Tm JOURNAL OF B I O ~ I C A L CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 5, Issue of February 4, pp. 3852-3857, 1994 Printed in U S A .

Regulation of Phosphatidylinositol 4-Kinase by the Protein Activator PIK-A49 ACTIVATION REQUIRES PHOSPHORYLATION OF PIK-A49*

(Received for publication, September 10, 1993, and in revised form, October 18, 1993)

Wannian Yang and Wendy E Boss$ From the Botany Department, North Carolina State University, Raleigh, North Carolina 27695-7612

PIK-A49 is a 49-kDa soluble protein that was isolated as an activator of the plasma membrane phosphatidy- linositol (PI) 4-kinase from carrot cells (Yang, W., Burkhart, W., Cavallius, J., Merrick, W. C., and Boss, W. F. (1993) J. Biol. Chem. 268, 392398). PIK-A49 is a multi- functional protein that binds and bundles F-actin and has translational elongation factor-la activity. In this paper, we have investigated the mechanism of activa- tion of PI 4-kinase by PIK-A49. PIK-A49 decreased the K,,, of PI 4-kinase for ATP from 0.40 to 0.19 m ~ . GTP and GDP, which affect the elongation factor-la function of the protein, inhibited the activation of PI 4-kinase by PIK-A49. Phosphorylation of purified PIK-A49 by a cal- cium-dependent protein kinase enhanced activation of PI 4-kinase. When dephosphorylated by alkaline phos- phatase, PIK-A49 no longer activated PI 4-kinase; how- ever, rephosphorylation of PIK-A49 by calcium-depend- ent protein kinase fully restored activation. Western blots using anti-PIK-A49 serum showed that PIK-A49 was associated with the plasma membrane and the F- actin fraction isolated from plasma membranes, indicat- ing that PIK-A49 would be in a position to regulate plasma membrane PI 4-kinase. Based on these data, we propose a mechanism for feed-forward regulation of polyphosphoinositide biosynthesis in response to in- creases in cytosolic calcium.

Polyphosphorylated inositol phospholipids are important in- termediates in transducing extracellular signals through the production of the second messengers inositol 1,4,5-trisphos- phate and diacylglycerol (Berridge and Irvine, 1984; Nishi- zuka, 1986; Majerus, 1992). In addition, they directly affect the activity of P-type ATPases (Missiaen et al., 1989; Chen and Boss, 1991), protein kinases (Chauhan et al., 1989; Sahyoun et al . , 1989), and actin-binding proteins (Lassing and Lindberg, 1985; Janmey and Stossel, 1987; Yonezawa et al., 1990). Al- though much emphasis has been placed on the study of phos- phatidylinositol-4,5-bisphosphate phospholipase C (Rhee and Choi, 1992; Fain, 1990), little is known about the regulation of the enzymes involved in PI-4,5-Pz biosynthesis (Carpenter and Cantley, 1990; Pike, 1992).

* This work was supported by Grant DCB-8812580 from the National Science Foundation. The costs of publication of this article were de- frayed in part by the payment of page charges. This article must there- fore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed: Botany Dept., Box 7612, North Carolina State University, Raleigh, NC 27695. “el.: 919-

1 The abbreviations used are: PI-4,5-P2, phosphatidylinositol 4,5-bi- sphosphate; PI, phosphatidylinositol; PI-4-P, phosphatidylinositol 4-monophosphate; EF-la, elongation factor-la; CDPK, calcium-depend- ent protein kinase; CHAPS, 3-[(3-cholamidopropyl)dimethylammoniol- 1-propanesulfonic acid.

515-3496; Fax: 919-515-3436.

PI 4-kinase (EC 2.7.1.67) catalyzes the first committed step in the biosynthesis of PI-4-P and PI-4,5-Pz. Regulation of PI 4-kinase is critical to maintaining the levels of PI-4-P and PI- 4,5-P2 within the cell. PI 4-kinase activity changes rapidly in response to external stimuli and growth factors (Walker and Pike, 1987; Memon and Boss, 1990; Chen and Boss, 1990; Cho et al . , 19931, and these changes persist in isolated membranes, suggesting that covalent modification of the enzyme or a regu- lator of the enzyme occurs. Although there is no evidence for phosphorylation of PI 4-kinase per se (Carpenter and Cantley, 1990; Pike, 19921, several laboratories have suggested that phosphorylation may indirectly regulate enzyme activity (Hol- land et al., 1988; Kat0 et al., 1989; Taylor et al., 1984; Van Dongen et al . , 1985).

We have purified a 49-kDa protein, denoted PIK-A49, from carrot cells that activates PI 4-kinase (Yang et al., 1993). PIK- A49 is a multifunctional protein that binds and bundles actin and has EF-la activity. Therefore, the potential exists for PIK- A49 to communicate the status of the cytoskeleton, protein synthesis, and PI-4-P levels within the cell. The question we address here is, what regulates the function of PIK-A49 as a PI 4-kinase activator?

