©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Properties of Type II Inositol Polyphosphate 5-Phosphatase (*)

Anne Bennett Jefferson , Philip W. Majerus

From the (1) From Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have isolated additional cDNA clones encoding type II inositol polyphosphate 5-phosphatase (5-phosphatase II) resulting in a combined cDNA of 3076 nucleotides encoding a protein of 942 amino acids. The 5-phosphatase II hydrolyzed both Ins (1, 4, 5) Pto Ins (1, 4) Pand the phospholipid PtdIns (4, 5) Pto PtdIns (4) P both in vitro and in vivo. There are two motifs highly conserved between types I and II 5-phosphatase and several other proteins presumed to be inositol phosphatases suggesting a possible role in catalysis. The type II 5-phosphatase also contains homology to several GTPase activating proteins although no such activity for 5-phosphatase II was found. The predicted protein ends with the sequence CNPL, suggesting that it is isoprenylated as a mechanism for membrane attachment. We found evidence for isoprenylation by demonstrating incorporation of [H]mevalonate into native but not C939S mutant 5-phosphatase II expressed in Sf9 insect cells. Furthermore, we showed that membrane localization and the activity of 5-phosphatase II toward its lipid substrate PtdIns (4, 5) Pis reduced by eliminating 5-phosphatase II isoprenylation in the mutant C939S relative to the native enzyme.


INTRODUCTION

The phosphatidylinositol signaling pathway influences numerous cellular responses including calcium ion mobilization, protein phosphorylation, and cell proliferation in response to various extracellular agonists (1, 2) . In one of the reactions of this system, phospholipase C acts on phosphatidylinositol (4, 5) -bisphosphate (PtdIns (4, 5) P)() to generate Ins (1, 4, 5) P. This metabolite is further converted to Ins (1, 3, 4, 5) Pby the action of a 3-kinase. These two soluble inositol phosphates are involved in calcium ion mobilization. Specific 5-phosphatase enzymes terminate calcium signaling by hydrolyzing Ins (1, 4, 5) Pand Ins (1, 3, 4, 5) Pto Ins (1, 4) Pand Ins (1, 3, 4) P, respectively (1, 2) .

Hansen et al. (17) first identified two types of soluble inositol phosphate 5-phosphatases from rat brain. Since that time, several forms of inositol polyphosphate 5-phosphatase have been identified in a variety of tissues, and these can be grouped according to molecular mass and affinity for Ins (1, 4, 5) Pand Ins (1, 3, 4, 5) P. The first group of 5-phosphatases of approximately 32-43 kDa have high affinity for both inositol phosphate substrates. This type of enzyme was first isolated from platelet cytosol and was named type I based on its position of elution from an anion exchange resin (3, 4, 5) . Enzymes with similar characteristics have been recognized and cloned from a variety of tissue sources (6, 7, 8, 9, 10, 11, 12, 13) . Immunoprecipitation of 5-phosphatase activity from platelets with antisera to the bovine brain and human placental isozymes suggest that these type I activities may be the same enzyme (10, 14) . A second group of 5-phosphatases have molecular masses of 69-75 kDa and lower affinities for Ins (1, 4, 5) Pand Ins (1, 3, 4, 5) P. Type II 5-phosphatase was originally isolated from human platelet cytosol (15) . Enzymes of similar size and substrate affinity have been observed in rat liver cytosol (6) and human erythrocyte membranes (9) . A partial cDNA encoding platelet 5-phosphatase II was obtained by screening pooled bacterial lysates from a gt11 cDNA library for ability to bind [H]Ins (1, 3, 4, 5) P(16) . Finally, 5-phosphatases have been reported of 115 kDa from bovine brain cytosol (7) and 160 kDa from rat brain cytosol (17) . These larger forms of 5-phosphatase act primarily on Ins (1, 4, 5) P.

There is also 5-phosphatase activity that hydrolyzes PtdIns (4, 5) Pto PtdIns (4) P. PtdIns (4, 5) P5-phosphatase activity has been identified in several tissues including chick, rat, and bovine brain (18, 19, 20) , rat kidney (21) , human erythrocytes (22) , and human platelets (23) . In most of these tissues, it is both cytosolic and membrane-associated, and in cases in which the enzyme has been most highly purified, it appears to be capable of hydrolyzing the 5-phosphate from both PtdIns (4, 5) Pand the soluble Ins (1, 4, 5) P(18, 20, 23) .

The PtdIns (4, 5) P5-phosphatase in platelets has been identified as 5-phosphatase II (23) . Polyclonal antibodies raised against recombinant 75-kDa Ins (1, 4, 5) P5-phosphatase II deplete PtdIns (4, 5) P5-phosphatase activity from the cytosolic and membrane fractions of platelets implying that this protein catalyzes both reactions. In this report, we have isolated additional cDNA clones encoding 5-phosphatase II and report corrections in the previously published sequence. We find that 5-phosphatase II has 2 regions of sequence conserved with 5-phosphatase I and several other proteins. We also find that 5-phosphatase II is isoprenylated on the carboxyl-terminal sequence CNPL, and this modification affects its ability to hydrolyze PtdIns (4, 5) P.


EXPERIMENTAL PROCEDURES

Materials

[H]Mevalonolactone was purchased from American Radiolabeled Chemicals, Inc. [P]PO, horseradish peroxidase-linked anti-rabbit IgG, and ECL Western blotting detection reagents were purchased from Amersham Life Sciences. PtdIns (4, 5) Pwas purchased from Boehringer Mannheim, and Silica Gel 60 TLC plates (20 20 cm, 0.2 mm) were from Merck. pVL1393 baculoviral expression vector and BaculoGold transfection kit were from PharMingen. pCB6 mammalian expression vector was from Jeffrey Milbrandt (Washington University, St. Louis). Compactin was from Sandra Hofmann (University of Texas Southwestern Medical Center), and mevinolin was from Alfred Alberts (Merck Research Laboratories). Purified CDC42Hs and GST-CDC42Gap and bacteria expressing GST-C-Ha-Ras, GST-Rac1, and GST-RhoA were from Richard Cerione (Cornell University). Purified GST-Rab5b was from David Wilson (Washington University, St. Louis). Most other materials were from Sigma.

