Activation of p21-Activated Kinase 2 by Human Immunodeficiency Virus Type 1 Nef Induces Merlin Phosphorylation

Abstract

The accessory human immunodeficiency virus type 1 (HIV-1) protein Nef activates the autophosphorylation activity of p21-activated kinase 2 (PAK2). Merlin, a cellular substrate of PAK2, is homologous to the ezrin-radixin-moesin family and plays a critical role in Rac signaling. To assess the possible impact on host cell metabolism of Nef-induced PAK2 activation, we investigated the phosphorylation of merlin in Nef expressing cells. Here we report that Nef induces merlin phosphorylation in multiple cell lines independently of protein kinase A. This intracellular phosphorylation of merlin directly correlates with in vitro assay of the autophosphorylation activity of Nef-activated PAK2. Importantly, merlin phosphorylation induced by Nef was also observed in human primary T cells. The finding that Nef induces phosphorylation of the key signaling molecule merlin suggests several possible roles for PAK2 activation in HIV pathogenesis.

Nef is a small, multifunctional protein encoded by human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus. The in vivo simian model indicated that Nef is a major determinant of viral pathogenicity (11). Four of Nef's numerous known functions include down-regulation of cell surface CD4 and major histocompatibility complex (MHC) class I, enhancement of the infectivity of viral particles and activation of p21-activated kinase 2 (PAK2) (3, 19, 22, 25). The significance of Nef's ability to activate PAK2 is controversial since it is not clear whether the level of activation is sufficient to alter cellular metabolism (14, 20).

PAK2 is a serine/threonine kinase activated by the GTPases Rac1 and Cdc42 through the CRIB (Cdc42-Rac1 interactive binding) domain. Merlin, the product of the neurofibromatosis 2 tumor suppressor gene (24, 29), is a key signaling protein involved in the Rac signal transduction pathway (27, 33). It has recently been shown to be phosphorylated at serine-518 by PAK2 and cyclic AMP-dependent protein kinase A (PKA) (1, 12, 23, 32). Merlin is a 595-amino-acid protein that resembles other members of the band 4.1 family, such as ezrin, radixin, and moesin (ERM), in its N-terminal half (24, 29). It is a negative regulator of cell proliferation, cell motility, and Rac1 signaling (33). Phosphorylation of merlin at serine-518 results in an inactive molecule thereby releasing its inhibitory effects (13, 34).

Nef induces merlin phosphorylation.

If Nef significantly alters metabolism of the infected cell by activating PAK2, then it would be expected that increased phosphorylation of merlin would result. We first looked for Nef-induced phosphorylation of merlin in human CEM (T) and U937 (monocytic) cells which express high levels of merlin and are often used in HIV research. We stably transduced these cell lines with either a control retroviral vector (LXSN) or a retroviral vector expressing HIV-1 SF2 Nef (LnefSNSF2) (4). Cells were lysed, and supernatants of whole cell lysates were analyzed by Western blotting (Fig. 1A). Merlin from control CEM and U937 cells migrates in sodium dodecyl sulfate-polyacrylamide gels as two bands with similar intensities. As reported by others, the slower migrating species represents a hyperphosphorylated form of merlin, and the faster migrating species represents a hypophosphorylated form (16, 27, 32). Interestingly, in the cells that expressed Nef, an increase in the amount of the slower migrating form (top band) was observed, and it became the dominant species (Fig. 1A). These experiments indicated that Nef expression induced merlin phosphorylation.

FIG. 1.

Nef-induced merlin phosphorylation is independent of protein kinase A. (A) Human CEM and U937 cells were stably transduced with a retroviral vector LXSN, as a control, and a Nef-expressing vector LnefSNSF2 (9). Cells were harvested, and cell lysates were analyzed by Western blot analysis (32) using rabbit anti-merlin polyclonal antibody (A-19; Santa Cruz) or sheep anti-Nef serum. Note the increase in the intensity of the hyperphosphorylated (upper) band in all the lanes containing Nef. (B) Nef-expressing and control CEM and U937 cells were treated with H89 (25 μM) for 5 h. Note that even in cells treated with H89, there is a shift in merlin mobility in Nef-expressing cells. (C) 293T cells were cotransfected with a vector expressing SF2 Nef and a vector expressing HA-tagged wild-type merlin or S518A mutant merlin, as indicated. Cells were treated with H89 (25 μM) for 5 h before harvesting in same transfections. Note that Nef expression does not change migration of the S518A mutant (lane 4). (D) 293T cells were transfected with constructs as indicated. Western blot analyses of cell lysates were conducted with either merlin antibody above or an affinity purified specific phosphoserine-518 merlin rabbit antibody (Sigma-Genosys). Ctrl, control.

