J Biol Chem, Vol. 274, Issue 49, 34765-34772, December 3, 1999

The HIV Nef Protein Alters Ca²⁺ Signaling in Myelomonocytic Cells through SH3-mediated Protein-Protein Interactions^*

Michelangelo Foti, Laetitia Cartier^§, Vincent Piguet^¶, Daniel P. Lew, Jean-Louis Carpentier, Didier Trono^¶, and Karl-Heinz Krause^§**

From the Departments of  Morphology, ^¶ Genetics and Microbiology, and ^§ Geriatrics and the  Division of Infectious Diseases, Geneva Medical School, University of Geneva, CH-1225 Geneva, Switzerland

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Human immunodeficiency virus Nef plays an important role in AIDS pathogenesis. In addition to the well known down-regulation of cell surface receptors (CD4, MHCI), Nef is able to alter cellular signaling. Of particular interest for this study is the ability of Nef to bind with a very high affinity to SH3 domains of myelomonocyte-specific protein-tyrosine kinases of the Src family (Src-like PTK). We have therefore investigated Ca²⁺ signaling in HL60 cells retrovirally transduced with wild type Nef or with a Nef mutant deficient in the SH3-interacting proline-rich motif (Nef_(PXXP)4). In differentiated HL60 cells, Nef markedly altered cellular Ca²⁺ signaling; the amount of intracellularly stored Ca²⁺ was increased, and as a consequence, store-operated Ca²⁺-influx was decreased. This effect was not observed in undifferentiated HL60 cells or in CEM T-lymphocytes and correlated with the differentiation-induced up-regulation of Src-like PTK. The Nef effect on Ca²⁺ signaling depended entirely on the integrity of its PXXP motif. The Src-like PTK p56/59^hck co-immunoprecipitated with both Nef and with the inositol 1,4,5-trisphosphate receptor, providing a possible mechanistic link between the viral protein and intracellular Ca²⁺ stores of the host cell. Collectively, our results demonstrate that the human immunodeficiency virus 1 Nef protein manipulates intracellular Ca²⁺ stores through SH3-mediated interactions in myelomonocytic cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Nef is a highly conserved 27-34-kDa myristoylated HIV¹ protein that belongs to a set of accessory proteins characteristic of primate lentiviruses. Nef is expressed early in the viral replication cycle (1, 2). Results of studies in rhesus macaques suggest that it is a key factor in the pathogenesis of HIV/simian immunodeficiency virus infection and that it plays a particularly important role for the persistence of infection (3, 4). An important role for Nef in HIV pathogenesis was also suggested by analysis of the HIV genome in long term surviving patients (5, 6).

The down-regulation of immunologically relevant cell surface proteins (CD4 and MHCI) is the best documented biological activity of Nef (7). Several studies indicate that Nef physically connects CD4 with components of the trafficking machinery, leading to the formation of endocytic structures that trigger the accelerated internalization and lysosomal sorting of CD4 (8-13). In addition, Nef exerts genetically and biochemically distinguishable effects on the biology of the cell and on viral growth (14, 15). Nef enhances virus replication by facilitating the early post-entry steps of infection, indirectly promoting the efficiency of reverse transcription (16). Nef also perturbs cellular activation pathways both in lymphoid and nonlymphocytic cells (17-20). The mechanisms and significance of these latter effects are poorly understood. The identification of cellular proteins interacting with Nef both in vitro and in vivo have strengthened the concept that Nef acts as a modulator of intracellular activation pathways. Indeed, tyrosine and serine/threonine protein kinases have been reported to interact with Nef (21). In particular, studies in vitro with recombinant proteins and crystal structure determinations have shown that the core region of Nef, via a proline-rich motif, binds with a high affinity to the SH3 domains of Src-like protein-tyrosine kinases (Src-like PTKs) (14, 22). Four members of the Src kinases family, p56^lck, p56/59^hck, p59^fyn, and p53/56^lyn have been shown to be able to interact physically with Nef (14, 22-25). Interestingly, the affinity of Nef-p56/59^hck interaction is the highest known for an SH3-mediated protein-protein binding (23).

