of overexpressed {delta}PKC. An average of six cells from three independent experiments is shown.

 
A Peptide Designed to Block {delta}PKC-Annexin V Complex Formation Blocks {delta}PKC Translocation—Because {delta}PKC-annexin V binding occurs prior to accumulation of {delta}PKC at the particulate fraction (Fig. 2C), we tested the hypothesis that annexin V is required for {delta}PKC translocation by designing a peptide to specifically block this interaction. We have previously shown that peptides derived from the PKC-binding proteins inhibit PKC translocation and function (10, 52). Therefore, we tested whether a peptide corresponding to the {delta}PKC homology site on annexin V will inhibit {delta}PKC translocation and function. (We have extended the length of the annexin-derived peptide (AnxV-(157-164)) by one amino acid at each side, to correspond to the length of the {psi}{delta}RACK peptide (7) (Fig. 1A) and to prevent potential effects of the free carboxyl and amino groups at the ends of the peptide.) The annexin V-derived peptide was synthesized and conjugated to TAT47-57 carrier to enable intracellular delivery of the peptide across biological membranes (34). If annexin V interaction with {delta}PKC is required for {delta}PKC translocation, we predicted that the annexin V-derived peptide (pAnxV) should block stimulation-induced translocation of {delta}PKC by binding to {delta}PKC and preventing its association to annexin V. When used in CHO cells, pretreatment with 1 µM pAnxV blocked PMA-induced translocation of {delta}PKC to the particulate fraction (Fig. 4A; measured after 5 min of PMA stimulation). As discussed earlier, there is a charge difference in the homologous sequence between annexin V and {delta}PKC (Arg161 to Glu78, respectively; see Fig. 1A), similar to that observed in all the homologous sequences between PKCs and their binding proteins (8). As expected, when the arginine in annexin V was replaced with the glutamate found in {delta}PKC (EpAnxV), the peptide lost its inhibitory effect on {delta}PKC translocation (Fig. 4A, left panel). Importantly, neither peptide affected PMA-induced translocation of {epsilon}PKC (Fig. 4A, right panel), indicating the selectivity of the annexin V-derived peptide effect on {delta}PKC translocation. The pAnxV peptide was also tested in the co-immunoprecipitation studies as described in Fig. 2A, and was seen to block {delta}PKC-annexin V complex formation (Fig. 4B). In addition, pAnxV inhibited {delta}PKC-annexin V binding in an ELISA assay, whereas EpAnxV did not affect it significantly (Fig. 4C). Together, these data suggest that pAnxV peptide corresponds to at least a part of the {delta}PKC binding site on annexin V, and that annexin V-{delta}PKC interaction is required for stimulation-induced {delta}PKC translocation from the cell soluble to the cell particulate fraction.


Figure 4
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  		FIGURE 4.
pAnxV blocks binding of {delta}PKC to annexin V and {delta}PKC translocation. A, pAnxV (1 µM, 15-min pretreatment) inhibits {delta}PKC (left panel), but not {epsilon}PKC (right panel), translocation in CHO cells. Data are percent of PKC present in the membrane fraction over total PKC after 10 nM PMA stimulation (5 min). Mutant peptide R161E (EpAnxV) does not inhibit translocation (n = 6, *, p < 0.001, - versus PMA; #, p < 0.001 PMA versus pAnxV+PMA). Representative blot of {delta}PKC translocation from soluble (S) to the particulate (P) fraction is also shown (bottom panel). B, pAnxV blocks {delta}PKC-annexin V complex formation in cells. 1 µM pAnxV (15-min pretreatment) blocks the association of {delta}PKC with annexin V as observed by cross-linking immunoprecipitation. This is representative of three experiments. C, pAnxV blocks association of {delta}PKC with annexin V in vitro. 0.5 mM pAnxV inhibited binding of {delta}PKC to immobilized annexin V, whereas EpAnxV did not show a significant inhibition of binding. n = 3, *, p < 0.006, pAnxV versus -;#, p < 0.007 pAnxV versus EpAnxV.

