Indian Journal of Biochemistry & Biophysics Vol. 36, February 1999, pp. 1-9

Purification and characterization of a type II phosphatidylinositol 4-kinase from rat spleen and comparison with a Ptdlns 4-kinase from lymphocytes

Mary Verghese, Aaron Z Fernandis and Gosukonda Subrahmanyam*

Biotechnology Centre, Indian Institute of Technology, Powai , Mumbai 400076, India

. Received 16 June 1998; revised 23 September 1998

A Ptdlns 4-kinase from rat spleen particulate fraction was purified to homogeneity and its molecular properties were compared with a Ptdlns 4-kinase from splenic lymphocytes. The enzyme acti vity was solubilized from spleen particulate fraction with Triton X-IOO and chromatographed sequentially on phosphocellulose, DEAE-sephacel , heparin acrylamide and hydroxyapatite columns. The purified enzyme preparation showed a 55 kDa band on SDS-PAGE with silver staining. Renaturati on of the enzyme activity from SDS-PAGE showed that it comigrated with the 55 kDa protein . Characterization of the enzyme showed that it was a type II Ptdlns 4-kinase. Polyclonal antibodies raised against Ptdlns 4-kinase inh ibited the enzyme activity in in vitro assays. Analysis of adult rat tissue particulate fraction s on immunoblots showed restricted immunoreacti vity among Ptdlns 4-kinases. However, the immunoreactivity is conserved in lymphoid tIssues from mouse to human , suggesting that lymphoid tissue has a distinct Ptdlns 4-kinase. Activation of rat splenocytes with Con A showed two fo ld in crease in Ptdlns 4-kinase activity. Comparison of Ptdlns 4-kinases from spleen and splenic lymphocytes showed identical chromatographi c behaviour, molecular mass, immunoreactivity, Km values for Ptdlns and inhibition by adenosine.

Occupancy of T cell receptor with an antigen in the context of MHC molecules results in rapid phospho­rylation of proteins at tyrosine, serine and threonine residues. Concomitant with protein phosphorylation, membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns( 4,5)P2) is hydrolyzed by phos­pholipase C to generate second messengers, inositol 1,4,5-trisphosphate (Ins (l,4 ,5)P)) and 1 ,2-diacylglycerol. These second messengers in turn trigger a cascade of biochemical events that result in interleukin-2 secretion and clonal proliferation of T­cells l

.). The hydrolysi s and synthesis of Ptdins (4 ,5)P2 is known as phosphatidylinositol (PtdIns) cy­cle4

• Activation of protein tyrosine kinases precedes phosphoinositich! breakdown and the mechanism by which it couples PtdIns( 4,5)P2 hydrolysis appe:>rs

*To whom correspondence may be addressed Phone No.: 091 -22-576 7777, Fax.: 091-22-5783480. E- mail : gsm@ btc.iitb.emet. in Abbreviations lIsed : TCR, T cell antigen receptor ; Ptdlns, phos­phatidylinositol ; Ptdlns4? , D-myo phosphatidylinositol 4-phosphate ; Ptdlns( 4 ,5)? 2 , D-myo phosphatidylinositol 4 ,5-bisphosphate ; Ins ( I A,5)? ], inositol 1,4,5-trisphosphate ; PM SF, phenylmethylsul fon yl fluoride ; Con A, concanavalin A ; SDS­PAGE, polyacrylamide gel electrophoresis in presence of sodium dodecyl su lphate ; NEMA , N-ethylmorpholine acetate buffer ; Pefabloc SC, 4-(2-aminoeth yl)-benzenesulfonyl fluoride hydro­chloride ; RPM I, Roswell Park Memori al Institute

through activation of phospholipase C-y by protein tyrosine phosphorylation5

-6

. While tyrosine phospho­rylation and its regulation during T cell activation are the subject for intense scientific inquiry very little is known about PtdIns cycle in lymphocytes .

