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More recent work has investigated the genetic interaction between STT4, a yeast PtdlnsPK M SS4 (see Section 1.5.2.d), PK Cl, and the yeast PLC, P L C l (Cutler et at., 1997). Briefly, overexpression of STT4 and M SS4 conferred resistance to wortmannin, an inhibitor of STT4p. Deletion of PLCl also conferred wortmannin resistance, suggesting that STT4p and MSS4p lie in a linear pathway and that PtdIns(4,5)P2 is an essential product of these enzymes. However, it is unlikely that STT4p is linked to PKC via PtdIns(4,5)P2 hydrolysis, since P L C l overexpression does not confer wortmannin resistance and the yeast PKC Ip is not activated by DAG in vitro (Kamada et al., 1996). It is possible that the STT4p-MSS4p Ptdlns(4,5)?2 biosynthetic pathway activates PKC Ip via an alternative pathway. A strong candidate is the ROM2p-RH01p pathway, whose components form a Rho-type GTPase switch that controls PKC Ip (Nonaka et al., 1995; Ozaki et al., 1996). PtdIns(4,5)P2 generated through the STT4p-MSS4p route may activate the R0M 2p-RH01p switch via the ROM2 PH domain in a manner analogous to the mammalian Pl-regulate exchange factors ARNO, cytohesin and G rpl (Section

1.5.4.b). It should be noted however that the PH domain of ROM2p has not been characterised with respect to phospholipid binding specificity.

The first metazoan Ptdlns 4K to be cloned was the 97 kDa Ptdlns 4K a, which has an identical domain organisation and displays a high degree of sequence identity (50% in the kinase domain) to STT4p (Figure 1.4b). The 230 kDa rat and bovine brain Ptdlns 4Ks have identical C-termini (Gehrmann et al., 1996; Nakagawa et al., 1996), indicating that Ptdlns 4-K a is probably a splice variant of the larger type III enzyme. It should also be noted that no purification studies have confirmed the existence of the 97 kDa Ptdlns 4-K a isoform. The type III Ptdlns 4Ks all contain a PH domain positioned between the LKH2 (lipid kinase unique) region and the C-terminal catalytic domain (Figure 1.4b). The PH domain of a plant homologue has recently been shown to bind PtdIns(4)P in vitro (Stevenson et al., 1998), and although the function of this is unclear, its position close to the kinase domain suggests a regulatory function. In addition to the PH domain, the mammalian p230 isoforms have a predicted N-terminal SH3 domain and a proline rich region (Nakagawa et al., 1996). Like the Ptdlns 4K(3 isozyme (see Section 1.5.1.C, below), the STT4p-related Ptdlns 4Ks are all inhibited, to various degrees, by wortmannin in vitro (Balia et al., 1997; Cutler et al., 1997; Woscholski et al., 1994).

1.5.1 c Pik1-re\ated Ptdlns 4 - k i n a se s

S. cerevisiae Pikl was purified as a soluble, 125 kDa Ptdlns 4K (Flanagan and Thomer, 1992) and was the first Ptdlns 4K to be cloned (Flanagan et al., 1993). The function of Piklp has not yet been elucidated but null mutation is lethal. Piklp has also been isolated as a component of the nuclear pore complex (Garcia-Bustos et al., 1994), a finding at

variance with the work described above. More recently, direct immunofluorescence has localised P ik lp exclusively to the cytosol (Thorner, 1996) and it is possible that differences in the preparation of yeast cell fractions accounts for the discrepancy.

As shown in Figure 1.4, the Ptdlns 4K family can be divided into two groups on the basis of primary stmcture homology within the C-terminal kinase domain. In addition, the domain organisation of these two groups (referred to here as STT4-like and Pikl-like) are significantly different. The Pikl-related Ptdlns 4Ks lack a PH domain but contain a region of homology (LKH3, for lipid kinase homology region 3) that is unique to this group. Also, in contrast to the STT4p-related proteins, the LKH2 region of the Piklp-related Ptdlns 4Ks is positioned close to their N-termini. The functional significance of these differences is not known but they may represent a dichotomy similar to that seen with the type I and type II PtdlnsPKs, or the subdivisions of PI 3-kinases, whose members have characteristic substrate specificities and modes of regulation (Sections 1.5.2 and 1.5.3). This is supported by the finding that STT4 overexpression cannot compensate P IK l function and vice versa (Cutler et al., 1997).