Cloning of Additional 5-Phosphatase II Sequence

A 2384-base pair clone originally isolated by Ross et al. (16) was used to obtain an additional 5` sequence of 5-phosphatase II. A human erythroleukemia cell gt11 cDNA library (Rodger McEver, University of Oklahoma) was subdivided and amplified as described (16) . The 5` end of 5-phosphatase II was amplified by PCR between an antisense oligonucleotide directed to the 5` sequence of this clone and a primer located 16-37 base pairs 5` of the EcoRI site of gt11. The amplified PCR product was found to contain an additional 656 nucleotides of 5` sequence. The original clone was labeled by random hexamer priming and used to screen a human fetal brain Lambda Zap II cDNA library (Stratagene). A single clone was obtained that contained all of the original plus an additional 330 nucleotides as confirmed by partial sequencing and restriction digestion. We used a 5`-Amplifinder RACE kit (CLONTECH) to obtain the most 5` 39 nucleotides with an antisense 5-phosphatase II oligonucleotide as primer to synthesize single-stranded cDNA from placental poly(A)RNA (CLONTECH). DNA was sequenced on both strands by the Sanger dideoxy sequencing method (Sequenase; U. S. Biochemical Corp.). The sequence corrected in this publication was sequenced from at least two cDNA clones, and the 3` region was also sequenced from a genomic clone.

Expression Vector Construction and Mutagenesis

pBlueSKII/5ptaseL was constructed by combining the two 5-phosphatase II cDNAs described above at a BamHI site at 5-phosphatase II nucleotide 499. This combined 5-phosphatase II cDNA contains a sequence encoding 5-phosphatase II amino acids 14-942 and was used to construct the pCB6 mammalian expression vectors and, subsequently, the pVL1393 transfer vector for expression of 5-phosphatase II in Sf9 insect cells.

Expression vectors beginning at methionine 250 were constructed by PCR between a sense oligonucleotide containing EcoRV, NheI, and NcoI restriction sites and the 5-phosphatase II nucleotides 749-759 and an antisense 5-phosphatase II oligonucleotide for nucleotides 1717-1733. This PCR product was attached to the 5-phosphatase II 3` cDNA at a SphI site at nucleotide 1156, and the PCR portion was sequenced to confirm that the plasmid was without mutations. Expression vectors beginning at this methionine are designated 5-PtaseS and encode 5-phosphatase II amino acids 250-942.

For studies comparing the native and C939S mutant 5-phosphatase II enzymes, the 3` untranslated region of the cDNA was removed by PCR between a sense oligonucleotide for 5-phosphatase II nucleotides 2175-2191 and an antisense oligonucleotide containing nucleotides 2810-2830 followed by an XbaI restriction site. The mutation of cysteine 939 to serine was accomplished by changing a single nucleotide 2817 to guanosine within the antisense PCR oligonucleotide. These 3` PCR portions of 5-phosphatase II were attached to the 5` portion of the clone at a NheI site at nucleotide 2714. The native and C939S PCR portions were confirmed to be without mutations by sequencing.

Baculovirus Expression and Labeling

Recombinant baculoviruses were constructed according to the manufacturers' instructions using the shuttle vector pVL1393 (PharMingen) containing either 5PtaseS, 5PtaseL, or 5PtaseS/C939S or a control construct (protein tyrosine phosphatase MEG-01) (24) . Sf9 insect cells were grown in TNM-FH medium with 10% heat-inactivated fetal calf serum and 100 µg of gentamicin/ml (25) .

For studies on P labeling of Sf9 cell phospholipids, approximately 3 10Sf9 insect cells were infected with baculovirus encoding either control MEG-01 (24) or 5PtaseS. Three days after infection, the cells were harvested from 60-mm dishes and resuspended in 450 µl of phosphate-free Grace's insect media containing 1 mCi of [P]POat room temperature. Samples of 100 µl were taken after various times and added to 100 µl of 1 N HCl. P-Labeled phosphatidylinositols were further extracted with chloroform/methanol, separated by TLC, and measured as described (26) .

For [H]mevalonate labeling of 5-phosphatase II, approximately 3 10Sf9 cells infected with baculovirus encoding either 5PtaseS or 5PtaseL were grown for 48 h in 60-mm dishes and then incubated with fresh media containing 100 µ M compactin for 4 h. [H]Mevalonolactone (0.75 mCi) was added (1.4 ml of media/dish) for an additional 18 h. Cells were pelleted and lysed by sonication in 200 µl of buffer containing 50 m M Mes/Tris, pH 6.5, 3 m M MgCl, 2 m M EGTA, 10 m M 2-mercaptoethanol, 1 m M phenylmethylsulfonyl fluoride, 10 µg of leupeptin/ml, 10 µg of benzamidine/ml, and 1 µ M pepstatin A. Supernatant and particulate fractions were obtained by centrifugation for 10 min at 16,000 g at 4 °C. These fractions were resolved by SDS-PAGE and transferred to nitrocellulose. Labeling with [H]mevalonate was detected by soaking the nitrocellulose in 1 M sodium salicylate for 30 min followed by fluorography for 17 days. The nitrocellulose was then washed and immunoblotted to identify 5-phosphatase II protein. For comparison of [H]mevalonate labeling of native and C939S mutant 5-phosphatase II, Sf9 cells were infected with baculovirus encoding either 5PtaseS or 5PtaseS/C939S. They were labeled essentially as described above except that the [H]mevalonate media was reused from the experiment above and the fluorography was for 20 days.

Membrane Localization of 5-Phosphatase II

COS-7 cells were maintained in Dulbecco's modified Eagle's medium, 10% fetal calf serum, 10 units of penicillin/ml, and 10 µg of streptomycin/ml. These cells (80-85% confluent on 60-mm dishes) were transiently transfected using 40 µg of Lipofectin (Life Technologies, Inc.) and 10 µg of pCB6/5PtaseL or pCB6/5PtaseL/C939S. After 3 days, cells were incubated in fresh media containing no addition, 100 µ M mevinolin, 100 µ M mevinolin, and 10 m M mevalonic acid, or 10 m M mevalonic acid. After 6 h of drug treatment, cells were harvested in phosphate-buffered saline and sonicated in 150 µl of buffer A containing 10 m M Tris, pH 7.4, 140 m M KCl, 1.5 m M MgCl, 1 m M phenylmethylsulfonyl fluoride, 10 µg of benzamidine/ml, 1 µ M pepstatin A, 10 µg of aprotinin/ml, and 10 µg of leupeptin/ml. Soluble and particulate fractions were prepared by centrifugation at 16,000 g at 4 °C for 10 min. The pellets were extracted in the same buffer with 1% Triton X-100 for 30 min with shaking, and a detergent supernatant and pellet fraction were prepared by centrifugation again at 16,000 g at 4 °C for 10 min. Half of each of these fractions was resolved by SDS-PAGE on 8% polyacrylamide gels and immunoblotted for 5-phosphatase II.