Activation of "Ptdins cycle" is sufficient for inter­leukin-2 production in T cells7

. PtdIns( 4,5)P2 turn­over is also found to be a key event in fas-induced apoptosis in human T cells8

. In addition to their role in signal transduction, phosphoinositides are shown to have pleiotrophic effects9

•1O

• Kinetic analysis of T cell activation showed that calcium levels have to be elevated for at least 1-2 hr for successful signal trans­duction II. However, the second messengers Ins(1,4,5)P) and diacylglycerol have very short bio­logical half lives, thereby necessitating continuous generation of these second messengers. Analysis of phosphoinositide levels during T cell activation sug­gest that there is a net increase in phosphoinositides12

.

This increase may be due to activation of two distinct phosphoinositide lipid kinases, known as phosphati­dylinositol 4-kinase (PtdIns 4-kinase) and phosphati­dylinositol 4-phosphate 5-kinase (Ptdins 4P 5-kinase)l3. PtdIns 4-kinase catalyzes the first com­mitted step in the biosynthesis of PtdIns( 4,5)P2. At least three types of PtdIns kinascs are reported from

2 INDIAN 1. BIOCHEM. BIOPHYS., VOL. 36, FEBRUARY 1999

mammalian cells. The properties of these enzymes were reviewed recentlyl4.

The early signal transduction pathways during T cell activation via TCR-CD3 or CD4 complex ap­pears to employ a Ptdlns 4-kinase than a Ptdlns 3-kinase l5.18 Further, Ptdlns 4-kinases are shown to be targets for rapamycin and FK506. In yeast, these pharmacological agents induce translocation of the enzyme from membrane organelles to cytoplasm re­su lting in physical separation of the enzyme from its substrate l9 20 A si milar mechanism may operate during their immunosuppressive action in lympho­cytes. These studies suggest that PtdIns 4-kinases playa pivotal role in T cell function and may be tar­get molecules for future immunomodulators. A criti­cal study of these enzymes in lymphocytes is neces­sary to understand their physiological significance and regulation during lymphocyte proliferation and apoptosi s.

Materials and Methods Materials

Phosphatidylinositol , Triton X-I 00, phenylmethyl­sulfonylfluoride (PMSF), heparin acrylamide were from Sigma Chemical Company, MO, USA. Phos­phocellulose was from Whatman BioSystems Lim­ited, Kent, England . DEAE-Sephacel was from Pharmac ia Fine Chemica ls. Sweden. Hydroxyapatite was from BioRad Laboratories. Silica coated alu­minium TLC plates were from E. Merck, Germany. Nitrocell ulose sheets were from Amersham Interna­tional. Protein A agarose beads, anti-rabbit IgG con­j ugated to alkaline phosphatase were from Boehringer Mannheim, Germany . [y_32p] ATP (3000 Ci/mmol) was from Board of Radiation and Isotopic Technol­ogy (BRIT), Bombay, India. Wistar rats and New Zealand white rabbits were purchased from Haffkine Biopharmaceuticals Corporation Ltd. , Bombay, India . All other chemicals were of analytical grade.

Preparation of rat spleen particulatefraction Spleen particulate fraction was prepared as de­

scribed earlier l.22 with minor modifications. Briefly, spleens were dissected out from Wistar rats and ho­mogenized in ice cold homogenization buffer (10 mllg) containing 50 mM Tris (PH 7.6), I mM EDTA, 2 mM MgCI 2, I mM PMSF, 0.1 mM pefabloc and 1 }.lg/ml soyabean trypsin inhibitor. All further steps were carried out at 4°C. The homogenate was centri-

fuged at 1000 g for 10 min. The pellet was rehomo­genized (5 mllg) and centrifuged at 1000 g for 10 min . The combined supernatants was centrifuged at 1000 g for 10 min . The membrane fractions were sedimented at 35000 g for 40 min . The pellet was washed with 0.1 M NaCI in homogenization buffer and suspended in 0.25 M sucrose in the same buffer to give particulate fraction . Identical procedure was followed for isolation of particulate fraction from different tissues. Aliquots of the particulate fraction were stored at -80°C for enzyme assays, protein esti­mation and for Western blots.

Ptdlns 4-kinase assay The Ptdlns 4-kinase activity was assayed as de­

scribed earlier23.24

. The enzyme showed linear kinet­ics up to 15 min and 2 flg of enzyme per assay. All assays were carried out in the linear part of the en­zyme kinetics.

