Alex Toker

Signalling through the lipid products of phosphoinositide-3-OH kinase

When a stimulatory agonist molecule binds at the exterior of the cell membrane, a second messenger transduces the signal to the interior of the cell. Second messengers can be derived from phospholipids in the membrane by the action of the enzymes phospholipase C or phosphoinositide-3-OH kinase (PI(3)K). PI(3)K is a key player in many cellular responses, including the movement of organelle membranes, shape alteration through rearrangement of cytoskeletal actin, transformation and chemotaxis. But how PI(3)K mediates these responses is only now becoming clear.

Sequence analysis of mutations and translocations across breast cancer subtypes

Breast carcinoma is the leading cause of cancer-related mortality in women worldwide, with an estimated 1.38 million new cases and 458,000 deaths in 2008 alone. This malignancy represents a heterogeneous group of tumours with characteristic molecular features, prognosis and responses to available therapy. Recurrent somatic alterations in breast cancer have been described, including mutations and copy number alterations, notably ERBB2 amplifications, the first successful therapy target defined by a genomic aberration. Previous DNA sequencing studies of breast cancer genomes have revealed additional candidate mutations and gene rearrangements. Here we report the whole-exome sequences of DNA from 103 human breast cancers of diverse subtypes from patients in Mexico and Vietnam compared to matched-normal DNA, together with whole-genome sequences of 22 breast cancer/normal pairs. Beyond confirming recurrent somatic mutations in PIK3CA, TP53, AKT1, GATA3 and MAP3K1, we discovered recurrent mutations in the CBFB transcription factor gene and deletions of its partner RUNX1. Furthermore, we have identified a recurrent MAGI3–AKT3 fusion enriched in triple-negative breast cancer lacking oestrogen and progesterone receptors and ERBB2 expression. The MAGI3–AKT3 fusion leads to constitutive activation of AKT kinase, which is abolished by treatment with an ATP-competitive AKT small-molecule inhibitor.

Thrombin receptor ligation and activated rac uncap actin filament barbed ends through phosphoinositide synthesis in permeabilized human platelets

Cells respond to diverse external stimuli by polymerizing cytoplasmic actin, and recent evidence indicates that GTPases can specify where this polymerization takes place. Actin assembly in stimulated blood platelets occurs where sequestered monomers add onto the fast-growing (barbed) ends of actin filaments (F-actin), which are capped in the resting cells. We report that D3 and D4 polyphosphoinositides, Pl(4)P, Pl(4,5)P2, Pl(3,4)P2, and Pl(3,4,5)P3, uncap F-actin in resting permeabilized platelets. The thrombin receptor-activating peptide (TRAP), GTP, and GTP gamma S, but not GDP beta S, also uncap F-actin in permeabilized platelets. GDP beta S inhibits TRAP-induced F-actin uncapping, and Pl(4,5)P2 overcomes this inhibition. Constitutively active mutant Rac, but not Rho, activates uncapping of F-actin. Pl(4,5)P2-binding peptides derived from gelsolin inhibit F-actin uncapping by TRAP, Rac, and GTP gamma S. TRAP and Rac induce rapid Pl(4,5)P2 synthesis in permeabilized platelets. The findings establish a signaling pathway for actin assembly involving Rac in which the final message is phosphoinositide-mediated F-actin uncapping.

Activation of Phosphoinositide 3-OH Kinase by the α6β4 Integrin Promotes Carcinoma Invasion

We demonstrate that the alpha6beta4 integrin promotes carcinoma invasion through a preferential and localized targeting of phosphoinositide-3 OH kinase (PI3K) activity. Stable expression of alpha6beta4 increased carcinoma invasion in a PI3K-dependent manner, and transient expression of a constitutively active PI3K increased invasion in the absence of alpha6beta4. Ligation of alpha6beta4 stimulated significantly more PI3K activity than ligation of beta1 integrins, establishing specificity among integrins for PI3K activation. Alpha6beta4-regulated PI3K activity was required for the formation of lamellae, dynamic sites of motility, in carcinoma cells. The small G protein Rac is required downstream of PI3K for invasion. These studies define a mechanism by which the alpha6beta4 integrin promotes carcinoma invasion and invoke a novel function for PI3K signaling.

