C2 Domain Research Articles

Arabidopsis MCTP family member QUIRKY regulates the formation of the STRUBBELIG receptor kinase complex.

Intercellular communication plays a central role in organogenesis. Tissue morphogenesis in Arabidopsis (Arabidopsis thaliana) requires signaling mediated by a cell surface complex containing the atypical receptor kinase STRUBBELIG (SUB) and the multiple C2 domains and transmembrane region protein QUIRKY (QKY). QKY is required to stabilize SUB at the plasma membrane. However, it is unclear what the in vivo architecture of the QKY/SUB signaling complex is, how it is controlled, and how it relates to the maintenance of SUB at the cell surface. We addressed these questions using a combination of genetics, yeast 2-hybrid assays, and Förster resonance energy transfer (FRET)/fluorescence lifetime imaging microscopy (FLIM) in epidermal cells of seedling roots. We found that QKY promotes the formation of SUB homooligomers in vivo. Homooligomerization of SUB appeared to involve its extracellular domain. We also showed that QKY and SUB physically interact and form a complex at the cell surface in vivo. In addition, the data showed that the N-terminal C2A-B region of QKY interacts with the intracellular domain of SUB. They further revealed that this interaction is essential to maintain SUB levels at the cell surface. Finally, we provided evidence that QKY forms homomultimers in vivo in a SUB-independent manner. We suggest a model in which the physical interaction of QKY with SUB mediates the oligomerization of SUB and attenuates its internalization, thereby maintaining sufficiently high levels of SUB at the cell surface required for the control of tissue morphogenesis.

Activation of Munc13-1 by Diacylglycerol (DAG)-Lactones.

Munc13-1 is a key protein necessary for vesicle fusion and neurotransmitter release in the brain. Diacylglycerol (DAG)/phorbol ester binds to its C1 domain in the plasma membrane and activates it. The C1 domain of Munc13-1 and protein kinase C (PKC) are homologous in terms of sequence and structure. In order to identify small-molecule modulators of Munc13-1 targeting the C1 domain, we studied the effect of three DAG-lactones, (R,Z)-(2-(hydroxymethyl)-4-(3-isobutyl-5-methylhexylidene)-5-oxotetrahydrofuran-2-yl)methyl pivalate (JH-131e-153), (E)-(2-(hydroxymethyl)-4-(3-isobutyl-5-methylhexylidene)-5-oxotetrahydrofuran-2-yl)methyl pivalate (AJH-836), and (E)-(2-(hydroxymethyl)-4-(4-nitrobenzylidene)-5-oxotetrahydrofuran-2-yl)methyl 4-(dimethylamino)benzoate (130C037), on Munc13-1 activation using the ligand-induced membrane translocation assay. JH-131e-153 showed higher activation than AJH-836, and 130C037 was not able to activate Munc13-1. To understand the role of the ligand-binding site residues in the activation process, three alanine mutants were generated. For AJH-836, the order of activation was wild-type (WT) Munc13-1 > R592A > W588A > I590A. For JH-131e-153, the order of activation was WT > I590 ≈ R592A ≈ W588A. Overall, the Z isomer of DAG-lactones showed higher potency than the E isomer and Trp-588, Ile-590, and Arg-592 were important for its binding. When comparing the activation of Munc13-1 and PKC, the order of activation for JH-131e-153 was PKCα > Munc13-1 > PKCε and for AJH-836, the order of activation was PKCε > PKCα > Munc13-1. Molecular docking supported higher binding of JH-131e-153 than AJH-836 with the Munc13-1 C1 domain. Our results suggest that DAG-lactones have the potential to modulate neuronal processes via Munc13-1 and can be further developed for therapeutic intervention for neurodegenerative diseases.

Factor VIII antibody immune complexes modulate the humoral response to factor VIII in an epitope-dependent manner.

