Inositol lipid phosphorylation in the cell nucleus

INOSITOLLIPID PHOSPHORYLATION THE CELL NUCLEUS

IN

S. CAPITANI*, L. COCCOt, N. M. MARALDI~t, G. MAZZOTI'I§, O. BARNABEI¶ and F. A. MANZOLI§ Institutes of Human Anatomy, Universities of Ferrara*, Chietit and Bologna§, Italy Institute of Cytomorphology, CNR, c/o Inst. Rizzoli, Bologna, Italy~ Department of Biology, University of Bologna, Italy¶ Istituto Superiore di Sanita', Roma, Italy§ INTRODUCTION

Cell signalling mediated by inositol lipids has recently been implicated in the response of the cell to a variety of stimuli generated by hormones, growth factors and neurotransmitters. A key step in this signalling pathway is the receptor-mediated breakdown of plasma membrane PIP 2 which generates IP 3 and DG, intracellular second messengers mobilizing Ca 2+ from cytoplasmic stores and activating PKC (EC 2.7.1.37), respectively (1, 2). A number of evidences indicate that inositol lipids and PKC can be found in other cell compartments, such as the nucleus (3--9), arguing against the restriction of cell signalling exclusively to the plasma membrane. These findings face the question of how signals proceed to the interior of the cell, and can provide a tentative answer to the problem of signal transmission to the nucleus, where key regulatory events take place for modulating genome activity. PKC has been found in nuclei of a number of cell types, even though conflicting results have been reported concerning its molecular weight and requirement for regulatory cofactors (4, 10-14). The lack of precise subcellular localization of PKC and full agreement on its molecular size reflect the complexity of the PKC system, which displays a number of isozymic forms and high sensitivity to proteolytic cleavage. Nevertheless, it seems well established that the enzyme translocates to the nuclear membrane following differentiating stimuli (8, 9), and part of it is Abbreviations used: PIP2, phosphatidylinositol-4,5-bisphosphate; IP3, inositol trisphosphate; DG, diacylglycerol; PKC, protein kinase C; PIP, phosphatidylinositol4-phosphate; PA, phosphatidic acid; PS, phosphatidylserine; PI, phosphatidylinositol; PI-TP, phosphatidylinositol transfer protein; PMSF, phenylmethylsulfonylfluoride; DTI', dithiothreitol; LS, low salt buffer; HS, high salt buffer; PC, phosphatidylcholine; SDS, sodium dodecyl sulfate; [3ME, ~-mercaptoethanol. 399

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retained by the nucleus and subnuclear fractions after removal of the nuclear membrane (5). Concomitantly, it has been reported that inositol lipid turnover takes place in the nucleus, following initial observations indicating that while synthesis of PI occurs essentially in the endoplasmic reticulum, its phosphorylation is more widely distributed throughout the cell, and is also accomplished in the nuclear envelope, which is able to incorporate 32p into PIP, PIP 2 and PA (15). More recent data indicate that isolated nuclei synthesize the same lipids in membrane-free systems, and that the synthesis of polyphosphoinositides is dependent on proliferative and/or differentiating events involving the cell (3, 5, 16, 17). Thus, it is conceivable that in the nucleus at least part of the inositol lipid-PKC signalling system is operative with a metabolic pathway similar to that described at the plasma membrane and cytoplasmic levels. If so, one crucial point concerns the availability in the nucleus of lipid cofactors and Ca 2÷. Ca 2+ may either freely exchange between nucleus and cytoplasm, or can be independently modulated in the nuclear compartment (18, 19). In addition, Ca 2÷ stores could exist in the nucleus from where IP 3 might exert its mobilization effect. Of the lipids required to optimize PKC activity, PS, which belongs to the pool of endogenous nuclear lipids (20), can be synthesized on the nuclear membrane (21). On the contrary, since PI synthesis does not occur in the nucleus, PI phosphorylation and related second messenger production at the nuclear level may indeed depend on transport systems capable of redistributing PI from the endoplasmic reticulum sites of synthesis. It is known that specific carrier proteins contribute to the intracellular lipid traffic, and for inositol lipid metabolism PI-TP (22, 23) should be considered to maintain appropriate levels of PI in the nucleus as well. Owing to previous findings indicating that membrane-depleted nuclei can synthesize in vitro polyphosphoinositides (3, 5, 16, 17), we describe here a fractionation study of rat liver nuclei devised to determine the subnuclear distribution of inositol lipid metabolism, with particular attention to nuclear matrix and lamina. We also report an analysis of the effect of PI-TP on the uptake of PI by isolated rat liver nuclei, and of the processing of exogenous PI by nucleus-associated enzymes. MATERIALS

AND METHODS

Materials. Myo-[2-3H]inositol (98 Ci/mmol), L-3-phosphatidyl[2-aH]inositol (16.6 Ci/mmol), tritium labelled inositol phosphates and [32p]ATP (5000 Ci/mmol) were obtained from Amersham, U.K. PC, PI, PIP and PIP 2 were purchased from Sigma, St. Louis, MO. ENaHANCE spray was

NUCLEARINOSITOLLIPIDS

401

obtained from NEN Research Products, Boston, MA, and Dowex AG 1 x 8 (100-200 mesh) from Bio Rad Laboratories, Richmond, CA. Silica gel plates were obtained from Merck, Darmstadt, and X-OMAT S Film from Kodak, France. All other reagents were analytical grade.

