No abstract

The central role of protein kinases in signal transduction pathways has generated intense interest in targeting these enzymes for a wide range of therapeutic indications. Here we report a method for identifying and quantifying protein kinases in any biological sample or tissue from any species. The procedure relies on acyl phosphate-containing nucleotides, prepared from a biotin derivative and ATP or ADP. The acyl phosphate probes react selectively and covalently at the ATP binding sites of at least 75% of the known human protein kinases. Biotinylated peptide fragments from labeled proteomes are captured and then sequenced and identified using a mass spectrometry-based analysis platform to determine the kinases present and their relative levels. Further, direct competition between the probes and inhibitors can be assessed to determine inhibitor potency and selectivity against native protein kinases, as well as hundreds of other ATPases. The ability to broadly profile kinase activities in native proteomes offers an exciting prospect for both target discovery and inhibitor selectivity profiling.

Micrometer-size patterned lipid bilayers containing liganded lipids are used to control the location and size of receptor clusters and enable direct visualization of structural reorganization of cellular components. Subsequent to concentration of Fc receptor I, the mast cell receptor for IgE, and colocalized tyrosine phosphorylation activity, Lyn kinase and other proteins anchored to the inner leaflet of the plasma membrane redistribute selectively with the receptor clusters in a process that depends on actin polymerization. Surprisingly, outer leaflet components characteristically associated with lipid rafts do not detectably coredistribute with these inner leaflet components. Cell activation using patterned surfaces provides unique insights into cell membrane structural organization, revealing dynamic, large-scale uncoupling of inner and outer leaflet components of lipid rafts.T he functional importance of plasma membrane heterogeneity and compartmentalization has become broadly appreciated since caveolae (1) and detergent-resistant membrane domains (2) were described and the ''lipid raft hypothesis'' was articulated several years ago. Rafts are considered to be membrane domains of ordered lipids, sphingomyelin, and cholesterol that selectively segregate proteins to participate in a range of cellular functions, including receptor-mediated signaling (3). Despite widespread attention, rafts have been elusive to experimental definition because of their compositional heterogeneity of lipids and proteins and their dynamic nature in the living cell (4). Crosslinking of proteins or lipid-based components that tend to associate with rafts causes their coalescence, allowing visualization in terms of coredistributing markers. Crosslinking in this manner often activates cells, and accumulating data support the view that coalesced rafts enhance selective association of protein and lipid components in the membrane, leading to targeting of the cellular response events (5-7).A prominent example of a process involving membrane domains is antigen-mediated crosslinking of IgE receptor complexes [IgE-Fc receptor I (FcRI)] that initiates signal transduction in rat basophilic leukemia (RBL) mast cells (8). Biochemical studies provide strong evidence that clustered IgE-FcRI associates with coalesced rafts containing active Lyn tyrosine kinase anchored to the inner leaflet of the plasma membrane (9). As a consequence of this association, cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) segments of the receptor are phosphorylated, creating new binding sites for Lyn and Syk tyrosine kinase, leading to Syk activation and phosphorylation of additional substrates and downstream signaling events (10). A wide range of questions remain regarding participation of such membrane domains in cell activation. A key issue is the nature of these domains in the outer and the inner leaflets of the plasma membrane and how they may be separately organized or coupled in sequential stages of signaling (2-4).Previous use of soluble antibo...

Recent work to characterize the roles of lipid segregation in IgE receptor signaling has revealed a mechanism by which segregation of liquid ordered regions from disordered regions of the plasma membrane results in protection of the Src family kinase Lyn from inactivating dephosphorylation by a transmembrane tyrosine phosphatase. Antigen-mediated crosslinking of IgE receptors drives their association with the liquid ordered regions, commonly called lipid rafts, and this facilitates receptor phosphorylation by active Lyn in the raft environment. Previous work showed that the membrane skeleton coupled to F-actin regulates stimulated receptor phosphorylation and downstream signaling processes, and more recent work implicates cytoskeletal interactions with ordered lipid rafts in this regulation. These and other results provide an emerging view of the complex role of membrane structure in orchestrating signal transduction mediated by immune and other cell surface receptors.

Cell-free reconstitution of membrane traffic reactions and the morphological characterization of membrane intermediates that accumulate under these conditions have helped elucidate the physical and molecular mechanisms involved in membrane transport1–3. Towards an improved understanding of endocytosis we have reconstituted vesicle budding and fission from isolated plasma membrane sheets and imaged these events. Electron and fluorescence microscopy, including sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) 4–6, revealed F-BAR (FBP17) domain coated tubules nucleated by clathrin-coated buds when fission was blocked (presence of GTPγS). Triggering fission by replacement of GTPγS with GTP led not only to separation of clathrin-coated buds, but also to vesicle formation by fragmentation of the tubules. These results suggest a functional link between FBP17 dependent membrane tubulation and clathrin-dependent budding. They also show that clathrin spatially directs plasma membrane invaginations that lead to the generation of endocytic vesicles larger than those enclosed by the coat.