PDK1:PKCα heterodimer association-dissociation dynamics in single-molecule diffusion tracks on a target membrane.

Previous studies have documented the formation of a heterodimer between the two protein kinases PDK1 and PKCα on a lipid bilayer containing their target lipids. This work investigates the association-dissociation kinetics of this PDK1:PKCα heterodimer. The approach monitors the two-dimensional diffusion of single, membrane-associated PDK1 molecules for diffusivity changes as PKCα molecules bind and unbind. In the absence of PKCα, a membrane-associated PDK1 molecule exhibits high diffusivity (or large diffusion constant, D) because its membrane-contacting PH domain binds the target PIP3 lipid headgroup with little bilayer penetration, yielding minimal frictional drag against the bilayer. In contrast, membrane-associated PKCα contacts the bilayer via its C1A, C1B, and C2 domains, which each bind at least one target lipid with significant bilayer insertion, yielding a large frictional drag and low diffusivity. The present findings reveal that individual fluor-PDK1 molecules freely diffusing on the membrane surface undergo reversible switching between distinct high and low diffusivity states, corresponding to the PDK1 monomer and the PDK1:PKCα heterodimer, respectively. The observed single-molecule diffusion trajectories are converted to step length time courses, then subjected to two-state, hidden Markov modeling and dwell time analysis. The findings reveal that both the PDK1 monomer state and the PDK1:PKCα heterodimer state decay via simple exponential kinetics, yielding estimates of rate constants for state switching in both directions. Notably, the PDK1:PKCα heterodimer has been shown to competitively inhibit PDK1 phosphoactivation of AKT1, and is believed to play a tumor suppressor role by limiting excess activation of the highly oncogenic PDK1/AKT1/mTOR pathway. Thus, the present elucidation of the PDK1:PKCα association-dissociation kinetics has important biological and medical implications. More broadly, the findings illustrate the power of single-molecule diffusion measurements to reveal the kinetics of association-dissociation events in membrane signaling reactions that yield a large change in diffusive mobility.

Binding of active Ras and its mutants to the Ras binding domain of PI-3-kinase: A quantitative approach to KD measurements.

Ras family GTPases (H/K/N-Ras) modulate numerous effectors, including the lipid kinase PI3K (phosphatidylinositol-3-kinase) that generates growth signal lipid PIP3 (phosphatidylinositol-3,4,5-triphosphate). Active GTP-Ras binds PI3K with high affinity, thereby stimulating PIP3 production. We hypothesize the affinity of this binding interaction could be significantly increased or decreased by Ras mutations at PI3K contact positions, with clinical implications since some Ras mutations at PI3K contact positions are disease-linked. To enable tests of this hypothesis, we have developed an approach combining UV spectral deconvolution, HPLC, and microscale thermophoresis to quantify the KD for binding. The approach measures the total Ras concentration, the fraction of Ras in the active state, and the affinity of active Ras binding to its docking site on PI3K Ras binding domain (RBD) in solution. The approach is illustrated by KD measurements for the binding of active H-Ras and representative mutants, each loaded with GTP or GMPPNP, to PI3Kγ RBD. The findings demonstrate that quantitation of the Ras activation state increases the precision of KD measurements, while also revealing that Ras mutations can increase (Q25L), decrease (D38E, Y40C), or have no effect (G13R) on PI3K binding affinity. Significant Ras affinity changes are predicted to alter PI3K regulation and PIP3 growth signals.

Single-molecule studies reveal regulatory interactions between master kinases PDK1, AKT1, and PKC.

