©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The pH-sensitive Actin-binding Protein Hisactophilin of Dictyostelium Exists in Two Isoforms Which Both Are Myristoylated and Distributed between Plasma Membrane and Cytoplasm (*)

(Received for publication, June 14, 1994; and in revised form, October 17, 1994)

Frank Hanakam Christoph Eckerskorn Friedrich Lottspeich Annette Müller-Taubenberger Wolfram Schäfer Günther Gerisch

From the Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The histidine-rich protein hisactophilin is known to be associated with the inner surface of the plasma membrane and to be present as a soluble protein in the cytoplasm of Dictyostelium discoideum cells. Mass spectrometry of hisactophilin from the cytosol or extracted from a membrane fraction showed that none of the hisactophilin purified from D. discoideum cells had the mass predicted from the known cDNA-derived amino acid sequence of the protein. Electrospray mass spectrometry and liquid secondary ion mass spectrometry of tryptic fragments separated by reversed-phase high performance liquid chromatography (HPLC) identified the most hydrophobic peptide as a myristoylated fragment from the N terminus of hisactophilin. Taken together the analytical data, it is concluded that all hisactophilin in D. discoideum cells is N terminally modified by myristoylation. By reversed-phase HPLC, two isoforms of hisactophilin, HsI and HsII, were recovered from the cytosolic as well as the membrane fraction of D. discoideum cells. Whereas the masses of HsI fragments produced by trypsin fit into the previously published sequence of hisactophilin (myristoylation considered), HsII is another protein distinguished from HsI by several amino acid exchanges. HsI and HsII can form homo- and heterodimers by disulfide bridges. Hisactophilin is phosphorylated in vivo. Both isoforms proved to be substrates of membrane-associated threonine/serine kinase from D. discoideum, which may regulate the interaction of hisactophilin with the plasma membrane.


INTRODUCTION

Highly motile amoeboid cells as those of Dictyostelium need sophisticated mechanisms for coupling of their actin skeleton to the plasma membrane. In order for a cell to move, it is essential to regulate this coupling locally at the leading edge and also at the sites of contact with a surface to which the cell adheres. In Dictyostelium cells, two proteins have been described that are capable of coupling actin filaments to membranes. One of them is ponticulin, a transmembrane glycoprotein that attaches laterally to actin filaments and binds them to the cytoplasmic phase of the cell envelope(1, 2) . The other protein is hisactophilin, which exists in a plasma-membrane bound and a free cytoplasmic state and binds in vitro to actin in a strictly pH-dependent manner(3) . The high content of 26% histidine residues is thought to be responsible for binding of the protein to actin at pH values below the pK of histidine and its dissociation from actin filaments beyond pH 7.0. NMR has shown that hisactophilin consists of a beta barrel, which forms a backbone around a 3-fold axis of symmetry, and of three major loops exposed on the surface of the molecule which contain nearly all of the histidine residues(4) .

In this paper modifications of hisactophilin are studied which might be implicated in reversible attachment to the plasma membrane. Earlier results have indicated that during metabolic labeling of D. discoideum cells with [^3H]palmitic acid radioactive label is incorporated into hisactophilin(5) . Here we demonstrate that all hisactophilin is myristoylated in D. discoideum cells. In addition, evidence is provided for the presence of two isoforms of hisactophilin and their phosphorylation by a membrane-associated kinase.


EXPERIMENTAL PROCEDURES

Cultivation of D. discoideum

Cells of Dictyostelium discoideum strain AX2-214 were cultivated axenically at 23 °C in liquid nutrient medium (6) on a gyratory shaker at 150 revolutions/min and harvested during exponential growth at a density of 5 times 10^6 cells/ml by three times washing in 17 mM potassium-sodium-phosphate buffer, pH 6.0(7) . Except for metabolic labeling, cells were lysed immediately in homogenization buffer (30 mM Tris-HCl, 4 mM EGTA, 2 mM EDTA, 2 mM DTT, (^1)5 mM benzamidine, 30% sucrose, pH 8.0) by nitrogen cavitation in a Parr bomb after equilibration at 950 pounds/square inch for 15 min.