Purification of Ptdlns 4-kinase from spleen particll­late fra ction

All steps involved with purification were carried out at 4°C. Rat spleen particulate fraction was solu­bilized in extraction buffer (10 mM Na2HP04 (PH 7.6), 1 mM (3-mercaptoethanol , 5% Triton X-IOO (v/v) , 20% glycerol (v/v) , 1 mM EDT A, 1 mM PMSF, 0.1 mM vanadate, 0.1 mM pefabloc and 1 flg/ml soy­abean trypsin inhibitor) on ice for 25 m in . The Triton X-IOO extract was centri fuged at 35000 g for 60 min . The supernatant was chromatographed on 30 ml of phosphoce llulose column pre-equilibrated with equilibration buffer (5 tw\,f Na2HP04 (PH 7.6), 1 mM (3-mercaptoethanol , 0.1 % Triton X-I00 (v/v), 10% glycerol (v/v), and 0.1 mM vanadate). The column was washed with 400 ml of equilibration buffer con­ta ining 0 .075 M NaCI followed by 20 ml of 0.1 M NaCI in equilibration buffer. The enzyme activity was eluted wi th a linear salt gradient of 0. 1 M to 0.4 M NaCI in equilibration buffer. Fractions (3.5 ml) were assayed for Ptdlns 4-kinase ac tivity and ana­lyzed on SDS-PAGE. The act ive fractions were pooled and dialyzed against storage buffer (50% glycerol (v/v) , 25 mM NEMA (PH 7.5), 1 rnfo.,f (3-mercaptoethanol , 0.1 % Triton X-I00 (v/v) and 0. 1 mM vanadate) overnight.

The enzyme from phosphocellulose pool was di­luted 2.5 times with 50 mM NEMA (PH 8.5), 0.1 % Triton X-IOO (v/v), 0 .1 mM vanadate and I ruM (3-mercaptoethanol and chromatographed on 5 ml

VERGHESE e/ at. : A TYPE" PHOSPHATIDYLINOSITOL 4-KINASE FROM RAT SPLEEN AND LYMPHOCYTES 3

DEAE-Sephacel column pre-equilibrated with 25 mM NEMA (PH 7.5), I mM f)-mercaptoethanol, 0.1% Triton X-IOO (v/v) , 0.1 mM vanadate and 10% g!yc'­erol (v/v) . . The column was washed with 50 m! of the above buffer. The enzyme activity was eluted with a discontinuous salt gradient of 0.02 M, 0.05 M and 0.0'8 M NaCI in the above buffer. Fractions (1 .5 ml) were assayed for PtdIns 4-kinase activity and ana­lyzed on SDS-PAGE. The active fractions were pooled and dialyzed against storage buffer.

The DEAE-Sephacel column pool was diluted 2.5 times with column equilibration buffer containing 10 mM HEPES (PH 7.4) , 10 mM MgCI2, I mM f)­mercaptoethanol , 0 .1 % Triton X-IOO (v/v), 10% glycerol (v /v) and 0.1 mM vanadate and chromato­graphed on 2 ml heparin acrylam ide column. The column was washed with 10 ml of above buffer. Ptdlns 4-k inase activity was eluted with a discontinu­ous salt gradient of 0 .15 M , 0.225 M , 0.35 M and 0.5 M NaCI (6 ml each) in the above buffer without MgCI2. Fractions (0.5 ml) were assayed for PtdIns 4-kina se act ivity and analyzed on SDS-PAGE. The active fract ions were pooled and dialyzed against storage buffer.

The heparin acrylamide column pool was diluted 2.5 ti mes with 10 mM Na2HP04 (PH 7.4), I mill EDT A, 0. 1 mM vanadate, ! mM l3-mercaptoethanol, 10% glycerol (v/v) and 0. 1% Triton X-IOO (v/v) and chromatographed on I ml hydroxyapatite column. Enzyme acti vity was eluted with a discontinuous gra­dient of 0.01 M, 0.04 M, 0.07 M and 0.09 M phos­phate (4 ml each) in the above buffer. Fractions (0.25 ml) were collected and assayed for PtdIns 4-kinase activity and analyzed on SDS-PAGE. The active fractions were pooled and dialyzed against storage buffer. The pooled enzyme was stored at -20°C for subsequent stud ies .