Leukocyte migration is controlled by a leading-edge chemosensory pathway that generates the regulatory lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3), a growth signal, thereby driving leading-edge expansion up attractant gradients toward sites of infection, inflammation, or tissue damage. PIP3 also serves as an important growth signal in growing cells and oncogenesis. The kinases PDK1, AKT1 or PKB, and PKCα are key components of a plasma-membrane-based PIP3 and Ca2+ signaling circuit that regulates these processes. PDK1 and AKT1 are recruited to the membrane by PIP3, whereas PKCα is recruited to the membrane by Ca2+. All three of these master kinases phosphoregulate an array of protein targets. For example, PDK1 activates AKT1, PKCα, and other AGC kinases by phosphorylation at key sites. PDK1 is believed to form PDK1-AKT1 and PDK1-PKCα heterodimers stabilized by a PDK1-interacting fragment (PIF) interaction between the PDK1 PIF pocket and the PIF motif of the AGC binding partner. Here, we present the first, to our knowledge, single-molecule studies of full-length PDK1 and AKT1 on target membrane surfaces, as well as their interaction with full-length PKCα. These studies directly detect membrane-bound PDK1-AKT1 and PDK1-PKCα heterodimers stabilized by PIF interactions formed at physiological ligand concentrations. PKCα exhibits eightfold higher PDK1 affinity than AKT1 and can competitively displace AKT1 from PDK1-AKT1 heterodimers. Ensemble activity measurements under matched conditions reveal that PDK1 activates AKT1 via a cis mechanism by phosphorylating an AKT1 molecule in the same PDK1-AKT1 heterodimer, whereas PKCα acts as a competitive inhibitor of this phosphoactivation reaction by displacing AKT1 from PDK1. Overall, the findings provide insights into the binding and regulatory interactions of the three master kinases on their target membrane and suggest that a recently described tumor suppressor activity of PKC isoforms may arise from its ability to downregulate PDK1-AKT1 phosphoactivation in the PIP3-PDK1-AKT1-mTOR pathway linked to cell growth and oncogenesis.

HPLC method to resolve, identify and quantify guanine nucleotides bound to recombinant ras GTPase.

The Ras superfamily of small G proteins play central roles in diverse signaling pathways. Superfamily members act as molecular on-off switches defined by their occupancy with GTP or GDP, respectively. In vitro functional studies require loading with a hydrolysis-resistant GTP analogue to increase the on-state lifetime, as well as knowledge of fractional loading with activating and inactivating nucleotides. The present study describes a method combining elements of previous approaches with new, optimized features to analyze the bound nucleotide composition of a G protein loaded with activating (GMPPNP) or inactivating (GDP) nucleotide. After nucleotide loading, the complex is washed to remove unbound nucleotides then bound nucleotides are heat-extracted and subjected to ion-paired, reverse-phase HPLC-UV to resolve, identify and quantify the individual nucleotide components. These data enable back-calculation to the nucleotide composition and fractional activation of the original, washed G protein population prior to heat extraction. The method is highly reproducible. Application to multiple HRas preparations and mutants confirms its ability to fully extract and analyze bound nucleotides, and to resolve the fractional on- and off-state populations. Furthermore, the findings yield a novel hypothesis for the molecular disease mechanism of Ras mutations at the E63 and Y64 positions.

Ras-guanine nucleotide complexes: A UV spectral deconvolution method to analyze protein concentration, nucleotide stoichiometry, and purity.

The many members of the Ras superfamily are small GTPases that serve as molecular switches. These proteins bind the guanine nucleotides GTP and GDP with picomolar affinities, thereby stabilizing on- and off-signaling states, respectively. Quantitative in vitro Ras studies require accurate determination of total protein, its fractional occupancy with guanine nucleotide, and spectroscopic purity. Yet the high nucleotide affinity of Ras and the overlapping UV spectra of the protein and bound nucleotide make such determinations challenging. Here we describe a generalizable UV spectral deconvolution method to analyze the total protein concentration, total nucleotide stoichiometry, and purity of Ras complexes.

The G-Protein Rab5A Activates VPS34 Complex II, a Class III PI3K, by a Dual Regulatory Mechanism.