Enzyme Assays and Western Blotting

Preparation of [P]Ins (1, 4, 5) Pand assay of 5-phosphatase activity using this substrate were as described (3) . Assay of 5-phosphatase activity using [H]PtdIns (4, 5) Pwas as described (23) except that PtdIns (4, 5) Pwas at a concentration of 50 µ M and the assay was conducted at room temperature. Under these conditions, the assay was linear for 1 min and about 70% of linear after 2 min. TLC was developed as described (26) . The enzyme concentration used in PtdIns (4, 5) Passays was determined by measuring the hydrolysis of Ins (1, 4, 5) P(assuming that 5-phosphatase II hydrolyzes 2.5 µmol of Ins (1, 4, 5) P/min/mg of protein). For experiments testing the effect of small GTP-binding proteins on 5-phosphatase II activity, the enzyme assays described above were done with the addition of GST-Ras, GST-Rac, or GST-Rho (at at least 1:1 G-protein:5-phosphatase) in the GDP- or GTPS-bound state. All immunoblotting was done with affinity-purified antibody to 5-phosphatase II (2 ng/ml) raised against a recombinant antigen containing amino acids 233-428 (16) . Secondary antibody was horseradish peroxidase-conjugated anti-rabbit IgG from Amersham (1:5000 dilution), and blots were developed using ECL (Amersham).

GAP Assays and Association of 5-Phosphatase II with GTP-binding Proteins

GAP assays were performed as described (27) with CDC42Hs, GST-c-Ha-Ras, GST-Rac1, GST-RhoA, or GST-Rab5b as the GTP-binding protein and Sf9 supernatants expressing 5PtaseS, 5PtaseL, or control MEG-01 or purified control CDC42GAP as the GTPase activating protein. 5-Phosphatase II was tested for association with GST-c-Ha-Ras, GST-Rac1, or GST-RhoA on glutathione-agarose beads exactly as described (28) .

As a second method of testing association, Sf9 cells infected with baculovirus encoding 5PtaseS, 5PtaseL, or control MEG-01 and labeled with [S]methionine and [S]cysteine (0.2 mCi/ml) for 20 h were sonicated in buffer A. GST-c-Ha-Ras, GST-Rac1, GST-RhoA or CDC42 (1 µg each) or whole platelets (50 µg) were resolved by SDS-PAGE on a 12.5% gel, transferred to nitrocellulose, and blocked in Tris-buffered saline with 3 g/100 ml of milk, 0.1% Tween 20, and 100 µg of GTPS/ml. A supernatant (centrifugation at 16,000 g for 10 min) containing S-labeled proteins was used to overlay the nitrocellulose (2 h at 4 °C), and binding was determined by autoradiography.


RESULTS

cDNA Cloning and Expression

The previously published cDNA sequence for 5-phosphatase II consisted of 2382 nucleotides and encoded amino-terminal amino acid sequence obtained from the protein purified from platelets. However, this cDNA sequence had an open reading frame upstream of the putative amino terminus, and a full length transcript appears to be about 4400 nucleotides based on Northern blot analysis. We therefore isolated additional cDNA clones to further define the structure of 5-phosphatase II. We have obtained clones containing 695 nucleotides 5` of the previously published sequence and that predict an additional 232 amino acids (Fig. 1). We have been unable to obtain cDNA clones with additional 5` sequences despite screening several million clones from HEL cell, human cerebellar, human fetal brain, human placenta, and MEG01 cDNA libraries and attempting 5`-rapid amplification of cDNA end PCR on several occasions. We also re-examined the previously published sequence and found several sequencing errors. These corrections altered the reading frame in two regions identified by underlined sequences in Fig. 1 . The nucleotide sequence of 5-phosphatase II predicts an open reading frame encoding 942 amino acids. Translation of the protein may begin at methionine 30 which has a surrounding nucleotide consensus sequence for initiator methionines (29) . However, it is possible that translation starts further 5` since there is no in-frame upstream stop codon in the cDNA, and the full length transcript is predicted to be 4.4 kilobases from Northern blot analysis (16) . The amino acid sequence obtained as an amino-terminal sequence of the purified protein from platelets begins at amino acid 270 (Fig. 1, arrow) and is preceded by a basic amino acid suggesting that the purified protein may be proteolyzed from a larger precursor.


Figure 1: Nucleotide and encoded amino acid sequence of the human inositol polyphosphate 5-phosphatase type II cDNA. Nucleotides are numbered on the left; amino acids are numbered on the right. The start of amino acid sequence obtained as the amino-terminal sequence of the purified protein from platelets is indicated by an arrow. Amino acid sequence altered by correction of nucleotide sequencing errors is underlined. Consensus amino acid sequence for isoprenylation is boxed.



The PtdIns (4, 5) P5-phosphatase activity in homogenates of platelets is depleted by an antibody to recombinant 5-phosphatase II (23) suggesting that the same enzyme hydrolyzes both the soluble and lipid substrate. We expressed 5PtaseS in Sf9 cells and showed directly that it is able to hydrolyze PtdIns (4, 5) Pto PtdIns (4) P in vitro (Fig. 2). The kinetics of this hydrolysis are complex. Note that Sf9 supernatant (0.4 µg) hydrolyzed 6.3% of added substrate (50 µ M) in 2 min while Sf9 supernatant (1.3 µg) hydrolyzed 21.5% of added substrate. The fact that the reaction plateaus after 2 min when there is still substrate present suggests that much of the PtdIns (4, 5) Pis not available for hydrolysis by 5-phosphatase II. One explanation for this finding is that the 5-phosphatase II binds strongly to PtdIns (4, 5) P/detergent micelles restricting substrate availability. A similar phenomenon has been found for the inositol polyphosphate 4-phosphatase hydrolysis of PtdIns (3, 4) P(26) . Consistent with this hypothesis, we found that mixing 5-phosphatase II with unlabeled PtdIns (4, 5) Por PtdIns (4) P containing detergent micelles prior to addition of radiolabeled substrate eliminated subsequent hydrolysis of H-labeled PtdIns (4, 5) P. Since the extent of substrate hydrolysis, but not the rate, is proportional to enzyme concentration over a wide range, it is difficult to compare the ability of 5-phosphatase II to hydrolyze the lipid- versus water-soluble substrate. However, it is clear that the lipid is a very good substrate. Thus, using the results at 2 min, 1.3 µg of Sf9 supernatant hydrolyzed 0.54 nmol of PtdIns (4, 5) Por 0.2 µmol/min/mg of protein compared to an activity of 0.05 µmol of Ins (1, 4, 5) Phydrolyzed/min/mg of protein.


