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.

Kinase inhibitors allosterically disrupt a regulatory interaction to enhance PKCα membrane translocation.

The eukaryotic kinase domain has multiple intrinsically disordered regions whose conformation dictates kinase activity. Small molecule kinase inhibitors (SMKIs) rely on disrupting the active conformations of these disordered regions to inactivate the kinase. While SMKIs are selected for their ability to cause this disruption, the allosteric effects of conformational changes in disordered regions is limited by a lack of dynamic information provided by traditional structural techniques. In this study, we integrated multiscale molecular dynamics simulations with FRET sensors to characterize a novel allosteric mechanism that is selectively triggered by SMKI binding to the protein kinase Cα domain. The indole maleimide inhibitors BimI and sotrastaurin were found to displace the Gly-rich loop (G-loop) that normally shields the ATP-binding site. Displacement of the G-loop interferes with a newly identified, structurally conserved binding pocket for the C1a domain on the N lobe of the kinase domain. This binding pocket, in conjunction with the N-terminal regulatory sequence, masks a diacylglycerol (DAG) binding site on the C1a domain. SMKI-mediated displacement of the G-loop released C1a and exposed the DAG binding site, enhancing protein kinase Cα translocation both to synthetic lipid bilayers and to live cell membranes in the presence of DAG. Inhibitor chemotype determined the extent of the observed allosteric effects on the kinase domain and correlated with the extent of membrane recruitment. Our findings demonstrate the allosteric effects of SMKIs beyond the confines of kinase catalytic conformation and provide an integrated computational-experimental paradigm to investigate parallel mechanisms in other kinases.

Identification, characterization, and comparison of n-alkanols and anesthetics binding to the C1b subdomain of protein kinase cα: similar function with different binding sites.

Protein kinase C (PKC) is a family of lipid-activated enzymes involved in anesthetic preconditioning signaling pathways. Previously, n-alkanols and general anesthetics have been found to activate PKC by binding to the kinase C1B subdomain. In the present study, we attempt to ascertain the molecular mechanism and interaction mode of human PKCα C1B subdomain with a variety of exogenous n-alkanols and volatile general anesthetics as well as endogenous activator phorbol ester (PE) and co-activator diacylglycerol (DG). Systematic bioinformatics analysis identifies three spatially vicinal sites on the subdomain surface to potentially accommodate small-molecule ligands, where the site 1 is a narrow, amphipathic pocket, the site 2 is a wide, flat and hydrophobic pocket, and the site 3 is a rugged, polar pocket. Further interaction modeling reveals that site 1 is the cognate binding region of natural PE activator, which can moderately simulate the kinase activity in an independent manner. The short-chain n-alkanols are speculated to also bind at the site to competitively inhibit PE-induced kinase activation. The long-chain n-alkanols and co-activator DG are found to target site 2 in a nonspecific manner, while the volatile anesthetics prefer to interact with site 3 in a specific manner. Since the site 1 is composed of two protein loops that are also shared by sites 2 and 3, binding of n-alkanols, DG and anesthetics to sites 2 and 3 can trigger a conformational displacement on the two loops, which enlarges the pocket size and changes the pocket configuration of site 1 through an allosteric mechanism, consequently enhancing kinase activation by improving PE affinity to the site.

Structural Basis of Protein Kinase Cα Regulation by the C-Terminal Tail.

Protein kinase C (PKC) isoenzymes are multi-modular proteins activated at the membrane surface to regulate signal transduction processes. When activated by second messengers, PKC undergoes a drastic conformational and spatial transition from the inactive cytosolic state to the activated membrane-bound state. The complete structure of either state of PKC remains elusive. We demonstrate, using NMR spectroscopy, that the isolated Ca2+-sensing membrane-binding C2 domain of the conventional PKCα interacts with a conserved hydrophobic motif of the kinase C-terminal region, and we report a structural model of the complex. Our data suggest that the C-terminal region plays a dual role in regulating the PKC activity: activating, through sensitization of PKC to intracellular Ca2+ oscillations; and auto-inhibitory, through its interaction with a conserved positively charged region of the C2 domain.

The Role of Regulatory Domains in Maintaining Autoinhibition in the Multidomain Kinase PKCα.