VPS34 complex II (VPS34CII) is a 386-kDa assembly of the lipid kinase subunit VPS34 and three regulatory subunits that altogether function as a prototypical class III phosphatidylinositol-3-kinase (PI3K). When the active VPS34CII complex is docked to the cytoplasmic surface of endosomal membranes, it phosphorylates its substrate lipid (phosphatidylinositol, PI) to generate the essential signaling lipid phosphatidylinositol-3-phosphate (PI3P). In turn, PI3P recruits an array of signaling proteins containing PI3P-specific targeting domains (including FYVE, PX, and PROPPINS) to the membrane surface, where they initiate key cell processes. In endocytosis and early endosome development, net VPS34CII-catalyzed PI3P production is greatly amplified by Rab5A, a small G protein of the Ras GTPase superfamily. Moreover, VPS34CII and Rab5A are each strongly linked to multiple human diseases. Thus, a molecular understanding of the mechanism by which Rab5A activates lipid kinase activity will have broad impacts in both signaling biology and medicine. Two general mechanistic models have been proposed for small G protein activation of PI3K lipid kinases. 1) In the membrane recruitment mechanism, G protein association increases the density of active kinase on the membrane. And 2) in the allosteric activation mechanism, G protein allosterically triggers an increase in the specific activity (turnover rate) of the membrane-bound kinase molecule. This study employs an in vitro single-molecule approach to elucidate the mechanism of GTP-Rab5A-associated VPS34CII kinase activation in a reconstituted GTP-Rab5A-VPS34CII-PI3P-PX signaling pathway on a target membrane surface. The findings reveal that both membrane recruitment and allosteric mechanisms make important contributions to the large increase in VPS34CII kinase activity and PI3P production triggered by membrane-anchored GTP-Rab5A. Notably, under near-physiological conditions in the absence of other activators, membrane-anchored GTP-Rab5A provides strong, virtually binary on-off switching and is required for VPS34CII membrane binding and PI3P production.

Rapid exposure of macrophages to drugs resolves four classes of effects on the leading edge sensory pseudopod: Non-perturbing, adaptive, disruptive, and activating.

Leukocyte migration is controlled by a membrane-based chemosensory pathway on the leading edge pseudopod that guides cell movement up attractant gradients during the innate immune and inflammatory responses. This study employed single cell and population imaging to investigate drug-induced perturbations of leading edge pseudopod morphology in cultured, polarized RAW macrophages. The drugs tested included representative therapeutics (acetylsalicylic acid, diclofenac, ibuprofen, acetaminophen) as well as control drugs (PDGF, Gö6976, wortmannin). Notably, slow addition of any of the four therapeutics to cultured macrophages, mimicking the slowly increasing plasma concentration reported for standard oral dosage in patients, yielded no detectable change in pseudopod morphology. This finding is consistent with the well established clinical safety of these drugs. However, rapid drug addition to cultured macrophages revealed four distinct classes of effects on the leading edge pseudopod: (i) non-perturbing drug exposures yielded no detectable change in pseudopod morphology (acetylsalicylic acid, diclofenac); (ii) adaptive exposures yielded temporary collapse of the extended pseudopod and its signature PI(3,4,5)P3 lipid signal followed by slow recovery of extended pseudopod morphology (ibuprofen, acetaminophen); (iii) disruptive exposures yielded long-term pseudopod collapse (Gö6976, wortmannin); and (iv) activating exposures yielded pseudopod expansion (PDGF). The novel observation of adaptive exposures leads us to hypothesize that rapid addition of an adaptive drug overwhelms an intrinsic or extrinsic adaptation system yielding temporary collapse followed by adaptive recovery, while slow addition enables gradual adaptation to counteract the drug perturbation in real time. Overall, the results illustrate an approach that may help identify therapeutic drugs that temporarily inhibit the leading edge pseudopod during extreme inflammation events, and toxic drugs that yield long term inhibition of the pseudopod with negative consequences for innate immunity. Future studies are needed to elucidate the mechanisms of drug-induced pseudopod collapse, as well as the mechanisms of adaptation and recovery following some inhibitory drug exposures.

A PKC-MARCKS-PI3K regulatory module links Ca2+ and PIP3 signals at the leading edge of polarized macrophages.

