The p53 isoform Δ133p53ß promotes cancer stem cell potential.

Cancer stem cells (CSC) are responsible for cancer chemoresistance and metastasis formation. Here we report that Δ133p53ß, a TP53 splice variant, enhanced cancer cell stemness in MCF-7 breast cancer cells, while its depletion reduced it. Δ133p53ß stimulated the expression of the key pluripotency factors SOX2, OCT3/4, and NANOG. Similarly, in highly metastatic breast cancer cells, aggressiveness was coupled with enhanced CSC potential and Δ133p53ß expression. Like in MCF-7 cells, SOX2, OCT3/4, and NANOG expression were positively regulated by Δ133p53ß in these cells. Finally, treatment of MCF-7 cells with etoposide, a cytotoxic anti-cancer drug, increased CSC formation and SOX2, OCT3/4, and NANOG expression via Δ133p53, thus potentially increasing the risk of cancer recurrence. Our findings show that Δ133p53ß supports CSC potential. Moreover, they indicate that the TP53 gene, which is considered a major tumor suppressor gene, also acts as an oncogene via the Δ133p53ß isoform.

Stability of GABAB receptor oligomers revealed by dual TR-FRET and drug-induced cell surface targeting.

The function of cell surface proteins likely involves the formation and dissociation of oligomeric complexes. However, the dynamics of this process are unknown. Here we examined this process for the GABA(B) receptors that assemble into oligomers of heterodimers through the association of their GABA(B1) subunit. We report a method to study oligomer dynamics based on a drug-controlled cell surface targeting of intracellularly retained receptors and a parallel measurement of two FRET signals in HEK293 cells. GABA(B1) subunits at the cell surface (4.0 ± 0.6 a.u.) are labeled with a pair of fluorophores (donor and red acceptor). New receptors are then targeted to the cell surface during 3h treatment with AP21967 such that the number of receptors is doubled (9.1 ± 0.7 a.u.). After labeling these new receptors with a second acceptor (green), the red FRET remained unchanged (5189 ± 36 vs. 4783 ± 32 cps), supporting the stability of the preformed oligomers. However, new oligomers are detected by the green FRET signal indicating both receptor populations are in the same microdomains. As a control, we confirmed the strict stability of the GABA(B) heterodimer itself. Herein, using a novel method to monitor the dynamics of cell surface complexes, we provide evidence for the stability of GABA(B) oligomers.

Ras is an indispensable coregulator of the class IB phosphoinositide 3-kinase p87/p110gamma.

Class I(B) phosphoinositide 3-kinase gamma (PI3Kgamma) elicits various immunologic and cardiovascular responses; however, the molecular basis for this signal heterogeneity is unclear. PI3Kgamma consists of a catalytic p110gamma and a regulatory p87(PIKAP) (p87, also p84) or p101 subunit. Hitherto p87 and p101 are generally assumed to exhibit redundant functions in receptor-induced and G protein betagamma (Gbetagamma)-mediated PI3Kgamma regulation. Here we investigated the molecular mechanism for receptor-dependent p87/p110gamma activation. By analyzing GFP-tagged proteins expressed in HEK293 cells, PI3Kgamma-complemented bone marrow-derived mast cells (BMMCs) from p110gamma(-/-) mice, and purified recombinant proteins reconstituted to lipid vesicles, we elucidated a novel pathway of p87-dependent, G protein-coupled receptor (GPCR)-induced PI3Kgamma activation. Although p101 strongly interacted with Gbetagamma, thereby mediating PI3Kgamma membrane recruitment and stimulation, p87 exhibited only a weak interaction, resulting in modest kinase activation and lack of membrane recruitment. Surprisingly, Ras-GTP substituted the missing Gbetagamma-dependent membrane recruitment of p87/p110gamma by direct interaction with p110gamma, suggesting the indispensability of Ras for activation of p87/p110gamma. Consequently, interference with Ras signaling indeed selectively blocked p87/p110gamma, but not p101/p110gamma, kinase activity in HEK293 and BMMC cells, revealing an important crosstalk between monomeric and trimeric G proteins for p87/p110gamma activation. Our data display distinct signaling requirements of p87 and p101, conferring signaling specificity to PI3Kgamma that could open up new possibilities for therapeutic intervention.

Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization.