Renaturation oj PtdfllS 4-kinaseJrom SDS-PAGE Ptdlns 4-kinase from DEAE-Sephacel step (- 10

Ilg) was electrophoresed on 12% SDS-PAGE at 4°C. In an adjacent lane - 2 Ilg of the enzyme was electro­phoresed and stained with Coomassie blue. For re.­naturation experiments the sample was not boiled in sample bu ffer. The lane used for renaturation was washed thr ice in ice-cold Tris-HCl (10 mM, pH 7.0) for 20 min . The gel was cut into 5 mm slices and assayed for Ptdlns 4-kinase activity at room tem­perature as described above except for the reaction volume (100 IJ.I) and incubation time (24 hr) with

high specific activity of [y_32p] A TP (1000-1500 cpm/pmole ).

Production oj polyclonal antisera against PtdIns 4-kinase

Immunization of New Zealand white rabbits was done as described25

. Primary immunization of rabbit was done with SDS-PAGE purified PtcPns 4-kinase from phosphocellulose pool. A part of the gel was assayed for Ptdlns 4-kinase activity as described above and the remaining part of the gel. was stained with Coomassie blue. The band corresponding to the PtdIns 4-kinase activity was excised and washed sev­eral times with 10% methanol to remove SDS and acetic acid. The gel slice was homogenized in phos­phate buffered saline in presence of liquid ni trogen. The homogenate was injected into rabbits subcutane­ously. The booster injections were also given by us­ing the electrophoretically purified Ptdlns 4-kinase. The rabbit was bled and anti sera was analyzed for immunoreactivity on Western blots. Antibodies were purified on Protein A agarose column.

Con A stimulation oj rat splenocytes A rat spleen was dissected into ice cold sterile

RPMI 1640 medium and teased to separate cells. The cell suspension was filtered through muslin cloth and cell s were collected by centrifugation at 349g for 10 min. Splenic RBCs were lysed with 0.84% NH4Cl.

Splenocytes were stimulated with Con A as de­scribed earlier4

. Briefly, splenocytes (5 x 106 cells, in 200 III of RPMI medium) were incubated at 37°C for 15 min before addition of Con A (5 Ilg/ml). The in­cubation was tenninated at di fferent time points with the addition of I ml of ice cold RPMI. Cells were lysed in 0.2 ml ot' lysis buffer (25 mM Tris-HCl (PH 7.4), 1% Triton X-IOO, 150 mM NaCl, 2 mM EDTA, 0.25 mM sodium orthovanadate, I mM PMSF and 10 Ilg/ml benzamidine). The lysate was centrifuged at 35000 g for 30 min. 10 III of the supernatant was as­sayed for PtdIns 4-kinase activity as described except for 0.3% Trit0l! X-IOO in the reaction mixture. Under these conditions Ptdlns 3-kinase activity will be in­hibited and only PtdIns 4-kinase activity will be measured.

Isolation oj PtdIns 4-kinase Jrom splenic lymphocytes Splenic lymphocytes were enriched from spleno­

cyt,es on Histopaque gradient as described earlier 6,27.

All adherent cells were removed by incubating the

4 INDIAN 1. BIOCHEM. BIOPHYS. , VOL. 36, FEBRUARY 1999

cells on glass surface at 3rC for 1 hr. The particu­late fraction was prepared from non adherent lym­phocytes as described above. The enzyme activity was solubilized and chromatography was carried out on analytical scale under identical conditions as de­scribed for splenic enzyme.

Immunoprecipitation of Ptdlns 4-kinase from lym­phocyte lysate

About 5 x 106 lymphocytes were lysed in 0.2 ml of lysis buffer and incubated with 20 )lg of antiPtdlns 4-kinase IgG at 4°C for 24 hr. The immunoprecipitate was collected by incubating with Protein A agarose beads precoated with bovine serum albumin at 4°C for I hr. The Protein A agarose beads were washed thrice with phosphate buffered saline and analyzed on SDS-PAGE in parallel lanes as described for renatu­ration assays.

Other techniques Gel electrophoresis was performed on 12% SDS­

PAGE as described by Laemmli 28• Immunoblots

were performed as described29. Protein concentra­

tion s were estimated by dye binding method30 or by bic inchoninic acid method J I

.