Soluble antigens complexed with immunoglobulin G (IgG) antibodies can induce robust adaptive immune responses in vitro and in animal models of disease. Factor VIII immune complexes (FVIII-ICs) have been detected in individuals with hemophilia A and severe von Willebrand disease following FVIII infusions. Yet, it is unclear if and how FVIII-ICs affect antibody development over time. In this study, we analyzed internalization of FVIII complexed with epitope-mapped FVIII-specific IgG monoclonal antibodies (MAbs) by murine bone marrow-derived dendritic cells (BMDCs) in vitro and antibody development in hemophilia A (FVIII-/-) mice injected with FVIII-IC over time. FVIII complexed with 2-116 (A1 domain MAb), 2-113 (A3 domain MAb), and I55 (C2 domain MAb) significantly increased FVIII uptake by BMDC but only FVIII/2-116 enhanced antibody titers in FVIII-/- mice compared to FVIII alone. FVIII/4A4 (A2 domain MAb) showed similar FVIII uptake by BMDC to that of isolated FVIII yet significantly increased antibody titers when injected in FVIII-/- mice. Enhanced antibody responses observed with FVIII/2-116 and FVIII/4A4 complexes in vivo were abrogated in the absence of the FVIII carrier protein von Willebrand factor. These findings suggest that a subset of FVIII-IC modulates the humoral response to FVIII in an epitope-dependent manner, which may provide insight into the antibody response observed in some patients with hemophilia A.

Effectiveness of the CinQASE Web Integrated Learning Model to Train Higher Order Thinking Skills

The lack of training in higher-order cognitive thinking skills in physics makes most students of class XI MIPA in a high school in Marabahan unable to solve problems that require the ability to analyze (C4) and evaluate (C5) well. This study was intended to describe the effectiveness of using the web-integrated CinQASE learning model to train higher-order thinking skills in the C4 and C5 domains. This study used the pre-experimental one-group pretest-posttest design. The efficacy of the web-integrated CinQASE learning model to train higher-order thinking skills is calculated with the eta-squared effect size for a paired sample t-test. In contrast, the description of students' achievement in higher-order thinking is viewed from the percentage of the average score of the student learning outcomes test on items that have C4 and C5 domains. The results showed that using the model had moderate effectiveness (η2 = 0.94) and helped train students’ ability to think at a higher level, as shown by an increase in students’ achievement in higher-level thinking in the good category. Thereby, the use of the web-integrated CinQASE learning model can supply considerable benefits in training students’ higher-order thinking skills. These results also write down a practical need for teachers and schools to implement learning approaches that use the potential of technology to enhance learners’ higher-order thinking skills in the 21st century.

Cooperative regulation of C1-domain membrane recruitment polarizes atypical protein kinase C.

Recruitment of the Par complex protein atypical protein kinase C (aPKC) to a specific membrane domain is a key step in the polarization of animal cells. While numerous proteins and phospholipids interact with aPKC, how these interactions cooperate to control its membrane recruitment has been unknown. Here, we identify aPKC's C1 domain as a phospholipid interaction module that targets aPKC to the membrane of Drosophila neural stem cells (NSCs). The isolated C1 binds the NSC membrane in an unpolarized manner during interphase and mitosis and is uniquely sufficient among aPKC domains for targeting. Other domains, including the catalytic module and those that bind the upstream regulators Par-6 and Bazooka, restrict C1's membrane targeting activity-spatially and temporally-to the apical NSC membrane during mitosis. Our results suggest that aPKC polarity results from cooperative activation of autoinhibited C1-mediated membrane binding activity.

Novel anti-PTEN C2 domain monoclonal antibodies to analyse the expression and function of PTEN isoform variants.

PTEN is a major tumor suppressor gene frequently mutated in human tumors, and germline PTEN gene mutations are the molecular diagnostic of PTEN Hamartoma Tumor Syndrome (PHTS), a heterogeneous disorder that manifests with multiple hamartomas, cancer predisposition, and neurodevelopmental alterations. A diversity of translational and splicing PTEN isoforms exist, as well as PTEN C-terminal truncated variants generated by disease-associated nonsense mutations. However, most of the available anti-PTEN monoclonal antibodies (mAb) recognize epitopes at the PTEN C-terminal tail, which may introduce a bias in the analysis of the expression of PTEN isoforms and variants. We here describe the generation and precise characterization of anti-PTEN mAb recognizing the PTEN C2-domain, and their use to monitor the expression and function of PTEN isoforms and PTEN missense and nonsense mutations associated to disease. These anti-PTEN C2 domain mAb are suitable to study the pathogenicity of PTEN C-terminal truncations that retain stability and function but have lost the PTEN C-terminal epitopes. The use of well-defined anti-PTEN mAb recognizing distinct PTEN regions, as the ones here described, will help to understand the deleterious effects of specific PTEN mutations in human disease.