Isolation of rat liver nuclei and subnuclear fractions. Nuclei were purified from a liver homogenate of adult male Sprague-Dawley rats (250-300 g) as previously described (4). Membrane-depleted nuclei were obtained by adding 0.2% Triton X-100 to the low and high density sucrose buffers employed for liver homogenization and density centrifugation. After the final wash, the nuclear pellet was resuspended in 0.25 M sucrose, 5 mM MgCI2, 0.5 mM D'I'T, 0.5 mM PMSF, 10 mM Tris-HCl, pH 7.4, at a concentration of 2 mg/ml DNA and, until use, stored at -85°C. Extraction of nuclear matrix involved the following steps: endogenous digestion of fresh nuclei at 37°C for 45 min, two extractions in 0.2 mM MgCI2, 1 mM PMSF, 10 mM Tris-HC1, pH 7.4 (LS), two extractions in 2 M NaCI, 0.2 mM MgCI2, 1 mM PMSF, 10 mM Tris-HCl, pH 7.4 (HS), one wash in LS containing 0.4% Triton X-100. The nuclear lamina was purified with the same procedure employed for the nuclear matrix except that all buffers contained 5 mM DTF and the isolated nuclei were digested with DNase I (14 units/mg DNA) and RNase A (80/~g/mg DNA) for 30 min in ice (24). Aliquots of pre-detergent lamina w e r e saved and the nuclear lamina was obtained with one final extraction in LS containing 0.4% Triton X-100. Criteria of nuclear purity. Nuclear preparations were checked for purity by electron microscopy and enzymological analysis. For ultrastruetural analysis, nuclear samples were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, for30 min, postfixed in 1% osmium tetroxide and embedded in plastic resin. Thin sections were stained with uranyl acetate and lead citrate, and observed with a Philips CM 10 electron microscope. To assess the levels of contamination by plasma membrane and microsomes, the nuclear fraction was assayed for 5'-nucleotidase and glucose-6-phosphatase activity, as previously described (4). Preparation of [3H]PI labelled rat liver microsomes. The microsomal fraction from rat liver was labelled with [3H]PI by incubation with 0.01 mCi/mg protein of [3H]myo-inositol (98 Ci/mmol) (25, 26). Prior to use, the [3H]PI labelled microsomes were sonicated up to optical clarity. Preparation of lipid vesicles. Vesicles were prepared by suspending [3H]PI (sp. act. 16.6 Ci/mmol) either pure or mixed with different amounts of egg

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PC, in 0.25 M sucrose, 10 mM Tris-HCl pH 7.4, followed by sonication (3 bursts of 30 see each in ice bath and under a stream of N2).

Purification ofPI-TP. PI-TP was purified from bovine brain as previously described (27) and stored in 50% glycerol at -200C. Incorporation of exogenous PL Unless otherwise stated, nuclei (0.8 mg DNA/ml) were incubated, with either rat liver microsomes or vesicles containing [3H]PI, in the absence or presence of PI-TP (4/.~g protein/ml) in 0.25 M sucrose, 10 mM Tris-HCl pH 7.4, 2 mM MgCI2, 0.45% NaCI, 3.5% glycerol, 0.2 mM DTI" and 0.2 mM PMSF. Incubations were at 37°C for the indicated times, and the vol were scaled up to ensure that the proper number of aliquots could be analyzed. At the end of incubation the nuclei were sedimented at 700 g for 10 min at 40C and washed twice in the same buffer to remove unincorporated label. After solubilization in 10% SDS, radioactivity in the nuclear pellet was determined by liquid scintillation spectrometry. Phosphorylation of exogenous PI. Nuclei were labelled with [3H]PI by incubation with vesicles consisting of [3H]PI-PC (1 : 99 mol%) for 10 min at 37°C as described above. After sedimentation and washing, the nuclear pellet was resuspended in the same vol of phosphorylation buffer (0.25 M sucrose, 15 m_M MgC12, 10 mM 13ME, 1 mM vanadate, 1 mM ATP, 0.45% NaCI, 0.2 mM PMSF, 10 mM Tris-HCl, pH 7.4). At the indicated times, aliquots (0.5 ml) of the nuclear suspension were taken and the nuclei sedimented at 700 g for 10 min. The supernatant was saved for analysis of water soluble compounds. The pellet was washed twice in the same buffer and extracted for inositol lipids as described by Shaikh and Palmer (28). Aliquots were taken for determination of D N A content. Characterization of inositides. The nuclear lipid extract was dissolved in ehloroforrrdmethanol/H20 (75 : 25 : 2, by vol) and analyzed on oxalateimpregnated thin layer plates. The running solvent was chloroform/methanol/NI-I4OH 3.43 M (45 : 35 : 10, by vol). The labelled spots were identified by fluorography after spraying the plates with ENaHANCE. Autoradiography was on Kodak X-OMAT S film (exposure time of 3--4 weeks). After comparison with authentic standards, the spots were scraped off the plates and counted by liquid scintillation spectrometry. All values were corr0cted for background levels, by counting blank areas equivalent to each spot in each single lane. Characterization of inositol phosphates. Nuclear supernatants were loaded on a Dowex AG-1 x 8 column (formate form) and eluted