The function of the cloned Ptdlns 4K isoforms is currently unclear, but they appear to localise to distinct subcellular membranes. p230 Ptdlns 4K localises to the endoplasmic reticulum (Nakagawa et al., 1996; Wong et al., 1997) whereas Ptdlns 4KP localises to the Golgi (Wong et al., 1997). A wortmannin-sensitive pool of PtdIns(4)P and Ptdlns(4,5)?2 has been described which is metabolised in response to both G-protein and RTK agonists (Nakanishi et al., 1995), but the finding that all cloned mammalian Ptdlns 4Ks can be inhibited by PI 3-kinase inhibitors precludes the determination of which Ptdlns 4K isoform is responsible for this pool. The type II Ptdlns 4K is not inhibited by wortmannin (Nakanishi et al., 1995; M. Waugh, unpublished data) and can therefore be excluded.

1 . 5 . 2 P t d l n s P kin ases

The mammalian PtdlnsPKs (sometimes referred to as PIPkins) were previously determined to be a family of at least three immunologically and chromatographically distinct isoforms with approximate molecular masses of 53 kDa, 68 kDa, and 98 kDa, which catalysed the formation of PtdIns(4,5)P2 from PtdIns(4)P (Bazenet et al., 1990; Jenkins et al., 1994). The PtdlnsPKs were classified as type I or type II based on their biochemical properties (described in more detail in Chapter 3). Evidence in the literature rapidly accumlated indicating that the type I and type II isoforms had profoundly different properties. Most notable was the finding that type I enzymes were active towards membrane PtdIns(4)P substrate and were activated by phosphatidic acid, whereas the type II isoform was not active toward membranes and was not stimulated by phosphatidic acid (Bazenet et al., 1990; Jenkins et a l, 1994; Moritz et al., 1992).

30

Figure 1.6 The P tdlnsP K fam ily

A. Similar to 1.5a, a dendrogram is shown of cloned PtdlnsPK family members' catalytic domains.

B. Schematic similar to Figure 1.5b, showing PtdlnsPK homology regions and proposed molecular interaction domains. The size of each PtdlnsPK is given in amino acids. Acession numbers/references: S.c. Fabl, S. cerevisiae FABl gene product (U01017; Yammamoto et a l, 1995); C.e. ORFl, C. elegans Fab Ip homologue (Z67879); H.s. Ila , (U14957; Borononkov and Anderson, 1995; Divecha et a l, 1995; Chapter 3), H.s. Up, human PtdlnsPK lip (U85245; Castellino et a l, 1997), H.s. Ily, human PtdlnsPK Ily (Itoh et a l, 1998); A.t. ORF 1, A. thalina putative PtdlnsPK (AF007269); A.t. ORF 2, A. thalina putative PtdlnsPK (AF019380); A.t. ORF 3, A. thalina putative PtdlnsPK (U95973); A.t. ORF 4, A. thalina putative PtdlnsPK (Y12776); D.m. skittles, D. melanogaster skittles gene product (U73490); H.s. Ip, human PtdlnsPK Ip, (Ishihara et a l , 1996); M.m. ly, murine PtdlnsPK ly (Shibasaki et a l, 1998); H.s. l a (STM7), human PtdlnsPK l a (Carvajal et a l, 1995), C.e. ORF2, C. elegans putative PtdlnsPK (AF003130); S.c. Mss4, S. cerevisiae MSS4 gene product (D 13716; Yoshida et a l,

s.c. Fab1 C.e. ORFl H.s. Ila H.s. lip H.s. Ily A.t. 0RF1 A.t. 0RF2 A.t. 0RF3 A.t. ORF 4 D.m. Skittles H.s.Ip M.m. ly H.s. la (STM7) C.e. 0R F2 S.c. Mss4

31 TCP-1 I Fab1p —//— , ’V c.e. ORFl --- H.s. type I l a H.s. type lip R.n. type Ily A.t. 0RF1 A.t. 0RF2 A.t. 0RF3--- --- A.t. 0RF4 --- D.m. Skittles--- H.s. type Ip M.m. ly --- STM7/type la C.e. 0RF2 ---- Mss4p ---