Resolving the conformational dynamics of large multidomain proteins has proven to be a significant challenge. Here we use a variety of techniques to dissect the roles of individual protein kinase Cα (PKCα) regulatory domains in maintaining catalytic autoinhibition. We find that whereas the pseudosubstrate domain is necessary for autoinhibition it is not sufficient. Instead, each regulatory domain (C1a, C1b, and C2) appears to strengthen the pseudosubstrate-catalytic domain interaction in a nucleotide-dependent manner. The pseudosubstrate and C1a domains, however, are minimally essential for maintaining the inactivated state. Furthermore, disrupting known interactions between the C1a and other regulatory domains releases the autoinhibited interaction and increases basal activity. Modulating this interaction between the catalytic and regulatory domains reveals a direct correlation between autoinhibition and membrane translocation following PKC activation.

Distinct structural mechanisms determine substrate affinity and kinase activity of protein kinase Cα.

Protein kinase Cα (PKCα) belongs to the family of AGC kinases that phosphorylate multiple peptide substrates. Although the consensus sequence motif has been identified and used to explain substrate specificity for PKCα, it does not inform the structural basis of substrate-binding and kinase activity for diverse substrates phosphorylated by this kinase. The transient, dynamic, and unstructured nature of this protein-protein interaction has limited structural mapping of kinase-substrate interfaces. Here, using multiscale MD simulation-based predictions and FRET sensor-based experiments, we investigated the conformational dynamics of the kinase-substrate interface. We found that the binding strength of the kinase-substrate interaction is primarily determined by long-range columbic interactions between basic (Arg/Lys) residues located N-terminally to the phosphorylated Ser/Thr residues in the substrate and by an acidic patch in the kinase catalytic domain. Kinase activity stemmed from conformational flexibility in the region C-terminal to the phosphorylated Ser/Thr residues. Flexibility of the substrate-kinase interaction enabled an Arg/Lys two to three amino acids C-terminal to the phosphorylated Ser/Thr to prime a catalytically active conformation, facilitating phosphoryl transfer to the substrate. The structural mechanisms determining substrate binding and catalytic activity formed the basis of diverse binding affinities and kinase activities of PKCα for 14 substrates with varying degrees of sequence conservation. Our findings provide insight into the dynamic properties of the kinase-substrate interaction that govern substrate binding and turnover. Moreover, this study establishes a modeling and experimental method to elucidate the structural dynamics underlying substrate selectivity among eukaryotic kinases.

Substrate Affinity Differentially Influences Protein Kinase C Regulation and Inhibitor Potency.

The overlapping network of kinase-substrate interactions provides exquisite specificity in cell signaling pathways, but also presents challenges to our ability to understand the mechanistic basis of biological processes. Efforts to dissect kinase-substrate interactions have been particularly limited by their inherently transient nature. Here, we use a library of FRET sensors to monitor these transient complexes, specifically examining weak interactions between the catalytic domain of protein kinase Cα and 14 substrate peptides. Combining results from this assay platform with those from standard kinase activity assays yields four novel insights into the kinase-substrate interaction. First, preferential binding of non-phosphorylated versus phosphorylated substrates leads to enhanced kinase-specific activity. Second, kinase-specific activity is inversely correlated with substrate binding affinity. Third, high affinity substrates can suppress phosphorylation of their low affinity counterparts. Finally, the substrate-competitive inhibitor bisindolylmaleimide I displaces low affinity substrates more potently leading to substrate selective inhibition of kinase activity. Overall, our approach complements existing structural and biophysical approaches to provide generalizable insights into the regulation of kinase activity.

Resveratrol inhibits phorbol ester-induced membrane translocation of presynaptic Munc13-1.

BACKGROUND: Resveratrol (1) is a naturally occurring polyphenol that has been implicated in neuroprotection. One of resveratrol's several biological targets is Ca2+-sensitive protein kinase C alpha (PKCα). Resveratrol inhibits PKCα by binding to its activator-binding C1 domain. Munc13-1 is a C1 domain-containing Ca2+-sensitive SNARE complex protein essential for vesicle priming and neurotransmitter release. METHODS: To test if resveratrol could also bind and inhibit Munc13-1, we studied the interaction of resveratrol and its derivatives, (E)-1,3-dimethoxy-5-(4-methoxystyryl)benzene, (E)-5,5'-(ethene-1,2-diyl)bis(benzene-1,2,3-triol), (E)-1,2-bis(3,4,5-trimethoxyphenyl)ethane, and (E)-5-(4-(hexadecyloxy)-3,5-dihydroxystyryl)benzene-1,2,3-triol with Munc13-1 by studying its membrane translocation from cytosol to plasma membrane in HT22 cells and primary hippocampal neurons. RESULTS: Resveratrol, but not the derivatives inhibited phorbol ester-induced Munc13-1 translocation from cytosol to membrane in HT22 cells and primary hippocampal neurons, as evidenced by immunoblot analysis and confocal microscopy. Resveratrol did not show any effect on Munc13-1H567K, a mutant which is not sensitive to phorbol ester. Binding studies with Munc13-1 C1 indicated that resveratrol competes with phorbol ester for the binding site. Molecular docking and dynamics studies suggested that hydroxyl groups of resveratrol interact with phorbol-ester binding residues in the binding pocket. CONCLUSIONS AND SIGNIFICANCE: This study characterizes Munc13-1 as a target of resveratrol and highlights the importance of dietary polyphenol in the management of neurodegenerative diseases.