Purification of Hisactophilin

The lysate was fractionated by centrifugation for 30 min at 10,000 times g into a crude membrane pellet fraction and for 3 h at 100,000 times g into the cytosolic supernatant fraction. From the latter, hisactophilin was purified by DE52 cellulose (Whatman, Biosystems Inc., Clifton, NJ) and either Sephacryl S-300 or Sepharose 6B (Pharmacia, Uppsala, Sweden) chromatography as described by Scheel et al.(3) . For purification from the membrane fraction, the 10,000 times g pellet was washed in TEDABP buffer (10 mM Tris-HCl, 1 mM EGTA, 1 mM DTT, 0.02% NaN(3), 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, pH 8.0), and adjusted in the buffer to a concentration of 5 mg protein/ml. For extraction, 150 mM NaCl and 0.5% Brij 35 (Pierce) were added, the pH was adjusted to 8.4, and the suspension was stirred for 1 h at 4 °C. After centrifugation at 40,000 times g for 30 min, the supernatant dialyzed against TEDABP buffer until conductivity was below 1 mS. Subsequent purification steps were the same as for the cytosolic fraction.

HPLC was performed using Kontron Instruments (München, Germany) pump 420 and detector 430 at a wavelength of 206 nm and a RP18 Lichrospher column (125 times 4 mm; Merck, Darmstadt, Germany). A gradient system of solvent A (0.1% trifluoroacetic acid (Sigma, Deisenhofen, Germany) in water) and solvent B (0.1% trifluoroacetic acid in acetonitrile; Lichrosolv, Merck) at a flow rate of 1 ml/min was used throughout. Linear gradients of 0-80% B in A in 80 min were used for final purification of the protein, and isoforms HsI and HsII were separated by a linear gradient of 0-35% B in A in 10 min, followed by an isocratic elution at 35% B. Fractions were collected manually and concentrated by evaporation in a Speed Vac centrifuge.

Reduction of Cysteine Residues and Proteolytic Cleavage

For preventing dimerization, hisactophilin purified by DE52 cellulose ion-exchange and Sepharose 6B gel filtration chromatography was denatured with 6 M guanidinium chloride and reduced with 5 mM DTT for 1 h at 37 °C. Subsequently, the cysteine residues were covalently modified with 4-vinylpyridine added at a 5-fold molar excess over total thiol groups for 2 h at 37 °C. The reaction was stopped by addition of 1% acetic acid, and the protein was subjected to HPLC.

For proteolytic cleavage 1 mg/ml of HPLC-purified, denatured, and reduced hisactophilin was digested with either trypsin or endoprotease Lys-C at 37 °C. Trypsin, sequencing grade (Boehringer Mannheim, Germany), was added at a concentration of 0.1 mg/ml in 100 mM Tris-HCl, pH 8.5, with 10% acetonitrile. For peptide sequencing endoprotease Lys-C, sequencing grade, was used for cleavage at a concentration of 0.01 mg/ml in buffer containing 50 mM Tris-HCl, 50 mM Tricine, pH 8.5, 1 mM EDTA, and 10% acetonitrile. The peptides were fractionated by HPLC using the same column and solvents as for uncleaved hisactophilin. A linear gradient of 0-80% B in A in 80 min was employed to separate tryptic fragments, and a gradient of 0-50% solvent B in A in 100 min for Lys-C cleavage products.

Mass Spectrometry and Amino Acid Sequencing

Masses were determined using an API-III electrospray mass spectrometer (Sciex, Perkin Elmer) either by direct infusion of HPLC-separated samples or by on-line reversed-phase HPLC on an Aquapore OD 300, 7-µm column, 50 times 1 mm (Applied Biosystems, Weiterstadt, Germany). The solvents were 0.1% trifluoroacetic acid in water (solvent A) and 0.1% trifluoroacetic acid in acetonitrile (solvent B). A linear gradient of 0-80% B in A in 80 min was applied at a flow rate of 50 µl/min with a split to the MS of 5 µl/min.

For LSI-MS analysis in a mass spectrometer MAT900 (Finnigan MAT, Bremen, Germany), the peptides were dissolved in acetonitrile/acetic acid/water (4:1:5) and mixed with the matrix (glycerol/monothioglycerol 1:1). The energy of the ionizing cesium ions was 20 keV.

For gas chromatography-mass spectrometry analysis the mass spectrometer CH7A (Finnigan MAT) was coupled with a gas chromatograph HRGC 5160 (Carlo Erba, Milano, Italy). The all-glass system consisted of a silica capillary coated with Durabond 1 (S & W. Scientific, Folsan, CA), which was introduced directly into the ion source (70 eV). The temperature program was 5 min at 100 °C, following 5 °C/min up to 250 °C with a helium flow of about 1 ml/min. The inlet temperature was 260 °C. For derivatization, the peptides were hydrolyzed as for amino acid analysis, evaporated, dried, and treated with hexamethyldisilazan/trimethyl-chlorosilan/pyridin (2:1:7) (Pierce) according to Sweeley et al.(8) .