Results Purification of Ptdlns 4-kinase from rat spleen

Most of the Ptdlns 4-kinase activity in spleen was found in particulate fraction with very little activity in cytosol. Non ionic detergent, Triton X-IOO was re­quired for solubili zation of the enzyme activity from particulate fraction . More than 90% of the activity was ~lubili zed with 5% Triton X-IOO . A small per­centage o f Ptdlns 4-kinase activity was still associ­ated with detergent insolub le pellet. The solubi lized enzyme was purified to homogeneity by sequential column chromatography on phosphocellulose, DEAE-Sephacel , heparin acrylamide and hydrox.y­apatite columns. SDS-PAGE analysis of the enzyme from different steps of purification showed enrich­ment of a 55 kDa protein (Fig. I) .

The enzyme activity was very labile after solubil i­zation and showed rapid loss of activity during phos­phocellulose column step with varying yields in dif­ferent preparations. However, the remaining enzyme activi ty was stabilized by dialysis against 50% glyc­ero l. Use of two ion-exchange chromatographic steps in tandem resu lted in greater purity of the enzyme (Fig. 2) . Heparin was used as an affinity matrix to

1 2 3 4 5

Fig. I--Protein profile of Ptdlns 4-kinase at different stages of purification on SDS-PAGE. [Ptdlns 4-kinase at different steps of purification was analyzed on 12 % SDS-PAGE and si lver stained . Lane I, phosphocellulose column pool ; lane 2, DEAE-Sephacel column pool; lane 3, heparin acrylamide column pool ; lane 4, hydroxyapatite col umn pool; lane 5, molecular weight markers (from top, bovine albumin : 66 kDa; egg albumin : 45 kDa; glycer­aldehyde 3-phosphate dehydrogenase: 36 kDa, carbonic anhy­drase: 29 kDa and trypsinogen: 24 kDa). The arrowhead indi­cates the position of Ptdlns 4-kinase].

purify Ptdlns 4-kinase from human erythrocytes and A431 cells23

.32

. Fractionation of Ptdlns 4-kinase on heparin acrylamide followed by hydroxyl apatite col­umn resulted in a homogeneous preparation of Ptdlns 4-kinase. The overall purification and yields of the enzyme preparation are summarized in Table I .

Characterization of the purified enzyme Earlier studies have shown that a part of Ptdlns 4-

kinase activity can be renatured from SDS-PAGE23.32

.

Renaturation of the splenic enzyme from SDS-PAGE showed that the act ivity comigrated with 55 kDa protein (Fig. 2) . The purified enzyme exhibited sub­strate specificity for Ptdlns and did not phosphorylate Ptdlns 4P and Ptdlns (4 ,5)P2 Cfable 2). The enzyme showed saturation kinetics with respect to Ptdlns and A TP. The Km for Ptdlns and A TP were 22 )lM and 50 )lM respectively. For Km determination for Ptdlns, a constant molar ratio of Triton X-IOO to PtdIns was maintained to avoid possible interference from deter­gene 3

. The enzyme was sti~ulated by Tri ton X-I OO (Table 2).

Magnesium ions (10-12 mM) were effective diva­lent cation activators of Ptdlns 4-kinase in in vitro assays. Manganese could not substitute for magne­sium ions. The enzyme activity was inhibited by cal­cium ions at non-physiological con.;entrations (Table 2).

Ptdlns kinase activity in membrane fractions were reported to be activated by polycations such as sper­mine and spermidine34

.35

. Spermine stimulated the

VERGHESE el at. : A TYPE II PHOSPHATIDYLINOSITOL 4-KINASE FROM RAT SPLEEN AND LYMPHOCYTES 5

N I I

I

-g I

I W

-

-

CPM X 10-3

I

... o I

... N I

... .. I

I

... CIt

Fig. 2- Renaturation of Ptdlns 4-kinase from SDS-PAGE. [Ptdlns 4-kinase from DEAE-Sephace\ step (10 Ilg) was electrophoresed on SDS-PAGE and the enzyme activity was renatured from the gel as described in Materials and Methods. In an adjacent lane 2 j..lg of the same enzyme was electrophoresed and stained with Coomassie blue. Lane I, molecular weight markers; lane 2, Ptdlns 4-kinase stained with Coomassie blue. Right panel represents renaturation of the enzyme activity from SDS-PAGEj.