NUCLEAR INOSITOL LIPIDS

403

according to Berridge et al. (29) with slight modifications. Briefly, stepwise elution included the following buffers: double distilled water (A), 5 mM Na tetraborate and 60 mM Na formate (B), followed by increasing concentrations of ammonium formate: 0.18 M (C), 0.4 M (D), 0.9 M (E) in 0.1 M formic acid. Elution peaks of inositol phosphates were identified by comparison with authentic tritiated standards. Endogenous lipid phosphorylation. Liver homogenate, whole nuclei and subnuclear fractions (200 pg protein) were preincubated for 10 min at 30°C in the presence or absence of 0.05% Triton X-100, and of 100 pM PI or PIP, and then incubated for 5 min with 20 mM MgCl,, 10 mM BME, lm~ vanadate, 100 pM ATP and 1 &i [3*P]ATP (5000 Ci/mmol), in a final vol of 200 ~1. The reaction was terminated by addition of 4 ml of chloroform/methanol/HCl (200 : 100 : 0.75, by vol) and the phosphoinositides were extracted and analyzed on TLC plates as described above. Otherprocedures. Protein content was determined as described by Lowry (30) and DNA was assayed according to Burton (31). RESULTS

Assessment of Nuclear Purity and Characterizationof Nuclear Subfractions Morphological and biochemical criteria were used to assess the purity of nuclei prepared from rat liver. As previously reported (4), negligible amounts of cytoplasmic marker enzymes were found. In particular, the contamination by plasma membrane, which displays maximum cell levels of enzymes of the inositol lipid cycle, was accurately evaluated by assaying the 5’nucleotidase activity. The specific activity, expressed as nmol of inorganic phosphate released/min/mg protein was: 447.0 for total homogenate, 49.8 for microsomes, 18.9 for intact nuclei and 12.3 for membrane depleted nuclei. These values were comparable to published data on rat liver nuclei isolated with similar procedures (7, 21, 32). Accordingly, ultrastructural analysis of thin sections embedded in plastic indicated a virtual absence of cytoplasmic membranes and organelles. Whole nuclei showed the nuclear membrane, with the presence of large blebs, while the inner structure appeared”wel1 preserved. Nuclei purified in the presence of Triton X-100 lacked the nuclear membrane (Fig. 1). In addition, nuclei isolated with the same procedure were shown to be free of redistributed cytoplasmic lipids labelled with tritiated glycerol and inositol, confirming that extranuclear membranes do not adventitiously stick to the nuclei during homogenization and subsequent purification

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(3). The protein composition of the nuclear fractions was analyzed on SDS-polyacrylamide gels. Figure 2 shows that many proteins were progressively lost from nuclei to matrix and lamina. After the high salt extraction, histones were virtually removed, while Lamins A, B and C were among the major components of the nuclear matrix, and largely predominated in lamina. Endogenous

Lipid Phosphorylation

The typical phosphorylation profiles of rat liver nuclei show three main products, which, according to co-migration with authentic internal standards, were identified as PIP, PIP, and PA. Scintillation counting of the spots (Table 1) indicates that the basal recovery of PIP and PIP, was larger in membrane-depleted nuclei than in whole nuclei. These incorporation levels, compared to total homogenate, were higher than predictable considering that most of the lipid kinase activities are located in the plasma membrane (15). The specific activity of PI-kinase expressed as cpm 32P incorporated/mg protein increased with purification, reaching maximum levels in pre-detergent matrix and lamina, and the same was true for PIP-kinase, except that it was reduced in pre-detergent lamina. In the corresponding membrane-free structures, nuclear matrix and lamina, the tendency of PI-kinase activity to increase was confirmed, while PIP-kinase was not detectable. DG-kinase was shown to be predominantly membrane-bound, since it was reduced in membrane-depleted nuclei in comparison to whole nuclei, and virtually absent in matrix and lamina after removal of the membrane. PI-kinase and PIP-kinase were activated with Triton X-100 in membranecontaining structures, except for the pre-detergent lamina, while DG-kinase was inhibited, in agreement with the role of non-ionic detergents in facilitating or inhibiting expression of maximum activity (15, 33). When PI or PIP was included in the assay, the recovery of PIP and PIP, was respectively increased, with maximum effect in membrane-depleted structures. In the presence of exogenous PIP and detergent the recovery of labelled PIP increased in membrane-depleted nuclei, matrix and lamina, suggesting a phosphomonoesteratic breakdown of PIP to PI, which could be then converted to PIP incorporating labelled ATP. Uptake of Exogenous

PI by Nuclei

Incorporation of exogenous PI by nuclei was determined by using two lipid donors, constituted by rat liver microsomes labelled in vitro with [sH]inositol, and artificial liposomes containing [3H]PI. In both cases a spontaneous uptake of labelled PI was observed. By including PI-TP, the

-PI

- Lyso PI -PIP

A

B

FIG. 5. Autoradiography of a thin layer chromatogram showing the labelled lipids extracted from nuclei incubated with [3H]PI-PC (1 : 99 mol%) either in the absence (A) or presence (B) of PI-TP. Incubation procedure was as described in Figure 4, with 10 ~g of PI-TP and a phosphorylation time of 20 min. (From 43.)