Figure 2: Hydrolysis of PtdIns(4,5)P by 5-phosphatase II. PtdIns(4,5)P( PIP) hydrolyzed by 0.4 µg () or 1.3 µg () of supernatant from Sf9 cells expressing 5-phosphatase II is plotted as a function of time. Results shown are typical of three experiments.



We also determined that 5-phosphatase II hydrolyzes PtdIns (4, 5) P in vivo. We used Sf9 cells infected for 3 days with baculovirus encoding 5PtaseS or encoding an irrelevant protein MEG-01. Cells were labeled with POfor 1-30 min and incorporation of isotope into inositol lipids was determined as described under ``Experimental Procedures.'' Under these non-steady state conditions, incorporation of POreflects turnover of inositol lipids. Cells infected with MEG-01 baculovirus had more P incorporated into PtdInsPthan into PtdInsP at all time points (Fig. 3) as did uninfected Sf9 cells (data not shown). Thus, Sf9 cells continue to maintain cellular inositol phospholipids despite infection with baculovirus. In contrast, cells expressing 5-phosphatase II had less radioactivity incorporated into PtdInsPthan into PtdInsP due to hydrolysis of the 5-phosphate group with the consequent increase in levels of PtdInsP. Expression of 5-phosphatase II did not affect the incorporation of P into total cellular phospholipid (61 cpm/nmol of cellular phospholipid/10 min versus 66 cpm/nmol of cellular phospholipid/10 min for 5-phosphatase II) indicating that the rate of labeling is not affected by 5-phosphatase II expression. This result shows that the recombinant 5-phosphatase II is able to function in vivo as a PtdIns (4, 5) P5-phosphatase, and that overexpression of the enzyme causes a measurable change in cellular inositol phospholipid levels.


Figure 3: Incorporation of PO into inositol lipids in Sf9 cells. Sf9 cells infected with baculovirus encoding 5-phosphatase II or control MEG-01 tyrosine phosphatase were labeled with POfor the indicated times. Radioactivity incorporated into PtdInsPor PtdInsP was determined after separation on TLC. The inset shows the ratio of radioactivity in PtdInsPdivided by that in PtdInsP in the infected cells. Results shown are typical of five experiments.



Homology Motifs

The two known 5-phosphatase enzymes I and II have no amino acid similarity (20.9% identity in 115 amino acids) except in two regions corresponding to 5-phosphatase II amino acids 472-483 and 545-570 (Fig. 4). The high degree of similarity in these regions suggests these regions may be involved in a function common to both of these proteins such as substrate binding or catalysis. Several other proteins show much greater overall similarity to 5-phosphatase II including the protein encoded by the gene mutated in Lowe's oculocerebrorenal syndrome (51.1% identity in 744 amino acids) and predicted proteins from Caenorhabditis elegans (25.5% identity in 255 amino acids), Arabadopsis thaliana (46% identity in 121 amino acids), and Saccharomyces cerevisiae (32.9% identity in 374 amino acids). The two motifs mentioned above are also conserved in these proteins, further suggesting similarity in function among these proteins (Fig. 4).


Figure 4: Two regions of amino acid similarity between human inositol polyphosphate 5-phosphatase type II and the amino acid sequence of related predicted proteins. The sequences compared were: C. elegans, predicted amino acid sequence of a protein encoded by C. elegans cosmid (GenBank accession number L14433); 5Ptase II, human 5-phosphatase II; 5Ptase I, human and dog 43-kDa inositol polyphosphate 5-phosphatase type I; OCRL, the protein encoded by the gene mutated in Lowe's oculocerebrorenal syndrome; A. thaliana, predicted amino acid sequence of a protein encoded by A. thaliana cDNA (GenBank accession number T04155); S. cerevisiae, predicted amino acid sequence of a protein encoded by S. cerevisiae cosmid (GenBank accession number Z38062). Residues showing conservation among at least four proteins are indicated as Consensus sequence. Amino acid sequence number is indicated in parentheses. Alignment was performed using the program ALIGN (59).



An additional region of 5-phosphatase II amino acid sequence is similar to several GTPase-activating proteins (Fig. 5). This sequence similarity was noted previously, but the corrected sequence for the 5-phosphatase II extends the region that can be aligned to the GAP proteins (30) . We have tested recombinant 5-phosphatase II for GTPase activating activity toward CDC42, Rac, Rho, Ras, and Rab5b and find none (data not shown). We also find no evidence for 5-phosphatase II binding to CDC42, GST-c-Ha-Ras, GST-Rac1, or GST-RhoA and found no effect of these GTP-binding proteins on 5-phosphatase II hydrolyzing activity as described under ``Experimental Procedures'' (data not shown). While these results provide no evidence for association of 5-phosphatase II with any GTP-binding protein, the apparent homology with binding domains of known GTPase activating proteins suggests that such an association may yet exist.


Figure 5: Amino acid similarity between human 5-phosphatase II and presumptive GAP domains of other proteins. The sequences compared were: 5Ptase II, human 5-phosphatase II; human -chimerin (RACGAP); human CDC42GAP; PI3K 85 kDa, bovine 85-kDa -subunit of phosphatidylinositol 3-kinase. Amino acid sequence numbers are indicated. Boxed amino acids are identical in at least two of the four aligned sequences. Percentage of identical amino acids among the sequences is indicated. Alignment was performed using the program ALIGN (59).



Protein Isoprenylation

The 5-phosphatase II sequence ends with CNPL. This sequence fits the C XXX consensus for modification of proteins by the addition of an isoprene group where C is cysteine and X is any amino acid. The terminal amino acid of the 5-phosphatase II is a lysine, further suggesting that the most likely modification is addition of a geranylgeranyl group (35, 36) . There are three lysines near the possible isoprenylation signal which may promote membrane association (31) .