Regulation of protein kinase C ζ by PI 3-kinase and PDK-1

BACKGROUND Protein kinase C zeta (PKC zeta) is a member of the PKC family of enzymes and is involved in a wide range of physiological processes including mitogenesis, protein synthesis, cell survival and transcriptional regulation. PKC zeta has received considerable attention recently as a target of phosphoinositide 3-kinase (PI 3-kinase), although the mechanism of PKC zeta activation is, as yet, unknown. Recent reports have also shown that the phosphoinositide-dependent protein kinase-1 (PDK-1), which binds with high affinity to the PI 3-kinase lipid product phosphatidylinositol-3,4,5-trisphosphate (Ptdins-3,4,5-P3), phosphorylates and potently activates two other PI 3-kinase targets, the protein kinases Akt/PKB and p70S6K. We therefore investigated whether PDK-1 is the kinase that activates PKC zeta. RESULTS In vivo, PI 3-kinase is both necessary and sufficient to activate PKC zeta. PDK-1 phosphorylates and activates PKC zeta in vivo, and we have shown that this is due to phosphorylation of threonine 410 in the PKC zeta activation loop. In vitro, PDK-1 phosphorylates and activates PKC zeta in a Ptdins-3,4,5-P3-enhanced manner. PKC zeta and PDK-1 are associated in vivo, and membrane targeting of PKC zeta renders it constitutively active in cells. CONCLUSIONS Our results have identified PDK-1 as the kinase that phosphorylates and activates PKC zeta in the PI 3-kinase signaling pathway. This phosphorylation and activation of PKC zeta by PDK-1 is enhanced in the presence of Ptdins-3,4-5-P3. Consistent with the notion that PKCs are enzymes that are regulated at the plasma membrane, a membrane-targeted PKC zeta is constitutively active in the absence of agonist stimulation. The association between PKC zeta and PDK-1 reveals extensive cross-talk between enzymes in the PI 3-kinase signaling pathway.

Determination of the Specific Substrate Sequence Motifs of Protein Kinase C Isozymes

Protein kinase C (PKC) family members play significant roles in a variety of intracellular signal transduction processes, but information about the substrate specificities of each PKC family member is quite limited. In this study, we have determined the optimal peptide substrate sequence for each of nine human PKC isozymes (α, βI, βII, γ, δ, ε, η, μ, and ζ) by using an oriented peptide library. All PKC isozymes preferentially phosphorylated peptides with hydrophobic amino acids at position +1 carboxyl-terminal of the phosphorylated Ser and basic residues at position −3. All isozymes, except PKCμ, selected peptides with basic amino acids at positions −6, −4, and −2. PKCα, -βI, -βII, -γ, and -η selected peptides with basic amino acid at positions +2, +3, and +4, but PKCδ, -ε, -ζ, and -μ preferred peptides with hydrophobic amino acid at these positions. At position −5, the selectivity was quite different among the various isozymes; PKCα, -γ, and -δ selected peptides with Arg at this position while other PKC isozymes selected hydrophobic amino acids such as Phe, Leu, or Val. Interestingly, PKCμ showed extreme selectivity for peptides with Leu at this position. The predicted optimal sequences from position −3 to +2 for PKCα, -βI, -βII, -γ, -δ, and -η were very similar to the endogenous pseudosubstrate sequences of these PKC isozymes, indicating that these core regions may be important to the binding of corresponding substrate peptides. Synthetic peptides based on the predicted optimal sequences for PKCα, -βI, -δ, -ζ, and -μ were prepared and used for the determination of Km and Vmax for these isozymes. As judged by Vmax/Km values, these peptides were in general better substrates of the corresponding isozymes than those of the other PKC isozymes, supporting the idea that individual PKC isozymes have distinct optimal substrates. The structural basis for the selectivity of PKC isozymes is discussed based on residues predicted to form the catalytic cleft.

Cellular Signaling: Pivoting around PDK-1

the discovery that PDK-1 is the Akt/PKB upstream ki-Harvard Medical School nase came the observation that PDK-1 also phosphory-Boston, Massachusetts 02215 lates a number of other kinases, including p70S6-kinase † Department of Pharmacology (p70S6-K) and protein kinase C (PKC) (Figure 1) (for a relieves autoinhibition of the ac-hormones. Deregulation of this pathway is associated tive site, allowing PDK-1 to access Thr308 on the activa-with human diseases such as cancer and diabetes. The tion loop (Stokoe et al., 1997). Consistent with this, an importance of this pathway in cell biology is under-Akt/PKB mutant lacking the PH domain no longer re-scored by the fact that PI3K signaling influences both quires PtdIns-3,4,5-P 3 for PDK-1-mediated phosphory-cell survival and death, in addition to other fundamental lation in vitro and is constitutively phosphorylated and cellular functions such as growth, motility, differentia-active in cells (Filippa et al., 2000). Thus, the PH domain tion and insulin action. It does so by activating multiple masks the activation loop site and its release is required distinct secondary signaling cascades, and consider-for PDK-1 phosphorylation. able information exists about the precise biochemical Similarly, access of PDK-1 to the activation loop of mechanisms by which PI3K mediates these events. One PKC is conformationally regulated. In this case, the au-group of enzymes that has emerged as a key mediator toinhibitory pseudosubstrate sequence of PKC must be of the PI3K signal is the AGC superfamily of serine/ removed from the substrate binding cavity in order for threonine protein kinases (so named because it includes PDK-1 to phosphorylate PKC. The phosphorylation by protein kinases A, G, and C), long known to be critical PDK-1 of PRK/PKN is also conformationally regulated: components of the signal transduction machinery. Most these kinases require interaction with the small GTPase members of this family require an activating phosphory-Rho to induce a conformational change which enables lation, setting off the search for a potential upstream PDK-1 binding and phosphorylation at the activation kinase that was linked to the PI3K pathway. loop. In addition, binding of sphingosine to PAK has The search for such a kinase culminated with the recently been proposed to alter PAK in a manner that discovery in 1997 of a novel member of the AGC fam-permits phosphorylation of this kinase by PDK-1. Lastly, ily, the phosphoinositide-dependent kinase-1 (PDK-1). p70S6-K requires prior phosphorylation on its autoinhib-PDK-1 has now been shown to stand at a pivotal point in itory sequence …