401 7025 635 774 22107 1893 199 517 25724 829 1342 -

PA PIP PIP* PA PIP PIP* PA PIP PIP* PA PIP PIP*

LS fraction

HS fraction

Nuclear matrix

Pre-detergent lamina

%

1.9 95.0 3.1 100.0 -

3.1 89.2 7.7 100.0 -

5.0 87.1 7.9

1.0 94.6 4.4

12.3 77.7 9.9

5:3

2.98

7676 158 1166 -

623 84989 10621 995 -

311 42005 919

890 105

10711 147

214

11585 361

Triton

98.0 2.0 100.0 -

0.6 88.3 11.1 100.0 -

0.7 97.2 2.1

2.9 95.3 1.8 98.6 1.4 89.4 10.6

%

216 88002 14125 23420 274 7688 294 13410 256

202 22748 672

271 15365 932

66 10441 118

340 10274 259

PI Triton

96; 3.7 98.1 1.9

0.2 86.0 13.8 98.8 1.2

0.9 96.3 2.8

1.6 92.8 5.6

0.6 98.3 1.1

3.1 94.5 2.4

%

110 9016 302 7272 340

721 57662 5394 2411 2000

617 42896 4263

117 5848 2133

129 10494 2264

415 10301 316

PIP Triton

9::: 3.2 95.5 4.5

1.1 90.4 8.5 54.7 45.3

1.3 89.8 8.9

1.4 72.2 26.4

1.0 81.4 17.6

3.7 93.4 2.9

%

Data expressed as cpm/mg of protein, and as internal percentages. Additions: 0.05% T&on, 100 @f PI and PIP. Mean values of 5 exneriments; (SD below 12%). (Renroduced and modified from 48.1

PA PIP PIP*

83 8050 376

PA PIP * PIP,

Membranedepleted nuclei

Lamina

249 1572 201

PA PIP PIP*

456 10914 638

Whole nuclei

Total homogenate

PA PIP PIP,

None

Additions

TABLE 1. IN VITRO SYNTHESIS OF PA, PIP AND PIP, BY RAT LIVER NUCLEI AND SUBNUCLEAR FRACTIONS

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uptake was greatly increased (Fig. 3), with similar features regardless of the source of labelled lipid. In the case of microsomes, after 30 min of incubation the PI-TP mediated uptake reached about 45% of the original lipid input, well above the 15% incorporated spontaneously. Since the lipid composition of the liposomal membrane could be easily modulated, the most suitable type of liposomes was selected for obtaining a lipid transfer as high as possible also compared to the spontaneous uptake. The vesicles were prepared with different molar ratios of [sH]PI and PC, and used either in the presence or absence of PI-TP. Despite the different amounts of PC included in the liposomes, the incorporation of PI into the nuclei was quite similar. Nevertheless, liposomes containing 99 mol % PC were used for most of the experiments, since under these conditions the spontaneous uptake of [sHIPI was distinctly reduced (Fig. 4). Comparison between whole and membrane-depleted nuclei indicated that the nuclear membrane was not essential for obtaining a spontaneous and PI-TP-mediated incorporation of PI (Table 2). In fact, the spontaneous incorporation proceeded more effectively in membrane-free nuclei, whilst the PI-TP-mediated incorporation was similar for both types of nuclei. Independent of whether intact or membrane-depleted nuclei were used as acceptors, Triton X-100 removed most of the [sHIPI incorporated (Table 3) regardless of the spontaneous or PI-TP mediated way of uptake. However,

FIG. 3. Time-dependent uptake of [SHIP1 by rat liver nuclei. Nuclei (1.6 mg DNA) were incubated with [3H]PI labelled microsomes (450 pg of protein, 1.8 x 106 dpm/mg protein) in the absence (0-O) or presence (+-+) of 8 pg of PI-TP. Aliquots containing 100 wg of DNA were taken at the indicated times and washed in incubation buffer. (From 43.)

407

NUCLEAR INOSITOL LIPIDS

FIG. 4. absence 30 pmol with the

Nuclear uptake of [sH]PI from liposomes of different lipid composition in the or presence of PI-TP. Nuclei (0.8 mg DNA) were incubated for 10 min with of [sHIPI ([sHIPI : nuclei ratio of 37.5 pmopmg DNA), either pure or mixed indicated lipids. (A) pure [sHIPI; (B) [‘HIPI-PC (10 : 90 mol%); (C) [sHIPI-PC (1 : 99 mol%). (From 43.)

TABLE 2. UPTAKE OF [3H]PI FROM LIPOSOMES COMPOSED OF [~H]PI-PC (1 :99MoL%) INmE ABSENCE 0~ PRESENCE 0~ PI-TP Condition

Whole nuclei

Control PI-TP

64,723 158,513

Membrane-depleted nuclei (+145%)

101,188 155,489

(+53%)

Incubation was for 30 min with a lipid : nuclei ratio of 37.5 pmol of [sH] PYmg DNA. Data are expressed as dpm/mg nuclear DNA, and the stimulation by PI-TP is given between the brackets (from 43).

the amount of label remaining with intact nuclei was about half of that remaining with membrane-depleted nuclei. Processing of PI by Nuclei

nuclei could As reported above, whole and membrane-deprived phosphorylate endogenous PI to PIP and PIP,. This capability was affected by substrate availability and detergent, and depended to some textent on the presence of nuclear membrane (Table 1).

15,368

3 10

20

3544

1829 4048 3992

13,139

15,157 17,358

20

1683 2270

9087 12,569

Triton washed

3 10

Uptake

Control

76.9

73.3 77.0

86.1

81.5 81.9

% loss

33,766

24,564 26,391

33,002

21,311 32,846

Uptake

TO DETERGENT OF LABELLED PI INCORPORATED SPONTANEOUSLY OR IN THE PRESENCE OF PI-TP

6007

3717 4204

1988

1810 2102

Triton washed

PI-TP

82.2

84.8 84.1

94.0

91.5 93.6

% loss

INTO NUCLEI EITHER

After incubation for the indicated times, the nuclear pellet, washed free of unincorporated [sHIPI, was resuspended in 0.3% Triton X-100, sedimented and then washed in detergent-free buffer. Data are given as dpm/mg DNA and represent mean values of 3 experiments (SD < 9%). (A) = whole nuclei; (B) = membrane depleted nuclei (reproduced and partly modified from 43).