To determine whether the presence of the C XXX motif in 5-phosphatase II results in isoprenylation of the protein, we carried out labeling studies using [H]mevalonate. Mevalonate incorporation into cellular proteins is considered to reflect isoprenylation of these proteins with either a farnesyl or a geranylgeranyl group (32) . We infected Sf9 cells with baculovirus encoding 5PtaseS which yields an 80-kDa protein and 5PtaseL which results in a prominent doublet of 100-103 kDa and a minor proteolysis product of 80 kDa. These two 5-phosphatase II species have identical enzyme activity toward soluble and lipid substrates (data not shown). Infected cells were labeled with [H]mevalonate as described under ``Experimental Procedures,'' and the proteins from lysates of the cells were separated by SDS-PAGE and transferred to nitrocellulose membranes prior to autoradiography. [H]Mevalonate was incorporated into proteins of the predicted sizes (Fig. 6 A). The H-labeled proteins were identified as 5-phosphatase II by Western blotting with an antibody to 5-phosphatase II (Fig. 6 A). There was insufficient isotope incorporation to further characterize the isoprenyl group. We constructed a 5-phosphatase II cDNA in which cysteine 939 is mutated to serine. This eliminates the site for attachment of an isoprene moiety. This mutant 5-phosphatase II has the same Ins (1, 4, 5) Phydrolyzing activity as the native 5-phosphatase II (data not shown). Sf9 cells expressing this C939S mutant 5-phosphatase did not incorporate [H]mevalonate into 5-phosphatase II even though equivalent amounts of recombinant protein were expressed (Fig. 6 B).


Figure 6: [H]Mevalonate labeling and Western blotting of 5-phosphatase II. A, membranes from Sf9 cells infected with baculovirus encoding an 80-kDa ( a) or 103-kDa ( b) 5-phosphatase II and labeled with [H]mevalonate were subjected to SDS-PAGE and transferred to nitrocellulose. The nitrocellulose was soaked in sodium salicylate and autoradiographed for 17 days to determine [H] mevalonate incorporation. The H-labeled protein was identified on the identical piece of nitrocellulose by immunoblotting with antibody to 5-phosphatase II. B, Sf9 cells infected with baculovirus encoding an 80-kDa wild type 5-phosphatase II ( c) or the C939S 5-phosphatase II mutant ( d) were assayed for [H]mevalonate incorporation and Western blotting as described in A.



We next determined whether isoprenylation affects the cellular localization of the 5-phosphatase II by examining the subcellular distribution of recombinant enzyme in COS-7 cells expressing 5-phosphatase II or the 5-phosphatase II C939S mutant. COS-7 cells express the longer 5-phosphatase at levels similar to those previously reported for the shorter 5-phosphatase (5ptaseL supernatant, 6.5 nmol/min/mg, versus control supernatant, 3.8 nmol/min/mg) (16) . In COS-7 cells expressing 5-phosphatase II, there was approximately equal distribution of recombinant 5-phosphatase II in supernatant and pellet fractions (Fig. 7). Extraction with Triton X-100 did not solubilize the bulk of membrane-associated 5-phosphatase II. The C939S mutant 5-phosphatase II was not isoprenylated and was found mostly in the supernatant fraction. Likewise, inhibition of cellular isoprenylation with mevinolin resulted in a shift of the native 5-phosphatase II to the supernatant. Addition of mevalonate to the media during mevinolin treatment overcame the mevinolin inhibition of isoprenylation and restored the equal distribution of 5-phosphatase II between the supernatant and pellet fractions (data not shown). Mevalonate alone had no effect on the distribution of 5-phosphatase II, and neither mevinolin nor mevalonate affected the localization of the C939S mutant 5-phosphatase II in the supernatant fraction. Results similar to those shown in Fig. 7were seen in three experiments. However, in some additional experiments, nearly all of the expressed recombinant protein was found in the pellet fraction in both native and C939S transfected cells, and, in those experiments, mevinolin treatment did not alter the distribution of enzyme. It is possible that the large amount of recombinant protein expressed transiently in COS-7 cells is denatured in some cases precluding the expression of soluble enzyme.


Figure 7: Subcellular fractionation of 5-phosphatase II. COS-7 cells expressing 5-phosphatase II or the 5-phosphatase II C939S mutant or COS-7 cells expressing 5-phosphatase II and treated for 6 h with 100 µ M mevinolin were separated into supernatant ( s), detergent-extracted supernatant ( ds), and pellet ( p) fractions. Equivalent proportions of each fraction were resolved by SDS-PAGE and transferred to nitrocellulose. The 5-phosphatase II was visualized by immunoblotting with antibody to 5-phosphatase II.



We next asked whether isoprenylation of 5-phosphatase II has a functional effect on its ability to act on its soluble or lipid substrates. We prepared native and C939S mutant 5-phosphatase II in Sf9 cells and assayed for activity using Ins (1, 4, 5) Pand PtdIns (4, 5) P. There are inherent difficulties in comparing the activity of an enzyme for a soluble substrate with the activity for PtdInsPin a two-dimensional environment, and, while the extent of hydrolysis of PtdInsPis proportional to protein concentration, this assay approaches linearity only at very short times (Fig. 2). Thus, the hydrolysis of PtdIns (4, 5) Pcan be expressed only as apparent specific activity with a defined length of assay and protein concentration. Therefore, we compared the ability of wild type and C939S mutant 5-phosphatase II to hydrolyze PtdIns (4, 5) Pboth as a function of time with constant amounts of Sf9 supernatant protein (, experiments 1 and 2) and at a fixed point with varying amounts of Sf9 supernatant protein (, experiment 3). With both of these assay conditions, the C939S mutant 5-phosphatase II showed a decreased ability to hydrolyze PtdIns (4, 5) P2 when compared to the wild type enzyme. The C939S mutant 5-phosphatase II had a 29-40% decreased 5-phosphatase activity toward PtdIns (4, 5) Prelative to Ins (1, 4, 5) P(). The native and C939S mutant 5-phosphatase II had the same activity using Ins (1, 4, 5) Pas indicated by assay of equivalent amounts of protein as determined by Western blotting (data not shown).


