Rab2 Utilizes Glyceraldehyde-3-phosphate Dehydrogenase and Protein Kinase C�� to Associate with Microtubules and to Recruit Dynein

Rab2 requires glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and atypical protein kinase Cι (aPKCι) for retrograde vesicle formation from vesicular tubular clusters that sort secretory cargo from recycling proteins returned to the endoplasmic reticulum. However, the precise role of GAPDH and aPKCι in the early secretory pathway is unclear. GAPDH was the first glycolytic enzyme reported to co-purify with microtubules (MTs). Similarly, aPKC associates directly with MTs. To learn whether Rab2 also binds directly to MTs, a MT binding assay was performed. Purified Rab2 was found in a MT-enriched pellet only when both GAPDH and aPKCι were present, and Rab2-MT binding could be prevented by a recombinant fragment made to the Rab2 amino terminus (residues 2-70), which directly interacts with GAPDH and aPKCι. Because GAPDH binds to the carboxyl terminus of α-tubulin, we characterized the distribution of tyrosinated/detyrosinated α-tubulin that is recruited by Rab2 in a quantitative membrane binding assay. Rab2-treated membranes contained predominantly tyrosinated α-tubulin; however, aPKCι was the limiting and essential factor. Tyrosination/detyrosination influences MT motor protein binding; therefore, we determined whether Rab2 stimulated kinesin or dynein membrane binding. Although kinesin was not detected on membranes incubated with Rab2, dynein was recruited in a dose-dependent manner, and binding was aPKCι-dependent. These combined results suggest a mechanism by which Rab2 controls MT and motor recruitment to vesicular tubular clusters.The small GTPase Rab2 is essential for membrane trafficking in the early secretory pathway and associates with vesicular tubular clusters (VTCs)² located between the endoplasmic reticulum (ER) and the cis-Golgi compartment (1, 2). VTCs are pleomorphic structures that sort anterograde-directed cargo from recycling proteins and trafficking machinery retrieved to the ER (3-6). Rab2 bound to a VTC microdomain stimulates recruitment of soluble factors that results in the release of vesicles containing the recycling protein p53/p58 (7). In that regard, we have previously reported that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and atypical PKC ι (aPKCι) are Rab2 effectors that interact directly with the Rab2 amino terminus and with each other (8, 9). Their interaction requires Src-dependent tyrosine phosphorylation of GAPDH and aPKCι (10). Moreover, GAPDH is a substrate for aPKCι (11). GAPDH catalytic activity is not required for ER to Golgi transport indicating that GAPDH provides a specific function essential for membrane trafficking from VTCs independent of glycolytic function (9). Indeed, phospho-GAPDH influences MT dynamics in the early secretory pathway (11).GAPDH was the first glycolytic enzyme reported to co-purify with microtubules (MTs) (12) and subsequently was shown to interact with the carboxyl terminus of α-tubulin (13). The binding of GAPDH to MTs promotes formation of cross-linked parallel MT arrays or bundles (14, 15). GAPDH has also been reported to possess membrane fusogenic activity, which is inhibited by tubulin (16). Similarly, aPKC associates directly with tubulin and promotes MT stability and MT remodeling at specific intracellular sites (17-21). It may not be coincidental that these two Rab2 effectors influence MT dynamics because recent studies indicate that the cytoskeleton plays a central role in the organization and operation of the secretory pathway (22).MTs are dynamic structures that grow or shrink by the addition or loss of α- and β-tubulin heterodimers from the ends of protofilaments (23). Their assembly and stability is regulated by a variety of proteins traditionally referred to as microtubule-associated proteins (MAPs). In addition to the multiple α/β isoforms that are present in eukaryotes, MTs undergo an assortment of post-translational modifications, including acetylation, glycylation, glutamylation, phosphorylation, palmitoylation, and detyrosination, which further contribute to their biochemical heterogeneity (24, 25). It has been proposed that these tubulin modifications regulate intracellular events by facilitating interaction with MAPs and with other specific effector proteins (24). For example, the reversible addition of tyrosine to the carboxyl terminus of α-tubulin regulates MT interaction with plus-end tracking proteins (+TIPs) containing the cytoskeleton-associated protein glycine-rich (CAP-Gly) motif and with dynein-dynactin (27-29). Additionally, MT motility and cargo transport rely on the cooperation of the motor proteins kinesin and dynein (30). Kinesin is a plus-end directed MT motor, whereas cytoplasmic dynein is a minus-end MT-based motor, and therefore the motors transport vesicular cargo toward the opposite end of a MT track (31).Although MT assembly does not appear to be directly regulated by small GTPases, Rab proteins provide a molecular link for vesicle movement along MTs to the appropriate target (22, 32-34). In this study, the potential interaction of Rab2 with MTs and motor proteins was characterized. We found that Rab2 does not bind directly to preassembled MTs but does associate when both GAPDH and aPKCι are present and bound to MTs. Moreover, the MTs predominantly contained tyrosinated α-tubulin (Tyr-tubulin) suggesting that a dynamic pool of MTs that differentially binds MAPs/effector proteins/motors associates with VTCs in response to Rab2. To that end, we determined that Rab2-promoted dynein/dynactin binding to membranes and that the recruitment required aPKCι.     