The Src family of nonreceptor tyrosine kinases consists of nine members (Src, Lck, Hck, Fyn, Fgr, Yes, Blk, Lyn, and Yrk) that share common structures and regulation (26). These kinases are widely expressed in cells of hematopoietic origin, with most cells expressing more than one family member (27). Src-family kinases have been implicated in multiple signaling events including cell growth (28), T cell antigen receptor signaling (29), Fc receptor signaling (30, 31), integrin signaling (32), glycosyl phosphatidylinositol-anchored protein signaling (33), phagocytosis (34), apoptosis (35), and tumor necrosis factor release (36). There is also growing evidence that Src-like tyrosine kinases are implicated in the control of cytosolic free Ca²⁺ concentration (37-42). In these studies, we used HL60 cells as a cellular model to investigate a possible effect of Nef on Ca²⁺-associated signal transduction pathways in myelomonocytic cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reagents-- Thapsigargin (Tg), ionomycin, 1,25(OH)₂D₃, phorbol 12-myristate 13-acetate (PMA), and Me₂SO were purchased from Sigma, fetal calf serum and geneticin disulfate (G418) were from Life Technologies, Inc., fura-2/AM was from Molecular Probes (Eugene, OR), Complement C tablets (mixture of protease inhibitors) was rom Roche Molecular Biochemicals. All other chemicals were of analytical grade and were obtained from Amersham Pharmacia Biotech, Calbiochem, Sigma, Merck, and Fluka (Buchs, Switzerland). The Ca²⁺-free medium contained 143 mM NaCl, 6 mM KCl, 1 mM MgSO₄, 0.1 mM EGTA, 5.6 mM glucose (0.1%), 20 mM Hepes; 0.1 mM EGTA, pH 7.4. The Ca²⁺ medium had the same ionic composition but was supplemented with 1 mM CaCl₂.

Cell Culture and HL60 Cell Differentiation-- The promyelocytic HL60 cell line stably expressing either Nef, Nef_(PXXP)4, or the empty vector was cultured in RPMI 1640 medium supplemented with 10% fetal calf serum and selected in the presence of 1 mg/ml G418. The CEM cell line expressing Nef was described elsewhere (9). CRE and CRIP cells (43) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% newborn calf serum. HL60 differentiation to the granulocyte phenotype was triggered by adding Me₂SO (final concentration 1.3% v/v for 3 days and then 0.65% for 2 days) to the culture medium. Differentiation toward the monocyte and macrophage phenotype was obtained by adding, respectively, 100 nM of 1,25(OH)₂D₃ for 5 days and 20 nM of PMA for 2 days to the culture medium (44-49). Nef expression did not affect the general morphology of differentiated cells, and cytofluorimetry analysis of specific cell surface markers such as CD14, CD11b, CD11c, CD18, HLA DR, HLA ABC, the fMet-Leu-Phe (fMLP) receptor, and CD71 confirmed that differentiation to each phenotype was efficient under these conditions (data not shown).

DNA Constructions-- The plasmid pLXSN (CLONTECH, Hampshire, UK) as well as pLNefSN were described elsewhere (9). The construct LNef_(PXXP)4-SN was created by ligation of a XhoI-DraIII fragment from pCMX Nef_(PXXP)4) (50) into the XhoI-DraIII-cleaved LNefSN plasmid.

HL60 Retroviral Transduction-- HL60 cell lines stably producing the various murine leukemia virus-based retroviral vectors were generated by infecting CRIP cells with the supernatant of transfected CRE cells in the presence of 8 µg/ml polybrene (43). Transduced CRIP clones were selected in the presence of 0.4 mg/ml G418 (Life Technologies Inc.). HL60 promyelocytes were infected with the various retroviral expression vectors by cocultivation with CRIP producer cell lines and selected in the presence of 1 mg/ml G418 (9).

Antibodies Used in This Study-- R-phycoerythrin-conjugated monoclonal antibodies against CD4, CD11b, CD11c, and fluorescein-5-isothiocyanate-conjugated monoclonal antibodies against CD18 were purchased from DAKO (Copenhagen), R-phycoerythrin-conjugated monoclonal antibodies against HLA DR and HLA (A, B, and C) were from Pharmingen (San Diego, CA), monoclonal antibodies to CD71 were from Roche Molecular Biochemicals, monoclonal antibodies to p56^lck and p59^fyn were from Santa Cruz Biotechnology (Santa Cruz, CA), monoclonal antibodies to p53/56^lyn were from Transduction Laboratories (Lexington, KY), and monoclonal antibodies to CD14 were kindly provided by Dr. J. Pugin (Hópital Cantonal, Geneva). Polyclonal antibodies against p56/59^hck were purchased from Santa Cruz Biotechnology. Polyclonal antibodies against the Ins(1,4,5)P₃-receptor type I Rbt04 were kindly provided by Dr. J. Parys (Leuven, Belgium). Polyclonal antibodies against the HIV-1 Nef were as described previously (50). Anti-mouse/rabbit horseradish peroxidase-conjugated IgG were purchased from Amersham Pharmacia Biotech, and anti-mouse/rabbit fluorescein-5-isothiocyanate-conjugated IgG was from DAKO (Copenhagen).

Flow Cytometry Analysis-- Flow cytometry was performed on a Becton Dickinson FACScalibur. Labeling was performed by incubating cells with fluorochrome-conjugated antibodies for 2 h in phosphate-buffered saline with 0.5% bovine serum albumin at 4 °C. Next cells were washed twice in cold phosphate-buffered saline and immediately analyzed on the flow cytometer. For fluorochrome-unconjugated antibodies, a second incubation with fluorescein-5-isothiocyanate-conjugated secondary antibodies was performed 1 h at 4 °C in phosphate-buffered saline, bovine serum albumin 0.5%. Time course of antibody binding showed that equilibrium was reached for all antibodies after 90 min at 4 °C, and binding was saturating at the concentration used (data not shown). Specificity of the binding was assessed by incubating the cells with a 10-fold excess of nonspecific immunoglobulins before the specific antibody (data not shown).

