Distinct, opposing functions for CFIm59 and CFIm68 in mRNA alternative polyadenylation of Pten and in the PI3K/Akt signalling cascade.

Precise maintenance of PTEN dosage is crucial for tumor suppression across a wide variety of cancers. Post-transcriptional regulation of Pten heavily relies on regulatory elements encoded by its 3'UTR. We previously reported the important diversity of 3'UTR isoforms of Pten mRNAs produced through alternative polyadenylation (APA). Here, we reveal the direct regulation of Pten APA by the mammalian cleavage factor I (CFIm) complex, which in turn contributes to PTEN protein dosage. CFIm consists of the UGUA-binding CFIm25 and APA regulatory subunits CFIm59 or CFIm68. Deep sequencing analyses of perturbed (KO and KD) cell lines uncovered the differential regulation of Pten APA by CFIm59 and CFIm68 and further revealed that their divergent functions have widespread impact for APA in transcriptomes. Differentially regulated genes include numerous factors within the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signalling pathway that PTEN counter-regulates. We further reveal a stratification of APA dysregulation among a subset of PTEN-driven cancers, with recurrent alterations among PI3K/Akt pathway genes regulated by CFIm. Our results refine the transcriptome selectivity of the CFIm complex in APA regulation, and the breadth of its impact in PTEN-driven cancers.

A Novel "Prebinding" Strategy Dramatically Enhances Sortase-Mediated Coupling of Proteins to Liposomes.

We have examined quantitatively the efficiency and the kinetics of sortase A-mediated coupling of model substrate proteins (derived from green fluorescent protein and the SNAP variant of O-alkylguanine-DNA alkyltransferase) to large unilamellar liposomes incorporating low levels of oligopeptide-modified acceptor lipids. Under normal reaction conditions, even using high concentrations of S. aureus or S. pyogenes sortase A and optimal protein coupling substrates and acceptor lipids, protein-liposome coupling is slow, gives at best modest coupling yields, and is markedly limited by the hydrolytic activity of sortase. We demonstrate, however, that these limitations can be overcome under "prebinding" conditions that promote initial reversible association of sortase and the substrate protein with the liposome surface. Using oligohistidine-tagged sortase and substrate proteins and liposomes incorporating an acceptor lipid together with a Ni(II)-chelating lipid derivative, high coupling rates and yields can be obtained at low sortase concentrations, while virtually eliminating adverse effects of sortase hydrolytic activity on protein coupling. The prebinding approach described here can readily be adapted, and if necessary rendered virtually "traceless", to accommodate diverse protein coupling substrates and end uses of the protein-modified liposomes.

Assessment of the roles of ordered lipid microdomains in post-endocytic trafficking of glycosyl-phosphatidylinositol-anchored proteins in mammalian fibroblasts.

We have used artificial phosphatidylethanolamine-polyethylene glycol (PE-PEG)-anchored proteins, incorporated into living mammalian cells, to evaluate previously proposed roles for ordered lipid 'raft' domains in the post-endocytic trafficking of glycosylphosphatidylinositol (GPI)-anchored proteins in CHO and BHK cells. In CHO cells, endocytosed PE-PEG protein conjugates colocalized strongly with the internalized GPI-anchored folate receptor, concentrating in the endosomal recycling compartment, regardless of the structure of the hydrocarbon chains of the PE-PEG 'anchor'. However, internalized PE-PEG protein conjugates with long-chain saturated anchors recycled to the plasma membrane at a slow rate comparable to that measured for the GPI-anchored folate receptor, whereas conjugates with short-chain or unsaturated anchors recycled at a faster rate similar to that observed for the transferrin receptor. These findings support the proposal (Mayor et al. Cholesterol-dependent retention of GPI-anchored proteins in endosomes. EMBO J 1998;17:4628-4638) that the slow recycling of GPI proteins in CHO cells rests on their affinity for ordered lipid domains. In BHK cells, internalized PE-PEG protein conjugates with either saturated or unsaturated 'anchors' colocalized strongly with simultaneously endocytosed folate receptor and, like the folate receptor, gradually accumulated in late endosomes/lysosomes. These latter findings do not support previous suggestions that the sorting of GPI proteins to late endosomes in BHK cells depends on their association with lipid rafts.