Exploring the influence of indololactone structure on selectivity for binding to the C1 domains of PKCα, PKCε, and RasGRP.

C1 domain-containing proteins, such as protein kinase C (PKC), have a central role in cellular signal transduction. Their involvement in many diseases, including cancer, cardiovascular disease, and immunological and neurological disorders has been extensively demonstrated and has prompted a search for small molecules to modulate their activity. By employing a diacylglycerol (DAG)-lactone template, we have been able to develop ultra potent analogs of diacylglycerol with nanomolar binding affinities approaching those of complex natural products such as phorbol esters and bryostatins. One current challenge is the development of selective ligands capable of discriminating between different protein family members. Recently, structure-activity relationship studies have shown that the introduction of an indole ring as a DAG-lactone substituent yielded selective Ras guanine nucleotide-releasing protein (RasGRP1) activators when compared to PKCα and PKCε. In the present work, we examine the effects of ligand selectivity relative to the orientation of the indole ring and the nature of the DAG-lactone template itself. Our results show that the indole ring must be attached to the lactone moiety through the sn-2 position in order to achieve RasGRP1 selectivity.

Dynamic Response of the C2 Domain of Protein Kinase Cα to Ca2+ Binding.

Ca2+-dependent conserved-region 2 (C2) domains target their host signaling proteins to anionic membranes. The Ca2+-binding event is a prerequisite for membrane association. Here, we investigate multiscale metal-ion-dependent dynamics of the C2 domain of protein kinase Cα (C2α) using NMR spectroscopy. Interactions with metal ions attenuate microsecond-timescale motions of the loop regions, indicating that preorganization of the metal-binding loops occurs before membrane insertion. Binding of a full complement of Ca2+ ions has a profound effect on the millisecond-timescale dynamics of the N- and C-terminal regions of C2α. We propose that Ca2+ binding allosterically destabilizes the terminal regions of C2α and thereby facilitates the conformational rearrangement necessary for full membrane insertion and activation of protein kinase Cα.

Curcumin Inhibits Protein Kinase Cα Activity by Binding to Its C1 Domain.

Curcumin is a polyphenolic nutraceutical that acts on multiple biological targets, including protein kinase C (PKC). PKC is a family of serine/threonine kinases central to intracellular signal transduction. We have recently shown that curcumin selectively inhibits PKCα, but not PKCε, in CHO-K1 cells [Pany, S. (2016) Biochemistry 55, 2135-2143]. To understand which domain(s) of PKCα is responsible for curcumin binding and inhibitory activity, we made several domain-swapped mutants in which the C1 (combination of C1A and C1B) and C2 domains are swapped between PKCα and PKCε. Phorbol ester-induced membrane translocation studies using confocal microscopy and immunoblotting revealed that curcumin inhibited phorbol ester-induced membrane translocation of PKCε mutants, in which the εC1 domain was replaced with αC1, but not the PKCα mutant in which αC1 was replaced with the εC1 domain, suggesting that αC1 is a determinant for curcumin's inhibitory effect. In addition, curcumin inhibited membrane translocation of PKCε mutants, in which the εC1A and εC1B domains were replaced with the αC1A and αC1B domains, respectively, indicating the role of both αC1A and αC1B domains in curcumin's inhibitory effects. Phorbol 13-acetate inhibited the binding of curcumin to αC1A and αC1B with IC50 values of 6.27 and 4.47 µM, respectively. Molecular docking and molecular dynamics studies also supported the higher affinity of curcumin for αC1B than for αC1A. The C2 domain-swapped mutants were inactive in phorbol ester-induced membrane translocation. These results indicate that curcumin binds to the C1 domain of PKCα and highlight the importance of this domain in achieving PKC isoform selectivity.

Selective Modulation of Protein Kinase C α over Protein Kinase C ε by Curcumin and Its Derivatives in CHO-K1 Cells.