DISCUSSION

The 5-phosphatase type II was purified and cloned as an enzyme that cleaves the phosphate from the 5-position of soluble Ins (1, 4, 5) P. However, an antibody to the recombinant protein depletes PtdIns (4, 5) P5-phosphatase activity in platelet cytosolic and membrane fractions (23) . We now report that recombinant 5-phosphatase II hydrolyzes PtdIns (4, 5) Pboth in vitro and in intact Sf9 cells. Whether 5-phosphatase II is the major enzyme that controls cellular PtdIns (4, 5) Plevels is unknown. Larger proteins have been identified in other tissues as PtdIns (4, 5) P5-phosphatases. Palmer et al. (20) have purified two immunologically related PtdIns (4, 5) P5-phosphatases of 155 and 115 kDa from bovine brain cytosol, and Roach and Palmer (22) identified a 105-kDa PtdIns (4, 5) P5-phosphatase in human erythrocyte cytosol. With the additional nucleotide sequence we report for the 5-phosphatase II in this paper, the open reading frame of 5-phosphatase II is sufficient to encode a protein of 107 kDa. It is possible that the full length cDNA of this protein encodes the larger forms of the 5-phosphatases from other tissues and that there is tissue-dependent proteolytic processing or artifactual proteolysis to account for the different sized proteins that have been identified. Of note we have expressed recombinant constructs of 80 kDa and 103 kDa and find that both are equally active. Alternatively, it is possible that the other forms of 5-phosphatase represent distinct isozymes. It is clear from the comparison of 5-phosphatase II to sequences in GenBank that there is a family of proteins that share at least two common motifs. One of these proteins, the 43-kDa 5-phosphatase I, hydrolyzes Ins (1, 4, 5) Pbut not PtdIns (4, 5) Pdespite its membrane localization (10, 23) . The protein encoded by the gene mutated in Lowe's oculocerebrorenal syndrome (OCRL) shares these conserved motifs with the two known 5-phosphatases, but no enzyme activity has yet been identified for this protein (33) . The OCRL protein is predicted to be as large as 112 kDa and thus could also account for larger 5-phosphatases that have been identified in other tissues. Likewise, there are homologs of the 5-phosphatase II in C. elegans, A. thaliana, and S. cerevisiae. While nothing is known about the 5-phosphatase activity of these organisms, in S. cerevisiae, antibodies to PtdIns (4, 5) Pinhibit cell proliferation (34) implying an importance for this lipid.

The 5-phosphatase II ends with a consensus sequence -CNPL for isoprenylation, and the enzyme is isoprenylated on cysteine 939 based on its incorporation of [H]mevalonate in Sf9 cells. It is interesting that the isoprenylation sequence for 5-phosphatase contains a proline. Generally, the two central amino acids of the carboxyl-terminal isoprenylation sequence are aliphatic amino acids, and proline in the third position of the 5-phosphatase isoprenylation motif is unusual in mammalian systems (35, 36) . However, the large hepatitis Delta antigen ends with the carboxyl-terminal isoprenylation sequence -CRPQ and becomes modified by the addition of a geranylgeranyl moiety. Site-specific mutagenesis studies have demonstrated that this sequence does not tolerate mutation of the proline to an aliphatic amino acid (37) . This suggests the possible existence of a novel prenyltransferase that isoprenylates the hepatitis Delta antigen and perhaps mammalian proteins with similar sequences such as the 5-phosphatase II (35) () .

The 5-phosphatase II is the first enzyme in the inositol phosphate pathway directly shown to be isoprenylated, and it is interesting to speculate on the possible effect this modification has on the function of the 5-phosphatase. One role for isoprenylation would be to promote the membrane association of 5-phosphatase II. Our experiments demonstrate that at least part of the recombinant 5-phosphatase II from COS-7 cells fractionates to the cell pellet, and, in platelets, 45% of the PtdIns (4, 5) Pactivity fractionates with the membrane or cytoskeleton (23) . The three basic amino acids preceding the isoprenylation motif may also combine with this isoprenylation to promote the membrane location of 5-phosphatase II as it does with K-Ras (31) . In K-Ras, a polybasic region containing 6 lysines is needed in addition to isoprenylation for complete association of K-Ras with the membrane. Mutation of the polybasic region to leave three lysines in addition to its isoprenylation signal results in approximately equal distribution of K-Ras between an S100 and P100 fraction (31) . 5-Phosphatase II which is not isoprenylated because of mutation of its isoprenylation motif or because of inhibition of isoprenylation by mevinolin does not localize to the cell pellet. However, it is apparent that membrane association is not absolutely necessary for 5-phosphatase II to hydrolyze PtdIns (4, 5) P. We find that the C939S mutant 5-phosphatase II is capable of hydrolyzing PtdIns (4, 5) Pin an in vitro assay, although the mutant enzyme is less active relatively than the wild type enzyme toward the lipid substrate.

Another possibility is that the isoprenylation of 5-phosphatase II serves to orient or stabilize the association of 5-phosphatase II with some other protein on membranes. A precedent for such a function is the report that isoprenylated Rho activates membrane phosphatidylinositol (4) P 5-kinase, whereas nonisoprenylated Rho has no effect (38) . Nonisoprenylated rhodopsin kinase is 4-fold less active toward Rho in an in vitro assay than the isoprenylated kinase (39) . Similarly, farnesylation and carboxylmethylation of the subunit of transducin leads to an increased association with membranes that can be accounted for by an improved interaction with other proteins in the transducin-metarhodopsin II complex (40) . There is as yet no direct evidence that 5-phosphatase II associates with any other cellular protein, although it cannot be extracted from a COS-7 cell pellet with Triton X-100, and, in platelets, 16% of 5-phosphatase activity is associated with cytoskeleton (23) . This raises the possibility of a secondary association with a cytoskeletal protein following recruitment to the membrane. The 5-phosphatase II also contains a region of apparent homology with other proteins that serve as GTPase activating proteins in the Rac and Rho families of GTP-binding proteins. The 5-phosphatase II used for GTPase activation and association experiments was produced in Sf9 cells and thus should have been isoprenylated, but it was not presented to any GTP-binding proteins in a lipid environment. The association of 5-phosphatase II with a GTP-binding protein may depend on orientation of 5-phosphatase II in a lipid bilayer. An isoprenylated and membrane-associated 5-phosphatase II would be ideally situated to terminate both GTP-Rho activity and the increased levels of PtdIns (4, 5) Pthat follow the 5-kinase activation.

Of the other proteins homologous to 5-phosphatase II, only the 43-kDa 5-phosphatase I has a consensus sequence for isoprenylation (12, 13) . Isoprenylation could account for its membrane association (10, 12) . Another possible 5-phosphatase from mammalian sources is the gene disrupted in Lowe's oculocerebrorenal syndrome (33) . The predicted amino acid sequence for this protein does not end with an isoprenylation consensus sequence and so it may be cytosolic.

Two other inositol polyphosphate phosphatases share the ability of the 5-phosphatase II to hydrolyze a soluble and corresponding lipid substrate. The 3-phosphatase that degrades Ins (1, 3) Pis the same enzyme that hydrolyzes PtdIns (3) P (41) . Similarly, the inositol polyphosphate 4-phosphatase has recently been shown to degrade PtdIns (3, 4) Pas well as Ins (1, 3, 4) P(26) . Both of these enzymes are found bound in part to cell membranes, and it will be interesting to find if they are also isoprenylated.