Peptides of HsII were sequenced using a pulsed liquid-phase sequencer 477 equipped with an on-line phenylthiohydantoin-analyzer 120A (Applied Biosystems).

Metabolic Labeling with [^3H]Fatty Acids

For fatty acid labeling, growth-phase cells were washed and adjusted to 10^7 cells/ml in 17 mM sodium-potassium phosphate buffer, pH 6.0. After starvation under shaking conditions for 2 h, 1 mCi of [^3H]myristic acid (New England Nuclear; NET-830) or [^3H]palmitic acid (New England Nuclear; NET-043) was added to 6-ml aliquots of cell suspension and shaking continued for another 6 h(9) . Then cells were washed with phosphate buffer, frozen in methanol/dry ice, and stored at -80 °C. Cell lysates corresponding to 5 times 10^7 cells were thawed and dissolved in 0.5 ml of PBS, pH 7.2, supplemented with 15 µl of protease inhibitor mix(10) . A crude membrane fraction was obtained by centrifugation for 30 min at 14,000 times g. The supernatant was further centrifuged in an airfuge (Beckman Instruments, Fullerton, CA) for 30 min at 100,000 times g, and the supernatant used as cytosolic fraction. The crude membranes were washed in TEDA (10 mM Tris-HCl, 1 mM EGTA, 1 mM DTT, 0.02% NaN(3), pH 8.0) and extracted with 0.5% Lubrol (Pierce) and 150 mM NaCl in a total volume of 750 µl for 30 min on ice. The extract was cleared by centrifugation for 20 min at 40,000 times g. Hisactophilin was immunoprecipitated with mAb 54-165-12 (3) using protein A-Sepharose Cl-4B beads (Pharmacia), essentially as described by Barth et al.(10) . The precipitate was dissolved in two-dimensional sample buffer, and proteins were separated by two-dimensional electrophoresis (11) using a 2:3 mixture of ampholines (Pharmacia) with pH ranges of 3-10 and 7-9, followed by electroblotting(12) . For fluorography the filters were dipped into 20% (w/v) of 2,5-diphenyloxazole in toluene and exposed to Hyperfilm-[^3H] (Amersham) at -70 °C for 4-20 days. Subsequently, filters were incubated with mAb 54-165-12 and I-sheep anti-mouse IgG (Amersham) and exposed to Hyperfilm-MP (Amersham).

Phosphorylation of Hisactophilin by Partially Purified Dictyostelium Kinase

Hisactophilin kinase was prepared from a crude membrane fraction of growth-phase cells by freezing, thawing, and centrifugation of the homogenate for 30 min at 14,000 times g. The pellet was washed with TEDA buffer and extracted with 0.5% Lubrol (Pierce) and 150 mM NaCl in TEDA buffer, and separated from endogenous substrates by DE52 cellulose and Sephacryl S-300 chromatography. For phosphorylation, kinase prepared from about 5 times 10^6 cells and hisactophilin purified from the cytosol were added to 150 µl of the incubation mixture (3 µg of hisactophilin for phosphoamino acid analysis, 10 µg for two-dimensional electrophoretic separation of isoforms). The mixture containing about 10 µCi of [-P]ATP (Amersham) in phosphorylation buffer (50 mM MES, pH 6.4, 0.5 mM DTT, 5 mM MgCl(2), 2.5 mM ATP, 0.5% aprotinin) was incubated for 10 min at 30 °C. For the identification of phosphorylated amino acids, hisactophilin was separated by SDS-PAGE, the band cut out of the gel, extracted, and hydrolyzed in 6 M HCl for 18 h at 100 °C. P-Labeled phosphoamino acids were separated by electrophoresis on cellulose thin layer sheets at pH 3.5(13) , and autoradiographs scanned on an Elscript-400 electrophoresis scanner (Hirschmann, Unterhaching, Germany). Two-dimensional electrophoresis was performed as described for [^3H]fatty acid-labeled protein.