The leukocyte chemosensory pathway detects attractant gradients and directs cell migration to sites of inflammation, infection, tissue damage, and carcinogenesis. Previous studies have revealed that local Ca2+ and PIP3 signals at the leading edge of polarized leukocytes play central roles in positive feedback loop essential to cell polarization and chemotaxis. These prior studies showed that stimulation of the leading edge Ca2+ signal can strongly activate PI3K, thereby triggering a larger PIP3 signal, but did not elucidate the mechanistic link between Ca2+ and PIP3 signaling. A hypothesis explaining this link emerged, postulating that Ca2+-activated PKC displaces the MARCKS protein from plasma membrane PIP2, thereby releasing sequestered PIP2 to serve as the target and substrate lipid of PI3K in PIP3 production. In vitro single molecule studies of the reconstituted pathway on lipid bilayers demonstrated the feasibility of this PKC-MARCKS-PI3K regulatory module linking Ca2+ and PIP3 signals in the reconstituted system. The present study tests the model predictions in live macrophages by quantifying the effects of: (a) two pathway activators-PDGF and ATP that stimulate chemoreceptors and Ca2+ influx, respectively; and (b) three pathway inhibitors-wortmannin, EGTA, and Go6976 that inhibit PI3K, Ca2+ influx, and PKC, respectively; on (c) four leading edge activity sensors-AKT-PH-mRFP, CKAR, MARCKSp-mRFP, and leading edge area that report on PIP3 density, PKC activity, MARCKS membrane binding, and leading edge expansion/contraction, respectively. The results provide additional evidence that PKC and PI3K are both essential elements of the leading edge positive feedback loop, and strongly support the existence of a PKC-MARCKS-PI3K regulatory module linking the leading edge Ca2+ and PIP3 signals. As predicted, activators stimulate leading edge PKC activity, displacement of MARCKS from the leading edge membrane and increased leading edge PIP3 levels, while inhibitors trigger the opposite effects. Comparison of the findings for the ameboid chemotaxis of leukocytes with recently published findings for the mesenchymal chemotaxis of fibroblasts suggests that some features of the emerging leukocyte leading edge core pathway (PLC-DAG-Ca2+-PKC-MARCKS-PIP2-PI3K-PIP3) may well be shared by all chemotaxing eukaryotic cells, while other elements of the leukocyte pathway may be specialized features of these highly optimized, professional gradient-seeking cells. More broadly, the findings suggest a molecular mechanism for the strong links between phospho-MARCKS and many human cancers.

Single-Molecule Study Reveals How Receptor and Ras Synergistically Activate PI3Kα and PIP3 Signaling.

Cellular pathways controlling chemotaxis, growth, survival, and oncogenesis are activated by receptor tyrosine kinases and small G-proteins of the Ras superfamily that stimulate specific isoforms of phosphatidylinositol-3-kinase (PI3K). These PI3K lipid kinases phosphorylate the constitutive lipid phosphatidylinositol-4,5-bisphosphate (PIP2) to produce the signaling lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3). Progress has been made in understanding direct, moderate PI3K activation by receptors. In contrast, the mechanism by which receptors and Ras synergistically activate PI3K to much higher levels remains unclear, and two competing models have been proposed: membrane recruitment versus activation of the membrane-bound enzyme. To resolve this central mechanistic question, this study employs single-molecule imaging to investigate PI3K activation in a six-component pathway reconstituted on a supported lipid bilayer. The findings reveal that simultaneous activation by a receptor activation loop (from platelet-derived growth factor receptor, a receptor tyrosine kinase) and H-Ras generates strong, synergistic activation of PI3Kα, yielding a large increase in net kinase activity via the membrane recruitment mechanism. Synergy requires receptor phospho-Tyr and two anionic lipids (phosphatidylserine and PIP2) to make PI3Kα competent for bilayer docking, as well as for subsequent binding and phosphorylation of substrate PIP2 to generate product PIP3. Synergy also requires recruitment to membrane-bound H-Ras, which greatly speeds the formation of a stable, membrane-bound PI3Kα complex, modestly slows its off rate, and dramatically increases its equilibrium surface density. Surprisingly, H-Ras binding significantly inhibits the specific kinase activity of the membrane-bound PI3Kα molecule, but this minor enzyme inhibition is overwhelmed by the marked enhancement of membrane recruitment. The findings have direct impacts for the fields of chemotaxis, innate immunity, inflammation, carcinogenesis, and drug design.