Regulation of the levels of cellular PtdIns (4, 5) Pis not understood. It is clear that a portion of the total pool of PtdIns (4, 5) Pcycles continuously between PtdIns (4) P and PtdIns (4, 5) Pand that only a portion of the total cellular PtdIns (4, 5) Pis hydrolyzed by phospholipase C in response to agonists (42, 43, 44) . PtdIns (4, 5) Phas other functions in addition to serving as the source of Ins (1, 4, 5) P. It regulates cellular enzymes including µ-calpain (45) , protein kinase C (46, 47, 48) , and ATPases (49, 50) and binds some proteins containing pleckstrin homology domains (51) . PtdIns (4, 5) Pbinds to components of the cytoskeleton to regulate actin assembly (52, 53, 54, 55, 56) , associates with clathrin assembly proteins (57) , and affects secretion (58) . Regulation of cellular PtdIns (4, 5) Plevels by 5-phosphatase II may affect a variety of cell functions in addition to calcium signaling.

  
Table: Wild type or mutant 5-phosphatase activity using Ins(1,4,5)Pand PtdIns (4,5)P



FOOTNOTES

*
This research was supported by National Institutes of Health Grants HL 14147 (Specialized Center for Research in Thrombosis), HL 16634, and Training Grant HL 07088. 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.

The abbreviations used are: PtdIns(4,5)P, phosphatidylinositol 4,5-bisphosphate; Ins(1,4,5)P, inositol 1,4,5-trisphosphate; Ins(1,3,4,5)P, inositol 1,3,4,5-tetrakisphosphate; Ins(1,4)P, inositol 1,4-bisphosphate; Ins(1,3,4)P, inositol 1,3,4-trisphosphate; GST, glutathione S-transferase; PCR, polymerase chain reaction; Mes, 4-morpholineethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; GTPS, guanosine 5`- O-(3-thiotriphosphate; GAP, GTPase activating protein.

P. Casey, personal communication.


ACKNOWLEDGEMENTS

We thank Theodora S. Ross for assistance in the cloning of 5-phosphatase II. We thank Richard Cerione, Patrick Casey, and F. Anderson Norris for helpful discussions; Cecil Buchanan for technical assistance; and J. Evan Sadler, Monita Wilson, and John D. York for critical review.