Metabolic Labeling with [P]Phosphate

Growth-phase cells were washed three times in 20 mM MES buffer, pH 6.5, and adjusted to 4 times 10^6 cells/ml in liquid nutrient medium that had been dialyzed against 20 mM MES, pH 6.5. The culture was shaken for 9 h at 20 °C with 100 µCi of [P]phosphate (Amersham)/ml. The labeled cells were washed with TN buffer (50 mM Tris, pH 8.0, 150 mM NaCl) and lysed in 1 ml of TN buffer containing 0.5% SDS. The lysate was heated for 5 min at 95 °C, centrifuged for 10 min at 10,000 times g, and the supernatant mixed with 0.9 ml of TN buffer containing 2% Nonidet (Nonidet P-40, Fluka) and 2 mg of bovine serum albumin. For immunoprecipitaton with mAb 54-11-10, 0.1 ml of hybridoma culture supernatant was added. After shaking for 2 h at 4 °C, 40 µl of protein A-agarose macrobeads (Sigma) were added, and shaking was continued for 2 h. The beads were washed twice with 1 ml of TN buffer, twice with 1 ml of H(2)O, and heated with 40 µl of SDS-sample buffer for 10 min at 95 °C. 20 µl of the samples were subjected to SDS-PAGE in 15% gels, blotted onto nitrocellulose, and exposed to X-Omat-film (Kodak) for 90 h. On the same blot the hisactophilin band was identified by labeling with mAb 54-165-12 and alkaline-phosphatase-conjugated anti-mouse IgG.

Molecular Modeling

Models were developed on a Silicon Graphics workstation using the ``Insight II 2.3.0'' software (Biosym, San Diego, CA). The lipid bilayer was built of phospholipids with C(18)-fatty acids constructed from the fragment library of Insight II without further energy minimization of the residues. The structural data of hisactophilin (HsI) were kindly provided by Dr. Tadeusz Holak, Martinsried, and that of rabbit skeletal muscle F-actin by Dr. Michael Lorenz, Heidelberg.


RESULTS

The N Terminus of Hisactophilin Is Modified by Myristoylation

In an attempt to search for modifications, hisactophilin was purified from Dictyostelium cells by DE52-cellulose and Sepharose 6B chromatography as described by Scheel et al.(3) , and finally analyzed by reversed-phase HPLC. As a reference for unmodified hisactophilin of known sequence(3) , protein expressed in Escherichia coli(3) was subjected to HPLC under the same conditions. Retention times in an acetonitrile gradient indicated that the hisactophilin from Dictyostelium cells was more hydrophobic than the bacterially expressed hisactophilin. As shown in the chromatogram of Fig. 1, hisactophilin from Dictyostelium eluted in two peaks clearly separated from the single peak of hisactophilin produced in E. coli cells. This result raised two questions: 1) what distinguishes hisactophilin from Dictyostelium cells from the bacterially produced one; and 2) what is the reason for the fractionation of hisactophilin from Dictyostelium cells into two peaks?


Figure 1: Comparison of hisactophilin purified from Dictyostelium cells (D.d.) with hisactophilin expressed in transformed E. coli cells (E.c.). The proteins were chromatographed under identical conditions on a reversed-phase column with a linear acetonitrile gradient. The bar below the curves indicates the fractions of Dictyostelium hisactophilin pooled for preparing fragments as shown in Fig. 2.




Figure 2: Separation of tryptic fragments of hisactophilin from Dictyostelium cells by reversed-phase HPLC. The most hydrophobic fragment eluting at 56-60% acetonitrile (bar) was analyzed by LSI-MS. The inset shows the presence of a single mass corresponding to that of the 3 N-terminal amino acids G-N-R plus one myristoyl moiety.



To answer the first question, modification of the hisactophilin by fatty acid was examined. Labeling by [^3H]fatty acids in vivo revealed that the label from palmitic acid as well as myristic acid was incorporated into hisactophilin. To determine the type of bond, the entire HPLC fraction of cytosolic Dictyostelium hisactophilin was treated with hydroxylamine at pH 7 or 10 and rechromatographed by HPLC. Presence of thioester or ester bonds should result in a shift in retention time toward the unmodified bacterially expressed hisactophilin. No shift was found and the two peaks did not merge, suggesting modification of hisactophilin from Dictyostelium cells by hydroxylamine stable amide bonds. Metabolic labeling with [^3H]myristic acid or [^3H]palmitic acid gave a similar result: the tritium label was not removed from hisactophilin by treatment with hydroxylamine at pH 10.