Regulation of a Coupled MARCKS-PI3K Lipid Kinase Circuit by Calmodulin: Single-Molecule Analysis of a Membrane-Bound Signaling Module.

Amoeboid cells that employ chemotaxis to travel up an attractant gradient possess a signaling network assembled on the leading edge of the plasma membrane that senses the gradient and remodels the actin mesh and cell membrane to drive movement in the appropriate direction. In leukocytes such as macrophages and neutrophils, and perhaps in other amoeboid cells as well, the leading edge network includes a positive feedback loop in which the signaling of multiple pathway components is cooperatively coupled. Cytoplasmic Ca2+ is a recently recognized component of the feedback loop at the leading edge where it stimulates phosphoinositide-3-kinase (PI3K) and the production of its product signaling lipid phosphatidylinositol 3,4,5-trisphosphate (PIP3). A previous study implicated Ca2+-activated protein kinase C (PKC) and the phosphatidylinositol 4,5-bisphosphate (PIP2) binding protein MARCKS as two important players in this signaling, because PKC phosphorylation of MARCKS releases free PIP2 that serves as the membrane binding target and substrate for PI3K. This study asks whether calmodulin (CaM), which is known to directly bind MARCKS, also stimulates PIP3 production by releasing free PIP2. Single-molecule fluorescence microscopy is used to quantify the surface density and enzyme activity of key protein components of the hypothesized Ca2+-CaM-MARCKS-PIP2-PI3K-PIP3 circuit. The findings show that CaM does stimulate PI3K lipid kinase activity by binding MARCKS and displacing it from PIP2 headgroups, thereby releasing free PIP2 that recruits active PI3K to the membrane and serves as the substrate for the generation of PIP3. The resulting CaM-triggered activation of PI3K is complete in seconds and is much faster than PKC-triggered activation, which takes minutes. Overall, the available evidence implicates both PKC and CaM in the coupling of Ca2+ and PIP3 signals and suggests these two different pathways have slow and fast activation kinetics, respectively.

Regulation of PI3K by PKC and MARCKS: Single-Molecule Analysis of a Reconstituted Signaling Pathway.

In chemotaxing ameboid cells, a complex leading-edge signaling circuit forms on the cytoplasmic leaflet of the plasma membrane and directs both actin and membrane remodeling to propel the leading edge up an attractant gradient. This leading-edge circuit includes a putative amplification module in which Ca(2+)-protein kinase C (Ca(2+)-PKC) is hypothesized to phosphorylate myristoylated alanine-rich C kinase substrate (MARCKS) and release phosphatidylinositol-4,5-bisphosphate (PIP2), thereby stimulating production of the signaling lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3) by the lipid kinase phosphoinositide-3-kinase (PI3K). We investigated this hypothesized Ca(2+)-PKC-MARCKS-PIP2-PI3K-PIP3 amplification module and tested its key predictions using single-molecule fluorescence to measure the surface densities and activities of its protein components. Our findings demonstrate that together Ca(2+)-PKC and the PIP2-binding peptide of MARCKS modulate the level of free PIP2, which serves as both a docking target and substrate lipid for PI3K. In the off state of the amplification module, the MARCKS peptide sequesters PIP2 and thereby inhibits PI3K binding to the membrane. In the on state, Ca(2+)-PKC phosphorylation of the MARCKS peptide reverses the PIP2 sequestration, thereby releasing multiple PIP2 molecules that recruit multiple active PI3K molecules to the membrane surface. These findings 1) show that the Ca(2+)-PKC-MARCKS-PIP2-PI3K-PIP3 system functions as an activation module in vitro, 2) reveal the molecular mechanism of activation, 3) are consistent with available in vivo data, and 4) yield additional predictions that are testable in live cells. More broadly, the Ca(2+)-PKC-stimulated release of free PIP2 may well regulate the membrane association of other PIP2-binding proteins, and the findings illustrate the power of single-molecule analysis to elucidate key dynamic and mechanistic features of multiprotein signaling pathways on membrane surfaces.