Measurements of Intracellular Free Ca²⁺ Concentrations-- [Ca²⁺]_c was measured with the fluorescent Ca²⁺ indicator fura-2. Cells (2 × 10⁷/ml) suspended in Ca²⁺ medium containing 0.1% bovine serum albumin were loaded for 45 min at 37 °C with 2 µM fura-2/AM, then diluted to 10⁷/ml and kept on ice. Just before use, a sample of loaded cells (5×10⁶/ml) was centrifuged and resuspended in the desired medium at 37 °C. Fluorescence measurements were performed on a Perkin-Elmer fluorometer (LS3, Perkin-Elmer) thermostated at 37 °C. Excitation and emission wavelengths were 340 and 505 nm, respectively. Calibration was performed for each cuvette by the sequential addition of 4-5 mM Ca²⁺ (to reach a 5 mM final Ca²⁺ concentration), 2 µM ionomycin to measure Ca²⁺-saturated fura-2 (F_max), followed by 24 mM EGTA, 60 mM Tris, pH 9.3, and 0.1% Triton X-100 (TX-100) to measure Ca²⁺-free fura-2 (F_min). Results are expressed as concentration (nm) of intracellular free Ca²⁺.

Protein Analysis-- For quantitative protein detection, cells were lysed in TX-100 1% buffer (25 mM Hepes, pH 7.4, 145 mM NaCl, 5 mM MgCl₂, 1% sucrose, 1% TX-100 and a mixture of protease inhibitors) or alternatively in radioimmune precipitation buffer (9.1 mM dibasic sodium phosphate, 1.7 mM monobasic sodium phosphate, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and a mixture of protease inhibitors) for 20 min on ice. Cell lysates were next centrifuged 20 min at 4 °C 1200 × g to discard aggregates, and protein concentrations were determined using the BCA assay from Pierce. The proteins were then separated by SDS-polyacrylamide gel electrophoresis and transferred on nitrocellulose membranes. Western blot analysis was performed using the ECL kit from Amersham Pharmacia Biotech.

For immunoprecipitations, cells were solubilized 20 min on ice with either solubilization buffer A (50 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl₂, 1 mM EGTA, pH 7.4, 1% TX-100, 10 mM orthovanadate, and a mixture of protease inhibitors) or alternatively with radioimmune precipitation buffer (9.1 mM dibasic sodium phosphate, 1.7 mM monobasic sodium phosphate, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM orthovanadate and a mixture of protease inhibitors). After a brief sonication, 400-500 µg of protein were adjusted to the same volume (800 µl) and precleared for 1 h with 3 mg/ml protein A-Sepharose. The beads were discarded by centrifugation, and 1.5 µg of anti-p56/59^hck antibodies were added to each sample overnight at 4 °C with gentle mixing. The immune complexes were then precipitated by adding 3 mg/ml protein A-Sepharose. The beads were washed three times with the corresponding solubilization buffer and boiled 5 min. The immunoprecipitates were finally separated by SDS-polyacrylamide gel electrophoresis and transferred on nitrocellulose membranes. Western blot analysis was performed using the ECL kit from Amersham Pharmacia Biotech.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Myelomonocytic cells (especially macrophages) are important targets of HIV. It therefore seems very relevant that the HIV Nef protein interacts with a high affinity with Src-like PTKs of myelomonocytic cells. However, the effects of Nef in these cells have so far not been examined. We therefore decided to use HL60 cells as a myelomonocytic cell model to investigate this question. Indeed, HL60 cells cannot only be used in their undifferentiated form as promyelocytes but can also be induced to differentiate major subtypes of myelomonocytic cells (44, 45). Differentiation with Me₂SO, 1,25(OH)₂D₃, and PMA will lead to a granulocyte (46), monocyte (47, 48), and macrophage phenotype (49), respectively. Retroviral transduction was used to generate HL60 cell lines stably expressing the wild type form of HIV1 Nef (Nef) or a mutated Nef protein (Nef_(PXXP)4), where prolines 69, 72, 75, and 78 were replaced by alanines. Previous in vitro studies have shown that, although Nef interacts with high affinity with four members of the Src-like PTK family (Lck, Fyn, Hck, and Lyn), no such interactions are observed for the Nef_(PXXP)4) mutant (14, 22-25).