REFERENCES
  1. Majerus, P. W. (1992) Annu. Rev. Biochem. 61, 225-250 [CrossRef][Medline] [Order article via Infotrieve]
  2. Berridge, M. J. (1993) Nature 361, 315-325 [CrossRef][Medline] [Order article via Infotrieve]
  3. Connolly, T. M., Bross, T. E., and Majerus, P. W. (1985) J. Biol. Chem. 260, 7868-7874 [Abstract/Free Full Text]
  4. Connolly, T. M., Lawing, W. J., Jr., and Majerus, P. W. (1986) Cell 46, 951-958 [Medline] [Order article via Infotrieve]
  5. Connolly, T. M., Bansal, V. S., Bross, T. E., Irvine, R. F., and Majerus, P. W. (1987) J. Biol. Chem. 262, 2146-2149 [Abstract/Free Full Text]
  6. Takimoto, K., Masato, O., and Nakagawa, H. (1989) J. Biochem. (Tokyo) 106, 684-690 [Abstract]
  7. Erneux, C., Lemos, M., Verjans, B., Vanderhaeghen, P., Delvaux, A., and Dumont, J. E. (1989) Eur. J. Biochem. 181, 317-322 [Abstract]
  8. Verjans, B., Lecocq, R., Moreau, C., and Erneux, D. (1992) Eur. J. Biochem. 204, 1083-1087 [Abstract]
  9. Hodgkin, M., Craxton, A., Parry, J. B., Hughes, P. J., Potter, B. V. L., Michell, R. H., and Kirk, C. J. (1994) Biochem. J. 297, 637-645 [Medline] [Order article via Infotrieve]
  10. Laxminarayan, K. M., Matzaris, M., Speed, C. J., and Mitchell, C. A. (1993) J. Biol. Chem. 268, 4968-4974 [Abstract/Free Full Text]
  11. DeSmedt, F., Verjans, B., Mailleux, P., and Erneux, C. (1994) FEBS Lett. 347, 69-72 [CrossRef][Medline] [Order article via Infotrieve]
  12. Laxminarayan, K. M., Chan, B. K., Tetaz, T., Bird, P. I., and Mitchell, C. A. (1994) J. Biol. Chem. 269, 17305-17310 [Abstract/Free Full Text]
  13. Verjans, B., DeSmedt, F., Lecocq, R., Vanweyenberg, V., Moreau, C., and Erneux, C. (1994) Biochem. J. 300, 85-90 [Medline] [Order article via Infotrieve]
  14. Verjans, B., Hollande, F., Moreau, C., Lejeune, C., and Erneux, C. (1990) Cell Signalling 2, 595-599 [Medline] [Order article via Infotrieve]
  15. Mitchell, C. A., Connolly, T. M., and Majerus, P. W. (1989) J. Biol. Chem. 264, 8873-8877 [Abstract/Free Full Text]
  16. Ross, T. S., Jefferson, A. B., Mitchell, C. A., and Majerus, P. W. (1991) J. Biol. Chem. 266, 20283-20289 [Abstract/Free Full Text]
  17. Hansen, C. A., Johanson, R. A., Williamson, M. T., and Williamson, J. R. (1987) J. Biol. Chem. 262, 17319-17326 [Abstract/Free Full Text]
  18. Palmer, F. B. St. C. (1990) Biochem. Cell Biol. 68, 800-803 [Medline] [Order article via Infotrieve]
  19. Nijjar, M. S., and Hawthorne, J. N. (1977) Biochim. Biophys. Acta 480, 390-402 [Medline] [Order article via Infotrieve]
  20. Palmer, F. B. St. C., Theolis, Jr., R., Cook, H. W., and Byers, D. M. (1994) J. Biol. Chem. 269, 3403-3410 [Abstract/Free Full Text]
  21. Cooper, P. H., and Hawthorne, J. N. (1975) Biochem. J. 150, 537-551 [Medline] [Order article via Infotrieve]
  22. Roach, P. D., and Palmer, F. B. St. C. (1981) Biochim. Biophys. Acta 661, 323-333 [Medline] [Order article via Infotrieve]
  23. Matzaris, M., Jackson, S. P., Laxminarayan, K. M., Speed, C. J., and Mitchell, C. A. (1994) J. Biol. Chem. 269, 3397-3402 [Abstract/Free Full Text]
  24. Gu, M., York, J. D., Warshawsky, I., and Majerus, P. W. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5867-5871 [Abstract]
  25. Summers, M. D., and Smith, G. E. (1987) Tex. Agric. Exp. Stn. Bull. 1555, 10-42
  26. Norris, F. A., and Majerus, P. W. (1994) J. Biol. Chem. 269, 8716-8720 [Abstract/Free Full Text]
  27. Hart, M. J., Shinjo, K., Hall, A., Evans, T., and Cerione, R. A. (1991) J. Biol. Chem. 266, 20840-20848 [Abstract/Free Full Text]
  28. Hart, M. J., Eva, A., Zangrilli, D., Aaronson, S. A., Evans, T., Cerione, R. A., and Zheng, Y. (1994) J. Biol. Chem. 269, 62-65 [Abstract/Free Full Text]
  29. Kozak, M. (1987) Nucleic Acids Res. 15, 8125-8148 [Abstract]
  30. Baldwin, G. S., and Zhang, Q.-X. (1993) Trends Biochem. Sci. 18, 378-380 [Medline] [Order article via Infotrieve]
  31. Hancock, J. F., Paterson, H., and Marshall, C. J. (1990) Cell 63, 133-139 [Medline] [Order article via Infotrieve]
  32. Mumby, S. M., and Buss, J. E. (1990) Methods 1, 216-220
  33. Attree, O., Olivos, I. M., Okabe, I., Bailey, L. C., Nelson, D. L., Lewis, R. A., McInnes, R. R., and Nussbaum, R. L. (1992) Nature 358, 239-242 [CrossRef][Medline] [Order article via Infotrieve]
  34. Uno, I., Fukami, K., Kato, H., Takenawa, T., and Ishikawa, T. (1988) Nature 333, 188-190 [CrossRef][Medline] [Order article via Infotrieve]
  35. Cox, A. D., and Der, C. J. (1992) Curr. Opin. Cell Biol. 4, 1008-1016 [Medline] [Order article via Infotrieve]
  36. Clark, S. (1992) Annu. Rev. Biochem. 61, 355-386 [CrossRef][Medline] [Order article via Infotrieve]
  37. Lee, C.-Z., Chen, P.-J., Lai, M. M. C., and Chen, D.-S. (1994) Virology 199, 169-175 [CrossRef][Medline] [Order article via Infotrieve]
  38. Chong, L. D., Traynor-Kaplan, A., Bokoch, G. M., and Schwartz, M. A. (1994) Cell 79, 507-513 [Medline] [Order article via Infotrieve]
  39. Inglese, J., Glickman, J. F., Lorenz, W., Caron, M. G., and Lefkowitz, R. J. (1992) J. Biol. Chem. 267, 1422-1425 [Abstract/Free Full Text]
  40. Ohguro, H., Fukada, Y., Takao, T., Shimonishi, Y., Yoshizawa, T., and Akino, T. (1991) EMBO J. 10, 3669-3674 [Abstract]
  41. Caldwell, K. K., Lips, D. L., Bansal, V. S., and Majerus, P. W. (1991) J. Biol. Chem. 266, 18378-18386 [Abstract/Free Full Text]
  42. Muller, E., Hegewald, H., Jaroszewicz, K., Cumme, G. A., Hoppe, H., and Frunder, H. (1986) Biochem. J. 235, 775-783 [Medline] [Order article via Infotrieve]
  43. King, C. E., Stephens, L. R., Hawkins, P. T., Guy, G. R., and Michell, R. H. (1987) Biochem. J. 244, 209-217 [Medline] [Order article via Infotrieve]
  44. King, C. E., Hawkins, P. T., Stephens, L. R., and Michell, R. H. (1989) Biochem. J. 259, 893-896 [Medline] [Order article via Infotrieve]
  45. Saido, T. C., Shibata, M., Takenawa, T., Murofushi, H., and Sujuki, K. (1992) J. Biol. Chem. 267, 24585-24590 [Abstract/Free Full Text]
  46. Lee, M.-H., and Bell, R. M. (1991) Biochemistry 30, 1041-1049 [Medline] [Order article via Infotrieve]
  47. Huang, F. L., and Huang, K. P. (1991) J. Biol. Chem. 266, 8727-8733 [Abstract/Free Full Text]
  48. Chauhan, A., Brockerhoff, H., Wisniewski, H. M., and Chauhan, V. P. S. (1991) Arch. Biochem. Biophys. 287, 283-287 [Medline] [Order article via Infotrieve]
  49. Memon, A. R., Chen, Q. Y., and Boss, W. F. (1989) Biochem. Biophys. Res. Commun. 162, 1295-1301 [Medline] [Order article via Infotrieve]
  50. Missiaen, L., Wuytack, F., Raeymaekers, L., DeSmedt, H., and Casteels, R. (1989) Biochem J. 261, 1055-1058 [Medline] [Order article via Infotrieve]
  51. Harlan, J. E., Hajduk, P. J., Yoon, H. S., and Fesik, S. W. (1994) Nature 371, 168-170 [CrossRef][Medline] [Order article via Infotrieve]
  52. Janmey, P. A., and Stossel, T. P. (1989) J. Biol. Chem. 264, 4825-4831 [Abstract/Free Full Text]
  53. Janmey, P. A., Lamb, J., Allen, P. W., and Matsudaira, P. T. (1992) J. Biol. Chem. 267, 11818-11823 [Abstract/Free Full Text]
  54. Lassing, I., and Lindberg, U. (1985) Nature 314, 472-474 [Medline] [Order article via Infotrieve]
  55. Goldschmidt-Clermont, P. J., Machesky, L. M., Baldassare, J. J., and Pollard, T. D. (1990) Science 247, 1575-1578 [Medline] [Order article via Infotrieve]
  56. Fukami, K., Furuhashi, K., Inagaki, M., Endo, T., Hatano, S., and Takenawa, T. (1992) Nature 359, 150-152 [CrossRef][Medline] [Order article via Infotrieve]
  57. Beck, K. A., and Keen, J. H. (1991) J. Biol. Chem. 266, 4442-4447 [Abstract/Free Full Text]
  58. Eberhard, D. A., Cooper, C. L., Low, M. G., and Holz, R. W. (1990) Biochem. J. 268, 15-25 [Medline] [Order article via Infotrieve]
  59. Dayhoff, M., Barker, W. C., and Hunt, L. T. (1983) Methods Enzymol. 91, 524-545 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.