Because PIK-A49 is highly homologous to the EF-la subunit from plant cells and has EF-la activity (Yang et al., 19931, we have based our studies on some of the known properties of EF-la. EF-1 is a heterotrimeric G-protein. It facilitates the binding of amino acyl-tRNA to ribosomes during protein bio- synthesis. The a-subunit (EF-la) has GTPase activity and binds both GTP and GDP (Riis et al., 1990). Homologs of EF-la also have been found to be associated with the actin cytoskel- eton (e.g. ABP-50 (Yang et al., 1990)), the mitotic apparatus (Kuriyama et al., 1990; Ohta et al., 19901, and ribosomes (Didi- chenko et al., 1991). In addition, ABP-50, like PIK-A49, was found to activate carrot PI 4-kinase (Yang et al., 1993). ABP-50 has EF-la activity (Yang et al., 19901, and the binding of ABP-50 to G-actin is decreased by GTP (Dharmawardhane et al . , 1991). In this work, we investigate the potential role of GTP or GDP in regulating the activation of PI 4-kinase by PIK-A49.

Covalent modification of EF-1 and EF-la by protein phos- phorylation has been studied in mammalian cells (Venema et al., 1991; Palen et al., 1990). EF-la can be phosphorylated by protein kinase C (Venema et a l . , 1991); however, phosphoryla- tion of EF-la did not affect its activity in vitro, and there are no reports of protein phosphorylation affecting the actin binding ability of ABP-50. In higher plants, calcium-dependent protein kinases (CDPKs) are the most common form of protein kinases found thus far (Roberts and Harmon, 1992; Roberts, 1993). CDPK purified from the cytosol of soybeans was found to be associated with the cytoskeleton and plasma membrane (Put- nam-Evans et al., 1989, 1990; Schaller et al., 1992). In addition, PI 4-kinase activity has been shown to be associated with iso- lated F-actin (Payrastre et al . , 1991; Tan and Boss, 1992). Be- cause PIK-A49 is an actin-binding and -bundling protein, there

3852

Phosphorylated PIK-A49 Activates PI 4-Kinase 3853

is a potential for modification of PIK-A49 (and thus of PI-4-P levels) by CDPK in response to changes in cytosolic calcium.

In this paper, we show that PIK-A49 can be phosphorylated in vitro by a CDPK and that activation of PI 4-kinase by PIK- A49 is dependent on phosphorylation of PIK-A49. PIK-A49 therefore provides a mechanism for calcium regulation of PI- 4-P biosynthesis as well as a mechanism for intracellular com- munication. Regulating the levels of PI-4-P is of particular importance in cells where the PI-4,5-P2 levels are relatively low and where alternative roles for the inositol phospholipids in signal transduction are receiving more attention (Rinc6n and Boss, 1990; Gross and Boss, 1993; Drabak, 1993).

EXPERIMENTAL PROCEDURES Materials-Wild carrot (Daucus carota L.) cells grown in suspension

culture were transferred weekly and used on the fourth day after trans- fer as previously described (Chen and Boss, 1990). Escherichia coli alkaline phosphatase, octyl-Sepharose CL-4B, CHAPS, Triton X-100, and bovine serum albumin were purchased from Sigma. DEAE-Sepha- rose CL-GB was purchased from Pharmacia LKB Biotechnology Inc. Hydroxylapatite was purchased from Calbiochem. GTP, GDP, and [y-32PlATP (7000 Ci/mmol) were purchased from ICN.

Isolation of Plasma Membrane and Plasma Membmne F-actin-All procedures were done at 4 "C except where specifically indicated. The cells (1 g) were collected on filter paper (Whatman No. 1) at room temperature and homogenized in 2 ml of buffer containing 50 m~ Tris, 95 nm LiCl, 2 ~ l l ~ EGTA, 10 nm KCl, 1.0 l ~ l ~ EDTA, 0.2 rn MgC12, 0.05 g of polyvinylpyrrolidone, and 8% (w/v) sucrose (pH 7.5). The homog- enate was centrifuged at 1000 x g for 10 min. The resulting supernatant was centrifuged at 40,000 x g for 45 min. The 40,000 x g pellet was resuspended and used to isolate plasma membrane by aqueous polymer two-phase partitioning as described previously (Wheeler and Boss, 1987). The final plasma membrane pellet was resuspended in 30 rn Tris-HC1 (pH 7.2) (buffer A), stored at -70 "C, and used for preparation of solubilized PI 4-kinase. The plasma membrane F-actin fraction was isolated by depolymerization and repolymerization of F-actin from plasma membranes isolated by aqueous two-phase partitioning as de- scribed by Tan and Boss (1992).