(B)

(A)

Incubation time

TABLE 3. RESISTANCE

z .!! B g

B z

v)

409

NUCLEAR INOSITOL LIPIDS

The fate of exogenously administered PI has indeed been studied by following its phosphorylation and hydrolytic cleavage. For these experiments the use of microsomes as [3H]PI donors was not allowed, since significant levels of [3H]PIP and [sH]PIP, were produced by incubation both in the absence of nuclei and in the presence. of heat-inactivated nuclei. A typical fluorography of the labelled lipids extracted from nuclei and analyzed by thin layer chromatography is reported in Figure 5. Three spots

la 20 Tim0 ol incubation (mrrr)

24

22

FIG. 6. Phosphorylation of [sHIPI by rat liver nuclei. Intact nuclei were labelled with vesicles consisting of [sHIPI-PC (1 : 99 mol%) for 10 min in the absence (0-O) or presence (+-+) of PI-TP, respectively. Phosphorylation was then allowed for the indicated times and the recovery of [sH]PI (panel A) and of [sH] PIP (panel B) was determined. Mean values of 3 separate experiments (SD c 11%). (From 43.)

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were present, corresponding to PI, Lyso PI and PIP, while PIP, was not detectable. A time-course analysis of PI utilization indicates that, in nuclei incubated in the presence of PI-TP, the higher level of PI accumulated rapidly disappeared during the first min of incubation with ATP, while a much lesser conversion of PI was observed in control nuclei. Time dependent formation of PIP was barely detectable in control nuclei, whilst it consistently increased with time in nuclei loaded with PI-TP (Fig. 6).

A

2.6,

I

B

1

C

I

D

I

E

2.2 2 1.6 1.6 1.4

i

GPI

I

I

PI -TP

Nuclei II

1.

1. 1. 1. 0. 0. 0. 0. 0

20 Fraction

40 number

60

FIG. 7. Recovery of water-soluble inositol-containing components released from nuclei. Nuclei were labelled for 10 min with vesicles consisting of [sHIPI-PC (1 : 99 mol%) in the absence (Control, Nuclei I) or presence of transfer protein (PI-TP, Nuclei II). After 30 min of phosphorylation the nuclei were sedimented and the supematants analyzed as described in Materials and Methods. I = free inositol, GPI = glycerophosphoinositol, IP = inositolphosphate, IPz= inositolbisphosphate, IPs = inositoltrisphosphate. (From 43.)

NUCLEAR INOSITOL LIPIDS

411

In order to establish whether water-soluble compounds were formed during incubation in the phosphorylation buffer, the nuclear supematants were analyzed by column chromatography, on Dowex AG l-X8 (Fig. 7). Glycerolphosphoinositol, inositolphosphate and inositol were clearly detectable in both loading conditions, suggesting a degradation of [3H]PI. Moreover, in the supematant of nuclei loaded with PI-TP a distinct peak of IP, was observed. This is compatible with a conversion of [sH]PIP to [3H]PIP, followed by degradation by phospholipase C. DISCUSSION The metabolic significance of nuclear lipids has long been investigated, even though clearcut definition of their roles in the control of nuclear functions awaits further work (34-36). A number of suggestions for the involvement of nuclear phospholipids in replication and transcription have accumulated, and changes of the nuclear lipid composition related to cell growth, differentiation and neoplastic transformation implicate these molecules in key regulatory events occurring at the nuclear level (37-41). Compared to other lipid classes, acidic phospholipids show a high relative abundance in nuclei with respect to the whole cell, suggesting that the nucleus is a cell compartment where anionic lipids like PS, PA and PI can exert their action, probably not related to mere structural roles. Interestingly, anionic phospholipids take part in a multifunctional cell signalling pathway which involves PI and its phosphorylation products, PIP and PIP,, as responsible for the production of second messengers, and PS as a lipid factor required for optimizing the activity of PKC (1, 2). It is indeed conceivable that nuclear PI and PS synergically participate in a sort of nuclear signalling system devised to modulate PKC similarly to what has been fully demonstrated in the cytoplasm. However, even though recent data have reported that nuclear inositol lipid turnover affects the PKC-dependent hyperphosphorylation of specific nuclear proteins (42), a comprehensive view of the relationship between phospholipid metabolism and protein kinase activity in the nucleus is lacking. Our present data have been obtained in the attempt of better characterizing the inositol lipid turnover in nuclei, considering both subnuclear localization of lipid kinases and related substrates and a PI-TP mediated transfer of PI to the nucleus. The results obtained indicate that rat liver total homogenate and intact nuclei do not respond properly to the addition of exogenous substrates, and this probably reflects a sufficient concentration of endogenous, substrates. In nuclear matrix and lamina, on the contrary, the lipid kinase activities clearly depend on the presence of the membrane and on the additions. In particular, the effect of exogenous substrates indicates that lipid kinases are also present in membrane depleted