Signaling and sensory adaptation in Escherichia coli chemoreceptors: 2015 update.

Motile Escherichia coli cells track gradients of attractant and repellent chemicals in their environment with transmembrane chemoreceptor proteins. These receptors operate in cooperative arrays to produce large changes in the activity of a signaling kinase, CheA, in response to small changes in chemoeffector concentration. Recent research has provided a much deeper understanding of the structure and function of core receptor signaling complexes and the architecture of higher-order receptor arrays, which, in turn, has led to new insights into the molecular signaling mechanisms of chemoreceptor networks. Current evidence supports a new view of receptor signaling in which stimulus information travels within receptor molecules through shifts in the dynamic properties of adjoining structural elements rather than through a few discrete conformational states.

Architecture and signal transduction mechanism of the bacterial chemosensory array: progress, controversies, and challenges.

Recent research has deepened our understanding of the ancient, conserved chemosensory array that detects small molecule attractants and repellents, and directs the chemotaxis of bacterial and archaeal cells towards an optimal chemical environment. Here we review advances towards a molecular description of the ultrastable lattice architecture and ultrasensitive signal transduction mechanism of the chemosensory array, as well as controversies and challenges requiring further research. Ultimately, a full molecular understanding of array structure and on-off switching will foster (i) the design of novel therapies that block pathogenic wound seeking and infection, (ii) the development of highly specific, sensitive, stable biosensors, and (iii) the elucidation of general functional principles shared by receptor patches in all branches of life.

Piston versus scissors: chemotaxis receptors versus sensor His-kinase receptors in two-component signaling pathways.

In this issue of Structure, Molnar and colleagues present a pair of important advances: (1) a method to analyze multiple signaling states in on-off switch proteins and (2) evidence for a scissors-type mechanism of on-off switching in a full-length, membrane-bound receptor of the sensor histidine-kinase class.

Increasing and decreasing the ultrastability of bacterial chemotaxis core signaling complexes by modifying protein-protein contacts.

The chemosensory signaling array of bacterial chemotaxis is composed of functional core units containing two receptor trimers of dimers, a homodimeric CheA kinase, and two CheW adaptor proteins. In vitro reconstitutions generate individual, functional core units and larger functional assemblies, including dimers, hexagons, and hexagonal arrays. Such reconstituted complexes have been shown to have both quasi-stable and ultrastable populations that decay with lifetimes of 1-2 days and ∼3 weeks at 22 °C, respectively, where decay results primarily from proteolysis of the bound kinase [Erbse, A. H., and Falke, J. J. (2009) Biochemistry 48, 6975-6987; Slivka, P. F., and Falke, J. J. (2012) Biochemistry 51, 10218-10228]. In this work, we show that the ultrastable population can be destabilized to the quasi-stable level via the introduction of a bulky tryptophan residue at either one of two essential protein-protein interfaces within the core unit: the receptor-kinase contact or kinase-adaptor interface 1. Moreover, we demonstrate that the quasi-stable population can be made ultrastable via the introduction of a disulfide bond that covalently stabilizes the latter interface. The resulting disulfide at least doubles the functional lifetime of the ultrastable population, to ≥5.9 weeks at 22 °C, by protecting the kinase from endogenous and exogenous proteases. Together, these results indicate that the ultrastability of reconstituted core complexes requires well-formed contacts among the receptor, kinase, and adaptor proteins, whereas quasi-stability arises from less perfect contacts that allow slow proteolysis of the bound kinase. Furthermore, the results reveal that ultrastability, and perhaps the size or order of chemosensory complexes and arrays, can be increased by an engineered disulfide bond that covalently cross-links a key interface. Overall, it appears that native ultrastability has evolved to provide an optimal rather than maximal level of kinetic durability, suggesting that altered selective pressure could either increase or decrease the functional lifetime of core complexes.