In the cell, merlin can be phosphorylated by either PKA or p21-activated kinases at S518 (1, 12, 32). To investigate whether PKA activity is involved in Nef-mediated merlin phosphorylation, we treated control and Nef-expressing CEM and U937 cells with H89 (25 μM), a selective PKA inhibitor, for 5 h (7). H89 eliminated the hyperphosphorylated (slower migrating) merlin species in the control samples (Fig. 1B). These results indicated that in the absence of Nef, PKA is responsible for the basal level of merlin phosphorylation. However, when Nef-expressing cells were treated with H89, merlin shifted to the slower migrating species (Fig. 1B). Similar results were obtained in 293T cells (Fig. 1C, lanes 5 and 6). We then determined if a mutant merlin replacing a serine at position 518 with alanine was phosphorylated in response to Nef in transient transfection experiments by using hemagglutinin (HA)-tagged merlin in 293T cells. The S518A merlin mutant migrated in the hypophosphorylated (faster migrating) form (Fig. 1C, lane 2). Nef expression did not alter its migration, suggesting that S518 is the main site of phosphorylation induced by Nef (Fig. 1C, lane 4). We further used an affinity-purified phosphoserine-518-merlin antibody to confirm that serine-518 is phosphorylated in the presence of Nef. We transfected 293T cells with a control vector, a vector expressing S518A mutant merlin, a vector expressing wild-type merlin, or vectors expressing wild-type merlin and SF2 Nef. The cell lysates were analyzed by Western blotting using either anti-merlin antibody or affinity purified anti-phosphoserine-518-merlin antibody. As shown in Fig. 1D, the S518A mutant merlin migrated in the hypophosphorylated form (lane 2, middle panel), while wild-type merlin migrated in both hyper- and hypophosphorylated forms (lane 3, middle panel). However, in the presence of Nef, wild-type merlin migrated mainly in the hyperphosphorylated form (lane 4, middle panel). The phosphoserine-518-merlin antibody detected only the hyperphosphorylated merlin (Fig. 1D, lane 3 and 4, top panel). Collectively, the experiments described above indicate that Nef-induced merlin phosphorylation at serine-518 is independent of PKA activity in CEM, U937, or 293T cells.

Phosphorylation of merlin is dependent on PAK2 activation.

We have previously demonstrated that a conserved phenylalanine in the C-terminal region of Nef (position 195 of SF2 Nef) is essential for PAK2 activation but not for CD4 downregulation, MHC class I downregulation, or the enhancement of viral infectivity (9). When the SF2 Nef F195 was mutated to isoleucine or arginine, the mutant lost its ability to activate PAK2 but retained the activities of CD4 or MHC class I downregulation and enhancement of infectivity (9) (data not shown). When SF2 Nef or the F195R mutant was coexpressed in 293T cells with merlin, Western blot analysis revealed that in cells expressing Nef F195R, there was no shift of the hypophosphorylated band, whereas in SF2 Nef expressing cells, there was a significant shift of this band (Fig. 2). Furthermore, HIV-1 D88-11 Nef, a primary isolate which was previously shown to be defective in PAK2 activation (9) did not alter merlin phosphorylation levels. However, replacing R189 with serine in D88-11 Nef, an alteration that restores the ability of D88-11 to activate PAK2 (9), also restored merlin phosphorylation (Fig. 2). These results strongly link PAK2 activation to Nef-induced merlin phosphorylation.

FIG. 2.

Activation of PAK2 is required for merlin phosphorylation induced by Nef. 293T cells were transfected with pcDNA expressing HA-tagged merlin alone or cotransfected with pcDNA vectors expressing Nef. The phosphorylation state of merlin and Nef expression levels were determined by anti-HA and sheep anti-Nef Western blots, respectively. PAK2 activation was determined on Nef immunoprecipitates by in vitro kinase assay (4). Note the increase in hyperphosphorylated merlin in lanes 2 and 5.

Nef-associated PAK2 phosphorylates GST-merlin in vitro.

In order to establish whether Nef-associated PAK2 phosphorylates merlin directly, we immunoprecipitated the Nef/PAK2 complex from 293T cells transfected with SF2 Nef or D88-11 Nef. We then incubated the immunoprecipitates with glutathione S-transferase (GST)-tagged merlin in the presence of [γ-³²P]ATP. The GST-merlin fusion protein contained amino acids 478 to 535 of merlin and was expressed in Escherichia coli and purified on glutathione-Sepharose beads (12). Phosphorylation of GST-merlin was visualized by autoradiography. As shown in Fig. 3A, only immunoprecipitates from SF2 Nef transfected cells showed PAK2 kinase activity and trans-phosphorylation of GST-tagged merlin. To determine if merlin S518 can serve as a substrate of PAK2 activated by SF2 Nef outside the context of the cellular milieu, we also used a recently described cell-free system to translate Nef mRNA in the presence of canine microsomal membranes to produce the Nef/PAK2 activated complex (21). As shown in Fig. 3B, wild-type GST-merlin, but not GST-merlin A518 was phosphorylated in immunoprecipitates from translation reactions containing Nef mRNA. Taken together, these in vitro results serve to substantiate the conclusion that Nef activated PAK2 phosphorylates merlin.

FIG. 3.