Members of the protein kinase C (PKC) family of serine/threonine kinases regulate various cellular functions, including cell growth, differentiation, metabolism, and apoptosis. Modulation of isoform-selective activity of PKC by curcumin (1), the active constituent of Curcuma L., is poorly understood, and the literature data are inconsistent and obscure. The effect of curcumin (1) and its analogues, 4-[(2Z,6E)-3-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-5-oxohepta-2,6-dien-1-yl]-2-methoxyphenyl oleate (2), (9Z,12Z)-4-[(2Z,6E)-3-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-5-oxohepta-2,6-dien-1-yl]-2-methoxyphenyl octadeca-9,12-dienoate (3), (9Z,12Z,15Z)-4-[(2Z,6E)-3-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-5-oxohepta-2,6-dien-1-yl]-2-methoxyphenyl octadeca-9,12,15-trienoate (4), and (1E,6E)-1-[4-(hexadecyloxy)-3-methoxyphenyl]-7-(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (5), and didemethylcurcumin (6) on the membrane translocation of PKCα, a conventional PKC, and PKCε, a novel PKC, has been studied in CHO-K1 cells, in which these PKC isoforms are endogenously expressed. Translocation of PKC from the cytosol to the membrane was measured using immunoblotting and confocal microscopy. 1 and 6 inhibited the TPA-induced membrane translocation of PKCα but not of PKCε. Modification of the hydroxyl group of curcumin with a long aliphatic chain containing unsaturated double bonds in 2-4 completely abolished this inhibition property. Instead, 2-4 showed significant translocation of PKCα but not of PKCε to the membrane. No membrane translocation was observed with 1, 6, or the analogue 5 having a saturated long chain for either PKCα or PKCε. 1 and 6 inhibited TPA-induced activation of ERK1/2, and 2-4 activated it. ERK1/2 is the downstream readout of PKC. These results show that the hydroxyl group of curcumin is important for PKC activity and the curcumin template can be useful in developing isoform specific PKC modulators for regulating a particular disease state.

Functional variations modulating PRKCA expression and alternative splicing predispose to multiple sclerosis.

The protein kinase C alpha (PRKCA) gene, encoding a Th17-cell-selective kinase, was repeatedly associated with multiple sclerosis (MS), but the underlying pathogenic mechanism remains unknown. We replicated the association in Italians (409 cases, 723 controls), identifying a protective signal in the PRKCA promoter (P = 0.033), and a risk haplotype in intron 3 (P = 7.7 × 10(-4); meta-analysis with previously published data: P = 4.01 × 10(-8)). Expression experiments demonstrated that the protective signal is associated with alleles conferring higher PRKCA expression levels, well fitting our observation that MS patients have significantly lower PRKCA mRNA levels in blood. The risk haplotype was shown to be driven by a GGTG ins/del polymorphism influencing the heterogeneous nuclear ribonucleoprotein H-dependent inclusion/skipping of a PRKCA alternative exon 3*. Indeed, exon 3* can be present in two different versions in PRKCA mRNAs (out-of-frame 61 bp or in-frame 66 bp long), and is preferentially included in transcripts generated through a premature polyadenylation event. The GGTG insertion downregulates 3* inclusion and shifts splicing towards the 66 bp isoform. Both events reduce the nonsense-mediated mRNA-decay-induced degradation of exon 3*-containing mRNAs. Since we demonstrated that the protein isoform produced through premature polyadenylation aberrantly localizes to the plasma membrane and/or in cytoplasmic clusters, dysregulated PRKCA 3* inclusion may represent an additional mechanism relevant to MS susceptibility.

Protein interacting with C-kinase 1 (PICK1) binding promiscuity relies on unconventional PSD-95/discs-large/ZO-1 homology (PDZ) binding modes for nonclass II PDZ ligands.

PDZ domain proteins control multiple cellular functions by governing assembly of protein complexes. It remains unknown why individual PDZ domains can bind the extreme C terminus of very diverse binding partners and maintain selectivity. By employing NMR spectroscopy, together with molecular modeling, mutational analysis, and fluorescent polarization binding experiments, we identify here three structural mechanisms explaining why the PDZ domain of PICK1 selectively binds >30 receptors, transporters, and kinases. Class II ligands, including the dopamine transporter, adopt a canonical binding mode with promiscuity obtained via differential packing in the binding groove. Class I ligands, such as protein kinase Cα, depend on residues upstream from the canonical binding sequence that are likely to interact with flexible loop residues of the PDZ domain. Finally, we obtain evidence that the unconventional ligand ASIC1a has a dual binding mode involving a canonical insertion and a noncanonical internal insertion with the two C-terminal residues forming interactions outside the groove. Together with an evolutionary analysis, the data show how unconventional binding modes might evolve for a protein recognition domain to expand the repertoire of functionally important interactions.