Isolation of PIK-A4%The isolation of PIK-A49 was the same as previously described Wang et al., 1993). The most purified fractions from the DEAE-Sepharose CL-GB column were stored at -20 "C and used as PIK-A49. For the experiments studying the effect of GDP and GTP, PIK-A49 was isolated in the presence of 25% (v/v) glycerol. The purity of PIK-A49 was >95% based on Coomassie Blue staining.

Isolation of CDPK-CDPK was partially purified from the 40,000 x g supernatant of a carrot cell homogenate by DEAE-Sepharose CL-GB, hydroxylapatite, and octyl-Sepharose chromatography. The purification scheme was a modification of that reported by Putnam-Evans et al. (1990). All procedures were done at 4 "C unless otherwise specified. Briefly, the 40,000 x g supernatant was prepared as previously de- scribed (Yang et al., 1993) and loaded onto a DEAE-Sepharose CL-GB column (2.5 x 16 cm) pre-equilibrated with 30 m Tris-HC1 (pH 7.2), 1 mM EGTA, 0.02% (w/v) NaN,, and 25% (v/v) glycerol (buffer B). A NaCl gradient (0-0.8 M, 300 ml) in buffer B was applied to the column. CDPK was eluted at 0.3 M NaCl. The CDPK fractions were pooled and loaded onto a hydroxylapatite column (1.5 x 10.5 c m ) pre-equilibrated with 100 ml of 30 nm Tris-HC1 (pH 7.2), 0.5 nm CaC12, 0.02% (w/v) NaN,, and 25% (v/v) glycerol (buffer C). The column was eluted with buffer C (25 ml), followed by buffer D (80 ml) containing 30 nm Tris-HC1 (pH 7.2), 5.5 m MgC12, 1 IM( EGTA, 0.02% (w/v) NaN3, and 25% (v/v) glycerol; a 0-0.6 M NaCl gradient in buffer D; and finally, a 0-0.3 M phosphate gradient in buffer E containing 30 nm Tris-HC1 (pH 7.2), 1 m EGTA, 0.02% (w/v) NaN3, and 25% (v/v) glycerol. CDPK eluted at 50 m phos- phate. The CDPK fractions were pooled and dialyzed against buffer E overnight a t 4 "C. The dialyzed CDPK fraction was loaded onto an octyl-Sepharose column (1 x 18 c m ) pre-equilibrated with buffer F con- taining 30 - Tris-HC1 (pH 7.2), 1 nm CaC12, 0.02% (w/v) NaN,, and 25% (v/v) glycerol. The column was eluted with buffer G (65 ml) con- taining 30 - Tris-HC1 (pH 7.2), 0.5 nm CaC12, 0.15 M NaCl, 0.02% (w/v) NaN3, and 25% (v/v) glycerol, followed by buffer E (27 ml); then buffer E (27 ml) plus 10 n m EDTA, and finally, buffer D plus 0.2% (w/v) CHAPS. CDPK eluted in buffer D (75 ml) plus 0.2% (w/v) CHAPS. The fraction containing the highest CDPK activity was dialyzed with 30 nm Tris-HC1 (pH 7.2), 0.02% (w/v) NaN,, and 10% (v/v) glycerol overnight and used as CDPK for protein phosphorylation.

Solubilization of PI 4-Kinase from Carrot Plasma Membranes by

niton X-100-The method used was the same as previously described (Yang et al., 1993). Briefly, 0.01% (v/v) Triton X-100 was added to the isolated carrot plasma membranes, and the membranes were centri- fuged at 40,000 x g for 30 min to remove the activator entrapped in the membrane vesicles. The pellet was resuspended in buffer A, then, Triton X-100 was added to 0.01% (v/v) final concentration, and the mixture was vortexed at room temperature for 30 s. The mixture was centrifuged at 40,000 x g for 30 min. The supernatant was used immediately after preparation for the PI 4-kinase assay.

PI 4-Kinase Assay-The solubilized PI 4-kinase fraction (20 pl from 18 pg of carrot cell membrane protein) and treatment solution (10 p l ) or buffer A alone were added to a disposable tube. Phosphorylation was started by adding 20 pl of reaction mixture to give a final volume of 50 pl containing 30 nm Tris-HC1 (pH 7.29, 7.5 nm MgC12, 0.6 nm PI, 1 nm sodium molybdate, 0.3% (v/v) Triton X-100, and 0.9 nm [y-32PlATP (0.2 pCi/nmol; 0.6 pCi/nmol for the enzyme kinetics assay). The reaction mixture was incubated at mom temperature for 10 min and stopped by adding 1.5 ml of ice-cold CHClJMeOH (1:2, v/v). The lipids were ex- tracted in acidic CHClJMeOH as previously described (Cho et al., 1992). [32PlPI-4-P was the only phosphorylated lipid recovered as veri- fied by thin-layer analysis on LK5D plates developed in CHClJMeOW NHlOwHZO (86:76:6:18). Thus, for routine analysis, the lipids were transferred into liquid scintillation counting vials for Cerenkov count- ing. The radioactivity was measured with a Beckman LS 7000 liquid scintillation counter.

