Hisactophilin is involved in osmoprotection in Dictyostelium
© Pintsch et al; licensee BioMed Central Ltd. 2002
Received: 19 December 2001
Accepted: 7 May 2002
Published: 7 May 2002
Dictyostelium cells exhibit an unusual stress response as they protect themselves against hyperosmotic stress. Cytoskeletal proteins are recruited from the cytosolic pool to the cell cortex, thereby reinforcing it. In order to gain more insight into the osmoprotective mechanisms of this amoeba, we used 1-D and 2-D gel electrophoresis to identify new proteins that are translocated during osmotic shock.
We identified hisactophilin as one of the proteins that are enriched in the cytoskeletal fraction during osmotic shock. In mutants lacking hisactophilin, viability is reduced under hyperosmotic stress conditions. In wild type cells, serine phosphorylation of hisactophilin was specifically induced by hypertonicity, but not when other stress conditions were imposed on cells. The phosphorylation kinetics reveals a slow accumulation of phosphorylated hisactophilin from 20–60 min after onset of the hyperosmotic shock condition.
In the present study, we identified hisactophilin as an essential protein for the osmoprotection of Dictyostelium cells. The observed phosphorylation kinetics suggest that hisactophilin regulation is involved in long-term osmoprotection and that phosphorylation occurs in parallel with inactivation of the dynamic actin cytoskeleton.
Cells steadily face changes of the external osmolarity, to which they have to adapt. To withstand a steep increase in osmolarity, eukaryotic cells activate responses like "regulatory volume increase", accumulation of compatible osmolytes and stimulated expression of stress proteins [1–4]. Recently, an exception from this scheme has been identified: Dictyostelium cells protect themselves against hyperosmolarity by largely rearranging cellular proteins, whereas no "regulatory volume increase", no accumulation of compatible osmolytes and no change of the expression pattern of the most abundant proteins were observed . Among the translocated proteins identified, cytoskeletal proteins appear to be predominant. In particular, the rearrangement of actin and myosin II to the cell cortex beneath the plasma membrane  was shown to constitute a pivotal element of osmoprotection in Dictyostelium. These two proteins form the core of a rigid network resembling a shell-like structure . Conversely, DdLIM, a cytoskeletal protein involved in the formation of protrusions , is depleted from the cytoskeletal fraction under hypertonic conditions, which is in consistence with the rounding up and the retraction of protrusions of cells . 30–60 min after onset of the shock condition, progressive actin phosphorylation is observed , which presumably leads to the inactivation of the dynamic actin system .
The pivotal role of the cytoskeleton in hyperosmotic stress response is also reflected by the fact, that the cytoskeletal proteins β-actinin and "120 kDa gelation factor" are essential to ensure survival under hyperosmotic conditions .
However, the signalling pathways mediating protein translocation and actin phosphorylation largely remain to be elucidated. The best understood signalling pathway involves myosin II translocation: upon hyperosmotic stress an increase in cGMP concentration was observed, which is due to guanylyl cyclase activation. This in turn results in the activation of the kinase, which eventually phosphorylates myosin II heavy chain [6, 12]. Thereby, disassembly of myosin II filaments is induced as a prerequisite for their reassembly at the cell cortex , which is accomplished within 10 min after onset of hyperosmotic shock .
In addition, DokA, a homologue of bacterial histidine kinases and cAMP are involved in hyperosmolar signal transduction [14, 15], however, molecular targets of these signalling pathways during osmoregulation have not been identified yet.
Hence, the translocation and phosphorylation of cytoskeletal proteins constitute pivotal osmo-responses in Dictyostelium. To further elucidate this unusual hyperosmolar stress response we attempted to identify additional proteins, which are translocated under hypertonic conditions and investigated, whether protein phosphorylation of a candidate protein is regulated in response to hyperosmolarity. We could identify hisactophilin as such a protein, which is translocated and phosphorylated during hyperosmotic shock. Hisactophilin is a 15 kDa protein in Dictyostelium, that consists of two highly identical isoforms Hisactophilin I and II, which are myristoylated at the N-terminus . The two isoforms exhibit 84% sequence identity and are both myristoylated and distributed between plasma membrane and cytoplasm . Due to this high degree similarity, both isoforms are in this manuscript referred to as hisactophilin, without distinguishing between them.