Nef Expression in HL60 Cell Lines-- As assessed by Western blot analysis, Nef and (Nef_(PXXP)4) were expressed to significant levels both in undifferentiated promyelocytes and differentiated cells (Fig. 1A). To verify whether the expressed Nef wild type and PXXP mutant were functional, we investigated the down-regulation of CD4 cell surface expression by cytofluorimetry. Indeed, it has been shown previously that the PXXP motif of Nef is not crucial for CD4 down-regulation (14, 15). As shown in Fig. 1B, CD4 surface expression was efficiently down-regulated by Nef in undifferentiated and differentiated HL60 cells showing that Nef was functional in this cellular background. The Nef_(PXXP)4) mutant was also able to down-regulate CD4, but, as described previously in other cells (14, 50), somewhat less efficiently than the wild type form. Thus, the expressed Nef and Nef_(PXXP)4) are functional with respect to CD4 down-regulation.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Nef expression and Nef-induced CD4 down-regulation in HL60 cells. HL60 promyelocytes stably transfected with Nef (LNefSN), the Nef(PXXP)4 mutant (LNef(PXXP)4-SN), or an empty vector as control (LXSN) were induced to differentiate toward various phenotypes by the addition of the appropriate inducer to the culture medium. A, Western blot analysis showing either the wild type form of Nef or the Nef(PXXP)4 mutant expression in HL60 cells. B, cytofluorimetric analysis showing down-regulation of CD4 cell surface expression in nef-expressing HL60 cells. DMSO, Me2SO.

Nef Decreases Agonist-induced Ca²⁺ Influx through an Increase in Stored Ca²⁺-- The cytosolic free Ca²⁺ concentration, [Ca²⁺]_c, is a crucial intracellular signaling mediator in myelomonocytic cells (51, 52). In response to specific agonists, [Ca²⁺]_c elevations occur in two different steps. First, Ca²⁺ is released from intracellular stores in response to the generation of inositol phosphate (=Ca²⁺ release). Second, the emptying of intracellular stores activates, by a yet unknown mechanism, Ca²⁺ channels in the plasma membrane (=Ca²⁺ influx). To investigate a potential effect of Nef on agonist-induced [Ca²⁺]_c elevations, fura-2-loaded HL60 cells were stimulated with the bacterial peptide fMLP. To separate the initial Ca²⁺ release phase from the subsequent Ca²⁺ influx phase, the following protocol was used. Cells were kept in a Ca²⁺-free medium. Under these conditions, the [Ca²⁺]_c increase in response to fMLP stimulation was used as a measure for Ca²⁺ release. Subsequently, Ca²⁺ was added to the extracellular medium, and the resultant [Ca²⁺]_c elevation was used as a measure of Ca²⁺ influx (Fig. 2). In control cells, basal [Ca²⁺]_c was around 70-80 nM. Ca²⁺ elevations due to Ca²⁺ release in response to 100 nM fMLP were 250-350 nM. Ca²⁺ influx, induced by Ca²⁺ readdition, raised [Ca²⁺]_c to 300-400 nM (Fig. 2B). Wild type Nef expression affected neither basal [Ca²⁺]_c nor Ca²⁺ release from intracellular stores. By contrast, Ca²⁺ influx was decreased by approximately 50% in the presence of the viral protein (Fig. 2B). The Nef inhibition of Ca²⁺ influx was also present when the lag time between fMLP addition and Ca²⁺ readdition was reduced to 1 min, suggesting that activation of Ca²⁺ channels is decreased by Nef already in the early phase of fMLP stimulation (not shown).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Nef inhibits the agonist-induced store-operated Ca2+ influx and increases Ca2+ store content. Control and nef-expressing cells were differentiated by Me2SO. [Ca2+]c was monitored in fura-2-loaded cells. A, typical traces showing [Ca2+]c elevations after stimulation in Ca2+-free medium by 100 nM fMLP followed by the readdition of 1 mM extracellular Ca2+after 5 min. B, [Ca2+]c values immediately before (Basal), 30 s after fMLP addition (fMLP-induced release), and 30 s after Ca2+ readdition (Ca2+ influx). C, typical traces showing [Ca2+]c elevations after stimulation in Ca2+-free medium by 100 nM fMLP followed by 1 µM ionomycin after 5 min. D, [Ca2+]c values immediately before (Basal), 30 s after fMLP addition (fMLP-induced release), and 30 s after ionomycin addition (ionomycin-induced Ca2+ release). Data are means ± S.E. of 3-11 independent experiments.

Since in myelomonocytic cells agonist-induced Ca²⁺ influx is regulated by the filling state of intracellular Ca²⁺ stores (53, 54), the decrease in agonist-induced Ca²⁺ influx in Nef-transfected cells might be due to an increase in residual Ca²⁺ store content after stimulation. To test this hypothesis, we performed experiments essentially similar to the Ca²⁺ influx protocol described above. However, instead of adding back Ca²⁺ in the medium to measure Ca²⁺ influx, the Ca²⁺ ionophore ionomycin was added to assess the amount of residual Ca²⁺ store content. Ionomycin-releasable Ca²⁺ from internal stores was approximately double in wild type nef-expressing cells as compared with control cells (Fig. 2, C and D). This would be compatible with the concept that an increased Ca²⁺ store content accounts for the decreased Ca²⁺ influx in Nef-transfected cells.