Representation of Some Ratios of Horn’s Hypergeometric Functions H7 by Continued Fractions

The paper deals with the problem of representation of Horn’s hypergeometric functions via continued fractions and branched continued fractions. We construct the formal continued fraction expansions for three ratios of Horn’s hypergeometric functions H7. The method employed is a two-dimensional generalization of the classical method of constructing a Gaussian continued fraction. It is proved that the continued fraction, which is an expansion of each ratio, uniformly converges to a holomorphic function of two variables on every compact subset of some domain of C2, and that this function is an analytic continuation of such a ratio in this domain. To illustrate this, we provide some numerical experiments at the end.

Genetically encoded phosphatidylserine biosensor for in vitro, ex vivo and in vivo labelling

BackgroundThe dynamics of phosphatidylserine in the plasma membrane is a tightly regulated feature of eukaryotic cells. Phosphatidylserine (PS) is found preferentially in the inner leaflet of the plasma membrane. Disruption of this asymmetry leads to the exposure of phosphatidylserine on the cell surface and is associated with cell death, synaptic pruning, blood clotting and other cellular processes. Due to the role of phosphatidylserine in widespread cellular functions, an efficient phosphatidylserine probe is needed to study them. Currently, a few different phosphatidylserine labelling tools are available; however, these labels have unfavourable signal-to-noise ratios and are difficult to use in tissues due to limited permeability. Their application in living tissue requires injection procedures that damage the tissue and release damage-associated molecular patterns, which in turn stimulates phosphatidylserine exposure.MethodsFor this reason, we developed a novel genetically encoded phosphatidylserine probe based on the C2 domain of the lactadherin (MFG-E8) protein, suitable for labelling exposed phosphatidylserine in various research models. We tested the C2 probe specificity to phosphatidylserine on hybrid bilayer lipid membranes by observing surface plasmon resonance angle shift. Then, we analysed purified fused C2 proteins on different cell culture lines or engineered AAVs encoding C2 probes on tissue cultures after apoptosis induction. For in vivo experiments, neurotropic AAVs were intravenously injected into perinatal mice, and after 2 weeks, brain slices were collected to observe C2-SNAP expression.ResultsThe biophysical analysis revealed the high specificity of the C2 probe for phosphatidylserine. The fused recombinant C2 proteins were suitable for labelling phosphatidylserine on the surface of apoptotic cells in various cell lines. We engineered AAVs and validated them in organotypic brain tissue cultures for non-invasive delivery of the genetically encoded C2 probe and showed that these probes were expressed in the brain in vivo after intravenous AAV delivery to mice.ConclusionsWe have demonstrated that the developed genetically encoded PS biosensor can be utilised in a variety of assays as a two-component system of C2 and C2m2 fusion proteins. This system allows for precise quantification and PS visualisation at directly specified threshold levels, enabling the evaluation of PS exposure in both physiological and cell death processes.

Genome-wide identification of papain-like cysteine proteases in Citrus sinensis and expression analysis in response to Candidatus Liberibacter asiaticus

ABSTRACT Papain-like cysteine proteases (PLCPs) play important roles in plant-pathogen interactions. In citrus, PLCPs are targeted by effectors from Candidatus Liberibacter asiaticus (CLas), the causal agent of Huanglongbing (HLB). To study interactions between Citrus sinensis and CLas, we identified 33 candidate CsPLCP genes by HMMsearch with the Peptidase_C1 domain (PF00112), and further confirmed them using local BLAST with PLCP sequences from Arabidopsis thaliana. The 33 CsPLCPs were classified into 9 subfamilies. Analysis of the cis-acting elements in their promoters indicated that CsPLCPs are responsive to various phytohormones and stressors. Further qPCR analysis indicated that several CsPLCPs were up-regulated during CLas infection. Subcellular localization analysis indicated that five CsPLCPs were localized to the cytoplasm and nucleus, while CsXBCP2 was localized only to the cytoplasm. In summary, this study provides a sound basis for understanding the roles of CsPLCPs in the Citrus-CLas interaction.

Otoferlin as a multirole Ca2+ signaling protein: from inner ear synapses to cancer pathways.