Quantitative experimental assessment of macromolecular crowding effects at membrane surfaces.

We examined how crowding of the surfaces of lipid vesicles with either grafted polyethyleneglycol (PEG) chains or bilayer-anchored protein molecules affects the binding of soluble proteins to the vesicle surface. Escherichia coli dihydrofolate reductase (DHFR, 18 kDa) or a larger fusion protein, NusA-DHFR (72 kDa), binds reversibly but with high affinity to a methotrexate-modified lipid (MTX-PE) incorporated into large unilamellar vesicles. Incorporation of phosphatidylethanolamine-PEG5000 into the vesicles strongly decreases the affinity of binding of both proteins, to a degree that varies roughly exponentially with the lateral density of the PEG chains. Covalently coupling maltose-binding protein (MBP) to the vesicle surfaces also strongly decreases the affinity of binding of NusDHFR or DHFR, to a degree that likewise varies roughly exponentially with the surface density of anchored MBP. Surface-coupled MBP strongly decreases the rate of binding of NusDHFR to MTX-PE-incorporating vesicles but does not affect the rate of NusDHFR dissociation. The large magnitudes of these effects (easily exceeding an order of magnitude for moderate degrees of surface crowding) support previous theoretical analyses and suggest that surface-crowding effects can markedly influence a variety of important aspects of protein behavior in membranes.

Multiple cellular proteins modulate the dynamics of K-ras association with the plasma membrane.

Although specific proteins have been identified that regulate the membrane association and facilitate intracellular transport of prenylated Rho- and Rab-family proteins, it is not known whether cellular proteins fulfill similar roles for other prenylated species, such as Ras-family proteins. We used a previously described method to evaluate how several cellular proteins, previously identified as potential binding partners (but not effectors) of K-ras4B, influence the dynamics of K-ras association with the plasma membrane. Overexpression of either PDEδ or PRA1 enhances, whereas knockdown of either protein reduces, the rate of dissociation of K-ras from the plasma membrane. Inhibition of calmodulin likewise reduces the rate of K-ras dissociation from the plasma membrane, in this case in a manner specific for the activated form of K-ras. By contrast, galectin-3 specifically reduces the rate of plasma membrane dissociation of activated K-ras, an effect that is blocked by the K-ras antagonist farnesylthiosalicylic acid (salirasib). Multiple cellular proteins thus control the dynamics of membrane association and intercompartmental movement of K-ras to an important degree even under basal cellular conditions.

K-ras4B and prenylated proteins lacking "second signals" associate dynamically with cellular membranes.

We have used fluorescence microscopy and the technique of rapamycin-regulated protein heterodimerization to examine the dynamics of the subcellular localizations of fluorescent proteins fused to lipid-modified protein sequences and to wild-type and mutated forms of full-length K-ras4B. Singly prenylated or myristoylated fluorescent protein derivatives lacking a "second signal" to direct them to specific subcellular destinations, but incorporating a rapamycin-dependent heterodimerization module, rapidly translocate to mitochondria upon rapamycin addition to bind to a mitochondrial outer membrane protein incorporating a complementary heterodimerization module. Under the same conditions analogous constructs anchored to the plasma membrane by multiply lipid-modified sequences, or by a transmembrane helix, show very slow or no transfer to mitochondria, respectively. Interestingly, however, fluorescent protein constructs incorporating either full-length K-ras4B or its plasma membrane-targeting sequence alone undergo rapamycin-induced transfer from the plasma membrane to mitochondria on a time scale of minutes, demonstrating the rapidly reversible nature of K-ras4B binding to the plasma membrane. The dynamic nature of the plasma membrane targeting of K-ras4B could contribute to K-ras4B function by facilitating redistribution of the protein between subcellular compartments under particular conditions.

Incorporation of Artificial Lipid-Anchored Proteins into Cultured Mammalian Cells.