Interactions of protein kinase C-α C1A and C1B domains with membranes: a combined computational and experimental study.

Protein kinase C-α (PKCα) has been studied widely as a paradigm for conventional PKCs, with two C1 domains (C1A and C1B) being important for the regulation and function of the kinase. However, it is challenging to explore these domains in membrane-bound environments with either simulations or experiments alone. In this work, we have combined modeling, simulations, and experiments to understand the molecular basis of the PKCα C1A and C1B domain interactions with membranes. Our atomistic simulations of the PKCα C1 domains reveal the dynamic interactions of the proteins with anionic lipids, as well as the conserved hydrogen bonds and the distinct nonpolar contacts formed with lipid activators. Corroborating evidence is obtained from additional simulations and experiments in terms of lipid binding and protein diffusion. Overall, our study, for the first time, explains with atomistic detail how the PKCα C1A and C1B domains interact differently with various lipids. On the molecular level, the information provided by our study helps to shed light on PKCα regulation and activation mechanism. The combined computational/experimental approach demonstrated in this work is anticipated to enable further studies to explore the roles of C1 domains in many signaling proteins and to better understand their molecular mechanisms in normal cellular function and disease development.

New insights into bacterial chemoreceptor array structure and assembly from electron cryotomography.

Bacterial chemoreceptors cluster in highly ordered, cooperative, extended arrays with a conserved architecture, but the principles that govern array assembly remain unclear. Here we show images of cellular arrays as well as selected chemoreceptor complexes reconstituted in vitro that reveal new principles of array structure and assembly. First, in every case, receptors clustered in a trimers-of-dimers configuration, suggesting this is a highly favored fundamental building block. Second, these trimers-of-receptor dimers exhibited great versatility in the kinds of contacts they formed with each other and with other components of the signaling pathway, although only one architectural type occurred in native arrays. Third, the membrane, while it likely accelerates the formation of arrays, was neither necessary nor sufficient for lattice formation. Molecular crowding substituted for the stabilizing effect of the membrane and allowed cytoplasmic receptor fragments to form sandwiched lattices that strongly resemble the cytoplasmic chemoreceptor arrays found in some bacterial species. Finally, the effective determinant of array structure seemed to be CheA and CheW, which formed a "superlattice" of alternating CheA-filled and CheA-empty rings that linked receptor trimers-of-dimer units into their native hexagonal lattice. While concomitant overexpression of receptors, CheA, and CheW yielded arrays with native spacing, the CheA occupancy was lower and less ordered, suggesting that temporal and spatial coordination of gene expression driven by a single transcription factor may be vital for full order, or that array overgrowth may trigger a disassembly process. The results described here provide new insights into the assembly intermediates and assembly mechanism of this massive macromolecular complex.

Single-molecule studies reveal a hidden key step in the activation mechanism of membrane-bound protein kinase C-α.

Protein kinase C-α (PKCα) is a member of the conventional family of protein kinase C isoforms (cPKCs) that regulate diverse cellular signaling pathways, share a common activation mechanism, and are linked to multiple pathologies. The cPKC domain structure is modular, consisting of an N-terminal pseudosubstrate peptide, two inhibitory domains (C1A and C1B), a targeting domain (C2), and a kinase domain. Mature, cytoplasmic cPKCs are inactive until they are switched on by a multistep activation reaction that occurs largely on the plasma membrane surface. Often, this activation begins with a cytoplasmic Ca(2+) signal that triggers C2 domain targeting to the plasma membrane where it binds phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP2). Subsequently, the appearance of the signaling lipid diacylglycerol (DAG) activates the membrane-bound enzyme by recruiting the inhibitory pseudosubstrate and one or both C1 domains away from the kinase domain. To further investigate this mechanism, this study has utilized single-molecule total internal reflection fluorescence microscopy (TIRFM) to quantitate the binding and lateral diffusion of full-length PKCα and fragments missing specific domain(s) on supported lipid bilayers. Lipid binding events, and events during which additional protein is inserted into the bilayer, were detected by their effects on the equilibrium bound particle density and the two-dimensional diffusion rate. In addition to the previously proposed activation steps, the findings reveal a major, undescribed, kinase-inactive intermediate. On bilayers containing PS or PS and PIP2, full-length PKCα first docks to the membrane via its C2 domain, and then its C1A domain embeds itself in the bilayer even before DAG appears. The resulting pre-DAG intermediate with membrane-bound C1A and C2 domains is the predominant state of PKCα while it awaits the DAG signal. The newly detected, membrane-embedded C1A domain of this pre-DAG intermediate confers multiple useful features, including enhanced membrane affinity and longer bound state lifetime. The findings also identify the key molecular step in kinase activation: because C1A is already membrane-embedded in the kinase off state, recruitment of C1B to the bilayer by DAG or phorbol ester is the key regulatory event that stabilizes the kinase on state. More broadly, this study illustrates the power of single-molecule methods in elucidating the activation mechanisms and hidden regulatory states of membrane-bound signaling proteins.