The biochemical properties of hisactophilin, namely actin- and membrane binding have recently shown to be strongly pH-dependent, which is due to the high content of 26–30% histidine residues [17–19]. Therefore, a role also as pH-sensor was postulated for this protein . In addition to myristoylation, phosphorylation was shown to be a posttranslational modification of hisactophilin ; the physiological role of hisactophilin phosphorylation however is unknown. Hisactophilin appears to play an essential role in osmoprotection, as hisactophilin null cells are osmosensitive.
Hisactophilin is enriched in the cytoskeletal fraction of wild type cells exposed to hyperosmotic shock
Hisactophilin is phosphorylated upon hyperosmotic stress
To prove that the phosphorylation of hisactophilin is dependent on osmolarity, but not on the osmolyte used, wild type cells were exposed to hypertonic stress conditions, using either NaCl, glucose or sorbitol as osmolyte. The concentrations of the osmolytes were adjusted to 400 mOsm in the case of sorbitol (400 mM) and NaCl (200 mM) and a slightly reduced osmolarity (350 mOsm) was chosen in the case of glucose. Analysis of hisactophilin phosphorylation in response to the hypertonic stress conditions revealed that phosphorylation occurred to the same extent in the presence of all three osmolytes, indicating that hisactophilin phosphorylation is a hyperosmolar stress response (Fig. 3B).
Serine phosphorylation of Hisactophilin
Hisactophilin-deficient cells exhibit reduced viability under hyperosmotic stress conditions
To investigate whether hisactophilin plays a role in osmoregulation, we determined whether survival of his--cells was affected under hypertonic conditions.
Dictyostelium was shown to employ an unusual mechanism to cope with the effects of osmotic stress by rearranging its cellular cortex [5, 22]. As the cells round up, actin and myosin moieties are being redistributed underneath the plasma membrane. In this process the distribution of the actin-associated protein Hisactophilin was investigated in order to gain a better insight into osmo-protective mechanism of the cell. Hisactophilin was found to be enriched in the cytoskeletal fraction of wild type cells exposed to hyperosmotic stress (Fig. 1A,1B). In addition, a thick layer of Hisactophilin II-GFP was formed at the cell cortex of cells, that were shocked for 10 minutes (data not shown), which correlates with the observation that actin and myosin II are translocated to the cell cortex within 10 minutes after onset of the stress condition . As hisactophilin was shown to enhance actin polymerization in vitro  and as cells overexpressing hisactophilin II exhibit an increased F-actin content , this protein could concur in the formation of the rigid filamentous network of actin and myosin II filaments.
The essential role of hisactophilin becomes evident when hisactophilin negative cells are investigated under osmotic stress conditions. They show a markedly reduced survival rate in a test were they are first exposed to high osmolarity for 2 hours and are then plated onto agar plates containing bacteria. This procedure involves a volume reduction and a subsequent volume increase caused by the re-dilution in the plating process. It therefore remains to be investigated whether hisactophilin deploys its function in the process of rearranging the cell's cortex upon volume reduction or is essential for reinforcing the cortex in its rounded up state.
A role comparably to the family of MARCKS (myristoylated alanine-rich protein kinase C substrate) proteins in mammalian cells was postulated recently . These proteins are also myristoylated at the N-terminus and crosslink actin into a rigid meshwork at the membrane. Upon phosphorylation, MARCKS proteins are rejected from the membrane by electrostatic interactions, which results in the spatial separation of the cytoskeleton from the membrane . In fact, hisactophilin phosphorylation was found to be specifically induced under hypertonic conditions (Fig. 3A, 4). However, the phosphorylation reaction is too slow to account for the translocation of the protein within 10 minutes (Fig. 3B, 3C): phospho-hisactophilin progressively accumulated within 60 min with a kinetics strikingly similar to the kinetics of actin phosphorylation under hyperosmotic conditions . This points out the possibility, that actin and hisactophilin cooperate in two distinct phases of osmoregulation. During the first phase, the proteins are translocated within the first 10 minutes of stress which is accompanied by cortical reinforcement. The second phase is characterized by actin and hisactophilin phosphorylation reinforced after about 30 min of hypertonic shock. Actin phosphorylation was shown to correlate with an inactivation of the dynamic actin system . The identical kinetics suggests a similar role for hisactophilin during this process. In addition, the coinciding phosphorylation kinetics raises the possibility of a common regulation of hisactophilin and actin phosphorylation. The signalling pathways regulating actin and hisactophilin phosphorylation are unknown, however, the second messengers involved in osmoregulation identified so far, namely cGMP and cAMP could be excluded as modulators of hisactophilin phosphorylation, as an increase or decrease in these messengers did not affect hisactophilin phosphorylation (data not shown). This suggests the presence of an additional signalling pathway concomitantly regulating a serine kinase specific for hisactophilin (Fig. 5) and, due to the identical phosphorylation kinetics, a tyrosine kinase specific for actin .