In order to determine the type of fatty acid linked to hisactophilin and to localize the bond, the protein was cleaved with trypsin, and the fragment with the highest retention time on a reversed-phase HPLC column was analyzed by LSI-MS (Fig. 2). The peak of the MH-ion was found at m/z = 556.1 (Fig. 2, inset). Amino acid analysis of this fragment resulted in Gly, Asx, and Arg in equimolar ratios, consistent with the sequence G-N-R of the N-terminal tryptic cleavage product predicted from the known hisactophilin sequence(3) . The fragment differed in mass from the tripeptide by 210 Da, which is exactly the mass of a myristoyl residue. In order to confirm that the N terminally bound fatty acid is myristic acid, an aliquot of the tryptic fragment was hydrolyzed and trimethylsilylated. Analysis by gas chromatography-mass spectrometry revealed only one major peak in the gas chromatogram, which coincided in retention time and mass spectrum with authentic trimethylsilylester of myristic acid.

Masses of Two Isoforms of Hisactophilin Purified from Both Cytosolic and Membrane Fractions

The double peak of hisactophilin purified from Dictyostelium cytosol (Fig. 1) suggested the presence of two isoforms. The mixture was separated into two distinct peaks by isocratic HPLC, and the masses of the separated proteins were determined by ES-MS. Peak A of Fig. 3revealed a single mass of 13,715 Da, and peak B of 13,536 Da. The mass of peak B corresponds to hisactophilin of the published sequence (3) (13,326 Da after removal of the N-terminal methione) plus one myristoyl residue (210 Da).


Figure 3: Separation of two isoforms of hisactophilin by isocratic HPLC at 35% acetonitrile. Peak A represents hisactophilin II, and peak B represents hisactophilin I.



Both the proteins of peak A and B reacted with antibodies mAb 54-11-10 and 54-165-12 previously used to label hisactophilin(3, 14) . In the following we refer to the protein of peak B as hisactophilin I (HsI), and to the protein of peak A as hisactophilin II (HsII). The same peptide of high retention time with the mass of myristoyl-G-N-R was found by LC-MS after trypsin treatment of both proteins, indicating that myristoylation is common to hisactophilins I and II.

Under non-reducing conditions, three different masses were detected in mixtures of purified HsI and HsII, which coincided with the predicted masses of homo- and heterodimers. Covalent modification of cysteine residues by 4-vinylpyridine resulted in masses of 13,641 and 13,820 Da, which correspond to the masses of HsI and HsII plus 105 Da, the mass of one ethylpyridine moiety/hisactophilin monomer. These results indicate 1 free cysteine residue to be present in each hisactophilin isoform.

A portion of hisactophilin is known to be attached in Dictyostelium cells to the inner face of the plasma membrane. To examine whether the same isoforms as from the cytosol can be isolated from a membrane fraction, hisactophilin was purified from a 10,000 times g pellet of a Dictyostelium cell homogenate. The hisactophilin was extracted with non-ionic detergent from the 10,000 times g pellet and purified essentially as the cytosolic one. HPLC of the extracted hisactophilin revealed two peaks with retention times coincident with those of HsI and HsII from the cytosol. ES-MS established that the masses of the two isoforms from the membrane fraction corresponded to the masses of HsI (13, 536 Da) and HsII (13, 715 Da) as purified from the cytosol (Fig. 4). However, the ratios of the two isoforms might differ in the soluble and detergent-extracted fraction: in the material purified from the cytosol an excess of HsI over HsII was found, in the membrane extract more HsII than HsI was recovered.


Figure 4: Comparison of hisactophilin isoforms from the cytosol and membrane fraction. Reconstructed masses of hisactophilins from the cytosolic fraction (A) and membrane extract (B) determined by ES-MS are shown.



The isoforms purified by HPLC (Fig. 3) could also be separated by isoelectric focusing in a shallow pH gradient. The less basic isoform proved to be HsI, and the slightly more basic form HsII (Fig. 5). Bacterially expressed hisactophilin focussed essentially as HsI. As judged from the [^3H]myristic acid label incorporated into the isoforms and from immunoblotting, the ratio of HsI to HsII was >1 in the cytosolic fraction and about 1 in an extract from membranes.