Regulation of conventional protein kinase C isozymes by phosphoinositide-dependent kinase 1 (PDK-1).

BACKGROUND Phosphorylation critically regulates the catalytic function of most members of the protein kinase superfamily. One such member, protein kinase C (PKC), contains two phosphorylation switches: a site on the activation loop that is phosphorylated by another kinase, and two autophosphorylation sites in the carboxyl terminus. For conventional PKC isozymes, the mature enzyme, which is present in the detergent-soluble fraction of cells, is quantitatively phosphorylated at the carboxy-terminal sites but only partially phosphorylated on the activation loop. RESULTS This study identifies the recently discovered phosphoinositide-dependent kinase 1, PDK-1, as a regulator of the activation loop of conventional PKC isozymes. First, studies in vivo revealed that PDK-1 controls the amount of mature (carboxy-terminally phosphorylated) conventional PKC. More specifically, co-expression of the conventional PKC isoform PKC betaII with a catalytically inactive form of PDK-1 in COS-7 cells resulted in both the accumulation of non-phosphorylated PKC and a corresponding decrease in PKC activity. Second, studies in vitro using purified proteins established that PDK-1 specifically phosphorylates the activation loop of PKC alpha and betaII. The phosphorylation of the mature PKC enzyme did not modulate its basal activity or its maximal cofactor-dependent activity. Rather, the phosphorylation of non-phosphorylated enzyme by PDK-1 triggered carboxy-terminal phosphorylation of PKC, thus providing the first step in the generation of catalytically competent (mature) enzyme. CONCLUSIONS We have shown that PDK-1 controls the phosphorylation of conventional PKC isozymes in vivo. Studies performed in vitro establish that PDK-1 directly phosphorylates PKC on the activation loop, thereby allowing carboxy-terminal phosphorylation of PKC. These data suggest that phosphorylation of the activation loop by PDK-1 provides the first step in the processing of conventional PKC isozymes by phosphorylation.

Signaling through protein kinase C.

Protein kinase C (PKC) comprises a large family of serine/threonine kinases which are activated by many extracellular signals. Inside the cell, PKCs are regulated by a variety of lipid second messengers, including the ubiquitous diacylglycerol and phosphatidylserine. Phosphorylation has also emerged as an important mechanism of regulation of all PKCs. Work in the last 20 years has provided evidence that these enzymes are involved in a multitude of physiological processes. Similarly, a number of proteins which are phosphorylated by PKCs have also been discovered and their role in cell biology has been investigated. More recently, there has been considerable interest in a number of specific PKC isoforms and their role in signaling pathways in the cell. This review will focus on recent findings on the mechanism of regulation of PKCepsilon, PKCmu and PKCzeta, and how these enzymes regulate cell growth, the actin cytoskeleton, apoptosis and other biological functions.

The role of NFAT transcription factors in integrin-mediated carcinoma invasion

Integrins, receptors for extracellular matrix ligands, are critical regulators of the invasive phenotype. Specifically, the α6β4 integrin has been linked with epithelial cell motility, cellular survival and carcinoma invasion, hallmarks of metastatic tumours. Previous studies have also shown that antagonists of the NFAT (nuclear factor of activated T-cells) family of transcription factors exhibit strong anti-tumour-promoting activity. This suggests that NFAT may function in tumour metastasis. Here, we investigate the involvement of NFAT in promoting carcinoma invasion downstream of the α6β4 integrin. We provide evidence that both NFAT1, and the recently identified NFAT5 isoform, are expressed in invasive human ductal breast carcinomas and participate in promoting carcinoma invasion using cell lines derived from human breast and colon carcinomas. NFAT1 and NFAT5 activity correlates with the expression of the α6β4 integrin. In addition, the transcriptional activity of NFAT5 is induced by α6β4 clustering in the presence of chemo-attractants, resulting in enhanced cell migration. These observations show that NFATs are targets of α6β4 integrin signalling and are involved in promoting carcinoma invasion, highlighting a novel function for this family of transcription factors in human cancer.