A conceivable signalling pathway regulating the hisactophilin translocation is a change in pH, as the biochemical properties of Hisactophilin are strongly pH-dependent: actin-binding is strongly increased in vitro when pH is lowered to = 7.0 . In fact, progressive cytosolic acification was observed during hyperosmotic stress, resulting in a pH drop from 7.5 to 6.8 15 min after onset of the stress condition . Hence, the translocation of hisactophilin to the actin-rich cytoskeleton can be attributed to the cytosolic acidification. Also, the enrichment of the elongation factor 1α in the cytoskeletal fraction of hyperosmotically shocked cells  can be explained by cytosolic acidification, as enhanced binding of this protein to F-actin at pH 6.5 in vitro has been described recently .
These examples are in accordance with the notion of pH changes as "synergistic messenger" . Changes in proton concentration affect the biochemical properties of proteins and can thereby act as signals. However, other signals have to concur to elicit the osmoprotective responses: the translocation of hisactophilin potentially caused by the drop in cytosolic pH is complemented by the relocation of myosin II mediated by cGMP and of actin  by a yet unknown mechanism. Therefore, the role of hisactophilin in osmoprotection also exemplifies the numerous interactions of the signalling pathways that are necessary for osmoprotection in Dictyostelium.
We could demonstrate that Hisactophilin is both translocated to the cytoskeleton and phosphorylated during hyperosmotic stress in Dicytostelium. The phosphorylation kinetics suggests a slow accumulation of phosphorylated hisactophilin resembling the phosphorylation kinetics for actin, which is known to eventual lead to cytoskeletal inactivation. Second messengers known to play an essential role during osmoprotection are not involved in hisactophilin phosphorylation suggesting a further signalling pathway being involved in the unusual osmoprotection mechanisms in Dictyostelium.
Materials and Methods
Growth of Dictyostelium cells
D. discoideum strain AX2-214 (AX-2), referred to as wild type, was cultivated axenically in shaken suspension at 21°C  and harvested during exponential growth. Clones of HsII-GFP  were cultivated as described for AX-2, but under the selection of 20 μg/ml Geneticin (G418). Clones of his--cells  were cultivated as described for AX-2, but under the selection of 7.5 μg/ml Geneticin.
Hyperosmotic shock in liquid culture
Hyperosmotic shock experiments were performed according to Schuster et al. (1996). Axenically grown Dictyostelium cells were harvested, washed twice in Soerensen Phosphate Buffer (SPB: 2.0 mM Na2HPO4, 14.6 mM KH2PO4, pH 6.0) and were then adjusted to 2*107 cells/ml with SPB buffer (osmolarity: 34 mOsm). Cell density was 1,6*107 cells/ml during the osmoshock, adjusted by the addition of SPB buffer/2 M sorbitol. To the control cells, SPB buffer was added until the same cell density was reached.
The cell suspension was shaken for 1 h. SPB buffer/2 M sorbitol was added to a final concentration of 400 mM sorbitol (osmolarity of the buffer: 434 mOsm). The cell suspension was incubated up to 2 hours according to the experiment. Viability of Dictyostelium cells after exposure of the cells to hyperosmotic stress was determined as described . In short, his--cells were shaken in SPB buffer for 1 h prior to the hyperosmotic shock. Samples were withdrawn at intervals and cell suspension corresponding to 200 cells was plated on agar plates together with Klebsiella aerogenes bacteria. Plaques in the bacterial lawn were counted after 2–4 days.
Exposure to stress conditions
To acidify Dictyostelium cells, axenically grown wild type cells were washed twice in SPB buffer and were resuspended in SPB buffer/5 mM propionic acid, pH 6.0. To impose oxidative stress, the cells were incubated in SPB buffer/3 mM H2O2 for 60 min. To study heat stress, the cells were exposed to 30°C for 30 min. Energy depletion was achieved by incubating the cells in SPB buffer/50 μM 2,4-dinitrophenol for 60 min.