Protein Kinase Assay-The protein kinase assay during the purifica- tion of CDPK was done as follows. The reaction mixture contained 30 m~ Tris-HC1 (pH 7.2), 25 pg of histone IIIS, 5 pl of the column fraction, 10 nm MgCl,, 1 nm sodium molybdate, 120 p [y-32PlATP (3.3 pCi/ nmol), and 1 m~ free calcium or 1 nm EGTAin a 25-pl total volume. The reaction was started by adding ATP. After incubation at 25 "C for 10 min, 10 pl of reaction mixture was spotted onto cellulose filter paper. The paper was kept at 25 "C for a few minutes and washed three times with 10% (w/v) trichloroacetic acid and 1% (w/v) pyrophosphate. The spots were cut and placed into glass scintillation vials. The radioactivity was measured using Cerenkov counting.

Determination of Calcium Dependence of Phosphorylation of PIK-A4%The reaction mixture contained 30 nm Tris-HC1 (pH 7.2), 0.0012 unit of CDPK, 0.8 pg of PIK-A49, 10 rn MgC12, 1 nm sodium molybdate, 120 p [y-32PlATP (3.3 pCi/nmol), and the indicated concen- trations of free calcium or 1 nm EGTA in a 25-p1 total volume. The free calcium concentration was adjusted using calcium EGTA (Putnam- Evans et al., 1990). The reaction was started by adding ATP. After incubation at 25 "C for 10 min, the reaction was stopped by adding 25 pl of electrophoresis sample buffer containing 20% (v/v) glycerol, 4% (w/v) SDS, 10% (v/v) /3-mercaptoethanol, a trace amount of bromphenol blue (dye), and 120 nm Tris-HC1 (pH 6.75). The phosphorylated protein was separated on a 10% SDS-polyacrylamide gel. Electrophoresis was performed according to Laemmli (1970). The radioactive bands were visualized by autoradiography. The PIK-A49 band on the autoradio- gram was determined by comparison with the purified PIK-A49 band on Coomassie Blue-stained gel.

Dephosphorylation of PIK-A49 by Alkaline Phosphatase fiom E. coli-PIK-A49 (9 p) was first phosphorylated with 0.027 unit of CDPK in a mixture (150 pl) containing 30 lll~ Tris-HC1 (pH 7.2), 10 n-m MgC12, 10 p CaC12, and 0.6 nm [y-32PlATP (2.4 pCi/nmol). After 20 min, the reaction was stopped by placing the sample on ice, and DEAE-Sepha- rose CG6B (30 pl) was added to remove CDPK. The mixture was incu- bated on ice for at least 20 min with occasional shaking. An aliquot of the supernatant (15 pl) was added to 10 pl of phosphatase solution of varying concentrations to give the final phosphatase concentrations indicated in a volume of 25 pl. After 20 min of incubation at 25 "C, 25 pl of electrophoresis sample buffer was added to each dephosphorylation mixture, and the entire sample (50 pl) was loaded onto a 10% SDS- polyacrylamide gel. Electrophoresis was performed according to Laemmli (1970). The radioactive bands were visualized by autoradiog- raphy.

For preparation of the DEAE-Sepharose CL-GB used to remove CDPK and alkaline phosphatase, 1.4 ml of DEAE-Sepharose CG6B stock gel (Pharmacia) was added to a disposable tube and centrifuged at 500 x g for 2 min. The supernatant was removed, and the gel pellet was washed twice with 4 ml of washing buffer (30 nm Tris-HC1 (pH 7.2)) by centrifugation at 500 x g for 2 min. Finally, the gel pellet was resus- pended in 0.7 ml of washing buffer and put on ice at least for 10 min prior to use.

Determination of Effect of Phosphorylation and Dephosphorylation of PIK-A49 on Activation of PI 4-Kinase-PIK-A49 was treated with CDPK isolated from carrot cells or with alkaline phosphatase from E.