Exogenous lipid-anchored proteins can be incorporated into the plasma membranes of living mammalian cells, allowing the chemical structure of the incorporated protein and its lipid anchor to be controlled (and varied) to a much greater degree than is possible for proteins expressed by the cells themselves. This technology offers a variety of potential applications, including incorporating novel and complex protein constructs into cell surfaces and exploring structure-function relationships for biologically important lipid-anchored proteins such as glycosylphosphatidylinositol-anchored proteins. Here we describe detailed methods for stable incorporation of artificial lipid-anchored proteins into cultured mammalian cells under mild, nonperturbing conditions.

Steric and not structure-specific factors dictate the endocytic mechanism of glycosylphosphatidylinositol-anchored proteins.

Diverse glycosylphosphatidylinositol (GPI)-anchored proteins enter mammalian cells via the clathrin- and dynamin-independent, Arf1-regulated GPI-enriched early endosomal compartment/clathrin-independent carrier endocytic pathway. To characterize the determinants of GPI protein targeting to this pathway, we have used fluorescence microscopic analyses to compare the internalization of artificial lipid-anchored proteins, endogenous membrane proteins, and membrane lipid markers in Chinese hamster ovary cells. Soluble proteins, anchored to cell-inserted saturated or unsaturated phosphatidylethanolamine (PE)-polyethyleneglycols (PEGs), closely resemble the GPI-anchored folate receptor but differ markedly from the transferrin receptor, membrane lipid markers, and even protein-free PE-PEGs, both in their distribution in peripheral endocytic vesicles and in the manner in which their endocytic uptake responds to manipulations of cellular Arf1 or dynamin activity. These findings suggest that the distinctive endocytic targeting of GPI proteins requires neither biospecific recognition of their GPI anchors nor affinity for ordered-lipid microdomains but is determined by a more fundamental property, the steric bulk of the lipid-anchored protein.

Artificially lipid-anchored proteins can elicit clustering-induced intracellular signaling events in Jurkat T-lymphocytes independent of lipid raft association.

We have incorporated artificial lipid-anchored streptavidin conjugates with fully saturated or polyunsaturated lipid anchors into the plasma membranes of Jurkat T-lymphocytes to assess previous conclusions that the activation of signaling processes induced in these cells by clustering of endogenous glycosylphosphatidylinositol-anchored proteins or ganglioside GM1 depends specifically on the association of these membrane components with lipid rafts. Lipid-anchored streptavidin conjugates could be incorporated into Jurkat or other mammalian cell surfaces by inserting biotinylated phosphatidylethanolamine-polyethyleneglycols (PE-PEGs) and subsequently binding streptavidin to the cell-incorporated PE-PEGs. Saturated dipalmitoyl-PE-PEG-streptavidin conjugates prepared in this manner partitioned substantially into the detergent-insoluble membrane fraction isolated from Jurkat or fibroblast cells, whereas polyunsaturated dilinoleoyl-PE-PEG-anchored conjugates were wholly excluded from this fraction, consistent with the differences in the affinities of the two types of lipid anchors for liquid-ordered membrane domains. Remarkably, however, antibody-mediated cross-linking of either dipalmitoyl- or dilinoleoyl-PE-PEG-anchored streptavidin conjugates in Jurkat cells induced elevation of cytoplasmic calcium levels and tyrosine phosphorylation of the scaf-folding protein linker of T-cell activation in a manner similar to that observed upon cross-linking of endogenous CD59 or ganglioside GM1. The amplitude of the cross-linking-stimulated elevation of cytoplasmic calcium moreover showed an essentially identical dependence on the level of incorporated streptavidin conjugate for either type of lipid anchor. Confocal fluorescence microscopy revealed that PE-PEG-streptavidin conjugates with saturated versus polyunsaturated anchors showed very similar surface distributions vis à vis GM1 or CD59 under conditions where one or both species were cross-linked. These results indicate that cross-linking of diverse proteins anchored only to the outer leaflet of the plasma membrane can induce activation of Jurkat T-cell-signaling responses, but they appear to contradict previous suggestions that this phenomenon rests specifically on the association of such species with lipid rafts.