412

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structures, suggesting that the nuclear membrane behaves as a source of components of the inositol lipid cycle, but its importance is higher for substrates than for enzymes. Lipid kinases are resistant to digestion, extensive salt extractions and detergent, supporting the contention that they are actual nuclear components tightly bound to nuclear structures. Under control conditions, the nuclear matrix-membrane complex (HS fraction) and the lamina-membrane complex (pre-detergent lamina) show by far the highest PI-kinase and PIP-kinase specific activity. This is consistent with the hypothesis that the periphery of the nucleus is a crucial site where key regulatory events take place and represents an important site of processing and exchange of information with the cytoplasm. Also phospholipid traffic occurs at the nucleus-cytoplasmic boundary utilizing transfer systems, especially for the lipids, like PI, which are synthesized exclusively at the cytoplasmic level (21). Lipid exchange and lateral diffusion can occur in the membrane continuity between nuclear envelope and endoplasmic reticulum. However, an important role can be played by specific transfer proteins, which have been reported to regulate phospholipid levels in other cell compartments (43). The results obtained indicate that isolated nuclei can accept phospholipids in vitro from different sources, according to previous data demonstrating that the interaction of lipid vesicles and nuclei results in lipid uptake which affects a variety of morphological and biochemical nuclear changes (34,44). The uptake of PI is greatly increased by PI-TP, and the incorporated lipid is utilized by the nucleus for phosphorylation and breakdown, indicating that in nuclei the endogenous PI pool available for lipid kinases and phosphoesterases is suboptimal. In this respect, the role of PI transfer mechanisms based on a specific carrier protein is of great importance, and ascribes to PI-TP modulatory properties based on the regulation of PI availability in the nucleus. Our data might correlate with the reported presence or translocation of PKC in nuclei and subnuclear fractions (4, g-10, 13, 14), and the PKC-directed phosphorylation of Lamin B in lymphocytes and HL 60 cells (45, 46). Additional modulatory events are likely to also take place in the inner nuclear compartment, reliant equally on the presence and accumulation of inositol-derived molecules (16, 47), phosphoinositide kinase (3,48) and PKC (4,6,7, 11). Taken together, these data support the hypothesis that a complete PI cycle is operative in the nucleus, similar to that present at the plasma membrane level (49). They also suggest an integrated signalling pathway which relies on PI redistribution in the cell towards different compartments, including the nucleus, followed by its phosphorylation to PIP and PIP, from which second messengers controlling Ca 2+ levels and PKC activity are generated.

NUCLEAR INOSITOL LIPIDS

413

SUMMARY

Inositol lipid metabolism has been analyzed in isolated rat liver nuclei and nuclear fractions, in order to determine the subcellular distribution of the sites of lipid phosphorylation and breakdown. Lipid kinases and phosphoesterases appear to be tightly bound nuclear components, and can utilize exogenous substrates administered to membrane-depleted structures. The possible involvement of specific carrier protein in the nuclear metabolism of inositol lipids has also been analysed by studying the uptake and processing of phosphatidylinositol transferred to the isolated nuclei by phosphatidylinositol transfer protein (PI-TP). PI-TP greatly stimulates the incorporation of phosphatidylinositol from microsomal membranes and synthetic vesicles, and the lipid taken up is available for phosphorylation and breakdown by enzymes associated to the nucleus. The results obtained support previous data on the metabolic and structural role of nuclear lipids, and suggest that the cell nucleus is a site of lipid phosphorylation, not necessarily involving enzymes and substrates located on the nuclear membrane. They also indicate that an integrated signalling pathway can exist at the nuclear level utilizing inositol lipid-derived second messengers and PKC to control replication and transcription.

ACKNOWLEDGEMENTS

The authors wish to express their appreciation to Bernd Helms and Karel W. A. Wirtz of the Center for Biomembranes and Lipid Enzymology, State University of Utrecht, for generous support and helpful discussion. They are also indebted to Valeria Bertagnolo, Maurizio Previati, Meri Mazzoni and Cinzia Carini for conceptual and skilled technical assistance in performing of the experiments and preparation of the manuscript. This research supported by grants from the Italian National Research Council (CNR 89.04123.04, 89.02470.04, PFIG and PFBBS) and Minister0 della Istruzione (40% and 60%). REFERENCES 1. M. J. BERRIDGE, Inositol trisphosphate, a novel second messenger in cellular signal transduction, Nature 312, 315-321 (1984). 2. Y. NISHIZUKA. The molecular heterogeneity of protein kinase C and its implications for cellular regulation, Nature 334, 66i-665 -(1988). 3. L. COCCO. R. S. GILMOUR. A. OGNIBENE. A. J. LETCHER. F. A. MANZOLI and R. F. ‘IRVINE, Synthesis of polyphosphbinositides in nuclei of Friend cell. Evidence for polyphosphoinositide metabolism inside the nucleus which changes with cell differentiation, Biochem. J. 248, 765-770 (1987). 4. S. CAPITANI, P. R. GIRARD, G. J. MAZZEI, J. F. KUO, R. BEREZNEY and F. A. MANZOLI, Immunochemical characterization of protein kinase C in rat liver nuclei and subnuclear fractions, Biochem. Biophys. Res. Commun. 142, 367-375 (1987).

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6.

S. CAPITANI, et al.

L. COCCO, A. M. MARTELLI, R. S. GILMOUR, A. OGNIBENE, F. A. MANZOLI and R. F. IRVINE, Rapid changes in phospholipid metabolism within nuclei of Swiss 3T3 cell induced by treatment of the cells with insulin-like growth factor I, Biochem. Biophys. Res. Commun. 154, 1266-1272 (1988). A. R. BUCKLEY, P. D. CROWE and D. H. RUSSELL, Rapid activation of protein kinase C in isolated rat liver nuclei by prolactin, a known hepatic mitogen, Proc. Nutl. Acad.

7.

8. 9. 10.

11. 12. 13. 14. 15. 16.

17. 18. 19. 20.

Reaul.

21. 22. 23.

Sci. USA 85, 8649-8653

(1988).

