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Important work has recently provided an explanation for the differences in type I and type II activities: investigations of in vitro substrate specificity using recombinant proteins and synthetic substrates has revealed that type II enzymes can phosphorylate PtdIns(5)P at the D-4 position to generate PtdIns(4,5)P2- The long-held belief that all PtdlnsPKs transferred phosphate exclusively to the D-5 position of the inositol headgroup was based on the assumption that the PtdIns(4,5)P2 product could only be generated by this reaction. It now appears that commercial preparations of PtdIns(4)P contain small amounts of the PtdIns(5)P isomer, a species which is presumably not found in erythrocyte membranes (Bazenet et a l, 1990). Interestingly, type Ila PtdlnsPK can also catalyse the formation of PtdIns(3,4)P2 from PtdIns(3)P, although with lower activity (Rameh et al., 1997b). The type I PtdlnsPKs are bona fide Ptdlns (4)P 5-kinases but can also convert PtdIns(3,4)P2 to PtdIns(3,4,5)P3, catalyse the phosphorylation of Ptdlns at the D-5 position, and PtdIns(3)P at both the D-4 and D-5 position to generate PtdIns(3,4,5)P3 (Rameh et a i,

1997b; Zhang et al., 1997). For both isoforms the formation of PtdIns(4,5)P2 is the most kinetically favoured reaction in vitro and, although the promiscuous substrate specificities of the type I isoforms are intriguing, the physiological relavence of these reactions is unclear at present.

1 . 5 . 2 . a Regulation of PtdlnsP ki n as e s

Little is known about the regulation of PtdlnsPK activity in intact cells. PtdIns(4,5)P2 is produced is response to both G-protein and RTK agonists (Stephens et al., 1993; Urumow and Wieland, 1988) and PtdIns(4,5)P2 levels may be regulated by the small GTPase Rho in an integrin-mediated pathway (Chong et al., 1994). Also, as a consequence of recent studies of the in vitro substrate specificities of the PtdlnsPKs, pathways which produce other PI species such as PtdIns(3,5)P2, PtdIns(3,4)P2 and PtdIns(3,4,5)P3 (produced from PtdIns(3)P) must now be considered potential products of the PtdlnsPKs (Rameh et al., 1997b; Zhang et a i, 1997).

1 . 5 . 2 . b Type I PtdlnsP kinase function

The regulation of PtdlnsPK activity by small G-proteins has been of particular interest to those working on signalling to the actin cytoskeleton because of the long-held hypothesis that PtdIns(4,5)P2 has profound and direct effects on cytoskeletal structures (Fukami et al., 1992; Janmey et al., 1987; Janmey and Stossel, 1987; Lassing and Lindberg, 1985). GTP-RhoA has been shown to stimulate PtdlnsPK activity in fibroblast lysates (Chong et al., 1994) and R a d has been shown to stimulate PtdIns(4,5)P2 biosynthesis in permeabilised platelets (Hartwig et al., 1995) leading to actin filament growth. Both Rac and Rho have been shown to form complexes with PtdlnsPK activity (Ren et al., 1996; Tolias et al., 1995), but in contrast to the results shown in Figure 1.7, neither of these

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P t d l n s P .

t

#e

s t d GST GDP GTPyS GDP GTPyS GDP GTPyS l y s s t d

%

R a c R h o C d c 4 2

Figure 1.7 GTP-dependeiit physical interaction of PtdInsPkinase activity with Racl and RhoA

T riton X -1 0 0 ly sa tes o f S w is s 3T 3 c e ils w ere prepared and incub ated w ith G D P - or G T P [y ]S - bou n d G S T fu s io n p ro tein s and a ss a y ed in d u p lica te for P td ln sP K a c tiv ity as in d ic a ted . Lanes: std; [32P]-PtdIns(4,5)P2 standard. G ST ; g lu ta h io n e-S -tra n sfer a se con trol, lys; 4 |il o f S w is s 3T 3 ly sa te. R e su lts sh o w n are ty p ica l o f three su ch e x p e r im e n ts. F ull e x p erim en ta l d etails can be found in S ec tio n 2 .5 .2 .f.

studies has shown GTP-dependence. There is currently no evidence for a direct interaction between PtdlnsPK and Rac and Rho and no PtdlnsPK has been isolated as the result of two-hybrid screening (a technique which has yielded numerous small G-protein targets). The binding of PtdlnsPK activity to Racl may require the Racl C-terminal basic region (Tolias et al., 1998). Furthemore, phosphatidic acid activation of the activities bound to Racl has been used to demonstrate that the PtdlnsPKs involved are type I in character, but exactly which isoforms are responsible remains to be determined.

A fragment of PtdlnsPK 1(3 has been isolated using a genetic screen where it was found to restore mitogenic signalling to a cell line expressing a mutant CSF-1 receptor with impaired tyrosine kinase activity. The catalytically inactive fragment was found to complement signalling by preventing receptor internalisation, thus PtdlnsPK 1(3 may have a role in receptor down regulation (Davis et at., 1997). Although this study did not address how PtdlnsPK 1(3 is involved in this process, it seems likely that the inactive PtdlnsPK 1(3 prevents endocytosis by inhibiting vesicle formation; a process in which the PtdOH-activated type I enzymes have been repeatedly implicated (see Section 1.11).

At least one direct link between lipid kinases and protein kinases has been demonstrated with the recent finding that PKCp (also known as PKD) interacts with a type U Ptdlns 4K and a type I PtdlnsPK (Nishikawa et al., 1998). PKCji is a phorbol ester and DAG- stimulated protein serine/threonine kinase which differs from the PKCs in that it contains a putative N-terminal transmembrane domain and a PH domain. Although PKCp has been included in the PKC family, the catalytic domain and several biochemical properties are more consistent with it being related to the CaMKII-like protein kinases (reviewed in (Mellor and Parker, 1998). Association of Ptdlns 4K and Ptdlns(4)P 5-kinase requires a region between residues 79-340, C-terminal to the PH domain and it is not known whether the interaction is direct or which PtdlnsPK isoform is involved (Nishikawa et al., 1998). The significance of this complex is unclear: PKCji is activated by GPCRs and RTKs via conventional and novel PKC isoforms (Sidorenko et al., 1996; Zugaza et al.,

1997), but no direct evidence for regulation by phosphoinositides has been reported.

1 . 5 . 2 . C Type II PtdlnsP kinase function

PtdlnsPK Ila translocates to the cytoskeletal fraction of thrombin stimulated platelets and is accompanied by an increase in PtdIns(4,5)P2 associated with the Triton X-100 insoluble fraction (Hinchliffe et al., 1996). Thrombin stimulation causes the secretion of fibrinogen (a ligand for the integrin tt2bp3) and stirring thrombin-stimulated platelets promotes collision which causes their rapid aggregation. Because PtdlnsPK Ila translocation was dependent on stirring and could be inhibited by an antagonist of a2bp3, regulation of P tdlnsP K I la by an integrin signalling pathway is clearly implicated. This work represents the first suggested role for a type II PtdlnsPK but the exact function of the

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