3854 Phosphorylated PIKA49 Activates PI 4-Kinase coli. For protein phosphorylation, the reaction mixture (100 pl) contain- ing 30 m~ Tris-HC1 (pH 7.2), 6 p~ PIK-A49,10 m~ MgC12,lO p~ CaCI2, 0.018 unit of CDPK, and 0.6 m~ ATP was incubated at 25 "C for 20 min. For protein dephosphorylation, the reaction mixture (100 pl) containing 30 m~ Tris-HCI (pH 7.2), 6 p~ PIK-A49,lO m~ MgC12, and 0.88 unit of phosphatase was incubated at 25 "C for 20 min. Samples were placed on ice. After PIK-A49 was dephosphorylated, CaC12 and ATP were added to the dephosphorylation mixture to final concentrations of 10 p~ and 0.6 m ~ , respectively. To remove CDPK and phosphatase, 20 pl of DEAE- Sepharose CL6B was added to the reaction mixture, and the mixture was incubated on ice for a t least 20 min with occasional shaking. The supernatant (20 phample) was used for the PI 4-kinase activation assay. The control samples were treated in the same manner, including adding DEAE-Sepharose CG6B.

Determination of Effect of Rephosphorylatwn of Dephosphorylated PIK-A49 on Activation of PZ 4-Kinase-PIK-A49 (11 p ~ ) was treated with 1.76 unit of phosphatase in a mixture (200 pl) containing 30 m~ Tris-HC1 (pH 7.2) and 10 m~ MgCl, a t 25 "C for 20 min. The reaction was stopped by incubating the sample on ice. DM-Sepharose CG6B (40 pl) was added to the mixture, and the mixture was incubated for at least 20 min with occasional shaking. The supernatant (91 galiquot) was transferred into disposable tubes. ATP, CaCl,, and CDPK or buffer (for the control) were added to the aliquot to give 100 pl of final volume containing 0.6 m~ ATP, 10 p~ CaCl,, and 0.018 unit of CDPK. The mixture was incubated at 25 "C for 20 min and placed on ice, and DM-Sepharose CL6B (20 pl) was added. After 20 min on ice, the supernatant (20 plhample) was used for the PI 4-kinase activation assay.

Phosphoamim Acid Analysis-For phosphorylation of PIK-A49, the phosphorylation mixture (50 pl) contained 4 pg of PIK-A49, 10 ng of CDPK, 10 p~ free calcium in 1 m~ CaClflGTAbuiTer, 10 m~ MgCI2, 0.3 m~ tyS2PIATP (4 pcihmol), and 30 m~ Tris-HCI (pH 7.2). Phosphor- ylation was started by adding ATP The mixture was incubated at 25 "C for 20 min and stopped by adding 0.5 ml of 10% (w/v) trichloroacetic acid. The sample tube was put on ice, and 80 pl of bovine serum albumin (1 mg/ml) was added. The sample was centrifuged at 2500 rpm for 10 min, and the supernatant was discarded. The pellet was washed twice with icecold ethyl ether and dried under vacuum (water aspiration). Hydrolysis of the phosphoprotein was performed as follows. 200 pl of 6 N HCl was added to the phosphoprotein pellet, and the mixture was incubated at 110 "C for 2 h until the mixture was dried. The dried sample was used for electrophoresis. -0-dimensional electrophoresis of phosphoamino acids on cellulose thin-layer plates and determination of phosphoamino acids with ninhydrin staining and autoradiography were performed as described by Huber and Huber (1992).

Zmmunoblot Assay-Immunoblot assays of proteins from the plasma membrane and F-actin fractions with anti-PIK-A49 serum were per- formed as previously described (Yang et al., 1993).

Protein Assay-Protein was determined according to Lowry et al. (1951) and Bradford (1976) with bovine serum albumin as a standard.

RESULTS PIKA49 Is Associated with the Plasma Membrane Fraction

and with F-actin Isolated from Plasma Membrane-PIK-A49 has F-actin binding and bundling ability in vitro (Yang et al., 1993). To confirm that PIK-A49 is associated with the F-actin fraction from carrot cells, we isolated F-actin from carrot cell plasma membranes and immunoblotted the proteins with anti- PIK-A49 serum (Fig. 1). A 49-kDa polypeptide in both F-actin fractions isolated from one cycle of actin depolymerization and repolymerization (lane 3) and two cycles of actin depolymeriza- tion and repolymerization (lane 2) cross-reacted with anti-PIK- A49. A 49-kDa polypeptide in the plasma membrane fraction also cross-reacted with anti-PIK-A49 (lane 4). Because PIK- A49 is not an integral membrane protein, it is most probable that PIK-A49 in the plasma membrane fraction is primarily associated with F-actin.