A. MASMOUDI, G. LABOURDETI’E, M. MERSEL, F. L. HUANG, K. P. HIJANG, G. VINCENDON and A. N. MALVIYA, Protein kinase C located in rat liver nuclei. Partial puritication and biochemical and immunochemical characterization, J. Biol. Chem. 264 1172~1179 (1988). K. L. LEACH, E. A. POWERS, V. A. RUFF, S. JAKEN and S. KAUFMANN, Type 3 protein kinase C localization to the nuclear envelope of phorbol ester-treated NIH/3T3 cells, J. Cell Biol. 109, 685-695 (1989). T. P. THOMAS, H. S. TALWAR and W. B. ANDERSON, Phorbol ester-mediated association of protein kinase C to the nuclear fraction in NIH 3T3 cells, Cancer Res. 48, 191&1919 (1988). S. AZHAR, J. BUTTE and E. REAVEN, Calcium-activated, phospholipid-dependent kinases from rat liver: subcellular distribution, purification, and characterization of multiple forms, Biochemisrry 26, 7047-7057 (1987). A. M. MARTELLI, R. S. GILMOUR, E. FALCERI, F. A. MANZOLI and L. COCCO, Mitogen-stimulated phosphorylation of nuclear proteins in Swiss 3T3 cells: evidence for a protein kinase C requirement, Exptl. Cell Res. 185,191-202 (1989). P. ROGUE, G. LABOURDE’ITE, A. MASMOUDI, Y. YOSHIDA, F. L. HUANG, K. P. HUANG. J. ZWILLER. G. V. VINCENDON and A. N. MALVIYA, Rat liver nuclei protein kinase C is the isoxyme type II, J. Biol. Chem. 265,4161-4165 (1990). M. M. GARCIA, C. L. WEILL and B. S. BECKMAN, Rapid activation by erythropoietin of protein kinase C in nuclei of erythroid progenitor cells, Biochem. Biophys. Res. Commun. 168,490-497 (1990). M. HAGIWARA, C. UCHIDA, N. USUDA, T. NAGATA and H. HIDAKA, {-related protein kinase C in nuclei of nerve cells, Biochem. Biophys. Res. Commun. 168,161-168(1990). C. D. SMITH and W. W. WELLS, Phosphorylation of rat liver nuclear envelopes, J. Biol. Chem. X18,9368-9373 (1983). S. CAPITANI, M. MAZZONI, V. BERTAGNOLO, M. PREVIATI, A. DADDONA, D. RICCI and F. A. MANZOLI, Nuclear inositol lipids in Friend erythroleukemia cells. Changes related to differentiation induced by hexamethylenebisacetamide, Cell Biol. Znt. Rep., 14,783-795 (1990). A. CATALDI. S. MISCIA. R. LISIO. R. RANA and L. COCCO, Transient shift of diacylglycerol and inositol lipids induced by interferon in Daudi cells. Evidence for a different pattern between nuclei and intact cells, FEBS Lett. 269, 465468 (1990). P. NICOTERA, D. J. McCONKEY, D. P. JONES and S. ORRIENIUS, ATP stimulates Ca++ uptake and increases the free Ca ++ concentration in isolated rat liver nuclei, Proc. -Natl. Acad. Sci. USA 86, 453-457 (1984). R. W. TUCKER and F. S. FAY. Distribution of intracellular free calcium in auiescent BALB/c 3T3 cells stimulated by’platelet-derived growth factor, Eur. J. Cell Viol. 51, 120-127 (1990). N. M. MARALDI, S. CAPITANI, E. CARAMELLI, L. COCCO, A BARNABEI and F. A. MANZOLI, Conformational changes of nuclear chromatin related to phospholipid induced modifications of the template availability, Advan. Enzyme 22. 447-464

(1984).

C.2ELSEMA and D: J. MORRE’, Distribution of phospholipid biosynthetic enzymes amone cell comoonents of rat liver. J. Biol. Chem. 253. 7960-7971 (1978). G. MYHELMKAMP, Phosphatidylinositol transfer protein: structure, catalytic activity and physiological function, Chem. Phys. Lipids 38, 3-16 (1985). P. VAN PARIDON, T. W. J. GADELLA, P. SOMERHARJU and K. W. A. WIRTZ, On the relationship of the dual specificity of the bovine brain phosphatidylinositol

NUCLEAR

INOSITOL LIPIDS

415

transfer protein and membrane phosphatidylinositol levels, Biochim. Biophys. Acfu 903,68-77 (1987). 24. H. C. SMITH, E. PUVION, L. A. BUCHOLTZ and R. J. BEREZNEY, Spatial distribution of DNA loop attachment and replicational sites in the nuclear matrix, .I. Cell Biol. 99, 1794-1802 (1984). 25. H. PAULUS and E. P. KENNEDY, The enzymatic synthesis of inositol mononhosohatide, .I. Biol. Chem. 235. 1303-1311 (19601. 26. C. D: SMITH and W. W. WELLS, Character&ation of a phosphatidylinositol 4-phosphate-specific phosphomonoesterase in rat liver nuclear envelopes, Arch. Biochem. Biophys. 235, 529-530 (1984). 27. P. A. VAN PARIDON, A. J. W. G. VISSER and K. W. A. WIRTZ, Binding of phospholipids to the phosphatidylinositol transfer protein from bovine brain as studied by steady-state and time-resolved fluorescence spectroscopy, Biochim. Biophys. Acta 298, 172-180 (1987). 28. H. A. SHAIKH and F. B. ST. C. PALMER. The composition of the lipid in the develooinpr central and oerinheral nervous system in the chicken. J. Neurochem. 26. 59743

29.

30. 31. 32. 33.

34. 35.

36. 37. 38. 39. 40. 41. 42.

(5976).

*

-

M. J. BERRIDGE, R. M. C. DAWSON, C. P. DOWNES, J. P. HESLOP and R. F. IRVINE, Changes in the levels of inositol phosphate after agonist-dependent hydrolysis of membrane phosphoinositides, B&hem. 1. 212,473-482 (1983). 6. H.-LOWRY, N. J. ROSEBROUGH, A. L. FARR and R. J. RANDALL, Protein measurement with the Folin ohenol reagent. J. Biol. Chem. 193. 265-275 (1951). K. BURTON, A study of ihe conditi&s ‘and mechanism of the diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid, Biochem. J. 62, 315-323 (1956). I. B. ZBARSKY, An enzyme profile of the nuclear envelope, Znt. Review of Cytol. 54, 295-363 (1978). W. W. WELLS, M. A. SEYFRED, C. D. SMITH and M. SAKAI, Measurement of subcellular sites of nolvohosnhoinositide metabolism in isolated rat hepatocytes, _ Methods in Enzymoloiy l%, 9%99 (1987). F. A. MANZOLI. N. M. MARALDI and S. CAPITANI. Effect of ohosoholioids on the control of nuclear’DNA template restriction, pp. 133-156 in The Phwm&ofo&cal Effect of Lipids,Vol. II (J. J. KABARA, ed.), AOCS Publisher, Champaign, IL (1985). P. B. GAHAN, M. P. VIOLA MAGNI and C. F. CAVE, Cytochemical and autoradiographic studies of the localization and turnover of chromatin and nucleolar associated phospholipids in plant and animal tissues, Bas. Appl. Histochem. 31, 343-353 (1987). F. A. MANZOLI, N. M. MARALDI and S. CAPITANI, Minor components of the chromatin and their role in the release of template restriction, pp. 443-486 in Cell Growth (C. NICOLINI, ed.), Plenum Press, New York (1983). H. G. ROSE and J. H. FRENSTER, Composition and metabolism of lipids within repressed and active chromatin of interphase lymphocytes, Biochim. Biophys. Actu 106, 577-591 (1965). F. A. MANZOLI, N. M. MARALDI, L. COCCO, S. CAPITANI and A. FACCHINI, Chromatin phospholipids in normal and chronic lymphocytic leukemia lymphocytes, Cancer Res. 37 8434349 (1977). F. A. MANZOLI, S. CAPITANI, N. M. MARALDI, L. COCCO and 0. BARNABEI, Chromatin lipids and their possible role in gene expression. A study in normal and neoplastic cells, Advan. Enzyme Regul. 17, 175-184 (1979). R. BERENZNEY and D. S. COFFEY, Identification of a nuclear protein matrix, Biochem. Biophys. Res. Commun. 60, 1410-1417 (1974). L. COCCO, N. M. MARALDI, F. A. MANZOLI, R. S. GILMOUR and A. LANG, Phospholipid interaction in rat liver nuclear matrix, Biochem. Biophys. Res. Commun. 96, 890-898 (1980). L. COCCO, S. CAPITANI, A. M. MARTELLI, R. F. IRVINE, R. S. GILMOUR, N. M. MARALDI, 0. BARNABEI and F. A. MANZOLI, Nuclear inositol lipids.

416

43.

44. 45. 46. 47.

48.

49.

S.CAPITANI, et al. Relationship between growth factor induced metabolic changes and protein kinase C activitv, Advan. Enzvme Reaul. 30. 155-172 (1990). s. ciiPmNr, 13.I-~ELMs,-M. MAZZONr, ‘M. +REvIATI, v. BERTAGNOL~, K. W. A. WIRTZ and F. A. MANZOLI, Uptake and phosphorylation of phosphatidvlinositol bv rat liver nuclei. Role of phosohatidvlinositol transfer nrotein. _ biocbn. B?;bphys.A& 1044,193-200 (1990). _ a S. CAPITANI, L. COCCO, N. M. MARALDI, S. PAPA and F. A. MANZOLI, Effect of phospholipids on transcription and ribonucleoprotein processing in isolated nuclei, Advan. Enzyme Regul. 25, 425-438 (1986). A. P. FIELDS, G. R. PETTE and W. S. MAY, Phosphorylation of lamin B at the nuclear membrane bv activated orotein kinase C. J. Biol. Chem. 263. 8253-8260 (1988). P. HORNBECK, K. P. HUANG and W. E. PAUL, Lamin B is phosphorylated in lymphocytes after activation of protein kinase C, Proc. iVatl. Acad. Sci. USA 85, 2279-2283 (19881. 0. TRUBIANI, A. M. MARTELLI, L. MANZOLI, E. SANTAVENERE and L. COCCO, Nuclear lipids in Friend cells. Shifted protile of DAG during erythroid differentiation induced by DMSO, CeN. Biof. Int. Rep. 14(6), 559-566 (1990). S. CAPITANI, V. BERTAGNOLO, M. MAZZONI, P. SANTI, M. PREVIATI, A. ANTONUCCI and F. A. MANZOLI, Lipid phosphoryiation in isolated rat liver nuclei. Synthesis of polyphosphoinositides at subnuclear level, FEBS Lett. 254, 194-198 (1989). F. A. MANZOLI, S. CAPITANI, L. COCCO, N. M. MARALDI, G. MAZZO’I’TI and 0. BARNABEI, Lipid mediated signal transduction in the cell nucleus, Advan. Enzyme Regul. 27, 83-91 (1988).