 
Annexin V Knockdown Prevents {delta}PKC Translocation—To further demonstrate that the interaction of {delta}PKC with annexin V is necessary for {delta}PKC translocation to the particulate fraction, we used an alternative approach; annexin V was knocked down using a pool of siRNA (Santa Cruz Biotechnology). When annexin V levels were reduced by ~80%, PMA-induced translocation of {delta}PKC was similarly inhibited, whereas {epsilon}PKC translocation was unaffected (Fig. 5A). We confirmed that the same selective inhibition of {delta}PKC translocation by annexin V knockdown is observed when cells are treated with a more physiologically relevant hormone stimulation. Using HeLa cells, the purinergic receptor, P2Y, was stimulated by UDP and the role of annexin V in {delta}PKC translocation was determined. Treatment with siRNA of these cells reduced annexin V levels by 80% without affecting the levels of other annexins (e.g. annexin IV, Fig. 5B). Importantly, {delta}PKC translocation was lost in the annexin V siRNA-treated cells, but not in the annexin IV siRNA-treated cells (Fig. 5C). Therefore, the presence of annexin V in cells is specifically required for activation-induced translocation of {delta}PKC to the particulate fraction in at least two cell types and as determined by at least two modes of PKC activation.

{delta}PKC-Annexin V Complex Formation Is Required for {delta}PKC Function—We next determined if {delta}PKC-annexin V complex formation is required for {delta}PKC-mediated function, in vivo. We previously found that, in models of cardiac ischemia, inhibition of {delta}PKC translocation with {delta}V1-1 inhibits cardiac damage (9, 44). Because we suggest that interaction of {delta}PKC with annexin prior to translocation is essential in the process of translocation, we hypothesized that the annexin V-derived peptide would also be cardioprotective when administered prior to {delta}PKC activation (Fig. 6A). When hearts were treated with the annexin V-derived peptide, pAnxV, prior to ischemia, there was a 70% reduction in infarct size (Fig. 6, B and C) and ~50% reduction in cell necrosis as measured by the leakage of the cardiac enzyme, CPK, into the heart perfusate (Fig. 6D). The extent of protection was similar to the effect observed with the {delta}PKC-RACK interaction inhibitor, {delta}V1-1, in the same model (7, 9). Note that EpAnxV, which did not inhibit {delta}PKC translocation (Fig. 3A), also did not inhibit ischemia-induced {delta}PKC-mediated damage to the heart (Fig. 6, B-D). These data indicate that {delta}PKC-annexin V interaction is an essential step in {delta}PKC function, and confirm the importance of {delta}PKC-annexin V complex formation as an initial step for {delta}PKC function in vivo.


Figure 5
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  		FIGURE 5.
Binding of {delta}PKC to annexin V is critical for {delta}PKC translocation. A, {delta}PKC translocation is inhibited in cells treated with annexin V siRNA, but not mock-transfected cells (left panel). {epsilon}PKC translocation was not affected by annexin V knock-down (right panel; n = 3, *, p < 0.0005, - versus PMA; #, p < 0.001 PMA versus pAnxV+PMA). B, knock-down levels of annexin V and annexin IV in HeLa cells using siRNA. Annexin IV and V total levels in cell lysate are shown following 48 h of transfection with 20 nM siRNA. Shown is a representative blot (bottom panel) and quantitation of three independent experiments (upper panel). *, p < 0.005, - versus anxIV siRNA; #, p < 0.001, - versus anxV siRNA. C, {delta}PKC translocation is inhibited in cells treated with annexin V siRNA (solid line), but not in cells treated with annexin IV siRNA (dashed line). HeLa cells transfected for 48 h with 20 nM annexin V or annexin IV siRNA were serum-starved for 24-h post-transfection, and stimulated by 100 µM UDP for times indicated. (n = 3, *^#, p < 0.005 for all time points except 0 and 5 min of stimulation).

 