Tabl e I- Purification of spleen Ptdlns 4-kinase [Purification from 20 g of rat spleen is represented . All assays were carried out at 25°C in a final volume of 50 j..ll of 50 mM Tris (PH 7.6), 10 mM MgCI2 0.25 mM EGTA, O. I mM vanadate, 20 Ilg/ml Ptdlns, 100 11M ATP (200-300 cpm/picomoles), 0.2% Triton X- I 00,2% glycerol and incubated for 6 minI] One unit of enzyme activity is defined as the

amount of enzyme which catalyzes the formation of I nmole of Ptdlns4P at room temp j.

Step Protein Total activity (mg) (Units)

Particulate fraction 645 37.53 Triton X-I 00 Extract 643 57.75 Phosphocellu lose 14.35 12.75 DEAE- Sephacel 0.87 6.38 Heparin Acrylamide 0.08 2.40 Hydroxyapatite 0.015 0.89

purified enzyme activity by two fold in in vitro as­says. The physiological significance of spennine ef­fect is not clear.

The enzyme activity was inhibited by adenosine Cfable 2). The molecular mass, substrate specificity, stimulation by non-ionic detergent, inhibition by adenosine, and the Kill for Ptdlns and A TP suggest that it is a type II Ptdlns 4-kinase36

.

Immunological studies with antiPtdIns 4-kinase anti­bodies

Affinity puri ficd antiPtdlns 4-kinase antibodies in­hibited the enzyme activity in in vitro assays in a

Sp.activity (Units.mg-I

)

0.057

0.089 0_88 7.81

30.7 59.94

Yield (%)

100 22.14 11 .08 4.17 1.56

Purification (-fold)

1.54 15 .33

135 .02 530.22 1035 .2

concentration dependent fashion (Table 2). Analysis of adult rat tissue particulate fractions on immu­nob lots showed that the antibodies readed with a 55 kDa protein in spleen and brain. No immunoreactiv­ity was observed in liver, lung, adrenal glands and heart particulate fractions (Fig. 3) . However, the en­zyme activity in lung, heart and liver are comparable to spleen, while in adrenal glands and brain the en­zyme activity is at least 2-3 fold higher than spleen (results not shown) . Analysis of spleen particulate fractions from goat, bovine, mice and human periph­eral blood lymphocyte membranes on immunobJots showed that these antibodies cross reacted with pro-

6 INDIAN 1. BIOCHEM. BIOPHYS., VOL. 36, FEBRUA RY 1999

teins in the range of 55-66 kDa (Fig. 4).

Stimulation oj a Ptdlns 4-kinase activity in spleno­cytes by Con A

To understand the role of Ptdlns 4-kinase during T cell activation, rat splenocytes were stimulated with Con A. Ptdlns 4-kinase activity in rat splenocytes was increased two fold in response to Con A stimula­tion. This stimulation of enzyme activity was an early biochemical event and maximal stimulation was observed in 2 min . The enzyme activity returned to basal level within 5-10 min (Fig. 5). The enzyme activity was inhibited by adenosine (Table 3) sug-

Table 2-Effect of activators and inhibitors on Ptdlns <f.:kinase activity [Various agents were assayed for their effect on Ptdlns 4-

kinase activity. The enzyme activity was assayed for 6 mi n at room temp (25 0 C)]

Additions

None Triton X-I 00 (v/v)·

IgG

Ptdlns 4P

Ptdlns (4,5)P2

Concentration

0.05 % · 0.1 % 0.2% 25 Jlg 7511g 10 JlM

300 JlM ImM

40 J1M 100 J1M 40 11M 100 J1M

Ptdlns 4-kinase activity (%)

100 250 250 170 50 30

130 120

18 89 78 93 90

• The initial concentration of Triton X-I 00 was calculated to be 0.02% and this was taken as base value.

1 2 3 4

kDa

66 ~ '\ .....

~ ''''--,",.