Cell-surface proteins are important in cell-cell communication. They assemble into heterocomplexes that include different receptors and effectors. Elucidation and manipulation of such protein complexes offers new therapeutic possibilities. We describe a methodology combining time-resolved fluorescence resonance energy transfer (FRET) with snap-tag technology to quantitatively analyze protein-protein interactions at the surface of living cells, in a high throughput-compatible format. Using this approach, we examined whether G protein-coupled receptors (GPCRs) are monomers or assemble into dimers or larger oligomers--a matter of intense debate. We obtained evidence for the oligomeric state of both class A and class C GPCRs. We also observed different quaternary structure of GPCRs for the neurotransmitters glutamate and gamma-aminobutyric acid (GABA): whereas metabotropic glutamate receptors assembled into strict dimers, the GABA(B) receptors spontaneously formed dimers of heterodimers, offering a way to modulate G-protein coupling efficacy. This approach will be useful in systematic analysis of cell-surface protein interaction in living cells.

Activation of a dimeric metabotropic glutamate receptor by intersubunit rearrangement.

Although many G protein-coupled receptors (GPCRs) can form dimers, a possible role of this phenomenon in their activation remains elusive. A recent and exciting proposal is that a dynamic intersubunit interplay may contribute to GPCR activation. Here, we examined this possibility using dimeric metabotropic glutamate receptors (mGluRs). We first developed a system to perfectly control their subunit composition and show that mGluR dimers do not form larger oligomers. We then examined an mGluR dimer containing one subunit in which the extracellular agonist-binding domain was uncoupled from the G protein-activating transmembrane domain. Despite this uncoupling in one protomer, agonist stimulation resulted in symmetric activation of either transmembrane domain in the dimer with the same efficiency. This, plus other data, can only be explained by an intersubunit rearrangement as the activation mechanism. Although well established for other types of receptors such as tyrosine kinase and guanylate cyclase receptors, this is the first clear demonstration that such a mechanism may also apply to GPCRs.

Assembly-dependent surface targeting of the heterodimeric GABAB Receptor is controlled by COPI but not 14-3-3.

Cell surface expression of transmembrane proteins is strictly regulated. Mutually exclusive interaction with COPI or 14-3-3 proteins has been proposed as a mechanism underlying such trafficking control of various proteins. In particular, 14-3-3 dimers have been proposed to "sense" correctly assembled oligomers, allowing their surface targeting by preventing COPI-mediated intracellular retention. Here we examined whether such a mechanism is involved in the quality control of the heterodimeric G protein-coupled GABAB receptor. Its GB1 subunit, carrying the retention signal RSR, only reaches the cell surface when associated with the GB2 subunit. We show that COPI and 14-3-3 specifically bind to the GB1 RSR sequence and that COPI is involved in its intracellular retention. However, we demonstrate that the interaction with 14-3-3 is not required for proper function of the GABAB receptor quality control. Accordingly, competition between 14-3-3 and COPI cannot be considered as a general trafficking control mechanism. A possible other role for competition between COPI and 14-3-3 binding is discussed.

Assigning functional domains within the p101 regulatory subunit of phosphoinositide 3-kinase gamma.

Phosphoinositide 3-Kinase (PI3K) gamma is a lipid kinase that is regulated by G-protein-coupled receptors. It plays a crucial role in inflammatory and allergic processes. Activation of PI3Kgamma is primarily mediated by Gbetagamma subunits. The regulatory p101 subunit of PI3Kgamma binds to Gbetagamma and, thereby, recruits the catalytic p110gamma subunit to the plasma membrane. Despite its crucial role in the activation of PI3Kgamma, the structural organization of p101 is still largely elusive. Employing fluorescence resonance energy transfer measurements, coimmunoprecipitation and colocalization studies with p101 deletion mutants, we show here that distinct regions within the p101 primary structure are responsible for interaction with p110gamma and Gbetagamma. The p110gamma binding site is confined to the N terminus, whereas binding to Gbetagamma is mediated by a C-terminal domain of p101. These domains appear to be highly conserved among various species ranging from Xenopus to men. In addition to establishing a domain structure for p101, our results point to the existence of a previously unknown, p101-related regulatory subunit for PI3Kgamma.

Rho GTPases and phosphoinositide 3-kinase organize formation of branched dendrites.