In vitro phosphorylation of GST-merlin at serine-518 by Nef-associated PAK2. (A) 293T cells were transfected with vectors expressing SF2 Nef or D88-11 Nef (PAK2 activation defective) and the cell extracts were immunoprecipitated with sheep anti-Nef serum. The immunoprecipitates were incubated with purified GST-merlin and [γ-³²P]ATP. (B) In vitro translation reactions were performed with no added RNA or SF2 Nef RNA. After incubation of the translation reactions, GTPγS (final concentration, 200 μM) was added and reactions were incubated an additional 10 min at 30°C. Reactions were immunoprecipitated with sheep anti-Nef serum. In vitro kinase assays were carried out in the presence of GST-merlin or the A518 mutant GST-merlin.

Nef induces merlin phosphorylation in human primary T cells.

We next investigated whether Nef induced activation of PAK2 in human primary T cells resulted in merlin phosphorylation. Three types of vesicular stomatitis virus G protein (VSV-G)-pseudotyped env-negative HIV-1 with an intact SF2 nef, a frameshift mutant nef, or nef F195R were used to infect activated primary human T cells. After infection cells were harvested and the expression of merlin, Nef, and Gag from the cell extracts was determined by Western blot analysis. As in the cell lines evaluated above, in activated human T cells, merlin was present as two species (Fig. 4, lane 1). Also, as observed with cell lines, the hyperphosphorylated merlin band was sensitive to the PKA inhibitor H89 (Fig. 4, lane 2). When T cells were infected with a virus containing an intact nef in the presence of H89, we observed the expected shift from the hypophosphorylated form to the hyperphosphorylated form (Fig. 4, lane 3). T cells infected by viruses with either a frameshift mutation (ΔNef) or Nef F195R resulted in no shift to the hyperphosphorylated form (Fig. 4, lanes 4 and 5). These results show that merlin is a bona fide target for Nef activated PAK2 in human primary T cells.

FIG. 4.

Merlin phosphorylation in human primary T cells infected with VSV-G-pseudotyped HIV-1 SF2. Human peripheral blood mononuclear cells were activated by phytohemagglutinin (2 μg/ml) stimulation prior to infection with VSV-G-pseudotyped virus. Cells (4 × 10⁶) were infected with HIV-1 SF2ΔE, SF2ΔEΔNef, or SF2ΔENefF195R for 6 h in the presence of DEAE-dextran (20 μg/ml). Cells were then washed twice with phosphate-buffered saline and resuspended in 2 ml complete RPMI 1640 supplemented with interleukin 2 (40 U/ml). After 48 h of incubation, H89 (25 μM) was added and cells were further incubated for 5 h. Then, cells were pelleted and lysed, and the cell lysates were centrifuged at 13,000 rpm for 15 min at 4°C. The expression of merlin, Nef, and Gag from the cells were determined by Western blotting using rabbit anti-merlin, sheep anti-Nef, and mouse anti-p24 (AG3.0), respectively. Note that merlin is hyperphosphorylated only in cells expressing wild-type Nef (lane 3).

The pathogenic significance of the activation of PAK2 by Nef has been difficult to establish. We attribute this situation, in part, to the extremely low level of the Nef/PAK2 activation complex in the cell. Here we report that merlin is strongly regulated by Nef through activation of PAK2. Our results suggest that the main site of phosphorylation on merlin in response to Nef is S518. These data are consistent with the results previously published by several other laboratories that identified S518 as the major site of merlin phosphorylation by PAK2 (12, 23, 27, 28, 32, 34). The multiple reported roles for merlin in cellular signaling, which include cell proliferation, cell motility, and Rac signaling (33), suggest several possible alterations of host cell metabolism that could be advantageous for viral replication.

Cytoskeletal remodeling is critical in several steps following the stimulation of T-cell receptors (2, 8, 31, 35). Since merlin interacts with actin and ERM proteins, some of these steps might involve activated merlin. In the presence of HIV-1 Nef, merlin was hyperphosphorylated. As a result, this modification may promote actin assembly and positively regulate T-cell activation (17). A possible connection between merlin and Nef's function in T-cell activation is also supported by the demonstration that PAK2 is a positive regulator for T-cell activation and that this effect is dependent on its kinase activity (6). Although the downstream networks of PAK2 leading to T-cell activation were not examined by these authors, it is conceivable that merlin plays a role as a downstream mediator after PAK2.

Nef increases T-cell activation by lowering the T-cell activation threshold (26). Furthermore, expression of Nef in primary resting cells following exposure to HIV-1 results in enhanced activation and enhanced viral production (26, 30). Nef-induced merlin phosphorylation may play a role in promoting T-cell activation and proliferation. Although HIV-1 preferentially infects activated/proliferating T cells, Johnson and Kaur (10) recently suggested that “resting” and “activated” may be oversimplification of the range of cellular states exhibited by T cells. Two recent studies found highly efficient depletion of memory CD4⁺ T cells from mucosal surfaces within a few days of infecting macaques with simian immunodeficiency virus even though these cells are noncycling (15, 18). Though these cells are immunophenotypically “resting,” their susceptibility to HIV-1 infection suggests that they are not truly resting T cells (15, 18). Enhancing viral production in these cells through the proliferative signal of merlin phosphorylation is a possible mechanism for enhancing viral production. Early, massive production of virus is thought to set the parameters of the subsequent chronic disease (5). Future studies will assess a possible role for Nef-induced phosphorylation of merlin at this early juncture of HIV-1 infection.