Leonard R. Stephens

Signalling through Class I PI3Ks in mammalian cells

It is now accepted that activation of Class I PI3Ks (phosphoinositide 3-kinases) is one of the most important signal transduction pathways used by cell-surface receptors to control intracellular events. The receptors which access this pathway include those that recognize growth factors, hormones, antigens and inflammatory stimuli, and the cellular events known to be regulated include cell growth, survival, proliferation and movement. We have learnt a great deal about the family of Class I PI3K enzymes themselves and the structural adaptations which allow a variety of cell-surface receptors to regulate their activity. Class I PI3Ks synthesize the phospholipid PtdIns(3,4,5)P3 in the membranes in which they are activated, and it is now accepted that PtdIns(3,4,5)P3 and its dephosphorylation product PtdIns(3,4)P2 are messenger molecules which regulate the localization and function of multiple effectors by binding to their specific PH (pleckstrin homology) domains. The number of direct PtdIns(3,4,5)P3/PtdIns(3,4)P2 effectors which exist, even within a single cell, creates an extremely complex signalling web downstream of PI3K activation. Some key players are beginning to emerge, however, linking PI3K activity to specific cellular responses. These include small GTPases for the Rho and Arf families which regulate the cytoskeletal and membrane rearrangements required for cell movement, and PKB (protein kinase B), which has important regulatory inputs into the regulation of cell-cycle progression and survival. The importance of the PI3K signalling pathway in regulating the balance of decisions in cell growth, proliferation and survival is clear from the prevalence of oncogenes (e.g. PI3Kalpha) and tumour suppressors [e.g. the PtdIns(3,4,5)P3 3-phosphatase, PTEN (phosphatase and tensin homologue deleted on chromosome 10)] found in this pathway. The recent availability of transgenic mouse models with engineered defects in Class I PI3K signalling pathways, and the development of PI3K isoform-selective inhibitors by both academic and pharmaceutical research has highlighted the importance of specific isoforms of PI3K in whole-animal physiology and pathology, e.g. PI3Kalpha in growth and metabolic regulation, PI3Kbeta in thrombosis, and PI3Kdelta and PI3Kgamma in inflammation and asthma. Thus the Class I PI3K signalling pathway is emerging as an exciting new area for the development of novel therapeutics.

PROTEIN KINASE B AND RAC ARE ACTIVATED IN PARALLEL WITHIN A PHOSPHATIDYLINOSITIDE 3OH-KINASE-CONTROLLED SIGNALING PATHWAY

The GTPase Rac and the protein kinase B (PKB) are downstream targets of phosphatidylinositide 3OH-kinase in platelet-derived growth factor-stimulated signaling pathways. We have generated PAE cell lines inducibly expressing mutants of Rac. Use of these cell lines suggests that Rac is involved in both platelet-derived growth factor-stimulated membrane ruffling and the activation of p70S6K but not in the activation of PKB. Furthermore, expression of constitutively active alleles of PKB in PAE cells suggests that PKB is able to regulate the activity of p70S6K but not the cytoskeletal changes underlying membrane ruffling. Thus, our results indicate that Rac and PKB are on separate pathways downstream of phosphatidylinositide 3OH-kinase in these cells but that both of these pathways are involved in the regulation of p70S6K.

Src Family Kinases Mediate Receptor-stimulated, Phosphoinositide 3-Kinase-dependent, Tyrosine Phosphorylation of Dual Adaptor for Phosphotyrosine and 3-Phosphoinositides-1 in Endothelial and B Cell Lines