Conserved modular domains team up to latch-open active protein kinase Cα.

Signaling proteins comprised of modular domains have evolved along with multicellularity as a method to facilitate increasing intracellular bandwidth. The effects of intramolecular interactions between modular domains within the context of native proteins have been largely unexplored. Here we examine intra- and intermolecular interactions in the multidomain signaling protein, protein kinase Cα (PKCα). We identify three interactions between two activated PKC molecules that synergistically stabilize a nanomolar affinity homodimer. Disruption of the homodimer results in a loss of PKC-mediated ERK1/2 phosphorylation, whereas disruption of the auto-inhibited state promotes the homodimer and prolongs PKC membrane localization. These observations support a novel regulatory mechanism wherein homodimerization dictates the equilibrium between the auto-inhibited and active states of PKC by sequestering auto-inhibitory interactions. Our findings underscore the physiological importance of context-dependent modular domain interactions in cell signaling.

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.

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.

Preferential star strand biogenesis of pre-miR-24-2 targets PKC-alpha and suppresses cell survival in MCF-7 breast cancer cells.

microRNAs (miRNA) are regulators of cellular pathways and alterations of normal miRNA expression levels have been shown to increase tumorigenesis. miR-24 has been demonstrated as having both tumor suppressive and oncogenic properties depending on cell context. Here, we demonstrate a possible role for pre-miR-24-2 as a tumor suppressor in the MCF-7 breast cancer cell line through the preferential processing of mature miR-24-2* over miR-24. Specifically, we show that the ectopic expression of miR-24-2* in MCF-7 breast cancer cells results in a suppression of cellular survival both in vivo and in vitro. Notably, the overexpression of miR-24-2* results in a dampening of cell survival through the targeted suppression of PKCα. In addition, a similar biological change is observed in vivo where MCF-7 cells overexpressing pre-miR-24-2 have decreased tumorigenicity and tumor incidence. Taken together our data demonstrate that when overexpressed biogenesis of the pre-miR-24-2 favors miR-24-2* in the MCF-7 breast cancer cell line and suggests a tumor suppressive role for miR-24-2* observed through the inhibition of PKCα-mediated cellular survival.

Role of calcium in membrane interactions by PI(4,5)P2-binding proteins.

Ca²âº and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] are key agents in membrane-associated signalling events. Their temporal and spatial regulation is crucial for activation or recruitment of proteins in the plasma membrane. In fact, the interaction of several signalling proteins with PI(4,5)P2 has been shown to be tightly regulated and dependent on the presence of Ca²âº, with co-operative binding in some cases. In these proteins, PI(4,5)P2 and Ca²âº binding typically occurs at different binding sites. In addition, several PI(4,5)P2-binding proteins are known targets of calmodulin (CaM), which, depending on the presence of calcium, can compete with PI(4,5)P2 for protein interaction, translating Ca²âº transient microdomains into variations of PI(4,5)P2 lateral organization in time and space. The present review highlights different examples of calcium-dependent PI(4,5)P2-binding proteins and discusses the possible impact of this dual regulation on fine-tuning of protein activity by triggering target membrane binding in the presence of subtle changes in the levels of calcium or PI(4,5)P2.

Cd2+ as a Ca2+ surrogate in protein-membrane interactions: isostructural but not isofunctional.

Due to its favorable spectroscopic properties, Cd(2+) is frequently used as a probe of Ca(2+) sites in proteins. We investigate the ability of Cd(2+) to act as a structural and functional surrogate of Ca(2+) in protein-membrane interactions. C2 domain from protein kinase Cα (C2α) was chosen as a paradigm for the Ca(2+)-dependent phosphatidylserine-binding peripheral membrane domains. We identified the Cd(2+)-binding sites of C2α using NMR spectroscopy, determined the 1.6 Å crystal structure of Cd(2+)-bound C2α, and characterized metal-ion-dependent interactions between C2α and phospholipid membranes using fluorescence spectroscopy and ultracentrifugation experiments. We show that Cd(2+) forms a tight complex with the membrane-binding loops of C2α but is unable to support its membrane-binding function. This is in sharp contrast with Pb(2+), which is almost as effective as Ca(2+) in driving the C2α-membrane association process. Our results provide the first direct evidence for the specific role of divalent metal ions in mediating protein-membrane interactions, have important implications for metal substitution studies in proteins, and illustrate the potential diversity of functional responses caused by toxic metal ions.