Thapsigargin, an inhibitor of the Ca²⁺ pumps of agonist-sensitive Ca²⁺ stores, is able to induce a depletion of intracellular Ca²⁺ stores and a subsequent store-operated Ca²⁺ influx independently from receptor activation and Ins(1,4,5)P₃ production. We therefore studied thapsigargin-induced [Ca²⁺]_c changes using protocols similar to those described above for fMLP. In contrast to fMLP, thapsigargin induced an equivalent Ca²⁺ influx in nef-expressing and in control cells (Fig. 3, A and B). The absence of inhibition of thapsigargin-induced Ca²⁺ influx by Nef might have two possible explanations. (i) Either the presence of a receptor agonist (i.e. fMLP) is necessary to unravel the inhibitory effect of Nef on Ca²⁺ influx, or (ii) after efficient depletion of intracellular Ca²⁺ stores by thapsigargin, a normal activation of store-operated Ca²⁺ influx occurs. When fMLP was coapplied with thapsigargin, no inhibition was observed (Fig. 3, C and D), excluding the former possibility. Instead, thapsigargin efficiently depleted intracellular Ca²⁺ stores both in nef-expressing and in control cells (Fig. 3, E and F), demonstrating that after efficient inhibition of the Ca²⁺-ATPase, (i) the Ca²⁺ store contents are down to similarly low levels in both nef-expressing and control cells, and (ii) normal size Ca²⁺ influx occurs.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   After efficient depletion of intracellular Ca2+ stores by thapsigargin, normal Ca2+ influx occurs in Nef-transfected cells. Control and nef-expressing cells were differentiated by Me2SO, and [Ca2+]c was monitored in fura-2-loaded cells. A, typical traces showing [Ca2+]c elevations after the addition of 50 nM Tg in Ca2+-free medium followed by the readdition of 1 mM extracellular Ca2+after 5 min. B, [Ca2+]c values immediately before (Basal), 30 s after Tg addition (Tg-induced release), and 30 s after Ca2+ readdition (Ca2+ influx). C, typical traces showing [Ca2+]c elevations after the concomitant addition of 50 nM Tg and 100 nM fMLP in Ca2+-free medium followed by the readdition of 1 mM extracellular Ca2+after 5 min. D, [Ca2+]c values immediately before (Basal), 30 s after Tg plus fMLP addition (fMLP+Tg-induced release), and 30 s after Ca2+ readdition (Ca2+ influx). E, typical traces showing [Ca2+]c elevations after the addition of 50 nM Tg in Ca2+ free medium followed by 1 µM ionomycin after 5 min. F, [Ca2+]c values immediately before (Basal), 5 min after Tg addition (Tg-induced release), and 30 s after ionomycin addition (ionomycin-induced release). Data are means ± S.E. of 3-7 independent experiments.

Taken together, these results demonstrate that, after agonist stimulation, an abnormally high residual Ca²⁺ store content prevents the normal activation of store-operated Ca²⁺ influx in nef-expressing cells. The normal activation of Ca²⁺ influx by thapsigargin excludes the possibility that Nef directly blocks store-operated Ca²⁺ channels. The residual Ca²⁺ after agonist stimulation is unlikely to be due to an inhibition of agonist-induced signaling to Ca²⁺ stores, as the amplitude of fMLP-induced Ca²⁺ release was identical in nef-expressing and in control cells. Thus, we considered the possibility that the Ca²⁺ content of intracellular stores is constitutively increased in nef-expressing cells. To test this hypothesis, we directly added the Ca²⁺ ionophore ionomycin to cells kept in a Ca²⁺-free medium without any prestimulation. As shown in Fig. 4A, nef-expressing cells had indeed a markedly increased ionomycin-releasable Ca²⁺ pool. Thus, we conclude that Nef is able to constitutively increase cellular Ca²⁺ store content in differentiated HL60 cells.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Nef-increased Ca2+ store content is a requirement for HL60 cell differentiation. Ionomycin-induced Ca2+ release from intracellular stores was measured in various fura-2-loaded cells stably transfected either with nef or control vector (undifferentiated HL60 cells, differentiated HL60 cells, and in the CEM T cells). A, typical traces showing [Ca2+]c elevations after the addition of 1 µM ionomycin in the absence of extracellular Ca2+ in both control and nef-expressing HL60 cells differentiated by Me2SO. B, effect of Nef on [Ca2+]c release 30 s after ionomycin addition in undifferentiated HL60, Me2SO/1,25(OH)2D3/PMA-differentiated HL60, and CEM T lymphocytes. Experiments were performed in the absence of extracellular Ca2+. Ionomycin-released [Ca2+]c value for cells expressing an empty vector (control) were 316 ± 65, 520 ± 19, 255 ± 33, 131 ± 13, and 272 ± 36 nM for undifferentiated HL60, Me2SO-differentiated HL60, 1,25(OH)2D3 -differentiated HL60, PMA-differentiated HL60, and CEM T cells, respectively. Data are means ± S.E. of 3-10 independent experiments.