2-D Gel electrophoresis
Preparation of the cytoskeletal fraction from hyperosmotically shocked cells and separation of the proteins by 2-D gel electrophoresis was performed as described recently . The cytoskeletal fraction was obtained by isolating the Triton X-100-insoluble fraction. The corresponding soluble fraction was defined as cytosolic fraction. 100 μg total protein was applied per gel.
Metabolic labeling with 32P-orthophosphate
50 ml cell suspension of axenically grown wild type cells were transferred to 300 ml phosphate-free medium buffered with 20 mM Mes, pH 6.0. The cells were shaken for 16 h and were subsequently washed twice in ice-cold 20 mM Mes, pH 6.0 (Mes buffer). The cells were adjusted to 2*107 cells/ml with Mes buffer. After incubating the cells for 30 min, 32P-orthophosphate (Amersham Pharmacia) was added to a final activity of 0.25 mCi/ml in the cell suspension. The cell suspension was shaken for 60 min, before the cells were exposed to hyperosmotic shock. Samples of 1 ml were withdrawn at intervals, the cells were washed twice with Mes buffer and were resuspended in 800 μl RIPA buffer (50 mM Tris/HCl, pH 7.5; 150 mM NaCl, 1% TritonX-100; 1% Natriumdeoxycholate; 0.1% SDS; 1 mM DTT; 1 mM EDTA; 100 μM Na-orthovanadate; Complete Protease Inhibitor Cocktail (Boehringer Mannheim)). The cell lysate was immediately frozen in liquid nitrogen. As control, samples were prepared from a cell suspension to which Mes buffer was added instead of Mes buffer/2 M sorbitol.
Immunoprecipitation of hisactophilin
Thawed cell lysate from 32P-labeled cells was centrifuged for 20 min at 10000 g (4°C). α-hisactophilin antibody 54-11-10  was added to the cleared lysate and was incubated for 3 h. Immunoprecipitation was performed by adding fixed Staphylococcus aureus cells (Pansorbin, Calbiochem) preincubated with rabbit-α-mouse IgG (Sigma). After 45 min incubation, the immunoprecipitate was washed twice with TBS and was analyzed by SDS-PAGE, Western blotting and autoradiography.
Axenically grown HisII-GFP cells were shaken in SPB buffer for 1 h at a cell density of 2*107 cells/ml. SPB buffer/2 M sorbitol was added to a final concentration of 400 mM sorbitol (cell density: 1,6*106 cells/ml). An aliquot of the living cells was allowed to adhere to glass cover slips for 10 min. Subsequently, fluorescence microscopic observations were carried out. As control, cells suspended in SPB buffer were analyzed.
The equipment consisted in a Leitz Aristoplan fluorescence microscope, set at 450–490 nm for excitation.
Phosphoamino acid analysis
Phosphoamino acid analysis was performed as described . In short, hisactophilin was immunoprecipitated from 32P-labeled cells exposed to hyperosmotic stress for 60 min. Acid hydrolysis of the immunoprecipitate was performed, followed by the separation of the phosphoamino acids by electrophoresis in two dimensions on TLC plates together with phosphoamino acid standards. The plates were analyzed by autoradiography and by the detection of amino acids with Ninhydrin.
Protein quantification, SDS-PAGE and Western blotting onto PVDF membrane were performed according to the published methods of Bradford , Laemmli  and Towbin et al.. Western blots were treated with the mouse monoclonal α-hisactophilin antibody 54-11-10 . Antibodies were detected using peroxidase-coupled rabbit-α-mouse IgG and the Renaissance® system (Du Pont). Prestained Seeblue marker (Novex) was used as a protein molecular weight standard.
We would like to thank Dr. G. Gerisch for the gift of the hybridoma culture expressing the monoclonal antibody 54-11-10 and the HsII-GFP-cells and Prof. Dr. M. Schleicher for the gift of his--cells. This work was supported by grants of the Boehringer Ingelheim Fonds to T.P. and of the Deutsche Forschungsgemeinschaft to S.C.S. (Schu778/3-1, Schu778/3-2, Schu778/5-1).
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