Figure 5: Separation of hisactophilins I and II from cytosolic and membrane fractions by two-dimensional-electrophoresis. Horizontal direction, isoelectric focussing; vertical direction, SDS-PAGE. Top panels, cells were metabolically labeled with [^3H]myristic acid during starvation until harvest at 8 h of development. Hisactophilins were immunoprecipitated and, after electrophoresis, fluorographed for ^3H incorporation. Bottom panels, proteins were separated under the same conditions and subjected to immunoblotting with hisactophilin-specific antibody.



In summary, the two hisactophilin isoforms separated by HPLC have different but uniform masses, they give rise to the same N-terminal peptide corresponding to the mass of myristoyl-G-N-R and, finally, hisactophilins purified from cytosolic and membrane fractions have the same masses. From these results we conclude that all hisactophilin in D. discoideum cells is myristoylated.

Hisactophilins I and II Are Distinguished by Amino Acid Sequence

To address the question whether the differences in mass and isoelectric point of HsI and HsII are due to a modification other than N-terminal myristoylation, or to differences in the amino acid sequence, HsI and HsII were separated and digested with trypsin for LC-MS. The results summarized in Fig. 6show that the two hisactophilins differed in all tryptic fragments except four: the two N-terminal fragments comprising 6 amino acids, the cysteine containing pentapeptide, and a tetrapeptide in the C-terminal half. Most of the mass differences did not fit to known modifications. In order to establish that alterations in amino acid sequences are responsible for these differences, HsII was digested with endoprotease Lys-C for the sequencing of fragments separated by HPLC (Fig. 7).


Figure 6: Comparison of the masses of tryptic fragments from hisactophilin I and II as detected by LC-MS with the known hisactophilin I sequence. Cleavage sites for trypsin as inferred from the HsI sequence are indicated and the corresponding masses of fragments shown on top of the sequence. All predicted masses of HsI fragments were detected by the electrospray MS (times = Cys blocked with ethylpyridine). The HsII fragments are provisionally aligned with HsI fragments according to similar mass and retention time in a reversed-phase column.




Figure 7: Reversed-phase HPLC of fragments obtained by digestion with endoprotease Lys-C for mass determination and amino acid sequencing. The peaks analyzed are indicated by bars, and masses are indicated below the curve. N-terminal sequences of the two larger peptides were determined, and 2 amino acid replacements as compared to HsI were detected (underlined). The mass of the small fragment with the largest retention time corresponded to the N-terminal myristoyl-hexapeptide predicted from the HsI sequence as a Lys-C cleavage product.



Two sharply separated fragments with masses of 3,706 and 2,766 Da, respectively, did not coincide with any of the fragments predicted from the HsI sequence. These fragments were N terminally sequenced. In the 3,706-Da fragment, 2 amino acid exchanges were discovered among the first 17 amino acid residues: instead of Ala and His of HsI, Val and Gly were found in HsII. In the 2,766-Da fragment, the first 14 amino acid residues fit to the HsI sequence, confirming that the two proteins designated as HsI and HsII are closely related to each other. In addition, the mass of the HsII fragment with the largest retention time, eluting from the HPLC column behind the uncleaved protein (Fig. 7), was determined to be 902 Da. This fits exactly with the myristoyl-GNRAFK fragment predicted from the HsI sequence. From the mass spectrometry and sequencing data, it can be inferred that HsI and HsII have the same N-terminal region, but that more than eight differences in sequence are distributed downstream along their polypeptide chains.

Hisactophilins I and II Are Phosphorylated by Dictyostelium Threonine/Serine Kinase

The identity of HsI and HsII purified from cytosolic and membrane fractions suggests that modifications which might be responsible for conversion of hisactophilins from a membrane-bound to a soluble state have been lost during the purification procedure. Since intracellular localization of other myristoylated proteins is determined by phosphorylation(15, 16) , we have examined purified hisactophilin as a substrate of kinases from Dictyostelium cells.

The membrane fraction proved to be an appropriate source of hisactophilin kinase. As a substrate, a mixture of HsI and HsII from the cytosol was employed. Kinase extracted with detergent cofractionated on a DE52-cellulose column with the hisactophilins, but could be separated on a Sephacryl S-300 column. The kinase eluted behind the excluded volume but in front of the hisactophilins, which can be easily separated due to their atypical elution behind the salt peak(3) . The kinase preparation was free of endogenous substrate and did efficiently phosphorylate both hisactophilins with a pH optimum of 6.4 and a temperature optimum at 30 °C. Activity of the kinase required Mg but not Mn ions, which suggested that the enzyme is a threonine/serine kinase. This was confirmed by total hydrolysis of hisactophilins phosphorylated in vitro with kinase from Dictyostelium cells and [-P]ATP (Fig. 8). 95% of the label was recovered in phosphothreonine and 5% in phosphoserine. No phosphotyrosine was detected.


