Pleckstrin homology (PH) domains may act as membrane localization modules through specific interactions with phosphoinositide phospholipids. These interactions could represent responses to second messengers, with scope for regulation by soluble inositol polyphosphates. A biosensor‐based assay was used here to probe interactions between PH domains and unilamellar liposomes containing different phospholipids and to demonstrate specificity for distinct phosphoinositides. The dynamin PH domain specifically interacted with liposomes containing phosphatidylinositol‐4,5‐bisphosphate [PI(4,5)P2] and, more weakly, with liposomes containing phosphatidylinositol‐4‐phosphate [PI(4)P]. This correlates with phosphoinositide activation of the dynamin GTPase. The functional GTPase of a dynamin mutant lacking the PH domain, however, cannot be activated by PI(4,5)P2. The phosphoinositide‐PH domain interaction can be abolished selectively by point mutations in the putative binding pocket predicted by molecular modelling and NMR spectroscopy. In contrast, the Bruton's tyrosine kinase (Btk)PH domain specifically bound liposomes containing phosphatidylinositol‐3,4,5‐trisphosphate [PI(3,4,5)P3]: an interaction requiring Arg28, a residue found to be mutated in some X‐linked agammaglobulinaemia patients. A rational explanation for these different specificities is proposed through modelling of candidate binding pockets and is supported by NMR spectroscopy.

Recent evidence has suggested that activation of phosphoinositide 3-kinase (PI 3-kinase) is required for the activation of Akt-1 by growth factors and insulin. Here we demonstrate by two independent methods that Akt-1 from L6 myotubes binds to PtdIns(3,4,5)P3, PtdIns(3,4)P2 and PtdIns(4,5)P2 when presented against a background of phosphatidylserine (PtdSer) or a 1:1 mixture of PtdSer and phosphatidylcholine (PtdCho). No binding was observed with the lipids PtdIns(3,5)P2, PtdIns4P and PtdIns3P or background lipids. Activated, hyperphosphorylated forms of Akt-1 from insulin-stimulated L6 myotubes bound to PtdIns(3,4,5)P3 in a similar manner as inactive Akt-1. Quantitative analysis using surface plasmon resonance showed that the equilibrium association constant for the binding of Akt-1 to PtdIns(3,4,5)P3 was submicromolar and that PtdIns(3,4)P2 and PtdIns(4,5)P2 bound to Akt-1 with 3- and 6-fold lower affinities respectively. Interaction of Akt-1 with PtdIns(3,4,5)P3 did not activate the protein kinase activity, either before or after incubation with MgATP. A model is presented in which PtdIns(3,4,5)P3 may prime Akt-1 for activation by another protein kinase, perhaps by recruiting it to the plasma membrane.

As potential targets for polyphosphoinositides, activation of protein kinase C (PKC) isotypes (␤ 1 , ⑀, , ) and a member of the PKC-related kinase (PRK) family, PRK1, has been compared in vitro. PRK1 is shown to be activated by both phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P 2 ) as well as phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P 3 ) either as pure sonicated lipids or in detergent mixed micelles. When presented as sonicated lipids, PtdIns-4,5-P 2 and PtdIns-3,4,5-P 3 were equipotent in activating PRK1, and, furthermore, sonicated phosphatidylinositol (PtdIns) and phosphatidylserine (PtdSer) were equally effective. In detergent mixed micelles, PtdIns-4,5-P 2 and PtdIns-3,4,5-P 3 also showed a similar potency, but PtdIns and PtdSer were 10-fold less effective in this assay. Similarly, PKC-␤ 1 , -⑀, and -were all activated by PtdIns-4,5-P 2 and PtdIns-3,4,5-P 3 in detergent mixed micelles. The activation constants for PtdIns-4,5-P 2 and PtdIns-3,4,5-P 3 were essentially the same for all the kinases tested, implying no specificity in this in vitro analysis. Consistent with this conclusion, the effects of PtdIns-4,5-P 2 and PtdIns-3,4,5-P 3 were found to be inhibited at 10 mM Mg 2؉ and mimicked by high concentrations of inositol hexaphosphate and inositol hexasulfate. The similar responses of these two classes of lipid-activated protein kinase to these phosphoinositides are discussed in light of their potential roles as second messengers.The phosphatidylinositol 3-kinase family of lipid kinases are responsible for the phosphorylation of inositol lipids at the 3-OH position (reviewed in Ref. 1). In response to various agonists, phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P 3 ) 1 accumulates and labeling studies suggest that this is the primary product and as such is the most likely second messenger candidate (2). While this remains an attractive hypothesis, in the absence of a defined intracellular target(s), the operation of the "PtdIns 3-kinase signaling pathway" will remain enigmatic.By their nature, lipid-dependent protein kinases are attractive candidates as targets for the postulated role of PtdIns-3,4,5-P 3 as a second messenger. Protein kinase C (PKC) isotypes constitute the major group of such enzymes, but several recent reports have indicated the existence of an additional class of lipid-activated protein kinases. These kinases, termed PKC-related kinases (PRKs) are closely homologous to PKC isotypes in the catalytic domain while retaining a distinct amino-terminal regulatory domain ((3, 4) see Fig. 6A). Unlike the PKCs which characteristically retain a cysteine-rich C1 domain responsible for effector binding, the PRKs do not encode a C1 domain but show two distinct conserved regulatory domains termed HR1 and HR2 (3). Consistent with the lack of a C1 domain, it has become clear that while the PRKs resemble PKCs in being activated by proteolysis, they differ from PKCs in not being activated by phorbol esters. However, like PKC, various fatty acids and phospholipids have bee...

No abstract

420s Biochemical Soclety Transactlons ( 1 995) 23 2-Hydroxyethyl a-D-glucopyranoside 2,3',4'-trisphosphate: a novel metabolieally resistant adenophostin A and myo-inositol 1,4,5-trispbosphate analogue potently interacts with tbe myoioositol 1,4,5-trispbospbate receptor.