Humans have six members of the ferlin protein family: dysferlin, myoferlin, otoferlin, fer1L4, fer1L5, and fer1L6. These proteins share common features such as multiple Ca2+-binding C2 domains, FerA domains, and membrane anchoring through their single C-terminal transmembrane domain, and are believed to play a key role in calcium-triggered membrane fusion and vesicle trafficking. Otoferlin plays a crucial role in hearing and vestibular function. In this review, we will discuss how we see otoferlin working as a Ca2+-dependent mechanical sensor regulating synaptic vesicle fusion at the hair cell ribbon synapses. Although otoferlin is also present in the central nervous system, particularly in the cortex and amygdala, its role in brain tissues remains unknown. Mutations in the OTOF gene cause one of the most frequent genetic forms of congenital deafness, DFNB9. These mutations produce severe to profound hearing loss due to a defect in synaptic excitatory glutamatergic transmission between the inner hair cells and the nerve fibers of the auditory nerve. Gene therapy protocols that allow normal rescue expression of otoferlin in hair cells have just started and are currently in pre-clinical phase. In parallel, studies have linked ferlins to cancer through their effect on cell signaling and development, allowing tumors to form and cancer cells to adapt to a hostile environment. Modulation by mechanical forces and Ca2+ signaling are key determinants of the metastatic process. Although ferlins importance in cancer has not been extensively studied, data show that otoferlin expression is significantly associated with survival in specific cancer types, including clear cell and papillary cell renal carcinoma, and urothelial bladder cancer. These findings indicate a role for otoferlin in the carcinogenesis of these tumors, which requires further investigation to confirm and understand its exact role, particularly as it varies by tumor site. Targeting this protein may lead to new cancer therapies.

Genetic mutations in smoking-associated prostate cancer.

Tobacco smoking is known to cause cancers potentially predisposed by genetic risks. We compared the frequency of gene mutations using a next generation sequencing database of smokers and nonsmokers with prostate cancer (PCa) to identify subsets of patients with potential genetic risks. Data from the American Association for Cancer Research Project Genomics Evidence Neoplasia Information Exchange (GENIE) registry was analyzed. The GENIE registry contains clinically annotated sequenced tumor samples. We included 1832 men with PCa in our cohort, categorized as smokers and nonsmokers, and compared the frequency of mutations (point mutations, copy number variations, and structural variants) of 47 genes with more than 5% mutation rate between the two categories and correlated with overall survival using logistic regression analysis. Overall, 1007 (55%) patients were nonsmokers, and 825 (45%) were smokers. The mutation frequency was significantly higher in smokers compared to nonsmokers, 47.6% and 41.3%, respectively (p = 0.02). The median tumor mutational burden was also significantly higher in the samples from smokers (3.59 mut/MB) compared to nonsmokers (1.87 mut/MB) (p < 0.001). Patients with a smoking history had a significantly higher frequency of PREX2, PTEN, AGO2, KMT2C, and a lower frequency of adenomatous polyposis coli (APC) and KMT2A mutations than compared to nonsmokers. The overall mortality rate (28.5% vs. 22.8%) was significantly higher among smokers (p = 0.006). On a multivariate logistic regression analysis, the presence of metastatic disease at the time of diagnosis (OR: 2.26, 95% CI: 1.78-2.89, p < 0.001), smoking history (OR: 1.32, 95% CI: 1.05-1.65, p = 0.02), and higher frequency of PTEN somatic gene mutation (OR: 1.89, 95% CI: 1.46-2.45, p < 0.001) were independent predictors of increased overall mortality among patients with PCa. Patients with PTEN mutation had poorer overall survival compared to men without PTEN mutations: 96.00 (95% CI: 65.36-113.98) and 120.00 (95% CI: 115.05-160.00) months, respectively (p < 0.001) irrespective of smoking history although the G129R PTEN mutation was characteristically detected in smokers. PCa patients with a tobacco smoking history demonstrated a significantly higher frequency of somatic genetic mutations. Whereas mutations of PREX2, KMT2C, AGO2, and PTEN genes were higher in smokers, the APC and KMT2A mutations were higher in nonsmokers. The PTEN somatic gene mutation was associated with increased overall mortality among patients with PCa irrespective of smoking history. We found that G129R PTEN mutation known to reduce the PTEN phosphatase activity and K267Rfs*9 a frameshift deletion mutation in the C2 domain of PTEN associated with membrane binding exclusively detected in smokers and nonsmokers, respectively. These findings may be used to further our understanding of PCa associated with smoking.