Figure 8: Phosphoamino acids of hisactophilin after phosphorylation in vitro. A mixture of HsI and HsII as purified from the cytosol was incubated with solubilized and partially purified kinase from a crude D. discoideum membrane fraction together with [-P]ATP. After total hydrolysis and electrophoretic separation of phosphoamino acids, an autoradiograph was scanned for P incorporation at the positions of phosphotyrosine (Y), phosphothreonine (T), and phosphoserine (S).



In order to explore whether hisactophilin is also phosphorylated in vivo, cells of D. discoideum were metabolically labeled during growth with [P]phosphate. Fig. 9shows that label is incorporated into hisactophilin, indicating that within the cells hisactophilin is not only modified by myristoylation but also subject to phosphorylation.


Figure 9: Phosphorylation of hisactophilin in vivo. After growth in the presence of [P]phosphate, cells were lysed with SDS. An aliquot of the cell lysate, hisactophilin immunoprecipitates, and controls were separated by SDS-PAGE and either stained with Coomassie Blue (lanes 1-4), or blotted, followed by indirect labeling with an anti-hisactophilin antibody, mAb 54-165-12, and anti-mouse IgG conjugated to alkaline-phosphatase (lanes 6-8). Lane 1, crude cell lysate. Lanes 2 and 6, hisactophilin precipitated from the lysate with mAb 54-11-10. For comparison purified hisactophilin (lanes 3 and 7) and protein-A precipitated mAb 54-11-10 (lanes 4 and 8) were applied. Lane 5, autoradiograph of lane 6 showing the P-label on hisactophilin. Although Coomassie Blue shows a single band of precipitated protein which coincides with the antibody-labeled hisactophilin band (lanes 2 and 6), the autoradiograph visualizes multiple phosphorylated bands in addition to the one of hisactophilin (lane 5). These traces of proteins coprecipitating with hisactophilin might represent phosphorylated kinases.




DISCUSSION

Attachment of Hisactophilin to the Inner Face of the Plasma Membrane

Previous data indicated that roughly half of the hisactophilin is attached to the plasma membrane of D. discoideum cells, and the other half is distributed in the cytoplasm as a soluble protein. The finding that hisactophilin as purified from Dictyostelium cells is myristoylated at its N terminus suggests that the C(14)-hydrocarbon chain of the fatty acid contributes to membrane binding. In Fig. 10we have modeled the size and shape of the hisactophilin protein as revealed by NMR (4) together with an N terminally linked myristoyl residue inserted into a phospholipid bilayer. It is assumed that the hisactophilin molecule faces the membrane with the flattened area of its surface that surrounds the N terminus.


Figure 10: Scheme of hisactophilin insertion into the inner lipid layer of the plasma membrane. For constructing the diagram, the structure of bacterially expressed, non-acylated hisactophilin I as determined by NMR (4) was used. To the N terminus a myristoyl moiety was attached by modeling. Dark areas represent histidine residues exposed on the protein surface, which are thought to be involved in pH-sensitive actin binding. Membrane phospholipids were assembled by computer modeling. Part of an actin filament representing four monomers is shown according to scale. The nucleating effect on actin polymerization (3) suggests that hisactophilin binds to F-actin at a site matched by two monomers.



Hisactophilin efficiently binds at pH 6.5 to actin filaments(3) . Bundling of actin filaments by hisactophilin has been observed by Urban (5) . This activity is possibly a function of hisactophilin dimers that are produced under non-reducing conditions by disulfide bridge formation. The bundling activity indicates that hisactophilin binds laterally to F-actin. Hisactophilin also nucleates actin polymerization (3) , which suggests that it stabilizes actin oligomers by binding to a cleft between two monomers. In Fig. 10four monomers of an actin filament, as constructed from refined x-ray fiber diffraction data (17) , are shown in scale with hisactophilin I to demonstrate that sizes and shapes would fit.