45

36 29

gesting that only a type II Ptdlns 4-kinase is activated by Con A. Immunoprecipitation of Ptdlns 4-kinase from splenic lymphocytes with antiPtdlns 4-kinase antibodies, followed by renaturation from SDS­PAGE showed that the enzyme activity migrated within 55 kDa region (resul ts not shown).

kDa

66

45

36 29 24

20

"0 c 0 Q) Q. Q)

0 Q. Q. en

." '0 c: .!! Q -

en to C)

c ~ c .. ! Q) c C'G l! :J "C Q) >

..J <C :c CD ::l

Fig. 1---Tissue specific expression of Ptd lns 4-kinase in adult rat. [Rat tissue particulate fractions from slPleen, lung, adrenal glands, heart, brain and liver were analyzed on Western Blots with anti Ptdlns 4-kinase antibodies. The immunoreactivity was detected using mouse antirabbit IgG monoclonal antibodies coupled to alkaline phosphatase. The colour products were visualised with BCIP-NBT as substrates].

5 6 7 8 9

•

Fig. 4--Expression of Ptdlns 4-kinase among lymphoid tissue from different mammals. [Particulate fractions (200 Jlg protein) from mouse thymus (lane I), mouse spleen (lane 2), human peripheral blood lymphocytes (lane 4), goat spleen (lane 6), rat thymus (lane 8) and rat spleen (lane 9) were analyzed on immunoblot with antiPtdlns 4-kinase antibodies as described in Fig. legend 3. Lanes 3, 5 and 7 are kept as blank lanes] .

VERGHESE et a/. : A TYPE II PHOSPHATIDYLINOSITOL 4-KINASE FROM RAT SPLEEN AND LYMPHOCYTES 7

240~-------------------4

~ '> ~

:4 200

: III c l160

I/) c l120

6--tt--B-----~

80 '-------r---....---¥-i

o 2 4 6

Time (min.)

Fig. 5----{;on A stimulation of splenocytes. [Splenocytes (5x I 06)

were incubated in presence, (e-e ) or absence, (0-0) of Con A for indicated time points at 37°e. At the end of incubation, cells were lysed in lysis buffer. The cell Iysates were centrifuged at 35000 g for 40 min and the solubilized enzyme was assayed for Ptdlns 4-kinase with exogenous Ptdlns in presence of 0.3% Triton X-I 00).

Table 3--Comparison of molecular properties of Ptdlns 4-kinase from spleen and lymphocytes

Properties Spleen Lymphocytes

M, on SDS-PAGE 55 kDa 55 kDa Km for Ptdlns 22 11M 231lM K; for adenosine 18 11M 181lM Detergent stimulation Yes Yes Activity towards Ptdlns 4P, Absent Absent Ptdlns(4,5)P1

NaCI required for elution from: Phosphocellulose column 0.22M 0.22M DEAE Sephacel column 0.08 M 0.08 M

Elution profile on S200 void vol ume void volume column

Comparison of Ptdlns 4-kinases from spleen and splenic lymphocytes

Presence of a 55 kDa Ptdlns 4-kinase in lympho­cytes suggests its similarity to splenic Ptdlns 4-kinase. Partial purification of PtdIns 4-kinase from lymphocytes on ion-exchange chromatography showed similar elution profiles to that of splenic Ptdlns 4-kinase (Table 3). On gel filtration column these enzymes were eluted in void volume. The en­zyme from lymphocyte is also specific for PtdIns and could not use Ptdlns 4P as substrate. The Km for Ptdlns is very simi lar if not identical, to that of sple­nic Ptdlns 4-kinase (Table 3). The enzyme was in­hibited by adenosine and inhibition pattern was iden-

tical to that of splenic Ptdlns 4-kinase (Table 3). The molecular mass, identical chromatographic behav­iour, kinetic parameters, inhibition by adenosine and immunoreactivity suggest that Ptdlns 4-kinases from lymphocytes and spleen are very similar.