The Dysferlin C2A Domain Binds PI(4,5)P2 and Penetrates Membranes

Dysferlin is a large membrane protein found most prominently in striated muscle. Loss of dysferlin activity is associated with reduced exocytosis, abnormal intracellular Ca2+ and the muscle diseases limb-girdle muscular dystrophy and Miyoshi myopathy. The cytosolic region of dysferlin consists of seven C2 domains with mutations in the C2A domain at the N-terminus resulting in pathology. Despite the importance of Ca2+ and membrane binding activities of the C2A domain for dysferlin function, the mechanism of the domain remains poorly characterized. In this study we find that the C2A domain preferentially binds membranes containing PI(4,5)P2 through an interaction mediated by residues Y23, K32, K33, and R77 on the concave face of the domain. We also found that subsequent to membrane binding, the C2A domain inserts residues on the Ca2+ binding loops into the membrane. Analysis of solution NMR measurements indicate that the domain inhabits two distinct structural states, with Ca2+ shifting the population between states towards a more rigid structure with greater affinity for PI(4,5)P2. Based on our results, we propose a mechanism where Ca2+ converts C2A from a structurally dynamic, low PI(4,5)P2 affinity state to a high affinity state that targets dysferlin to PI(4,5)P2 enriched membranes through interaction with Tyr23, K32, K33, and R77. Binding also involves changes in lipid packing and insertion by the third Ca2+ binding loop of the C2 domain into the membrane, which would contribute to dysferlin function in exocytosis and Ca2+ regulation.

Biological roles of plant synaptotagmins

Plant synaptotagmins (SYTs) are resident proteins of the endoplasmic reticulum (ER). They are characterized by an N-terminal transmembrane region and C2 domains at the C-terminus, which tether the ER to the plasma membrane (PM). In addition to their tethering role, SYTs contain a lipid-harboring SMP domain, essential for shuttling lipids between the ER and the PM. There is now abundant literature on Arabidopsis SYT1, the best-characterized family member, which link it to biotic and abiotic responses as well as to ER morphology. Here, we review the current knowledge of SYT members, focusing on their role in stress, and discuss how these roles can be related to their tethering and lipid transport functions. Finally, we contextualize this information about SYTs with their homologs, the yeast tricalbins and the mammalian extended synaptotagmins.

1770-P: Islet ß-Cells Shed DOC2B-Laden EVs via Sorting Signals Present in DOC2B Protein

Loss of β-cell function occurs early in type 1 diabetes (T1D). However, C-peptide is insufficient to detect early pre-type 1 diabetes-related β-cell dysfunction. Thus, the discovery of clinically accessible β-cell health biomarkers may enable early therapeutic interventions, mitigate T1D, and prevent complete loss of β-cell mass. The exocytosis regulatory protein double C2-containing protein β (DOC2B) abundance is closely related to β-cell health and function. Our recent publication demonstrated the correlation between the decline in DOC2B abundance in blood-derived platelets and the loss of β-cell function in early-onset T1D and pre-diabetic nonobese diabetic (NOD) mice. Indeed, preliminary data show that DOC2B reduction in plasma precedes C-peptide reduction in prediabetic mice, suggesting that reduced DOC2B levels in plasma may serve as an early biomarker of β-cell dysfunction. However, the mechanisms by which β-cells export DOC2B into the circulation remain unknown. We hypothesize that DOC2B is uniquely sorted into β-cell extracellular vesicles (EVs). Preliminary data show DOC2B is detected in EVs isolated from human plasma, and that DOC2B-laden EVs can stem from β-cells. Because DOC2B is a broadly expressed protein, we questioned whether DOC2B-laden EVs are predominantly released by β-cells, relative to that of other cell types known to express DOC2B protein. Biochemical analyses reveal that DOC2B largely exists as luminal cargo in β-cell-derived EVs and is largely absent from EVs released by liver, brain, and skeletal muscle cells. Mechanistically, the research has delineated that the sufficient structural domain enabling DOC2B sorting into β-cell-derived EVs is that which encompasses the tandem C2 domains at the C-terminus of DOC2B. Taken together, these data indicate that DOC2B in circulation could originate from DOC2B-laden EVs released from β-cells, and that the mechanism by which DOC2B is sorted into those EVs involves one or both C2 domains of the DOC2B protein. Disclosure D.Esparza: None. E.Oh: None. I.Ghaeli: None. T.Jovanovic-talisman: None. D.C.Thurmond: Consultant; Immutics, Inc. Funding National Institute of Diabetes and Digestive and Kidney Diseases (DK067912, DK112917, DK102233); Wanek Family Project for Type 1 Diabetes (to D.C.T); Ford Foundation (to D.E.)