Figure 6
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  		FIGURE 6.
Association of {delta}PKC with annexin V is critical for downstream signaling in an ex vivo model of ischemia/reperfusion. A, isolated rat hearts were perfused with pAnxV or EpAnxV (1 µM) for 10 min prior to a 30-min no-flow ischemia and for 10 min at the onset of 60 min of reperfusion. B, upon completion of experiment, hearts were sliced and stained with TTC to visualize live tissue red and dead tissue white. Representative heart slices showing TTC staining. C, infarct size was measured by taking the average of the white area on both sides of the tissue slices. pAnxV treatment reduced infarct size by ~70%, whereas EpAnxV had no effect (n = 3, *, p < 0.05). D, CPK release was assessed in the heart perfusate during the duration of reperfusion. Treatment with pAnxV resulted in a >50% reduction in necrosis, whereas treatment with EpAnxV did not have statistical difference from control heart (n = 3, *, p < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we provide evidence that translocation of {delta}PKC to the cell particulate fraction is not simply a diffusion-driven process. Our study provides evidence that {delta}PKC activation is a complex process involving selective and transient binding of the activated isozyme to annexin V. We found that {delta}PKC-annexin V interaction precedes {delta}PKC translocation to the cell particulate fraction and that microtubule integrity and energy are required for the dissociation of the complex. We showed that {delta}PKC binding to annexin V is an essential step in {delta}PKC translocation; annexin V knockdown with siRNA selectively inhibited stimulation-induced {delta}PKC translocation, but not translocation of other PKC isozymes. In addition, a peptide, derived from annexin V and corresponding to the interaction site of {delta}PKC on annexin V, acted as a selective inhibitor of {delta}PKC translocation. This peptide also selectively inhibited {delta}PKC function in vivo; treating hearts with the annexin-derived peptide prior to ischemic insult inhibited {delta}PKC-mediated cardiac damage. Together, our data demonstrate that {delta}PKC-annexin V interaction precedes PKC translocation, and is an essential step in the function of {delta}PKC, thus modifying the paradigm for PKC activation (Fig. 7). The exact role of annexin V in {delta}PKC function is still not clear, but this work suggests that annexin V may serve as a shuttle protein on microtubules, moving {delta}PKC to its subcellular destination. Because no direct interaction of annexins has been reported with microtubules, we hypothesize that this movement might be facilitated through annexin V-mediated membrane vesicle binding that is transported on the microtubules. The suggestion that microtubules transport activated PKC to the particulate fraction appears plausible in view of data that translocation of another PKC isozyme, {alpha}PKC, is also dependent on microtubule integrity (53). As annexin V translocates to membranes upon increase in intracellular calcium levels (54, 55), annexin V may act as an early sensor in the {delta}PKC activation cascade, thus initiating {delta}PKC translocation and activation (Fig. 7). Though the RACK for {delta}PKC has yet to be identified, unique {delta}PKC signaling suggests the presence of an isozyme-specific anchoring protein ({delta}RACK) that determines proper subcellular location and access to specific substrate for phosphorylation. However, the details of the final steps of the translocation process for {delta}PKC remain to be elucidated.

There are multiple reports that PKC translocation is independent of cytoskeletal elements (56, 57). However, most of this work was conducted with overexpressed PKCs. Here we demonstrate that overexpressed {delta}PKC no longer requires microtubules for translocation to the cellular membrane, thus emphasizing the importance of studies at endogenous levels for identification of physiologically relevant cellular mechanisms. In addition, unlike the endogenous {delta}PKC-annexin V association, the interaction of the overexpressed {delta}PKC and annexin V was not transient, likely because of the fact that the levels of these proteins exceed the endogenous "final" {delta}PKC binding partner, the {delta}RACK. This observation is not surprising. We have observed that overexpressed activated GFP-{epsilon}PKC is not localized to the same cell compartment as endogenous {epsilon}PKC,3 and Ping and co-workers (58) showed that when {epsilon}PKC is overexpressed, it binds not only to {epsilon}RACK following activation, but also to the betaIIRACK. Together, these discrepancies emphasize the importance of studies at the endogenous levels when PKC activation mechanisms are investigated.


Figure 7
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  		FIGURE 7.
Scheme of {delta}PKC activation and translocation. Inactive cytoplasmic {delta}PKC (Step 1) binds to annexin V (in red) as the initial step after {delta}PKC activation (Step 2) through, at least in part, the {psi}{delta}RACK-like site on annexin V (highlighted in blue), thus displacing the inhibitory intramolecular interaction within {delta}PKC mediated by the {psi}RACK site (dark gray on PKC). Annexin V-{delta}PKC complex then translocates in a microtubule- and ATP-dependent manner, possibly on vesicles carried by kinesin motor (Step 3). Disassociation of the complex allows complete translocation and anchoring of the active {delta}PKC to the particulate fraction, where it binds to its isozyme-specific anchoring RACK (Step 4), thus facilitating substrate (S) phosphorylation and downstream signaling (Step 5). Inactivation of the PKC (Step 6) may return the system to the basal state.