PIKA49 Enhances the Binding Afjinity of PI 4-Kinase for ATP"T0 investigate the mechanism of activation of PI 4-ki- nase, we determined the effects of PIK-A49 on the K,,, of PI Ckinase for ATP. As shown in Fig. 2A, without PIK-49, the K,,, of PI 4-kinase for ATP was -0.40 m ~ . The K, for ATP de- creased to 0.19 m~ if PIK-A49 was added, indicating that PIK- A49 enhanced the binding affinity of the enzyme for ATP. PIK-

-PIK-A49

1 2 3 4 FIG. 1. PIK-A49 is present in the plasma membrane and F-actin

fraction isolated from plasma membrane. Shown is a Western blot of the proteins from the plasma membrane and the F-actin fractions using anti-PIK-A49 serum. Lune 1, PIK-A49 (0.8 pg of protein); lane 2, the F-actin fraction (10 pg of protein) isolated from the second repoly- merization cycle; lane 3, the F-actin fraction (10 pg of protein) isolated from the first repolymerization cycle; lane 4, the plasma membrane (55 pg of protein).

-0.0003 J

- 0.003 - B . 0 Plus Cardlotoxin P

B 0 Control

0.002 - 0

E a

= 0.001 - P . > . c

1

10 20 30 40

-0001 J 1lIATPI IllmL(1 FIG. 2. PIK-A49 decreases the K, of PI 4-kinase for ATP. Car-

diotoxin did not affect the K, of PI 4-kinase for ATP. A, PIKA49 (22 pg/ml); B, cardiotoxin (20 pg/ml).

A49 did not affect the V,, of PI 4kinase. PIK-A49 is a positively charged protein. Many positively charged compounds can activate PI 4-kinases (Vogel and Hoppe, 1986L2 To compare the effect of PIK-A49 with that of other positively charged polypeptides on PI 4-kinase, we determined the effect of car- diobxin on carrot PI 4-kinase (Fig. 2 8 ) . Cardiotain has been shown to activate PI Ckinase (Walker and Pike, 1990). With solubilized carrot PI 4kinase, cardiotoxin did not affect the K,,, for ATP, but rather enhanced V,, (Fig. 2 8 ) . These data indi- cate that the activation of carrot PI 4-kinase by PIK-A49 is different from that by cardiotoxin.

W. Yang and W. F. Boss, unpublished results.

Phosphorylated PIKA49 Activates PI 4-Kinase 3855

250 1 A

- PIK-A49

Frc. 3. GTP or GDP inhibits activation. of PI 4kinase by PIE- A49. Treatments were as follows: Control, buffer alone; GTP, 0.5 = GTP; GDP, 0.5 m~ GDP; PIK-A49,1 w PIK-A49; GTP + PIK-A49,0.5 ~ l l ~ GTP and 1 w PIK-A49; GDP + PIK-A49, 0.5 m~ GDP and 1 w PIK-A49.

Effect of Guanine Nucleotides on PIK"A49-induced Activatwn of PI 4-Kime-PIK-A49 is an isoform of carrot EF-la (Yang et al., 1993), and the EF-la activity of PIK-A49 required milli- molar concentrations of GTP. PIK-A49 is also a GTPase be- cause EF-lPy, which are factors required for GTPIGDP ex- change, stimulated the EF-la activity of PIK-A49. When we studied the effect of GTP and GDP on PIK-A49-induced acti- vation of PI 4-kinase, we found that GTP alone did not affect PI Ckinase activity and that GDP alone slightly increased PI Ckinase activity. However, both GTP and GDP inhibited the activation of PI Ckinase by PIK-A49 (Fig. 3). These data sug- gest that when the protein binds GTP or GDP, the tertiary structure is altered so that it can function as EF-la, but not as an activator of PI 4-kinase. Changes observed in the crystal structure of EF-Tu when GTP or GDP binds are consistent with this hypothesis (Berchtold et al., 1993).

PIK-A49 Is Phosphorylated by CDPK-Phosphorylation of PIK-A49 by CDPK was calcium-dependent and saturated a t 1

calcium (Fig. 4A). High concentrations of calcium (10 II~M) strongly inhibited phosphorylation. When PIK-A49 was phos- phorylated by CDPK, the major phosphorylated amino acid was phosphoserine (data not shown).

Phosphorylation of PIK-A49 by CDPK could be reversed with E. coli alkaline phosphatase (Fig. 4B). For these experiments, we took advantage of the fact that PIKA49 does not bind to DEAE-Sepharose CGGB and that CDPK and alkaline phospha- tase do. PIK-A49 was phosphorylated by CDPK in the presence of [y-32P]ATP, and CDPK was removed by adding DEAE- Sepharose CL-GB. When the supernatant containing P2P1PIK- A49 was treated with alkaline phosphatase, PIK-A49 was de- phosphorylated (lanes 2-5). If alkaline phosphatase was added to DEAE-Sepharose CGGB, it bound the DEAE, and the result- ing supernatant had little effect on the phosphorylation state of PIK-A49 (lane 6).