Impaired factor V-related anticoagulant mechanisms and deep vein thrombosis associated with A2086D and W1920R mutations.

Factor V (FV) plays pivotal roles in both procoagulant and anticoagulant mechanisms. Genetic mutations, FV-W1920R (FVNara) and FV-A2086D (FVBesançon), in the C1 and C2 domains of FV light chain, respectively, seem to be associated with deep vein thrombosis. However, the detailed mechanism(s) through which these mutations are linked to thrombophilia remains to be fully explored. The aim of this study was to clarify thrombotic mechanism(s) in the presence of these FV abnormalities. Full-length wild-type (WT) and mutated FV were prepared using stable, human cell lines (HEK293T) and the piggyBac transposon system. Susceptibility of FVa-A2086D to activated protein C (APC) was reduced, resulting in significant inhibition of APC-catalyzed inactivation with limited cleavage at Arg306 and delayed cleavage at Arg506. Furthermore, APC cofactor activity of FV-A2086D in APC-catalyzed inactivation of FVIIIa through cleavage at Arg336 was impaired. Surface plasmon resonance-based assays demonstrated that FV-A2086D bound to Glu-Gly-Arg-chloromethylketone active site-blocked APC and protein S (P) with similar affinities to that of FV-WT. However, weakened interaction between FVa-A2086D and phospholipid membranes was evident through the prothrombinase assay. Moreover, addition of FVa-A2086D to plasma failed to inhibit tissue factor (TF)-induced thrombin generation and reduce prothrombin times. This inhibitory effect was independent of PC, PS, and antithrombin. The coagulant and anticoagulant characteristics of FV(a)-W1920R were similar to those of FV(a)-A2086D. FV-A2086D presented defects in the APC mechanisms associated with FVa inactivation and FV cofactor activity, similar to FV-W1920R. Moreover, both FV proteins that were mutated in the light chain impaired inhibition of TF-induced coagulation reactions. These defects were consistent with congenital thrombophilia.

Composition operators between Bergman spaces on infinite type convex domains in ℂ2

ABSTRACT We provide characterizations of boundedness and compactness for composition operators between Bergman spaces of smoothly bounded convex domains in C 2 admitting the so-called geometric F-type. Such geometric type contains all domains of finite type and many cases of infinite type in the sense of Range.

Toll-interacting protein participates in immunity and development of the lepidopteran insect Antheraea pernyi.

Toll-interacting protein (Tollip) participates in multiple biological processes. However, the biological functions of Tollip proteins in insects remain to be further explored. Here, the genomic sequence of tollip gene from Antheraea pernyi (named Ap-Tollip) was identified with a length of 15,060 bp, including eight exons and seven introns. The predicted Ap-Tollip protein contained conserved C2 and CUE domains and was highly homologous to those tollips from invertebrates. Ap-Tollip was highly expressed in fat body compared with other determined tissues. As far as the developmental stages were concerned, the highest expression level was found at the 14th day in eggs or the 3rd day of the 1st instar. Ap-Tollip was also obviously regulated by lipopolysaccharide, polycytidylic acid or 20E in different tissues. In addition, the interaction between Ap-Tollip and ubiquitin was confirmed by western blotting and pull-down assay. RNAi of Ap-Tollip significantly affected the expression levels of apoptosis and autophagy-related genes. These results indicated that Ap-Tollip was involved in immunity and development of A. pernyi.