Cytoplasmic Versus Membrane-bound Hisactophilin

In vertebrate cells N-terminal myristoylation occurs cotranslationally and is a permanent modification of the respective protein(18) . Our data concerning a D. discoideum protein are in line with these results in that apparently all hisactophilin molecules carry the modification. Immunogold labeling has shown that hisactophilin recognizes selectively the plasma membrane as a site of attachment (14) . The question is then how the myristoylated hisactophilin redistributes during membrane internalization. This question has been addressed previously by following endocytosis of membrane glycoproteins after their capping and labeling with fluorescent concanavalin A(19) . These experiments have provided evidence that hisactophilin disappears in D. discoideum cells from the internalized membrane. One reason for this depletion might be exclusion of the protein from membrane areas that are in the process of being invaginated. Another possibility is that hisactophilin is detached from the membrane during or shortly after invagination and is released into the cytoplasm.

Three mechanisms by which an equilibrium between cytoplasmic and membrane-bound hisactophilin can be maintained in Dictyostelium cells will be discussed here. 1) As in the alpha subunits of heterotrimeric G proteins, membrane binding might be supported by reversible, covalent linkage of palmitic acid to a cysteine residue. In vivo radiolabel from myristic acid as well as palmitic acid is incorporated into hisactophilin. However, since hisactophilin I contains only 1 cysteine residue, which is free in the purified protein, it is more likely that palmitic acid is converted by the cells into myristic acid which is then linked to the N terminus of hisactophilin. 2) Binding to negatively charged membrane lipids might be enhanced by positively charged amino acids as in the adhesion plaque protein MARCKS (20) and in proteins of the Src family (21) , where lysine residues are clustered in the N-terminal region. Phosphorylation by protein kinase C relieves MARCKS from its membrane-bound state(15) . Since hisactophilin is phosphorylatable by membrane-associated threonine/serine kinase, and is also phosphorylated in vivo, this modification might be responsible for dissociation of the protein from the membrane. 3) In contrast to other myristoylated proteins(22) , the hisactophilin I sequence does not show any clustering of lysine residues in the N-terminal region. In HsI only 1 lysine but several histidine residues are located in the polypeptide chain close to the N terminus(3) . The pK of histidine of 6.8 makes the net charge of hisactophilin sensitive to small changes in the cytoplasmic pH. The presence of proton pumps in the membrane of endosomes (23) might be responsible for a rise of pH at the cytoplasmic surface of these vesicles, which may cause hisactophilin to detach from the endosomal membrane.

Actin Coupling to the Plasma Membrane

Coupling of the actin cortex to the cell membrane has important functional implications in cell motility and cytokinesis(24, 25) . Two proteins described in Dictyostelium have the potential of binding actin filaments to membranes. Ponticulin has been described as a transmembrane protein considered to be the major high affinity link between the plasma membrane and the cortical actin network in Dictyostelium(2) . Since the elimination of ponticulin by gene disruption has no dramatic effects on cell movement and development(2) , other proteins must be involved in actin coupling to the membrane. The second protein is hisactophilin, which exists in a membrane-associated and a cytoplasmic state. The finding that two isoforms of hisactophilin that differ in amino acid sequence coexist in D. discoideum cells opens the possibility that the activity of hisactophilin is fine tuned in these highly motile cells by a pair of proteins with slightly diverging properties. Our results are in accord with the notion that the two isoforms of hisactophilin differ in their capacity of binding to the membrane. However, since changes in the ratio of HsI to HsII during their extraction from a membrane fraction or during purification have to be taken into account, this point needs further investigation.


FOOTNOTES

*
This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 266/D7 (to G. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: DTT, dithiothreitol; ES-MS, electrospray mass spectrometry; HPLC, high performance liquid chromatography; HsI, hisactophilin I; HsII, hisactophilin II; LC-MS, liquid chromatography mass spectrometry; LSI-MS, liquid secondary ion mass spectrometry; MS, mass spectrometry; Tricine, N-tris(hydroxymethyl)methylglycine; MES, 4-morpholineethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Dr. Michael Schleicher, München, for providing recombinant hisactophilin I from E. coli as a reference, Richard Albrecht, Martinsried, for the support in hard- and software handling, and Gisela Maniak for expert technical assistance. We are grateful to Dr. Tadeusz Holak, Martinsried, for the data on hisactophilin I structure and to Dr. Michael Lorenz, Heidelberg, for the data on reconstructed rabbit skeletal muscle F-actin.

Note Added in Proof-The cDNA sequence of hisactophilin II (EMBL/Genbank accession number U13671) and its genomic sequence (A. A. Noegel, personal communication) confirm the mass-spectrometric data presented in this paper.


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