Protein Phosphorylation Is Required for Activation of PI 4-Ki- nase by PIKA49-The effect of phosphorylation on the ability of PIKA49 to activate PI Ckinase was tested by comparing the phosphorylated and dephosphorylated forms of the protein. CDPK and alkaline phosphatase were removed by adding DEAE-Sepharose CGGB prior to testing for the ability of PIK- A49 to activate PI Ckinase. Phosphorylated (CDPK-treated) PIK-A49 activated solubilized PI 4-kinase -40% more than nontreated PIK-A49 (Fig. 5 4 ) . However, dephosphorylated (al- kaline phosphatase-treated) PIK-A49 did not activate PI Cki- nase. The supernatant from DEAE-Sepharose CGGB plus

1 2

6

".

3 4 5 6 7

- PIK-A49

1 2 3 4 5 6 Fro. 4. PIE-A49 can be phoephorylated by CDPK and dephos-

phorylated by alkaline phosphatase. A, shown is an autoradiogram of phosphorylation of PIK-A49 by a calcium-dependent protein kinase isolated from carrot cells. CDPK alone (0.0012 unit) was used as a control for autophosphorylation (lune 1; 10 w calcium). Lanes 2-7, PIK-A49 (0.8 &lane) and CDPK (0.0012 unifflane) were added, and the indicated concentrations of free calcium were used in the phosphoryla- tion mixture. The free calcium concentrations were 0, 1, 10, 100,1000, and 10,000 w for lanes 2-7, respectively. One unit of CDPK transferred 1 nmol of 32Pi from [v.~~P]ATP to histone IIISlmin. B, [32PlPIK-A49 was obtained by phosphorylation of purified PIK-A49 by CDPK. CDPK was removed by adding DM-Sepharose C1-6B, and E. coli alkaline phos- phatase was added a t the concentrations indicated, except in lune 6, where alkaline phosphatase was mixed with DEAE-Sepharose to re- move the phosphatase, and the supernatant was added to [32PlPIK-A49. The proteins were separated on a 10% SDS-polyacrylamide gel, and 32P was monitored by autoradiography. Lane 1, no phosphatase; lane 2,0.74 unit of phosphatase; lane 3,0.15 unit of phosphatase; lune 4,0.15 unit of phosphatase plus 500 w sodium vanadate; lane 5,0.49 unit of phos- phatase; lane 6, 0.49 unit of DEAE-Sepharose-treated phosphatase.

CDPK alone or alkaline phosphatase alone had no effect on PI 4kinase activity. Because dephosphorylation completely elimi- nated the ability of PIK-A49 to activate PI 4kinase and yet phosphorylation by CDPK increased activation by 40%, phos- phorylation appeared to be essential for activation. Further- more, these data suggest that the purified PIK-A49 is partially phosphorylated.

If phosphorylation of PIK-A49 is truly required for activa- tion, then rephosphorylation of dephosphorylated PIK-A49 should restore its ability to activate PI Ckinase. As shown in Fig. 5B, dephosphorylated PIK-A49 did not activate PI 4ki- nase; however, rephosphorylation by CDPK of dephosphor- ylated PIK-A49 restored full activation. These data demon-

3856 Phosphorylated PIK-A49 Activates PI 4-Kinase

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n I

B

FIG. 5. Activation of PI 4-kinase is regulated by phosphoryla- tion and dephosphorylation of PIK-A49.A, phosphorylation of PIK- A49 by CDPK enhanced its ability to activate PI 4-kinase, and dephos- phorylation of PIK-A49 by alkaline phosphatase eliminated activation of PI 4-kinase by PIK-A49. Treatments were as follows: Control, buffer alone; CDPK, CDPK alone; Ptase, alkaline phosphatase alone; PIK-A49, PIK-A49 alone; CDPK + PIK-A49, PIK-A49 phosphorylated by CDPK Ptase + PIK-A49, PIK-A49 dephosphorylated by alkaline phosphatase. DM-Sepharose CL-GB was added to each solution to remove CDPK

B , phosphorylation of dephosphorylated PIK-A49 recovered its ability to and alkaline phosphatase prior to assaying for activation of PI 4-kinase.

activate PI 4-kinase. PIK-A49 was dephosphorylated with alkaline phosphatase. Dephosphorylated PIK-A49 was rephosphorylated with CDPK. Control, buffer only; Ptase + PIK-A49, PIK-A49 treated with alkaline phosphatase only; Ptase,CDPK + PIK-A49, PIK-A49 treated first with alkaline phosphatase and then with CDPK

strate that phosphorylation of PIK-A49 is required for its acti- vation.

DISCUSSION

PIK-A49 was characterized previously (Yang et al., 1993). In this paper, we investigated the mechanism by which PIK-A49 activated PI 4-kinase. PIK-A49 lowered the K, of PI 4-kinase for ATP, suggesting that it enhanced the binding affhity of the enzyme for ATP. When GTP and GDP were added alone, they had little effect on PI 4-kinase activity; however, when added in the presence of PIK-A49, GTP and GDP decreased the activa- tion of PI 4-kinase, suggesting that when PIK-A49 functions as EF-la (binding GTP and GDP), it may not function as an ac- tivator of PI 4-kinase.