Identification of protein kinase C domains involved in its translocation induced by propofol

Propofol is widely used for general anesthesia and sedation; however, the mechanisms of its anesthetic and adverse effects are not fully understood. We have previously shown that propofol activates protein kinase C (PKC) and induces its translocation in a subtype-specific manner. The purpose of this study was to identify the PKC domains involved in propofol-induced PKC translocation. The regulatory domains of PKC consist of C1 and C2 domains, and the C1 domain is subdivided into the C1A and C1B subdomains. Mutant PKCα and PKCδ with each domain deleted were fused with green fluorescent protein (GFP) and expressed in HeLa cells. Propofol-induced PKC translocation was observed by time-lapse imaging using a fluorescence microscope. The results showed that persistent propofol-induced PKC translocation to the plasma membrane was abolished by the deletion of both C1 and C2 domains in PKCα and by the deletion of the C1B domain in PKCδ. Therefore, propofol-induced PKC translocation involves the C1 and C2 domains of PKCα and the C1B domain of PKCδ. We also found that treatment with calphostin C, a C1 domain inhibitor, abolished propofol-induced PKCδ translocation. In addition, calphostin C inhibited the propofol-induced phosphorylation of endothelial nitric oxide synthase (eNOS). These results suggest that it may be possible to modulate the exertion of propofol effects by regulating the PKC domains involved in propofol-induced PKC translocation.

ERK3 and DGKζ interact to modulate cell motility in lung cancer cells.

Extracellular signal-regulated kinase 3 (ERK3) promotes cell migration and tumor metastasis in multiple cancer types, including lung cancer. The extracellular-regulated kinase 3 protein has a unique structure. In addition to the N-terminal kinase domain, ERK3 includes a central conserved in extracellular-regulated kinase 3 and ERK4 (C34) domain and an extended C-terminus. However, relatively little is known regarding the role(s) of the C34 domain. A yeast two-hybrid assay using extracellular-regulated kinase 3 as bait identified diacylglycerol kinase ζ (DGKζ) as a binding partner. DGKζ was shown to promote migration and invasion in some cancer cell types, but its role in lung cancer cells is yet to be described. The interaction of extracellular-regulated kinase 3 and DGKζ was confirmed by co-immunoprecipitation and in vitro binding assays, consistent with their co-localization at the periphery of lung cancer cells. The C34 domain of ERK3 was sufficient for binding to DGKζ, while extracellular-regulated kinase 3 bound to the N-terminal and C1 domains of DGKζ. Surprisingly, in contrast to extracellular-regulated kinase 3, DGKζ suppresses lung cancer cell migration, suggesting DGKζ might inhibit ERK3-mediated cell motility. Indeed, co-overexpression of exogenous DGKζ and extracellular-regulated kinase 3 completely blocked the ability of ERK3 to promote cell migration, but DGKζ did not affect the migration of cells with stable ERK3 knockdown. Furthermore, DGKζ had little effect on cell migration induced by overexpression of an ERK3 mutant missing the C34 domain, suggesting DGKζ requires this domain to prevent ERK3-mediated increase in cell migration. In summary, this study has identified DGKζ as a new binding partner and negative regulator of extracellular-regulated kinase 3 in controlling lung cancer cell migration.

Control of atypical PKCι membrane dissociation by tyrosine phosphorylation within a PB1-C1 interdomain interface

Atypical PKCs are cell polarity kinases that operate at the plasma membrane where they function within multiple molecular complexes to contribute to the establishment and maintenance of polarity. In contrast to the classical and novel PKCs, atypical PKCs do not respond to diacylglycerol cues to bind the membrane compartment. Until recently it was not clear how aPKCs are recruited; whether aPKCs can directly interact with membranes or whether they are dependent on other protein interactors to do so. Two recent studies identified the pseudo-substrate region and the C1 domain as direct membrane interaction modules, however their relative importance and coupling are unknown. We combined molecular modelling and functional assays to show that the regulatory module of aPKCι, comprising the PB1 pseudo-substrate and C1 domains, forms a cooperative and spatially continuous invariant membrane interaction platform. Furthermore, we show the coordinated orientation of membrane-binding elements within the regulatory module requires a key PB1-C1 interfacial β-strand (BSL). We show this element contains a highly conserved Tyr residue that can be phosphorylated and that negatively regulates the integrity of the regulatory module, leading to membrane release. We thus expose a hitherto unknown regulatory mechanism of aPKCι membrane binding and release during cell polarization.