The Nef-induced Increase in Cellular Ca²⁺ Store Content Requires Factors Expressed upon Differentiation of Myelomonocytic Cells-- To investigate the mechanisms underlying the Nef-induced increase in cellular Ca²⁺ content, we analyzed the Nef effect in undifferentiated HL60 cells, in three different types of differentiated HL60 cells, and in CEM T-lymphocytes. As shown in Fig. 4B, an increase of Ca²⁺ content ranging from 30 to 60% was observed in all types of differentiated HL60 cells, i.e. HL60 granulocytes (Me₂SO), HL60 monocytes (1,25(OH)₂D₃), and HL60 macrophages (PMA). In contrast, in undifferentiated HL60 promyelocytes and in CEM lymphocytes, no increase in Ca²⁺ store content was observed.

Thus Nef appears to require the presence of differentiation-induced cofactors not present in T-lymphocytes to exert its action on intracellular Ca²⁺ stores. Importantly, the two myelomonocytic cell types, which are thought to be relevant for HIV infection, namely monocytes and macrophages, clearly are sensitive to the Nef effect on Ca²⁺ store content.

SH3-mediated Protein-Protein Interactions via the PXXP Motif in the Nef Core Is Required for the Nef Effect on Ca²⁺ Stores-- Myelomonocyte-specific Src-like PTKs would be good candidates as differentiation-induced cofactors that mediate the Nef effect on Ca²⁺ signaling. Nef binds to four members of this PTK family (p56^lck, p56/59^hck, p59^fyn, and p53/56^lyn (14, 22-25) with a high affinity via interactions between a proline-rich motif in the Nef core and SH3 domains of Src-like PTKs. The mutation of the Nef proline motifs prevents the binding of Nef to Src-like PTKs and their subsequent activation but does not abolish other Nef effects such as cell surface CD4 down-regulation (Fig. 1B). We therefore studied Ca²⁺ signaling in HL60 cells expressing a proline mutant of Nef (Nef_(PXXP)4). As shown above, this mutant was well expressed and functional with respect to CD4 down-regulation (Fig. 1). As shown in Fig. 5, cells expressing the Nef_(PXXP)4 mutant behaved like control cells. (i) fMLP-induced Ca²⁺ influx was not diminished (Fig. 5A), and ii) the amount of ionomycin-releasable Ca²⁺ was not increased either in unstimulated cells or after fMLP stimulation (Fig. 5B).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Mutation of a proline motif in the Nef core abolished the Nef-mediated increase of intracellular Ca2+ store content and the subsequent inhibition of agonist-induced store-operated Ca2+ influx. [Ca2+]c elevations after various stimulation protocols were measured in Me2SO-differentiated HL60 cells expressing either the wild type form of Nef or the Nef(PXXP)4 mutant. A, effect of Nef and Nef(PXXP)4 expression on the agonist-induced Ca2+ release from internal stores and the subsequent store-operated Ca2+ influx. In control cells, 277 ± 43 nM of Ca2+ were released by 100 nM fMLP from internal stores, and the [Ca2+]c elevation subsequent to Ca2+ influx was of 333 ± 62 nM. Data are mean ± S.E. of 3-5 independent experiments. B, effect of Nef or Nef(PXXP)4 expression on the ionomycin-releasable intracellular Ca2+ with or without a prior stimulation by 100 nM fMLP. In control cells, 520 ± 19 nM of Ca2+ were released by ionomycin in unstimulated cells; 5 min after fMLP addition, 157 ± 27 nM of Ca2+ were released by ionomycin. Data are means ± S.E. of 5-10 independent experiments.

Up-regulation of Src-like PTKs during of Myelomonocytic Differentiation-- Among the four Src-like PTKs able to interact with Nef, p56/59^hck, p53/56^lyn, and p56^lck are restricted in their expression to cells of the hematopoietic lineage; p56/59^hck expression is restricted to myelomonocytic cells (55), whereas p53/56^lyn is found either in myelomonocytic or in B-lymphocytic cells (56), and p56^lck is specifically expressed in lymphocyte. By contrast p59^fyn is more ubiquitously expressed. We thus investigated the expression of these four Src-like PTKs under our conditions of HL60 cell differentiation by Western blots analysis of total cell extracts. We did not detect p56^lck and p59^fyn in undifferentiated or differentiated HL60 cells (data not shown). By contrast p56/59^hck and p53/56^lyn were detected to varying amounts in HL60 cells depending upon the differentiation type (Fig. 6). p56/59^hck and p53/56^lyn were not expressed to detectable levels in CEM T cells (data not shown). The p56/59^hck- and p53/56^lyn-associated signals were further quantitated by densitometry in wild type and nef- or nef_(PXXP)4 -expressing HL60 cells (Fig. 6, B and C). In undifferentiated HL60 cells, only a very weak p56/59^hck-associated signal was detected. However, upon differentiation, the p56/59^hck signal strongly increased, reaching the highest level in Me₂SO-differentiated cells. By contrast, the p53/56^lyn-associated signal was already relatively high in undifferentiated cells and showed only relatively small increases with differentiation as previously reported by others (57).