Discussion Ptdlns 4-kinase from rat spleen particulate fraction

requires detergent for solubilization suggesting that it is an integral membrane protein. The enzyme activity was purified to apparent homogeneity from Triton X-100 extract of particulate fraction on sequential col­umn chromatography with a specific activity of 60 nmoles/mg/min. The enzyme suffered rapid loss of activity during first chromatography step. A similar observation was made by the earlier workers while puri fying the enzyme from human erythrocytes and A43l cells23.J2. This loss of activity was believed to be an intrinsic property of the enzyme. However, the recent studies suggest that 'the enzyme activity was regulated by protein tyrosine phosphorylation24. These results prompt us to suggest that the loss of activity during first chromatographic step may be either due to dephosphorylation of the enzyme and/or separation of Ptdlns 4-kinases from protein tyrosine kinases rather than inactivation of the enzyme. Ad­dition of orthovanadate in column buffers, signifi ­cantly increased the PtdIns 4-kinase activity during purification of the enzyme from spleen suggesting a role for protein phosphotyrosine dephosphorylation.

The purified PtdIns 4-kinase preparation showed a 55 kDa protein on SDS-PAGE with co-migrating en ­zyme activity. These results indicate that the enzyme is a monomeric protein. However, on Sephacryl S200 column the enzyme activity was recovered in void volume (results not shown) suggesting that it could be a multimeric protein. An alternate explana­tion is that the enzyme may be entrapped in Triton X-100 micelles and is eluting as a Triton X-IOO-PtdIns 4-kinase aggregate32

. The purified enzyme showed the characteristics of a type II Ptdlns 4-kinase. In addition to inhibition by adenosine and stimulation by detergent, the most convincing evidence was its in­ability to use PtdIns 4P as a substrate.

Earlier studies have reported a type II Ptdlns 4-kinase activity in numerous membrane structures in different tissues37

.4o . It is not known whether same or

different isoforms of enzyme are present in various tissues. Analysis of adult rat tissue particulate frac­tions on immunoblots showed restricted immunore-

8 INDIAN 1. BIOCHEM. BIOPHYS., VOL. 36, FEBRUARY 1999

activity among PtdIns 4-kinases. Absence of immu­nocrossreactivity in i-iver, heart, adrenal glands, and lung tissue indicates heterogeneity in primary struc­ture of PtdIns 4-kinases in different tissues. These results:sypport the hypothesis that PtdIns 4-kinases are a family of related enzymes expressed in a tissue specific manner. To the best of our knowledge it is the first report of tissue distribution of a 55 kDa PtdIns 4-kinase in mammalian cells. Molecular cloning of type II PtdIns 4-kinase genes from differ­ent tissues can suggest the origin and function of this heterogeneity. In contrast to type II PtdIns 4-kinases, northern blot analysis of type III PtdIns 4-kinase mRNA transcripts in adult rat tissues showed that they are ubiquitously present. These enzymes are speculated to have a role in vesicular trafficking rather than in signal transduction process4

1.4

2.

The conserved immunocrossreactivity of PtdIns 4-kinase among lymphoid tissue from mouse to human suggests a fundamental role for PtdIns 4-kinase in these tissues. The role of PtdIns 4-kinases in these ti ssues were addressed by stimulating splenocytes with Con A. Con A is a known mitogen to T cells and its activity towards T cells is potentiated by macrophages43

.44, A transient increase in type II PtdIns 4-kinase activity in Con A stimulated splenic cells suggests that the enzyme is part of T cell early signal transduction machinery and its activity is stringently regulated. This increase in enzyme activ­ity may not be due to removal of product inhibition (Table 2). An alternate explanation is that it may be regulated by phosphorylation45

.46.

Characterization of PtdIns 4-kinase from particu­late fraction of highly enriched lymphocytes showed identical molecular properties to that of splenic en­zyme. The immunoreactivity of the enzyme in lym­phocytes and thymus suggests that T cells express this enzyme. This suggestion does not exclude the possibility that this enzyme is also present in other cell types. The identical biochemical properties and immunoreactivity strongly suggest that splenic PtdIns 4-kinase can be a paradigm to understand regulation of PtdIns 4-kinase from lymphocytes.

Acknowledgement This work was supported by Board of Research in

Nuclear Sciences 4/7/93-GI736 and Department of Science and Technology, Govt. of India SP/SO/D-76/93 . A Z Fernandis is the recipient of CSIR-Senior Research Fellowship.

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