Interplay between phosphoinositide lipids and calcium signals at the leading edge of chemotaxing ameboid cells.

The chemotactic migration of eukaryotic ameboid cells up concentration gradients is among the most advanced forms of cellular behavior. Chemotaxis is controlled by a complex network of signaling proteins bound to specific lipids on the cytoplasmic surface of the plasma membrane at the front of the cell, or the leading edge. The central lipid players in this leading edge signaling pathway include the phosphoinositides PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3), both of which play multiple roles. The products of PI(4,5)P2 hydrolysis, diacylglycerol (DAG) and Ins(1,4,5)P3 (IP3), are also implicated as important players. Together, these leading edge phosphoinositides and their degradation products, in concert with a local Ca(2+) signal, control the recruitment and activities of many peripheral membrane proteins that are crucial to the leading edge signaling network. The present critical review summarizes the current molecular understanding of chemotactic signaling at the leading edge, including newly discovered roles of phosphoinositide lipids and Ca(2+), while highlighting key questions for future research.

Structure, function, and on-off switching of a core unit contact between CheA kinase and CheW adaptor protein in the bacterial chemosensory array: A disulfide mapping and mutagenesis study.

The ultrasensitive, ultrastable bacterial chemosensory array of Escherichia coli and Salmonella typhimurium is representative of the large, conserved family of sensory arrays that control the cellular chemotaxis of motile bacteria and Archaea. The core framework of the membrane-bound array is a lattice assembled from three components: a transmembrane receptor, a cytoplasmic His kinase (CheA), and a cytoplasmic adaptor protein (CheW). Structural studies in the field have revealed the global architecture of the array and complexes between specific components, but much remains to be learned about the essential protein-protein interfaces that define array structure and transmit signals between components. This study has focused on the structure, function, and on-off switching of a key contact between the kinase and adaptor proteins in the working, membrane-bound array. Specifically, the study addressed interface 1 in the putative kinase-adaptor ring where subdomain 1 of the kinase regulatory domain contacts subdomain 2 of the adaptor protein. Two independent approaches, disulfide mapping and site-directed Trp and Ala mutagenesis, were employed (i) to test the structural model of interface 1 and (ii) to investigate its functional roles in both stable kinase incorporation and receptor-regulated kinase on-off switching. Studies were conducted in functional, membrane-bound arrays or in live cells. The findings reveal that crystal structures of binary and ternary complexes accurately depict the native interface in its kinase-activating on state. Furthermore, the findings indicate that at least part of the interface becomes less closely packed in its kinase-inhibiting off state. Together, the evidence shows the interface has a dual structural and signaling function that is crucial for incorporation of the stable kinase into the array, for kinase activation in the array on state, and likely for attractant-triggered kinase on-off switching. A model is presented that describes the concerted transmission of a conformational signal among the receptor, the kinase regulatory domain, and the adaptor protein. In principle, this signal could spread out into the surrounding array via the kinase-adaptor ring, employing a series of alternating frozen-dynamic transitions that transmit low-energy attractant signals long distances.