View larger version (22K): [in this window] [in a new window]   Fig. 6.   p56/59hck and p53/56lyn expression in HL60 cells. Expression of p56/59hck and p53/56lyn tyrosine kinases was investigated by quantitative Western blot analysis of lysates from HL60 cells differentiated toward each phenotype. A, typical Western blot revealing the expression of p56/59hck and p53/56lyn in undifferentiated and Me2SO (DMSO)-differentiated HL60 cells expressing either an empty vector (control), the wild type form of Nef, or the Nef(PXXP)4 mutant. B, relative increase of p56/59hck expression with cell differentiation as compared with undifferentiated HL60 cells. Data are means ± S.E. of 4-7 independent experiments. C, relative increase of p53/56lyn expression with cell differentiation as compared with undifferentiated HL60 cells. Data are means ± S.E. of 3-6 independent experiments.

Note that, although the expression of Src-like PTKs was clearly differentiation-dependent, it was not influenced by the expression of Nef or the Nef mutant. Thus, although Nef might possibly modulate activity or cellular localization of Src-like PTKs, it does not influence protein levels of these kinases.

P56/59^hck Interacts in Vivo with Nef and the Ins(1,4,5) Receptor-- Interactions of HIV1 Nef and Src-like PTKs have been clearly demonstrated in vitro (14). In vivo Nef-p56/59^hck interaction has been observed only in reconstituted cell systems overexpressing both Nef and p56/59^hck (58). The results presented so far would be compatible with a mediation of the Nef effect on Ca²⁺ signaling by p56/59^hck and p53/56^lyn. To study whether Nef is indeed able to interact with these Src-like PTKs in myelomonocytic cells, we immunoprecipitated p56/59^hck and p53/56^lyn and then examined the immunocomplexes by blotting with Nef-specific antibodies. We first performed immunoprecipitations with lysates from differentiated HL60 cells using anti p56/59^hck antibodies. Coimmunoprecipitation of Nef was observed with cells expressing wild type Nef but not the Nef_(PXXP)4 mutant (Fig. 7). Using anti-p53/56^lyn antibodies, we were unable to detect any Nef-associated signal in the immunoprecipitates (data not shown). Thus, Nef clearly interacts with p56/59^hck through its proline-rich motifs. The negative results concerning p53/56^lyn should be taken with caution. They might suggest that Nef does not interact in vivo with p53/56^lyn. Alternatively however, the interaction between Nef and p53/56^lyn might be of too low affinity to be detected by immunoprecipitation, or the antibody used could have disrupted the interaction.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Src-like PTK p56/59hck interacts in vivo with Nef and the inositol 1,4,5-trisphosphate receptor. p56/59hck was immunoprecipitated from Me2SO (DMSO)-differentiated HL60 cells, and Western blot analysis of the immune complexes were performed. Upper lane, Western blot analysis with antibodies specific for the Ins(1,4,5)P3 receptor. Middle lane, Western blot analysis with antibodies specific for p56/59hck. Lower lane, Western blot analysis with antibodies specific for Nef. The result is representative of three independent experiments.

Previous studies in lymphocytes have implicated nonreceptor tyrosine kinases in the control of Ca²⁺ store regulation. More specifically, the Src-like PTK p59^fyn regulates the activity of the Ins(1,4,5)P₃ receptor in lymphocytes through direct interaction with this Ca²⁺ release channel (42). We could not detect p59^fyn in HL60 cells (not shown). It is, however, conceivable that myelomonocyte-specific Src-like PTKs play a role in the regulation of Ca²⁺ store function. We therefore examined whether p56/59^hck is able to interact with the Ins(1,4,5)P₃ receptor in HL60 cells. As shown in Fig. 7, the Ins(1,4,5)P₃ receptor coimmunoprecipitated with p56/59^hck. This observation provides a possible link between the known regulation of Src-like PTKs activity by Nef and the SH3-dependent regulation of myeloid cell Ca²⁺ signaling demonstrated in our study. Note, however, that the amounts of Ins(1,4,5)P₃ receptor, which coimmunoprecipitated with p56/59^hck, were comparable in control and nef-transfected cells. This would be compatible with a concept where Nef regulates in a qualitative, rather than a quantitative manner, the association between p56/59^hck and the Ins(1,4,5)P₃ receptor.