 
It was previously suggested that PMA is not necessarily sufficient to induce proper translocation of PKCs. For example, Vallentin et al. (59) showed that PMA did not include translocation of {alpha}PKC in single, isolated cells, whereas it caused translocation to cell-cell contacts when cells were in a cluster. This result is an example that PMA is an initiator of PKC activation, but does not itself determine translocation of PKC to the proper physiological locations. Though the mechanism underlying this observation is still not fully understood, our work adds to the hypothesis that additional steps involving annexin V, energy and microtubules are required for {delta}PKC translocation.

The effect of annexin V-derived peptide described above merits further discussion. It represents an example for rational design of a pharmacological agent with interesting therapeutic potential, which we show here to prevent ischemic damage to hearts in a model of myocardial infarction. The annexin V-derived peptide was identified by a simple sequence homology search between two unrelated gene products, {delta}PKC and annexin V, which we suspected and now confirmed, to interact in cells in a stimulation-dependent manner. The homology between the two proteins was on a short stretch of six consecutive amino acids (Fig. 1A) and was rather limited: only three amino acids were identical and two other represent relatively conservative substitutions. However, one amino acid of these six represents a difference in charge, from glutamate in {delta}PKC to arginine in annexin V (Fig. 1A). It is because of this charge change that we focused our attention on this sequence; we have previously found that sequence homologies between two other PKC-binding proteins and their corresponding PKCs have such a charge change (8). We suggested that the charge present on the PKC-binding protein, in this case arginine 161 in annexin V, is critical for a high affinity interaction with {delta}PKC (13). As predicted, when a single amino acid substitution in the annexin V-derived peptide (pAnxV) from arginine to glutamate was made, the new peptide (EpAnxV) was inactive as a {delta}PKC inhibitor (Figs. 4 and 6). Therefore, this study demonstrates a potential method to rationally identify sites of protein-protein interactions without extensive mutagenesis analysis or structural analysis of the complex. Furthermore, this method provides an easy and fast approach to make pharmacological agents to determine the role of protein-protein interactions of interest.

A role for annexins in PKC activation may be a common theme in PKC signaling. Previous reports suggest that individual PKC isozymes interact with unique members of the annexin family (e.g. betaPKC-annexin I, {epsilon}PKC-annexin II, and {alpha}PKC-annexin VI) (30, 52, 60-62). It remains to be determined whether, as for {delta}PKC and annexin V, the biological activity of other PKC isozymes requires a step of association with other members of the annexin family and whether association with these annexins also precedes translocation of the corresponding PKC isozymes. Regardless of whether the other annexins have a role in regulating PKC activation and function, our finding that the {delta}PKC-annexin V complex is required for {delta}PKC function alters the paradigm of {delta}PKC activation process. This study further illustrates the importance of understanding protein-protein interactions in signal transduction for the development of potential pharmacological modulators.


   FOOTNOTES
 
* This research was supported by National Institutes of Health Grant HL52141. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Molecular Pharmacology, Stanford University School of Medicine, CCSR, Rm. 3145A, 269 Campus Dr., Stanford, CA 94305-5174. Tel.: 650-725-7720; Fax: 650-723-2253; E-mail: mochly{at}stanford.edu.

2 The abbreviations used are: RACK, receptor for activated C kinase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; pAnxV, peptide designed from annexin V sequence; siRNA, small interfering RNA; CHO, Chinese hamster ovary cells; GST, glutathione S-transferase; ELISA, enzyme-linked immunosorbent assay; CPK, creatine phosphokinase; FRET, fluorescence resonance energy transfer; MBP, myelin basic protein. Back

3 V. Kheifets, D. Schechtman, and D. Mochly-Rosen, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Adrienne Gordon and Dr. Yasuki Kihara for important discussions and insight. FRET expertise was provided by Dr. Tobias Meyer and Dr. Marc Fivaz. The authors also thank Melissa Wong and Philip Vitorino for assistance with the binding studies. Dr. Mochly-Rosen is the founder of KAI Pharmaceuticals, Inc., a company that plans to bring PKC regulators to the clinic. However, none of the work described here is based on or supported by the company.



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