Most important, phosphorylation of PIK-A49 was essential for activation of PI 4-kinase. Regulation of PI 4-kinase activa- tion by phosphorylation and dephosphorylation of PIK-A49 im- plies that PI-4-P biosynthesis will be responsive to external signals that are perceived by a CDPK or protein phosphatase. The sensitivity of the activation to calcium via CDPK means that an increase in cytosolic calcium (e.g. from inositol 1,4,5- trisphosphate-induced release from intracellular stores or from other intracellular stores or extracellular sources) could result in an increase in PI-4-P and PI-4,5-Pz. In addition, rabbit re- ticulocyte EF-la can be phosphorylated by protein kinase C (Venema et al., 1991). There is no evidence for a diacylglycerol- regulated C-type kinase in higher plants (Roberts and Harmon, 1992; Cote and Crain, 1993); however, regulation by either diacylglycerol (phosphorylation by protein kinase C) or inositol 1,4,5-trisphosphate (phosphorylation by CDPK) could provide a mechanism of feed-forward control and a means of recovering PI-4-P and PI-4,5-Pz levels after activation of phospholipase C.

EF-la is known to be a relatively abundant protein within all cells (Riis et al., 1990). One question has been, what regulates this multifunctional protein? One mechanism of regulation is the binding of GTP or GDP, both of which are essential for EF-la activity, but which decreased the ability of PIK-A49 to activate PI 4-kinase. Another mechanism of regulation would be via phosphorylation. Although phosphorylation of the iso- lated EF-la subunit did not affect its activity as an elongation factor (Venema et al., 19911, phosphorylation was essential for PIK-A49 to activate plasma membrane PI 4-kinase. Thus, pro- tein phosphorylation is at least one means of covalent modifi- cation of PIK-A49 that could affect its functional distribution and the levels of PI-4-P in vivo.

Most higher plant responses to external stimuli involve growth. Enhanced protein synthesis and changes in cytoskel- eta1 structure are necessary for cell elongation, cell division, and cell differentiation. Because PIK-A49 is a multifunctional protein, it provides a means of communicating the status of the plasma membrane phosphoinositides to the cytoskeleton and the protein synthesis machinery of the cell. Furthermore, as a substrate for CDPK, PIK-A49 provides a mechanism for sens- ing transient changes in cytosolic calcium. Calcium is the most accepted second messenger for plants and changes rapidly in response to touch and light (Knight et al., 1992; Shacklock et al., 1992). To give some perspective as to how PIK-A49 might affect PI-4-P levels, actin structure, and protein synthesis in stimulated cells, we propose the following scenario, which is based on our data and those of others and on the assumption that activated phospholipase C can hydrolyze polyphosphory- lated phosphatidylinositol and release the bound actin-capping and -severing proteins in vivo as has been shown in vitro (Gold- Schmidt-Clermont et al., 1991). Phospholipase C is activated and hydrolyzes PI-4-P and PI-4,5-Pz. Cytosolic calcium in- creases, and phospholipase A2, CDPK, and actin-severing and -capping proteins such as profilin and gelsolin are activated. The actin-severing proteins cut F-actin into actin oligomers. PIK-A49 no longer bundles the F-actin and is phosphorylated by CDPK. Phosphorylated PIK-A49 activates PI 4-kinase that is released from the membrane by phospholipase A2 (Gross et al., 1992) or that is present in the F-actin fraction (Payrastre et al., 1991; Tan and Boss, 1992). The levels of PI-4-P increase. Actin-severing and -capping proteins will dissociate from actin oligomers and bind PI-4-P (Lassing and Lindberg, 1985; Jan- mey and Stossel, 1987). Actin polymerization occurs, and new F-actin is formed. PIK-A49 is dephosphorylated, binds to F- actin, and enhances the formation of new actin bundles. Phos- phorylated PIK-A49 also can function as EF-la and affect pro- tein synthesis. As the cell recovers, the cytosolic free calcium is either pumped out of the cell or into intracellular stores. Phos-

Phosphorylated PIK-A49 Activates PI 4-Kinase 3857

pholipases C and A,, actin-severing proteins, and CDPK are no longer activated. The cell returns to its resting stage and is ready to respond to the next signal.

Acknowledgments-We thank Dr. Alice Harmon (University of Florida) for sending us purified CDPK from soybean and for advice on the purification of CDPK from carrot cells, Dm. Joan and Steve Huber (North Carolina State University) for helping us with the phos-

brane F-actin fractions. phoamino acid analysis, and Zheng Tan for preparing the plasma mem-

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