	DISCUSSION

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In this study we show a novel mechanism whereby the HIV Nef protein manipulates cellular signaling; Nef expression in myelomonocytic cells leads to an increased Ca²⁺ content of intracellular stores. The Nef PXXP motif was crucial for the observed effect. This suggests that proteins bearing SH3 domains are able to interact with Nef, such as Src-like PTKs, might act downstream to Nef to alter the regulation of intracellular Ca²⁺ signaling pathways.

What Are the Downstream Mediators of the Nef Effect on Ca²⁺ Signaling in Myelomonocytic Cells?-- Our results allowed assembly of the following information concerning elements downstream of Nef involved in the increase in Ca²⁺ store content.

The absence of the Nef effect in lymphocytes and undifferentiated HL60 cells suggests that factors induced by myelomonocytic differentiation are involved in the mediation of this effect.

The absolute requirement for the proline-rich regions of Nef suggests the involvement of SH3 domain-containing proteins, in particular Src-like PTKs.

The coimmunoprecipitation of Nef and the Ins(1,4,5)P₃ receptor with p56/59^hck makes the latter Src-like PTK a particularly attractive candidate.

Thus, our results are compatible with the following scheme. Nef interacts with Src-like PTKs (in particular p56/59^hck), which in turn interact with elements of intracellular Ca²⁺ stores (in particular the Ins(1,4,5)P₃ receptor). Further experiments will however be necessary to definitively prove this hypothesis.

Possible Molecular Mechanisms of the Nef-induced Increase in Ca²⁺ Storage-- The amount of Ca²⁺ stored within intracellular Ca²⁺ stores is essentially a function of two parameters, the activity of Ca²⁺ uptake and the permeability of the Ca²⁺ store membrane. Thus, either an increased Ca²⁺-ATPase activity or a decreased Ins(1,4,5)P₃ receptor activity are the most likely mechanisms accounting for the increase Ca²⁺ storage observed in nef-expressing cells. The observation that p56/59^hck is not only coimmunoprecipitating with Nef but also with the Ins(1,4,5)P₃ receptor together with the known regulation of the Ins(1,4,5)P₃ receptor by Src-like PTKs in lymphocytes suggest the latter possibility. However, a modulation of the Ca²⁺-ATPase activity and thereby of the Ca²⁺ uptake mechanisms of intracellular stores is an attractive alternative or additional hypothesis to explain the Nef effect. Note that Nef has also been shown to interact with a cellular H⁺-ATPase (59).

Potential Physiological Role of Increased Ca²⁺ Storage-- What could be the potential physiological role of the observed increase in intracellular Ca²⁺ storage? A most obvious explanation might be to allow cells to release increased amounts of Ca²⁺ during stimulation, although we could not observe such an effect in nef-expressing cells. Indeed, after fMLP stimulation, an increased residual Ca²⁺ store content rather than an increased Ca²⁺ release, was observed. Thus, the relevant effect of the Nef-induced Ca²⁺ increase should reside within other cellular events, which are controlled by the content of Ca²⁺ stores. Indeed, there is a decrease in store-operated Ca²⁺ influx in response to receptor activation in nef-expressing cells. As Ca²⁺ influx is involved in a variety of crucial cellular functions, a decreased Ca²⁺ influx might be a pathophysiologically relevant Nef effect. In addition, Nef could alter other cellular functions independent of an extracellular Ca²⁺ influx but which may be regulated by the Ca²⁺ content of the endoplasmic reticulum. Indeed, the Ca²⁺ content of the endoplasmic reticulum plays an important role in protein synthesis (60, 61), regulation of apoptosis (62, 63), gene expression (64), and regulation of the nuclear pore activity (65). Future studies will have to address the question of how the Nef-induced increase in intracellular Ca²⁺ storage and the subsequent potential effects relate to the specific properties of HIV infection in myelomonocytic cells.

	ACKNOWLEDGEMENTS

We thank A. Monod for her helpful and excellent technical support and Dr. W. Kelley for critical reading of the manuscript.

	FOOTNOTES

^* This work was supported by Swiss National Science Foundation (FNRS) Grants 3139 055226.98 (to D. P. L.) and 31.50560.37 (to D. T.), by National Institutes of Health Grant R37 AI34306 (to D. T.), and by a professorship from the Giorgi-Cavalieri Foundation (to D. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: Biology of Aging Laboratory, Dept. of Geriatrics, 2, Chemin Petit Bel-Air, CH-1225 Chêne-Bourg/Genève, Switzerland. Tel.: 41-22-3055451 or 41-22-3056521; Fax: 41-22-3055455 or 41-22-3056611; E-mail: kkrause@cmu.unige.ch.

	ABBREVIATIONS

The abbreviations used are: HIV, human immunodeficiency virus; HLA, human major histocompatibility; Src-like PTK, Src family protein-tyrosine kinase; TG, thapsigargin; 1, 25(OH)₂D, 1,25 dihydroxy vitamin D₃; PMA, phorbol 12-myristate 13-acetate; fMLP, fMet-Leu-Phe.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES