Phosphatidylinositol 4-kinases

Eur. J. Biochem.253, 3572370 (1998) FEBS1998

Review

Phosphatidylinositol 4-kinases

Thor GEHRMANN and Ludwig M. G. HEILMEYER Jr

Ruhr-Universität Bochum, Institut für Physiologische Chemie, Abteilung für Biochemie Supramolekularer Systeme, Bochum, Germany

Received10 October/12 December1997 2 EJB 971439/0

Polyphosphoinositides are involved in many signal transduction pathways in eukaryotic cells. Thefirst committed step is catalysed by phosphatidylinositol 4-kinase leading to the formation ofphosphatidylinositol 4-phosphate. In the last four years, ten cDNA molecules have been cloned whichcode isoforms of phosphatidylinositol 4-kinase; some of which are highly related. Characteristically, theycontain a C-terminal catalytic domain which is similar to that of (poly)phosphoinositide 3-kinases and tothat of more distantly related lipid/protein kinases. Alignment has characterised cDNAs fromChaenorab-ditis, Dictyosteliumand Schizostaphyloccus pombeas those of phosphatidylinositol 4-kinases also. Allthese lipid kinases are related to the superfamily of protein kinases. Several amino acids are highlyconserved in catalytic domains of lipid and protein kinases. Employing the catalytic subunit of the cAMP-dependent protein kinase as template, these residues can be assigned functionally. On the basis of thealignment, a phylogenetic tree of the superfamily of phosphatidylinositol kinases has been constructed.Three families, the phosphatidylinositol 4-kinases, phosphoinositide 3-kinases, and the phosphatidylinosi-tol related lipid/protein kinases, can be recognised. Each family comprises two subfamilies. The involve-ment of the phosphatidylinositol 4-kinases in signal transduction processes is summarised and a newhypothesis for the function of their isoforms in polyphosphoinositide signalling is presented. The involve-ment of phosphatidylinositol 4-kinases in formation of lipid2protein interactions with cytoskeleton pro-teins and the metabolism of polyphosphoinositide in the nucleus is discussed.

Keywords: phosphatidylinositol 4-kinase; types and genes of phosphatidylinositol 4-kinases; domainstructure; phylogeny; inositide phospholipid signaling; phosphatidylinositol 4,5-bisphosphate; phosphati-dylinositol 4-phosphate ; vesicular traffic; phosphatidylinositol transfer protein.

The phosphatidylinositol cycle (Hokin and Hokin,1953), an the hydrophobic backbone, diacylglycerol, activates protein ki-nase C (Mitchell et al.,1991 ; Clapham,1995). In eukaryotesincrease in receptor-stimulated polyphosphoinositide turnover,

has been in the focus of attention for more than four decades. A this is one of the most commonly used signalling pathways in awide range of cells and may be surpassed only by cAMP whichhighlight was the discovery of two second messengers, inositol

1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol, which are is used also in prokaryotes (de Gunzburg,1985).In the last decade, it has become increasingly clear that poly-produced from phosphatidylinositol 4,5-bisphosphate (PtdInsP2)

by phospholipase C (PLC). The hydrophilic headgroup, phosphoinositides cover a wider variety of functions other thanjust being second-messenger precursors. In addition to beingIns(1,4,5)P3, mobilises Ca21 from intracellular stores whereasphosphorylated at the D4 and D5 positions of the inositol ring,

Correspondence toL. M. G. Heilmeyer, Ruhr-Universität Bochum, they can also be phosphorylated at the D3 position. Thus, threeInstitut für Physiologische Chemie, Abteilung für Biochemie Supramo-types of polyphosphoinositide are formed: phosphatidylinositollekularer Systeme, D-44780 Bochum, Germany

3-phosphate, phosphatidylinositol 3,4-bisphosphate and phos-E-mail : [email protected] 3,4,5-trisphosphate. These lipids are not sub-Abbreviations.PtdIns, phosphatidylinositol ; PtdInsP, phosphatidyl-strates for PLC but play essential roles in cytoskeleton re-inositol 4-phosphate; PtdInsP2, phosphatidylinositol 4,5-bisphosphate ;arrangement (Whiteman et al.,1988; Wennstrom et al.,1994;PLC, phospholipase C; Ins(1,4,5)P3, inositide 1,4,5-trisphosphate;

Ins(1,4)P2, inositol 1,4-bisphosphate ; Ins(1,3,4,5)P4, inositol 1,3,4,5- Hartwig et al.,1996), intracellular vesicle transport (Shu et al.,tetrakisphosphate; MAPK, mitogen-activated protein kinase; cAPK, ca-1993) and mitogenesis (Cantley et al.,1991). A family of en-talytic subunit of cAMP-dependent protein kinase; EGF, epidermalzymes, phosphoinositide 3-kinases (PI3K), is known today cata-growth factor; PtdIns TP, phosphatidylinositol transport protein; PH,lysing these phosphorylations (for review see Carpenter andpleckstrin homology; PI3K, phosphoinositide 3-kinase; PI4K, phospha-Cantley, 1996). Wortmannin, a potent inhibitor of PI3K, hastidylinositol 4-kinase. been employed to clarify that these lipids act upstream of theEnzymes.Phosphatidylinositol 3-kinase (EC 2.7.1.137); phosphati-

mitogen-activated protein kinase (MAPK) cascade as mem-dylinositol 4-kinase (EC 2.7.1.67) ; phosphatidylinositol-4-phosphate 5-brane-bound effectors (for review, see Ui et al.,1995). Theirkinase (EC 2.7.1.68); phospholipase C (EC 3.1.4.3); phospholipase Dexact role, however, is still unclear.(EC 3.1.4.4); protein kinase C (EC 2.7.1.37).

The existence and tightly controlled formation of polyphos-Note.This Review will be reprinted inEJB Reviews 1998which willbe available in April1999. phoinositides phosphorylated at the D3 position extended the

358 Gehrmann and Heilmeyer Jr (Eur. J. Biochem. 253)

view that both phosphatidylinositol 4-phosphate (PtdInsP) and Downes and MacPhee,1990; Carpenter and Cantley,1990).Type II is a 55-kDa enzyme present in all animal cells, whereasphosphatidylinositol 4,5-bisphosphate (PtdInsP2) can serve more

functions than being solely second-messenger precursors. As ex- the type-III isoform expresses high activity in bovine and ratbrain as well as in bovine uterus. The type-III enzyme is nowpected, both acidic phospholipids can be found in plasma mem-

branes, where signalling systems are located, responding to ex- identified as PI4K230 (for nomenclature see Table1) (Gehr-mann et al.,1996). These two PI4K, type II and III, differ fromtracellular neurotransmitters, hormones and growth factors. Seen

with doubt, initially, they are indeed also found in intracellular type-I PI3K in their requirement of non-ionic detergent for en-zyme activity and in their resistance to Triton X-100 inhibition,membranes (Jergil and Sundler,1983; Collins and Wells,1983;

Milting et al., 1994). Now it is firmly established that they serve up to 3%. Moreover, the type-II enzyme is inhibited by adeno-sine about 20-fold more strongly than the type-III enzyme;as effectors of enzymes like Ca21-transport ATPases (Varsa´nyi

et al., 1983; Vrolix et al.,1988; cf. Starling et al.,1995) and furthermore, theKm values for the substrates PtdIns and ATP/Mg21 are 327-fold lower for the type-II than for the type-IIIprotein kinases (Gross et al.,1995; Palmer et al.,1995; Bazenet,

1990), as components of the vesicular fusion process (for review enzyme (Endemann et al.,1987; Downes and MacPhee,1990;Carpenter and Cantley,1990). In S. cerevisiae, multiple formssee Liscovitch and Cantley,1995) and as anchors for cytoskele-

tal proteins (for review see Isenberg and Goldmann,1995). of 45 kDa and 55 kDa have also been demonstrated and havebeen proven to be membrane-bound (Belunis et al.,1988;Finally, a polyphosphoinositide cycle entirely separate from

the one in plasma membranes exists within the nucleus, pointing Nickels et al.,1992) ; in addition, a soluble PI4K of125 kDaexists (Flanagan et al.,1993).to a role of PtdInsP and PtdInsP2 in cell division and growth

(Divecha et al.,1993a). Inhibition of PtdIns kinases by wortmannin has now beenemployed as an alternative mode of classification, namely intoIn the reaction sequence from PtdIns to PtdInsP2, PtdIns 4-

kinase (PI4K) catalyses the first committed step resulting in wortmannin-sensitive and wortmannin-insensitive PtdIns ki-nases. Generally wortmannin inhibits PI3K very stronglyPtdInsP formation. PtdInsP formation, as well as PtdInsP2 for-

mation, is under tight control in plasma membranes. Generally, (Arcaro and Wymann,1993; Yano et al.,1993). PI4K are inhib-ited half-maximally either at submicromolar concentrations (Na-upon stimulation of PLC, a drop in PtdInsP and PtdInsP2 con-

centrations is followed by rapid resynthesis. Little is known kanishi et al.,1995; Meyers and Cantley,1997) or not at all(Downing et al.,1996). The wortmannin-sensitive PI4K are theabout the regulatory mechanisms involved. There are indications

that the PtdInsP/PtdInsP2 ratio can be shifted by activation of type-III enzymes, PI4K230 and PI4K92, whereas the type-II en-zyme, the 55-kDa form, is the insensitive one.protein kinase C (Apgar,1995) or, in correlation, to increased

cAMP levels. More direct evidence for a regulation of PI4K andphosphatidylinositol 4-phosphate 5-kinase (PtdIns4P 5-kinase) Genes.Table 1 summarises the PI4K genes known today.

Three orthologous genes of both PI4K230 and PI4K92 werecame from several cAMP mutants ofSaccharomyces cerevisiae(Kato et al.,1989). However, the complex metabolism of the cloned from human, rat and bovine tissues. Two are known from

yeast, PI4K120 and PI4K200. Whether these two genes are alsopolyphosphoinositides does not allow one to assign these alter-ations unequivocally to specific enzymatic steps (for review see functionally equivalents and/or orthologous to the mammalian

PI4K230 and PI4K92 enzymes remains to be established. APike, 1992). More recently evidence has accumulating that G-proteins are involved in the stimulation of PtdInsP2 synthesis. PI4K97 form was cloned indeed as the first mammalian PI4K

but was found to be a partial clone of the PI4K230. One mam-This is based on the original observation that non-hydrolysableGTP analogues can cause a large activation of PtdInsP2 synthe- malian PI4K55 has not yet been cloned but it is well character-

ised enzymatically. There are three other PI4K fromSchizo-sis (Stephens et al.,1993; for the mechanism see below).Regarding the wide variety of functions that PtdInsP is insaccharomyces pombe, Dictyostelium and Chaenorabditisfor

which sequence but no enzymological data have been reported.volved in, it is expected that multiple isoforms of PI4K exist.On the basis of their sensitivity to adenosine inhibition and non- According to their sequence similarity (Fig. 3), they have been

assigned tentatively as PI4K also.ionic detergents, early enzymology has characterised PI3K astype-I phosphatidylinositol kinase and two isoforms of PI4K as The complete yeast genome was searched for similarities in

the catalytic domain of PI4K. Genes for the PI4K120 andtype II and III (Carpenter and Cantley,1990). However, it wasnot until 1994 that, in yeast, two genes encoding PI4K were PI4K200 could be identified but no others corresponding to the

45-kDa or 55-kDa forms were found. Thus, either these yeastisolated (Flanagan et al.,1993; Garcia-Bustos et al.,1994;Yoshida et al.,1994) and the first mammalian cDNA for PI4K enzymes are proteolytically processed forms of either the

PI4K120 or PI4K200 or these smaller forms could belong towas cloned and sequenced (Wong and Cantley,1994). Sincethen, a variety of cDNAs for PI4K have become known. This another family of PI4K which perhaps might be similar to the

PtdIns4P 5-kinases (Boronenkov and Anderson,1995; Loijenssequence information, reviewed here, allows some sequences indata banks to be assigned as PI4K so that a phylogentic tree of et al.,1996).PI4K could be assigned, as well as of their relatives, the PI3Kand PtdIns-related lipid/protein kinases. Finally, a survey of theTissue distribution of PI4K by northern blot analysisPI4K known so far may help to clarify the role of PtdInsP inthe many diverse functions mentioned above. The expression pattern of PI4K was tested for human, bo-

vine, and rat PI4K230 (Nakagawa et al.,1996a; Balla et al.,1997), human, bovine, and rat PI4K92 (Nakagawa et al.,1996b ;Enzymology and genes of PI4KMeyers and Cantley,1997; Balla et al.,1997) and the humanPI4K97 (Wong and Cantley,1994).Enzymology. In earlier times, based on the recognition of

the PtdIns cycle as a major signalling pathway in all eukaryotic A hybridisation signal of 7.0 kb consistent with the size ofan mRNA coding for human PI4K230 (Fig.1) exhibits a widecells, the enzymology of PtdIns phosphorylation was thought to

be simple. As an initiating step, phosphorylation of PtdIns oc- tissue distribution. A cDNA probe composed of approximatelythe whole sequence of PI4K97 yields an identical 7.0-kb signal ;curs on the D4 position of the inositol ring catalysed by PI4K.

At least two PI4K isoforms have been characterised in mamma- thus, PI4K97 represents a partial clone of the PI4K230 (Naka-gawa et al.,1996a). This signal is intense in heart, brain, pla-lian tissues enzymatically (Endemann et al.,1987, 1991 ;

359Gehrmann and Heilmeyer Jr (Eur. J. Biochem. 253)

Table 1. Recently cloned PI4K.

Subfamily Name Previous name Source Size Reference Accession no.

residues

1.1 PI4K92 PI4KIIIβ bovine 801 Balla et al.,1997 2198789PI4K92 PI4Kβ human 801 Meyers and Cantley,1997 1894947PI4K92 2 rat 816 Nakagawa et al.,1996b 1906794PI4K68 2 Chaenorabditis 604 Wilson,1994 U41540PI4K122 DdPIK4 Dictyostelium 1093 Zhou et al.,1995 2120376DPI4K95 2 S. pombe 851 http://sanger.ac.uk/ Z70043PI4K120 PIK1 S. cerevisiae 1066 Flanagan et al.,1993; Garcia-Bustos et al.,1994 S39245

1.2 PI4K230 type 3 PI4K human 2044 2326227PI4K97 PIKA human 854 Wong and Cantley,1994 1172504PI4K230 type 3 PI4K rat 2041 Nakagawa et al.,1996a D83538PI4K230 PI4K200, PI4KIIIA bovine 2043 Gehrmann et al.,1996; Balla et al.,1997 2136690

and 2198791PI4K200 STT4 S. cerevisiae 1900 Yoshida et al.,1994 D13717

PI4K55 type 2 PI4K 2 not cloned

Fig. 1. Tissue distribution of mammalian PI4K by northern blot analysis. Lanes :1 heart, 2 brain, 3 placenta, 4 lung, 5 liver, 6 skeletal muscle,7 kidney, 8 pancreas, 9 spleen,10 thymus,11 prostate,12 testis,13 ovary,14 small intestine,15 colon,16 leukocytes. At the right margin the sizesof an RNA standard are indicated. mRNAs of PI4K were detected employing multiple tissue blots (Clontech) with 2µg human poly(A)-rich RNAin each lane. As probe a cDNA-fragment coding for amino acids181222044 (see Fig. 2A) was used. Probes were labelled with DIGdUTP byPCR. Hybridisation and chemiluminescence detection with CDP-STAR (Tropix) as substrate were performed following the Boehringer DIG hybrid-isation manual.

centa, skeletal muscle, kidney, spleen, thymus, prostate, ovary, of 2.8 kb. It is strong in ovaries, placenta, skeletal muscle andmuch weaker in spleen, thymus, and heart; in all other tissues,small intestine, and colon. In lung, liver, pancreas, testis, and

leucocytes the signal was faintly visible or absent. In many brain it is nearly undetectable (Fig.1).The smallest signal of 2.8 kb is compatible in size with anareas, such as amygdala, nucleus caudatus, corpus callosum, hip-

pocampus, substantia nigra, nucleus subthalamus and thalamus, mRNA coding for the type II PI4K55. Enzymes correspondingin size to the weak signal at 6.5 kb clearly seen in placenta ora strong 7.0-kb signal can also be detected exhibiting only slight

differences in the mRNA abundance for PI4K230. 4.4 kb observable also in heart, skeletal muscle and colon areunknown.An mRNA of 3.5 kb coding for PI4K92 also shows a wide

tissue distribution similar to that of the 7.0-kb signal of thePI4K230 (Fig.1). Indeed, it is only in brain and kidney that theStructure of PI4K3.5-kb signal is very faint or not observable, whereas in testisthe 7.0-kb signal is almost absent. Domain structure. It is a very characteristic feature that all

PI4K, PI3K, as well as several more distantly related lipid/pro-The nearly ubiquitous expression pattern of PI4K230 andPI4K92, as well as the high degree of conservation of both en- tein kinases, contain a highly conserved catalytic domain in the

C-terminal part of the molecule and a more variable N-terminalzymes among different mammals (see below), strongly indicatesthat these enzymes exert specialised functions in the cell and part.

In the amino acid sequence of the high-molecular-massplay essential roles.These messages can be detected employing a hybridisation human PI4K230 (Fig. 2A), shown as a representative of these

lipid kinases; the highly conserved C-terminal part is high-probe which derives from the catalytic domain of the humanPI4K230 (Fig.1). A probe containing this highly conserved do- lighted. The organisation of the N-terminal part is more variable

and is characterised by a so-called lipid kinase unique domainmain is able, particularly under non-stringent conditions, to de-tect in multiple adult human tissues even more than the above- (bold letters, Fig. 2A); this is not, however, a clearly defined

motif, stretching over approximately 70 amino acid residues ofmentioned mRNAs namely, at 4.4, 6.5, and 2.8 kb. In a similarway, Balla et al. (1997) reported several smaller transcripts hy- which 25 are conserved or conservative replacements (Wong and

Cantley,1994). The function of this domain has been unclearbridising with a partial clone of PI4K230. The signals at 4.4 kband/or 6.5 kb are observed in heart, placenta, skeletal muscle until now. PI4K230, PI4K97 and PI4K200 contain, as a charac-

teristic domain, a PH domain (bold underlined letters, Fig. 2A).and colon. A different distribution pattern is shown by an mRNA


Fig. 2. Structure and domain organisation of PI4K.(A) Amino acid sequence of human PI4K230 shown in single-letter code. Residues in motifsare: bold and underlined, (1) SH3 domain, (2) Pro rich region, (3) nuclear targeting signal, (4) leucine zipper, (5) helix-loop-helix motif, (6) lipid-kinase unique domain, (7) PH domain, (8) catalytic domain; residues identical in all recently known PI4K are highlighted. (B) Domain organisationof PI4K conserved motifs and domains. These motifs and domains are described in Gehrmann et al.,1996; Nakagawa et al.,1996a, b; Wong andCantley,1994; Meyers and Cantley,1997; Balla et al.,1997.


The alignment of the lipid kinases mentioned above showsThe function of this domain is controversial (reviewed by Cohenet al.,1995). It has been shown that several PH domains bind to a high degree of similarity between all of these catalytic do-

mains: 50 out of the approximately 260 amino acid residues arethe βγ subunit of heterotrimeric G proteins. When humanPI4K97 is tested with recombinantβγ subunit, no significant identical in all12 PI4K catalytic domains, while15 are invariant

among all known PI4K and PI3K and, finally,10 residues arechange of PI4K activity occurs (Wong and Cantley,1994). Alter-natively, the PH domain has been described as a phospholipid- invariant in all listed sequences including those which are very

distantly related (Fig. 3). The similarity is less pronounced ifbinding domain and, thus, may be important for membrane asso-ciation of the enzyme. Furthermore, PI4K230 is characterised these catalytic domains are compared to those of protein kinases.

It is interesting to note that minimally four out of these10 resi-by several motifs involved in protein2protein interactions (boldunderlined letters, Fig. 2A). The SH3 domain and Pro-rich se- dues are involved in the catalytic mechanism of phosphate

transfer from ATP to the substrate. Therefore, the catalyticquences, acting as SH3 ligands, are localised at the far N-termi-nal end of PI4K230 and are characteristic motifs in proteins in- mechanism of lipid and protein kinases seems to be very similar,

if not identical, in both lipid and protein kinases.volved in signal transduction (Cohen et al.,1995). Interestingly,in the N-terminal part of PI4K230, two leucine-zipper motifs, a Differences between lipid and protein kinases may be best

recognised if highly conserved residues of both superfamilieshelix-loop-helix motif and two nuclear-targeting signals are alsolocated (Fig. 2A). Leucine zippers, as well as helix-loop-helix are compared. Those residues involved in the catalytic mecha-

nism should be identical in both superfamilies. Non-identicalmotifs, are found in a number of eukaryotic DNA-binding pro-teins that act as transcription factors. In both cases binding to residues, highly conserved in either lipid or protein kinases, can

indicate potential differences in binding the substrate or formingDNA requires dimerisation and adjacent basic regions. Basicstretches, however, are weak or absent in PI4K230. A whole the catalytic cleft. Therefore the best understood protein kinase,

the catalytic subunit of cAMP-dependent protein kinase (cAPK)class of proteins contains the helix-loop-helix motif but lacks anadjacent basic domain. They are thought to inhibit transcrip- may serve as template to characterise the function of some of

the highly conserved residues in the lipid and protein kinasetional control by forming heterodimers with proteins containingthe helix-loop-helix motif and the adjacent basic DNA-binding catalytic domains and additionally may help to assign functions

to those residues highly conserved in the lipid kinase domainsregion (Clark and Docherty,1993). The nuclear targeting signalsindicate that this enzyme could be transported into the nucleus. only (for a correlation of structural elements with sequence and

with function of highly conserved residues in protein kinases,Maybe the enzyme shuttles between membranes, possibly alsothe nuclear envelope and the inner space of the nucleus where see Taylor and Radzio-Andzelm,1994). The catalytic core re-

gion of cAPK consists of two lobes with the small lobe associ-it could bind to transcription factors. A possible function ofPI4K230 in the nucleus is an involvement in the development ated primarily with ATP binding and the large lobe with cataly-

sis. The two lobes are held together by a short linker regionof the rat brain during embryogenesis because its expression infetal brain is much higher than in adult animals (Nakagawa et which is also important in positioning ATP as well as the peptide

substrate (Zheng et al.,1993; Taylor and Radzio-Andzelm,al., 1996a).The domain organisation of two pairs of mammalian and1994).

From the alignment of the lipid kinase catalytic domains, ayeast PI4K is shown in Fig. 2B. For the PI4K92 a Pro-rich re-gion similar to that of the PI4K230 has been described, in addi- separation into three parts can be clearly recognised (Fig. 3).

There is a first block of<70 residues nearly identical in sizetion to the catalytic and lipid kinase unique domains (Fig. 2B).In comparison to PI4K230, the lipid-kinase-unique domain has with the small lobe of cAPK which contains several highly con-

served residues. The second part is quite variable in sequencea more N-terminal position. Solely, the mammalian PI4K92shares a common region with the yeast PI4K120 but not as well as in length and comprises 20270 residues. This part

corresponds to the linker region and comprises only a few, fourPI4K230.or five, residues in the protein kinase family. The third part con-tains even more conserved residues than the first part and isCatalytic domain of phosphatidylinositol kinases and re-

lations to protein kinases.Sequences of catalytic domains were bigger, comprising<170 residues as in cAPK.Adopting the term small lobe for the first part of the lipidaligned from all known PI4K including yeast PI4K200 and

PI4K120, as well as those of the putative PI4K fromS. pombe, kinases also, indicates that this small lobe has the same functionin both kinds of kinases, namely to harbour residues involved inDictyosteliumandChaenorabditis.For comparison, catalytic do-

mains of all known related PI3K were included as well as those fixing the non-transferableA and β phosphates as well as theadenine moiety of ATP. In the small lobe of cAPK, a Gly-richof the more distantly related PI3K like DRR1, TOR1 and 2,

FRAP and very distantly related proteins like RAD, MEI-41, loop and a Lys residue (a characteristic feature of all proteinkinases) bind, via hydrogen bonds and by forming a salt bridge,ESR-1 and Mec1p which may be lipid or protein kinases.

It has been well established that the110-kDa catalytic sub- these two phosphates. A Glu residue positions the Lys residuecorrectly. The corresponding Lys residue in the lipid kinase cata-unit of the mammalian PI3K and of the yeast PI3K VPS34, are

dual-specificity kinases which catalyse phosphotransfer to lytic domains can be easily identified (Lys6 in Fig. 3). In a PI3Kthis residue binds wortmannin (Wymann et al.,1996; cf. ScholzPtdIns as well as to proteins like the 85-kDa regulatory subunit

of PI3K (Carpenter et al.,1993; Dhand et al.,1994). Mutation et al.,1992 for FSBA binding). Moreover, this residue is con-served in all lipid kinase domains (Fig. 3). In analogy to cAPK,of crucial residues within the catalytic centre eliminates both the

lipid kinase and protein kinase activity, establishing that both the correct positioning of this absolutely required Lys residuemay be taken in the lipid kinases by an Asp (Asp10 Fig. 3)substrates are phosphorylated by one and the same catalytic

centre (Stack and Emr,1994). Much earlier, it was shown that which is conserved in all lipid kinase catalytic domains.In cAPK, <20 residues N-terminally of this crucial LysPI4K is associated with phosphorylase kinase (Georgoussi and

Heilmeyer, 1986) and that FRAP (Sabatini et al.,1995) and residue, the Gly-rich loop is located connecting twoβ strands.No lipid kinase contains a cluster of Gly residues at an equiva-TOR2 (Cardenas and Heitmann,1995) possess associated PI4K

activity as well. However, it has yet to be shown that these latter lent position. However, 48 residues C-terminally a single Gly,Gly54, is conserved in all lipid kinase catalytic domains. Inkinases contain an intrinsic lipid kinase activity, e.g. by muta-

tional analysis as demonstrated for PI3K. the PI4K230 family1.2 (for nomenclature see Fig. 4) it is a


Fig. 3. Alignment of PI3K and PI4K catalytic domains. The sequences of the catalytic domains are shown in single-letter code. The alignmentwas calculated using the clustal algorithm with the PAM250-residue mass table (Megalign, DNAstar Inc.). The whole sequences with referencesare available via internet in the nr-database at EMBL, NIH or GenomNet. Accession numbers are as follows: for PI4K, see Table1 ; for PI3K:PI3K soybean nodule,1171966; PI3K soybean root,1171965; PI3K ATVPS34Arabidopsis, U10669; PI3KChlamydomonas, 2109289; PI3KDrosophila, 1854505; PI3K VPSP34 human, S57219; PI3K Candida, 1653974; PI3K VPSP34S. cerevisiae, A36369; PI3K VPSP34S. pombe,U32583; PI3K110a bovine,1171955; PI3K110d human, 2104840; PI3K110 Drosophila, 1707448; PI3KDictyostelium, 1709526; PI3K cpk mouse,U52193; PI3K human, 2143260; PI3K p170 mouse, U55772; PI3K human, 2076604; PI3K 68DDrosophila, X92892; PI3K cpkDrosophila,U52192; PI3K Chaenorhabditis, 1272625; for PtdInsP2 3-kinases: PI4,5P?3KDictyostelium, P54673 and 2120376C; other PtdIns kinases: PIK?DRR1 S. cerevisiae, P35169; PIK? TOR1 S. cerevisiae, 1174744; PIK? TOR2S. cerevisiae, P32600; PIK? FRAP human,1169735; PIK? FRAPrat, 1169736; ?FRAP-related human,1235902 ; ?RAD3S. cerevisiae, 1654096; ?MEI-41 Drosophila, 998353; ?ESR1 S. cerevisiae, 586545;?Mec1p S. cerevisiae, 950173

GlyXaaGly motif as in cAPK. In the PI3K family, it seems to involvement of the linker region in substrate binding in thesesubfamilies of lipid kinases.be a GlyXaaXaaXaaGly motif ; in others, additionally one of

the Gly residues is replaced by Ser as in phosphorylase kinase Quite a few residues are highly conserved in the large lobeof both lipid and protein kinases which are essential in catalys-(Johnson,1997). Therefore, one could tentatively assign the

Gly-rich vicinity of Gly54 to be the equivalent of the Gly-rich ing the phosphotransfer; thus, they are parts of the catalytic loopand the activation loop in cAPK (Madhusudan et al.,1994).loop in cAPK. The C-terminal location could, but need not nec-

essarily, mean that positioning of ATP is different in lipid and These are two Asp and an Asn residue; they are found at corre-sponding positions and in the same distance in the lipid kinaseprotein kinase catalytic domains. Altogether, it seems plausible

that indeed the small lobe of the lipid kinase domain may also large lobe, namely Asp175, Asp194, and Asn180 (see Fig. 3).In analogy to their function in cAPK, Asp175 would be the cata-have the function of positioning ATP correctly, even though dif-

ferently than in cAPK. lytic base and Asp194 would chelate Mg21, as does Asn180. Inthe large lobe of cAPK another Lys residue is crucial whichIt is evident in Fig. 3 that the linker region in the PI4K and

in the subfamily 2.1 of the PI3K family (Fig. 4) is, with a few binds theγ-phosphate of ATP before and after transfer to thesubstrate (Madhusudan et al.,1994). In the lipid kinase largeexceptions, very similar. These three subfamilies are indeed

PtdIns kinases employing PtdIns as substrate and phosphorylat- lobe an equivalent position is occupied by a His residue(His177) which indeed is conserved in all lipid kinase catalyticing either the hydroxyl group at position D4 or D3 of the inositol

ring. The linker region is larger in the polyphosphoinositide ki- domains. In cAPK and other protein kinases the activation loopis located<30 residues C-terminally from the catalytic basenases represented in subfamily 2.2 (compare Fig. 4). Again the

linker region is characteristically different in the PtdIns-kinase- (Taylor et al.,1993). It is a region of regulation which is consti-tutively phosphorylated at Thr197 in cAPK or, for example, atrelated lipid/protein kinase family (subfamilies 3.1 and 3.2 see

Fig. 4). These differences most probably reflect, therefore, the Tyr and Thr in MAPK. This region is more variable in the pro-


Fig. 3. (Continued).

tein kinase family and it is therefore more difficult to assign lipid kinase catalytic domains. Such a switch is found fromLys216 in the PI4K to the Arg212 in PI3K.equivalent residues in the lipid kinase family. However, there is

a highly conserved Glu211 residue in the majority of the lipid Taken together, the similarity of protein and lipid kinases issignificantly lower in the small lobe than in the large lobe. Thekinase large lobes which could be equivalent to a phosphorylated

residue. A Glu residue also replaces a phosphorylated residue in switch of motifs in the small lobe, i.e. the change of position ofthe Gly-rich loop in relation to the absolutely required Lys, maythe activation loop of the catalytic subunit of phosphorylase ki-

nase (Johnson,1997). TheP-Thr residue in cAPK forms salt even indicate that these lobes, present in the lipid and proteincatalytic domains, respectively, may not be homologues. In con-bridges with Arg and Lys residues in order to keep the loop in

an active conformation. In the same way, Arg residues form salt trast, the large lobes are almost certainly homologues. Thus,lipid and protein kinases may have evolved from a commonbridges with phosphorylated residues in the activation loop of

cyclin-dependent kinase 2 and of MAPK. Thus to keep the acti- ancestor and subsequently have lost all or most traces of se-quence similarity in the linker region and in the small lobes,vation loop in the lipid kinases also in an active conformation,

basic residues should be in equivalent positions in the lipid ki- respectively, due to divergent evolution. In a different scenario,in one of the two lineages leading to protein kinase or lipidnase catalytic domain. The high degree of conservation of two

residues, Arg176 and His192, points towards this function in kinase, the small lobe has been exchanged by domain shuffling.Resolving the three dimensional structure of a lipid kinase maylipid kinase catalytic domains.

If Asp175 is the catalytic base, then the hydroxyl group of answer this question.the inositide ring to be phosphorylated must be positioned withina distance of 223 A. This is the D3 OH in PI3K and the D4 Phylogenetic tree of phosphatidylinositol kinases.On theOH in PI4K. Besides hydrogen bonds, the negative charge ofbasis of the alignment of the conserved C-terminal catalytic do-the phosphodiester group would be an ideal point of fixing amains, a phylogenetic tree for the superfamily of PtdIns kinaseshydroxyl group near the Asp catalytic base. On the surface ofhas been constructed (Fig. 4). Three families, the PI4K, the PI3Kthe large lobe, maybe near or in the activation loop, then thereand the PtdIns-related lipid/protein kinases, can easily be recog-should be a positively charged residue which would position thenised, each family comprising two subfamilies. Momentarily,inositol ring. Switching from PI4K to PI3K catalytic domains, only the (poly)phosphoinositide 3-kinase subfamily is furtherthe inositide ring must be flipped over by180° and must be subdivided into two subgroups. The structure of the phyloge-turned by 60° relative to the catalytic base, Asp. Thus, the pos- netic tree implies that these families and subfamilies existed be-

fore the eukaryotes diverged because members within the dif-tulated positive charge must be moved also within these two


Fig. 4. Phylogenetic tree of PtdIns-kinase superfamily.The phylogenetic tree was constructed on the basis of the alignment in Fig. 3. The scalebeneath the tree measures the distance between sequences. Units indicate the number of substitution events. Enzyme families are shown in a greybox, subfamilies in a light grey box. The whole sequences with references are available via the internet in the nr-database at EMBL, NIH orGenomNet. Accession numbers as in Table1 and Fig. 3.

ferent families are more conserved across species than are dif- whether PI3K fromChaenorabaditisbelongs to a new subfamilyor must be integrated into one of the others.ferent family members of the same species.

The PI4K clearly form two subfamilies (1.1 and1.2, Fig. 4) The more distantly PtdIns-kinase-related proteins (for reviewsee Lavin et al.,1995; Keith and Schreiber,1995) are also subdi-comprising PI4K92, yeast PI4K120 and the tentatively assigned

uncharacterised PI4K, on the one hand, and the PI4K230 and vided into at least two subfamilies, 3.1 and 3.2. These enzymesshow in the catalytic domain a higher similarity to PI3K thanthe yeast PI4K200, on the other hand.

The PI3K are also divided into two subfamilies. These sub- PI4K. Nevertheless, the substrate specificity is unclear. None ofthem has been shown to have endogenous PtdIns kinase activity.families are not only characterised by sequence similarity but

they can also be defined by substrate specificity and physiologi- It has only been reported that TOR2 is associated with PI4Kactivity by immunoprecipitation (Cardenas and Heitman,1995).cal roles. Enzymes of the first subfamily 2.1 phosphorylate only

PtdIns, i.e. they are PI3K; functionally, they are important for These kinases are more often thought to be protein kinases ratherthan lipid kinases since TOR and FRAP can undergo self-phos-intracellular vesicle traffic (Shu et al.,1993). Enzymes of the

second subfamily 2.2 (PI3K110) phosphorylate additionally phorylation. As discussed above, none of these proteins, how-ever, contain a Gly-rich loop C-terminally of the absolutely re-polyphosphoinositides, PtdInsP and PtdInsP2, with increasing

affinity. The enzymes of the subgroups 2.2.1 are located at the quired Lys residue in the small lobe. In contrast, they containthe conserved Gly54 of the lipid kinase proteins (see Fig. 3).plasma membrane and are involved in signal transduction. En-

zymes of the second subgroup of PI3K, subfamily 2.2.2, have Therefore, they are more likely to be lipid rather than proteinkinases.been described only recently and for this reason are not well

characterised. For PI3K68 (MacDougall et al.,1995) and It is well recognised that all these lipid kinases are relatedto the much bigger family of protein kinases (see above). IndeedPI3K170 (Virbasius et al.,1996) it is reported thatin vitro these

enzymes utilise PtdIns and to a limited extend PtdInsP as sub- for some of the PI3K a dual specificity has been documented(reviewed by Hunter,1995). It remains to be shown if proteinstrates. In contrast to the enzymes of subgroup 2.2.1, they do

not phosphorylate PtdInsP2. Probably these members of the fam- kinases branched off from lipid kinases orvice versa. Alterna-tively both lipid and protein kinases must have had a commonily are also located at the plasma membrane and may be in-

volved in a new signal transduction mechanism. It is unclear ancestor which had a different catalytic property.


Fig. 5. Possible role of vesicular transport in providing PtdInsP2 to plasma membranes for the sustained phase of agonist-stimulatedIns(1,4,5)P3 release.The immediate release of Ins(1,4,5)P3 involves a complex of receptor tyrosine kinase (RTK), PtdIns transfer protein (PITP),55-kDa PI4K (PI4K55) and PLCγ. Suppression of the sustained phase of Ins(1,4,5)P3 release by wortmannin (WT) indicates involvement of PI4K230(PI4K230) and/or PI4K92 (PI4K92) in PtdInsP2 (PIP2) synthesis required for production of Ins(1,4,5)P3 in the sustained phase. Other abbreviations:ER, endoplasmic reticulum; PIP, PtdInsP; PIS, PtdIns synthase; DAG, diacylglycerol; IP3R, inositide(1,4,5)trisphosphate receptor.

Physiological roles and intracellular location of PI4K by platelet-derived growth factor. In a similar situation, it hasbeen shown previously that profilin-bound PtdInsP2 is not avail-Intracellular and plasma membranes. Many aspects ofable for PLCγ. It becomes, however, a substrate when PLCγ is

PtdInsP and PtdInsP2 function have been dealt with in excellentphosphorylated by the EGF receptor, then profilin-boundreviews (Pike,1992; Liscovitch and Cantley,1994; Divecha andPtdInsP2 can be hydrolysed (Goldschmidt-Clermont et al.,1991).Irvine, 1995) so only a few newer aspects in relation to PI4K

Similarly, in sickle-red-cell membranes, evidence was pre-will be summarised here. Epidermal growth factor (EGF) hassented that the activity of type-II PI4K55 is enhanced and that along been known to stimulate PtdInsP2 hydrolysis in epithelial56-kDaP-Tyr-containing protein can be detected (Rhoda-Hardy-and fibroblast cells (for review see Rhee,1991), concomitantly,Dessources et al.,1993; de Neef et al.,1996). In accordance,PI4K activity increases (Pike and Eakes,1987; Walker and Pike,several other systems have been shown to involve type-II1987; Payrastre et al.,1990; Cochet et al.,1991). It has beenPI4K55 in plasma-membrane-located polyphosphoinositide sig-clearly shown that the type-II PI4K55 is involved and that analling. Enzymatic and immunochemical assays have convinc-50-kDa protein is phosphorylated on Ser and Thr as well as oningly demonstrated that type-II PI4K55 is complexed to theTyr residues in parallel. Due to the lack of knowledge about theCD63 and CD81 proteins of the transmembrane 4 superfamilystructure of this enzyme, a definitive proof that type-II PI4K55(TMS4F) and an integrin (A3β1). Interestingly, aµA3β1CD63-itself can be phosphorylated is still missing (Kauffmann-Zeh etCD81-PI4K complex is located at the cell periphery indicatingal., 1994). This EGF-stimulated PI4K activation involves thethat phosphoinositide signalling plays a role in cell motility viaformation of a multi-protein complex. Recently it has beena completely new pathway distinct from that through focal adhe-shown that the PtdIns transfer protein (TP) is required for PI4Ksion kinase (Berditchevski et al.,1997). Again the type-IIstimulation and that PtdIns TP, PI4K55 and PLCγ are coprecipi-PI4K55 has been shown to associate in T-cells with the CD42tated following EGF binding to its receptor (Kauffmann-Zeh et56-kDa-protein-tyrosine-kinase complex upon crosslinking withal., 1995). This suggests that PtdInsP and PtdInsP2 are synthe-anti-CD4 (Prasad et al.,1993; Pertile and Cantley,1995). Takensised on the transfer protein and PLC (activated by Tyr phos-together, it seems that type-II PI4K55 is the lipid kinase in-phorylation) may act directly on PtdInsP2 bound to PtdIns TPvolved in agonist-stimulated polyphosphoinositide signalling in(Cunningham et al.,1995). An alternative immediate source ofplasma membranes.substrate for PLCγ can be PtdInsP2 bound to actinin or vinculin

However, the situation seems to be more complex and other(Fukami et al.,1994). The amount of PtdInsP2 bound to theseproteins decreases in Balb/c 3T3cells in response to stimulation PI4K might be additionally involved. It is a well documented


observation that in some cells, upon agonist stimulation, might involve the same system coupled to PtdIns TP but dif-ferent PI4K in different locations, namely PI4K55, PI4K92 andIns(1,4,5)P3 is released in a biphasic manner, e.g. in rat pancre-

atic acini (Matozaki and Williams,1989), and in adrenal glomer- PI4K230.Hence the following hypothesis can be formulated. Uponulosa cells (Nakanishi et al.,1994; cf. Nakanishi et al.,1992 for

smooth muscle cells). An initially rapid release of Ins(1,4,5)P3 agonist stimulation, PtdInsP2, present in the plasma membrane,is employed for the immediate release of Ins(1,4.5)P3 and poly-is observed within about1 min which is followed by a longer

sustained phase up to10 min. Interestingly, wortmannin inhibits phosphoinositide resynthesis involves type-II PI4K55. The sus-tained phase of Ins(1,4,5)P3 release would employ PtdInsP2the slower sustained phase but not the initial phase (Nakanishi

et al.,1994). These changes are almost mirrored in intracellular brought into the plasma membrane by the fusion process ofPtdInsP2-containing vesicles. Their synthesis, in accordance,[Ca21] changes, i.e. wortmannin suppresses the plateau phase

but not the rapid initial rise of [Ca21] i. PtdInsP and PtdInsP2 would require a different PI4K, probably the PI4K92 located inthe Golgi. This hypothesis would solve the discrepancy that asynthesis in intact adrenal glomerulosa cells are also inhibited

by wortmannin (Nakanishi et al.,1995); this was also found wortmannin-insensitive as well as a wortmannin-sensitive PI4Kis involved in agonist-stimulated phosphoinositide signalling.in a variety of cells, e.g. adrenal glucomerulosa cells, NIH3T3

fibroblasts, and Jurkat lymphoblasts. Thus, its seems that a wort- Furthermore, this hypothesis is easily tested: for example, themodel imagines that PtdIns TP can bind polyphosphoinositidesmannin-sensitive PI4K is involved in this agonist-stimulated

process. as well as PtdIns. There is no evidence for this as yet. It isalso proposed that adenosine-sensitive PI4K55 is involved in theThe type-II PI4K55, as discussed above, constitutes a wort-

mannin-insensitive PI4K (Downing et al.,1996). Wortmannin- synthesis of the existing pool of polyphosphoinositides requiredfor the wortmannin-insensitive phase of Ins(1,4,5)P3 production.sensitive PI4K are the PI4K230 (Downing et al.,1996) and

PI4K92 (Meyers and Cantley,1997; Nakagawa et al.,1996b; This could be tested in permeabilised cells exposed to adenosinelevels that could block PI4K55. Furthermore, the involvementBalla et al.,1997). Therefore, both wortmannin-insensitive and

sensitive PI4K might both be involved in agonist-stimulated of PI4K230 or PI4K92 in these processes could be tested inknock-out experiments.polyphosphoinositide turnover.

Recently, it has been shown that over-expressed PI4K97 islocated at the endoplasmic reticulum (Wong et al.,1997). This Cytoskeletonis probably also true for PI4K230 since the anti-polypeptide an-tibody employed in these localisation studies would not discrim- It has been well established that many actin-binding proteins

bind to phospholipids and some very specifically to PtdInsP andinate between them. Endogenous PI4K92 is present in the Golgiregion and the cytosol (Wong et al.,1997). Thus, the wortman- PtdInsP2. On this basis, it has been speculated that PtdInsP2 in

general may regulate lipid2protein interactions (for a recent re-nin sensitivity of agonist-sensitive polyphosphoinositide poolssuggests that one or both of these membrane compartments, en- view see Isenberg and Goldmann,1995). PtdInsP2-binding sites

have been mapped at the N-terminus of S2 in gelsolin and villindoplasmic reticulum and/or Golgi, are involved in agonist-stim-ulated phosphoinositide signalling to produce the sustained (Jamney et al.,1992) and a second PtdInsP2-binding site has

been identified near the C-terminus in the gelsolin moiety S1phase but not the immediate Ins(1,4,5)P3 release. Consequently,replenishment of PtdInsP2 for the sustained phase could involve (Yu et al.,1992). PLCδ1 also contains an N-terminal stretch of

14 residues which is similar to the consensus motif proposed forvesicular traffic of PtdInsP2-containing vesicles. It has been dis-cussed above that PtdInsP is synthesised in these intracellular the PtdInsP2-binding sites of actin-binding proteins (Yagisawa

et al.,1994; Milting et al.,1996). It has been proposed that thismembranes. Therefore a wortmannin-sensitive PI4K could beinvolved, possibly PI4K92, from which PtdInsP2 is produced cluster, rich in basic amino acids, might form the core of an

Ins(1,4,5)P3-binding site. This stretch of amino acids is part ofand which is required for the fusion process. There is a growingbody of evidence that vesicular traffic depends on synthesis of a larger segment which shows partial similarities to the PH do-

main (Burgoyne,1994) containing positively charged Lys resi-polyphosphoinositide. Recently, PtdIns TP was also identified asa factor which is required for exocytotic release of norepineph- dues. This domain seems to be responsible for the binding of

the protein to PtdInsP2 (Harlan et al.,1994) which might berine from PC12 cells, thus for vesicular traffic (Hay et al.,1995).It has been shown also that inhibition of PI4K activity with phe- regulated in a competitive manner by Ins(1,4,5)P3. Thus

Ins(1,4,5)P3 formation might be intrinsically involved in thesenylarsine oxide inhibits secretion in chromaffin cells (Wiede-mann, 1996). Other factors are PtdIns4P 5-kinase and ATP, lipid2protein interactions. Although these studies suggest an

important role for PtdInsP2 and Ins(1,4,5)P3 in regulating thesewhich suggests that, maybe similarly to phosphoinositide signal-ling in plasma membranes, PtdInsP2 synthesis occurs on PtdIns protein2lipid interactions, it has yet to be shown that phospho-

inositide turnover actually exerts this effect. Assuming this isTP involving PI4K and PtdIns4P 5-kinase in this cellular loca-tion as well. the case, Ins(1,4,5)P3 has a much wider regulatory potential than

just being a Ca21-mobilising agent and, indeed, in skeletal mus-In addition, phopholipase D, stimulated by the small myris-toylated G protein ARF (ADP ribosylating factor), seems to play cle Ins(1,4,5)P3 does not open the Ca21 release channel, the rya-

nodine receptor (Penner et al.,1989) and accordingly must havea central role in vesicular traffic. Both ARF and PtdIns TP arerequired to promote PtdInsP2 synthesis in cytosol-depleted another function (Mayr and Thieleczek,1991). Be that as it may,

the PI4K of the subfamily1.2 all contain a PH domain whichHL60 cells and thus secretory function (Fensome et al.,1996).ARF activates phospholipase D (Brown et al.,1993) which in could serve such a function, namely the dislocation of these

PI4K from the membrane by Ins(1,4,5)P3 creating a negative-turn produces the PtdIns4P-5-kinase activator phosphatidic acid.Phosphatidic acid then turns on PtdInsP2 synthesis from PtdInsP feedback loop.which involves PtdIns TP and PI4K. Thus, in vesicular transportthe identical components are required as for hormone-stimulatedNucleusPtdInsP and PtdInsP2 synthesis in the plasma membrane (seeabove). In conclusion, polyphosphoinositide synthesis, stimu- There are many lines of evidence that, in the nucleus, a sepa-

rate phosphoinositide cycle exists (for review see Divecha et al.,lated either by hormones in conjunction with plasma-membrane-located receptors or by agonists enhancing vesicular traffic,1993a). Nuclei purified from rat liver (Smith and Wells,1984)


4-phosphate and phosphatidylinositol 4,5-bisphosphate: correlationor Friend cells (Cocco et al.,1987) incorporate radioactivity intowith actin polymerization,Mol. Biol. Cell 6, 972108.PtdInsP and PtdInsP2 incubated with [γ-32P]ATP. Another case

Arcaro, A. & Wymann, M. P. (1993) Wortmannin is a potent phos-for a distinct nuclear PtdIns-cycle came from data on Swiss 3T3phatidylinositol 3-kinase inhibitor: the role of phosphatidylinositolcells (Divecha et al.,1991). When these cells are treated with3,4,5-trisphosphate in neutrophil responses.Biochem, J. 296, 2972insulin-like growth factor1, a rapid decrease of PtdInsP and 301.

PtdInsP2 occurs within the nucleus, with a concomitant increaseBalla, T., Downing, G. J., Jaffe, H., Kim, S., Zolyomi, A. & Catt, K. J.in nuclear diacylglycerol and a translocation of protein kinase C (1997) Isolation and molecular cloning of wortmannin-sensitive bo-into the nucleus. The enzymes involved in this PtdIns metabo- vine type III phosphatidylinositol 4-kinases,J. Biol. Chem. 272,lism have been shown to be present in different parts in the 18353218366.

Barell, B. G., Rajandream, M. A. & Walsh, S. V. (1996)Schizosaccharo-nucleus (Payrastre et al.,1992). In mouse NIH 3T3 fibroblastsmyces pombe chromosome I sequencing project, Sanger Centre,and rat liver cells, a PI4K is located exclusively in the peripheralCambridge.matrix (lamina-pore complex). The isoform has not yet been

Bazenet, C. E., Brockman, J. L., Lewis, D., Chan, C. & Anderson, R.differentiated. In contrast, the PtdIns4P 5-kinase, identified asA. (1990) Erythroid membrane-bound protein kinase binds to athe C isoform (Divecha et al.,1992), was found to be associatedmembrane component and is regulated by phosphatidylinositol 4,5-with internal matrix structures. Diacylglycerol kinase and PLCγ bisphosphate,J. Biol. Chem. 265, 736927376.

activities were also preferentially detected in the internal matrix.Belunis, C. J., Bae-Lee, M., Kelley, M. J. & Carman, G. M. (1988)The PtdIns concentration in the nucleus from rat liver is sub- Purification and characterization of phosphatidylinositol kinase from

optimal for the PI4K activity, therefore participation of the Saccharomyces cerevisiae, J. Biol. Chem. 263, 18897218903.PtdIns TP, which provides PtdIns to the nucleus, was suggestedBerditchevski, F., Tolias, K. F., Wong, K., Carpenter, C. L. & Hemler,(Capitani et al.,1990). Recently, it could be shown that isoforms M. E. (1997) A novel link between integrins, transmembrane-4 su-

perfamily proteins (CD63 and CD81), and phosphatidylinositol 4-of PtdIns TP are located at distinct intracellular sites (de Vrieskinase,J. Biol. Chem. 272, 259522598.et al.,1996). In fetal bovine heart endothelial cells PtdIns TPA

Boronenkov, I. V. & Anderson, R. A. (1995) The sequence of phosphati-is predominantly present in the nucleus and the cytoplasm,dylinositol-4-phosphat-5-kinase defines a novel family of lipid ki-whereas PtdIns TPβ is located at the perinuclear Golgi system.nases,J. Biol. Chem. 270, 288122884.A possible mechanism is a rapid phosphorylation of PtdIns pro-

Brown, H. A., Gutowski, S. Moomaw, C. R., Slaughter, C. & Sternweis,vided by PtdIns TPA to the PI4K at the peripheral matrix and aP. C. (1993) ADP-ribosylation factor, a small GTP-dependent regula-

subsequent production of PtdInsP2 in the internal matrix. This tory protein, stimulates phospholipase D activity,Cell 75, 11372compound is hydrolysed to Ins(1,4,5)P3 and diacylglycerol by 1144.PLC (Martelli et al.,1992; Divecha et al.,1993b). Diacylglyc- Burgoyne, R. D. (1994) Phosphoinositides in vesicular traffic,Trendserol may modulate protein kinase C or may be phosphorylated Biochem. Sci. 19, 55257.to phosphatidate by diacylglycerol kinase which is also preferen-Cantley, L. C., Auger, K. R., Carpenter, C., Duckworth, B., Graziani,

A., Kapeller, R. & Soltoff, S. (1991) Oncogenes and signal transduc-tially located in the internal nuclear matrix.tion, Cell 64, 2812302.The regulation of the nuclear PI4K and PtdIns4P 5-kinase is

Capitani, S., Helms B., Mazzoni, M., Previati, M., Bertagnolo, V., Wirtz,unclear. There is some evidence that these enzymes are regulatedK. W. & Manzoli, F. A. (1990) Uptake and phosphorylation of phos-by GTP-binding proteins (Martelli et al.,1996).phatidylinositol by rat liver nuclei. Role of phosphatidylinositolA crucial point is the molecular basis of the nuclear PI4Ktransfer protein,Biochim. Biophys. Acta 1044, 1932200.activity; in mammals, no enzyme isoform has been identified

Cardenas, M. E. & Heitman, J. (1995) FKBP12-rapamycin target TOR2yet. Only in yeast has it been shown that PI4K120 (Garcia- is a vacuolar protein with an associated phosphatidylinositol-4-ki-Bustos et al.,1994) is part of a nuclear phosphoinositide cycle. nase activity,EMBO J. 14, 589225907.

The role of the compounds of the phosphoinositide cycle inCarpenter, C. L. & Cantley, L. C. (1990) Phosphoinositide kinases,Bio-the nucleus has been obscure until now. It has been shownin chemistry 29, 11147211156.vitro that PtdInsP and Ins(1,4)P2 activate DNA polymeraseA Carpenter, C. L., Auger, K. R., Duckworth, B. C., Hou, W. M., Schaff-

hausen, B. & Cantley, L. C. (1993) A tightly associated serine/threo-(Sylvia et al.,1988). In contrast, a recent study reported an inhi-nine protein kinase regulates phosphoinositide 3-kinase activity,Mol.bition of DNA polymerases with the greatest effect on DNACell. Biol. 13, 165721665.polymeraseε by PtdIns and PtdInsP (Shoji-Kawaguchi et al.,

Carpenter, C. L. & Cantley, L. C. (1996) Phosphoinositide 3-kinase and1995). Ins(1,4,5)P3 may act as a second messenger involved inthe regulation of cell growth,Biochim. Biophys. Acta 1288, M112nuclear Ca21 homeostasis. In the inner nuclear membrane aM16.220-kDa Ins(1,4,5)P3 receptor was identified (Malviya,1994)

Clapham, D. E. (1995) Calcium signaling,Cell 80, 2592268.and the Ca21-release can be enhanced by phosphorylation ofClark, A. R. & Docherty, K. (1993) Negative regulation of transcriptionPLCγ. Also there are two kinds of Ins(1,3,4,5)P4-binding sites in eukaryotes,Biochem. J. 296, 5212541.located at the nuclear envelope. The high-affinity Ins(1,3,4,5)P4 Cocco, L., Gilmour, R. S., Ognibene, A. J., Letcher, A., Manzoli, F. A. &receptor is associated with the outer membrane and mediatesIrvine, R. F. (1987) Synthesis of polyphosphoinositides in nuclei ofthe calcium uptake stimulated by Ins(1,3,4,5)P4. A low-affinity Friend cells. Evidence for polyphosphonositide metabolism insideIns(1,3,4,5)P4 receptor is present in the inner membrane and is the nucleus which changes with cell differentiation,Biochem. J. 248,

7652770.not involved in the Ins(1,3,4,5)P4-mediated calcium entry intoCochet, C., Filhol, O., Payrastre, B., Hunter, T. & Gill, G. N. (1991)the nucleus.

Interaction between the epidermal growth factor receptor and phos-This work was supported by theMinister für Wissenschaft und phoinositide kinases,J. Biol. Chem. 266, 6372644.

Forschung des Landes NRW, the Deutsche ForschungsgemeinschaftCohen, G. B., Ren, R. & Baltimore, D. (1995) Modular binding domains(SFB 394/B3) and theFonds der Chemischen Industrie.We thank H. in signal transduction proteins,Cell 80, 2372248.Gülkan, M. Böttcher, F. Herberg, M. Varsanyi and G. Vereb for readingCollins, C. A. & Wells, W. W. (1983) Identification of phosphatidylinosi-the manuscript and offering valuable suggestions. tol kinase in rat liver lysosomal membranes,J. Biol. Chem. 258,

213022134.Cunningham, E., Thomas, G. M., Ball, A., Hiles, I. & Cockcroft, S.

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Note added in proof. During preparation of this manuscript, a reviewwas published [Wirtz, K. A. (1997)Biochem. J. 324, 3532360] in whicha model very similar to that presented here was presented to explain howvesicular transport participates in PtdInsP2 supply to plasma membranes.

Phosphatidylinositol 4-kinases

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