Neurons receive information from other neurons via their dendritic tree. Dendrites and their branches result from alternating outgrowth and retraction. The Rho GTPases Rac and Cdc42 (cell division cycle 42) facilitate the outgrowth of branches, whereas Rho attenuates it. The mechanism of neurite retraction is unknown. Because the adenylyl cyclase activator forskolin causes numerous branched extensions in NG108-15 cells, we have investigated the underlying mechanism in this cell line. In additional studies, we used cultured hippocampal neurons in which forskolin induces branched dendrites. In both cell types, forskolin enhanced the activity of Cdc42, but not that of Rac, although both GTPases were necessary for the formation of branched extensions. Time lapse microscopy showed that forskolin did not increase the rate of addition of new extensions or branches, but it reduced the rate of the retraction so that more branched extensions persisted. Inhibition of phosphoinositide 3-kinase activity by wortmannin or LY294002 also reduced the rate of retraction and thus facilitated dendritic arborization. Forskolin diminished the activity of phosphoinositide 3-kinases. Inhibitors of phosphoinositide 3-kinases not only reduced the retraction but also the addition of new dendrites and branches. This reduction was no longer present when Rho kinase was simultaneously inactivated, suggesting an interaction of phosphoinositide 3-kinases and Rho kinase. The present results show a central role of phosphoinositide 3-kinases in dendrite formation. In neuronal cells, increased levels of cAMP can support dendritic arborization by modulating the activity of the lipid kinase.

Roles of G beta gamma in membrane recruitment and activation of p110 gamma/p101 phosphoinositide 3-kinase gamma.

Receptor-regulated class I phosphoinositide 3-kinases (PI3K) phosphorylate the membrane lipid phosphatidylinositol (PtdIns)-4,5-P2 to PtdIns-3,4,5-P3. This, in turn, recruits and activates cytosolic effectors with PtdIns-3,4,5-P3-binding pleckstrin homology (PH) domains, thereby controlling important cellular functions such as proliferation, survival, or chemotaxis. The class IB p110 gamma/p101 PI3K gamma is activated by G beta gamma on stimulation of G protein-coupled receptors. It is currently unknown whether in living cells G beta gamma acts as a membrane anchor or an allosteric activator of PI3K gamma, and which role its noncatalytic p101 subunit plays in its activation by G beta gamma. Using GFP-tagged PI3K gamma subunits expressed in HEK cells, we show that G beta gamma recruits the enzyme from the cytosol to the membrane by interaction with its p101 subunit. Accordingly, p101 was found to be required for G protein-mediated activation of PI3K gamma in living cells, as assessed by use of GFP-tagged PtdIns-3,4,5-P3-binding PH domains. Furthermore, membrane-targeted p110 gamma displayed basal enzymatic activity, but was further stimulated by G beta gamma, even in the absence of p101. Therefore, we conclude that in vivo, G beta gamma activates PI3K gamma by a mechanism assigning specific roles for both PI3K gamma subunits, i.e., membrane recruitment is mediated via the noncatalytic p101 subunit, and direct stimulation of G beta gamma with p110 gamma contributes to activation of PI3K gamma.

Identification and characterization of the autophosphorylation sites of phosphoinositide 3-kinase isoforms beta and gamma.

Class I phosphoinositide 3-kinases (PI3Ks) are bifunctional enzymes possessing lipid kinase activity and the capacity to phosphorylate their catalytic and/or regulatory subunits. In this study, in vitro autophosphorylation of the G protein-sensitive p85-coupled class I(A) PI3K beta and p101-coupled class I(B) PI3K gamma was examined. Autophosphorylation sites of both PI3K isoforms were mapped to C-terminal serine residues of the catalytic p110 subunit (i.e. serine 1070 of p110 beta and serine 1101 of p110 gamma). Like other class I(A) PI3K isoforms, autophosphorylation of p110 beta resulted in down-regulated PI3K beta lipid kinase activity. However, no inhibitory effect of p110 gamma autophosphorylation on PI3K gamma lipid kinase activity was observed. Moreover, PI3K beta and PI3K gamma differed in the regulation of their autophosphorylation. Whereas p110 beta autophosphorylation was stimulated neither by G beta gamma complexes nor by a phosphotyrosyl peptide derived from the platelet-derived growth factor receptor, autophosphorylation of p110 gamma was significantly enhanced by G beta gamma in a time- and concentration-dependent manner. In summary, we show that autophosphorylation of both PI3K beta and PI3K gamma occurs in a C-terminal region of the catalytic p110 subunit but differs in its regulation and possible functional consequences, suggesting distinct roles of autophosphorylation of PI3K beta and PI3K gamma.