DAPP-1 (dual-adaptor for phosphotyrosine and 3-phosphoinositides-1) is a broadly distributed pleckstrin homology (PH) and Src homology 2 domain containing protein that can bind phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) and can be phosphorylated on tyrosine 139 and internalised in response to activation of type I phosphoinositide 3-kinases (PI3K). Tyrosine phosphorylation of DAPP-1 appears important for appropriate intracellular targeting and creates a potential binding site for Src homology 2 domain-containing proteins. In endothelial cells overexpressing wild-type platelet-derived growth factor β (PDGFβ) receptors, which express Bmx and Src as their major Btk (Brutons tyrosine kinase) family and Src family tyrosine kinases, respectively, PDGF can stimulate PI3K-dependent tyrosine phosphorylation of DAPP-1. Transient overexpression of Src most effectively, compared with Bmx and Syk, augments basal and PDGF-stimulated tyrosine phosphorylation of DAPP-1, whereas overexpression of dominant-negative Src, but not dominant-negative Bmx, inhibits PDGF-stimulated phosphorylation of DAPP-1. Cells expressing mutant PDGFβ (Y579F/Y581F) receptors (which fail to bind and activate Src-type kinases) fail to tyrosine phosphorylate DAPP-1 in response to PDGF. We show that in DT40 chicken B cell lines, antibody stimulation leads to PI3K-dependent tyrosine phosphorylation of DAPP-1 that is lost in Lyn- or Syk-deficient cell lines but not Btk-deficient cell lines. PI3K-dependent activation of PKB is only lost in Syk-deficient lines. Finally, in vitro we find lipid-modified Src to be the most effective DAPP-1 tyrosine kinase (versus Syk, Lyn, Btk, and Bmx); phosphorylation of DAPP-1 but not Src autophosphorylation is stimulated ∼10-fold by PtdIns(3,4,5)P3 (IC50 = 150 nm) and phosphatidylinositol 3,4-bisphosphate but not by their nonbiological diastereoisomers and depends on PH domain mediated binding of DAPP-1 to PtdIns(3,4,5)P3-containing membranes. We conclude that Src family kinases are responsible for tyrosine phosphorylation of DAPP-1 in vivo and that PI3K regulation is at the level of PH domain-mediated translocation of DAPP-1 to PI3K products in the membrane.

An inositol 1,4,5-trisphosphate-6-kinase activity in pea roots

A soluble extract from pea (Pisum sativum L.) roots, when incubated with ATP and inositol 1,4,5-trisphosphate, produced an inositol tetrakisphosphate. The chromatographic properties of this inositol tetrakisphosphate, and of the products formed by its chemical degradation, identify it as inositol 1,4,5,6-tetrakisphosphate. No evidence was obtained for a 3-phosphorylation of inositol 1,4,5-trisphosphate. The importance of these observations with respect to inositol phosphates and calcium signalling in higher plants, is discussed.

General synthesis of 3-phosphorylated myo-inositol phospholipids and derivatives

The D-3-phosphorylated myo-inositol phospholipids PtdIns(3)P, PtdIns(3,4)P2, PtdIns(3,4,5)P3 and PtdIns(3,5)P2 were synthesised from myo-inositol orthoformate 8. Key transformations included the regioselective DIBAL- and trimethylaluminium-mediated cleavages of myo-inositol orthoformate intermediates and a resolution–protection protocol using the camphor acetals 17. The final reductive debenzylation was effected with Pearlmans catalyst [Pd(OH)2] in the presence of sodium hydrogen carbonate. The biological properties of the phospholipids were evaluated against various protein kinases (PKB and PDK-1) in which they played an important activation role.

Synthesis and biological evaluation of aPtdIns(3,4,5)P3 affinity matrix

New PtdIns(3,4,5)P3 binding proteins have been identified utilising PtdIns(3,4,5)P3 modified affinity matrix 1 which was synthesised from myo-inositol derivative 2, phosphoramidite 9 and an agarose based solid support.

Synthesis of dipalmitoyl phosphatidylinositol 3,4-bis(phosphate)and 3,4,5-tris(phosphate) and their enantiomers

The dipalmitoyl derivatives 4 and 5 of 3-phosphorylated myo-inositol phospholipids 2 and 3 and their enantiomers are synthesised from homochiral myo-inositol precursors 6 and 11; they serve as biological probes for cell signal transduction.

The roles of PI3Ks in cellular regulation

The term phosphoinositide 30H kinase (PI3K) is given to a family of enzymes which can phosphorylate one or more of the conventional inositol phospholipids found in cells in the 3-position of their inositol headgroup (Fig. 1). It is now clear that these lipids act as regulators of intracellular metabolism and at least one of them, PtdIns(3,4,5)P3, shows all the credentials of being a major ‘second-messenger’ in signalling pathways used by cell-surface receptors for growth factors, inflammatory stimuli and antigens (Stephens et al., 1993; Cantley et al., 1991).