Identification of the Gene Encoding the Tryptophan Synthase β-Subunit from Chlamydomonas reinhardtii

We report the isolation of a Chlamydomonas reinhardtii cDNA that encodes the β-subunit of tryptophan synthase (TSB). This cDNA was cloned by functional complementation of a trp-operon-deleted strain of Escherichia coli. Hybridization analysis indicated that the gene exists in a single copy. The predicted amino acid sequence showed the greatest identity to TSB polypeptides from other photosynthetic organisms. With the goal of identifying mutations in the gene encoding this enzyme, we isolated 11 recessive and 1 dominant single-gene mutation that conferred resistance to 5-fluoroindole. These mutations fell into three complementation groups, MAA2, MAA7, and TAR1. In vitro assays showed that mutations at each of these loci affected TSB activity. Restriction fragment-length polymorphism analysis suggested that MAA7 encodes TSB. MAA2 and TAR1 may act to regulate the activity of MAA7 or its protein product.     

siah-1 Protein Is Necessary for High Glucose-induced Glyceraldehyde-3-phosphate Dehydrogenase Nuclear Accumulation and Cell Death in M��ller Cells

The translocation and accumulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the nucleus has closely been associated with cell death induction. However, the mechanism of this process has not been completely understood. The E3 ubiquitin ligase siah-1 (seven in absentia homolog 1) has recently been identified as a potential shuttle protein to transport GAPDH from the cytosol to the nucleus. Previously, we have demonstrated that elevated glucose levels induce GAPDH nuclear accumulation in retinal Müller cells. Therefore, this study investigated the role of siah-1 in high glucose-induced GAPDH nuclear translocation and subsequent cell death in retinal Müller cells. High glucose significantly increased siah-1 expression within 12 h. Under hyperglycemic conditions, siah-1 formed a complex with GAPDH and was predominantly localized in the nucleus of Müller cells. siah-1 knockdown using 50 nm siah-1 small interfering RNA significantly decreased high glucose-induced GAPDH nuclear accumulation at 24 h by 43.8 ± 4.0%. Further, knockdown of siah-1 prevented high glucose-induced cell death of Müller cells potentially by inhibiting p53 phosphorylation consistent with previous observations, indicating that nuclear GAPDH induces cell death via p53 activation. Therefore, inhibition of GAPDH nuclear translocation and accumulation by targeting siah-1 promotes Müller cell survival under hyperglycemic conditions.     

Different Thermostability of Skeletal Muscle Glyceraldehyde-3-phosphate Dehydrogenase from Hibernating and Euthermic Jerboa （Jaculus orientalis）

D Glyceraldehyde 3 phosphatedehydrogenase(GAPDH ,EC 1.2 .1.12 )isakeyenzymeoftheglycolyticpathwaythatispresentinthecytosolofallorganismssofarstudied[1] .TheglycolyticGAPDHhasbeenremarkablyconservedduringevolution ,havingahomotetramericstructurewithsubunitsof 35 - 37kD[1] .GAPDHhasbeenisolatedfromavarietyofspecies[2 ] ,includingmesophilic ,moderatelythermophilicandhyperthermophilicmicroorganisms[3 ] .Theseenzymes ,whichdifferinthermalstability ,havebeenshowntobehighlysimilarinaminoacidse…     

The Cytoskeletal Protein ��-Actinin Regulates Acid-sensing Ion Channel 1a through a C-terminal Interaction

The acid-sensing ion channel 1a (ASIC1a) is widely expressed in central and peripheral neurons where it generates transient cation currents when extracellular pH falls. ASIC1a confers pH-dependent modulation on postsynaptic dendritic spines and has critical effects in neurological diseases associated with a reduced pH. However, knowledge of the proteins that interact with ASIC1a and influence its function is limited. Here, we show that α-actinin, which links membrane proteins to the actin cytoskeleton, associates with ASIC1a in brain and in cultured cells. The interaction depended on an α-actinin-binding site in the ASIC1a C terminus that was specific for ASIC1a versus other ASICs and for α-actinin-1 and -4. Co-expressing α-actinin-4 altered ASIC1a current density, pH sensitivity, desensitization rate, and recovery from desensitization. Moreover, reducing α-actinin expression altered acid-activated currents in hippocampal neurons. These findings suggest that α-actinins may link ASIC1a to a macromolecular complex in the postsynaptic membrane where it regulates ASIC1a activity.Acid-sensing ion channels (ASICs)² are H⁺-gated members of the DEG/ENaC family (1–3). Members of this family contain cytosolic N and C termini, two transmembrane domains, and a large cysteine-rich extracellular domain. ASIC subunits combine as homo- or heterotrimers to form cation channels that are widely expressed in the central and peripheral nervous systems (1–4). In mammals, four genes encode ASICs, and two subunits, ASIC1 and ASIC2, have two splice forms, a and b. Central nervous system neurons express ASIC1a, ASIC2a, and ASIC2b (5–7). Homomeric ASIC1a channels are activated when extracellular pH drops below 7.2, and half-maximal activation occurs at pH 6.5–6.8 (8–10). These channels desensitize in the continued presence of a low extracellular pH, and they can conduct Ca²⁺ (9, 11–13). ASIC1a is required for acid-evoked currents in central nervous system neurons; disrupting the gene encoding ASIC1a eliminates H⁺-gated currents unless extracellular pH is reduced below pH 5.0 (5, 7).Previous studies found ASIC1a enriched in synaptosomal membrane fractions and present in dendritic spines, the site of excitatory synapses (5, 14, 15). Consistent with this localization, ASIC1a null mice manifested deficits in hippocampal long term potentiation, learning, and memory, which suggested that ASIC1a is required for normal synaptic plasticity (5, 16). ASICs might be activated during neurotransmission when synaptic vesicles empty their acidic contents into the synaptic cleft or when neuronal activity lowers extracellular pH (17–19). Ion channels, including those at the synapse often interact with multiple proteins in a macromolecular complex that incorporates regulators of their function (20, 21). For ASIC1a, only a few interacting proteins have been identified. Earlier work indicated that ASIC1a interacts with another postsynaptic scaffolding protein, PICK1 (15, 22, 23). ASIC1a also has been reported to interact with annexin II light chain p11 through its cytosolic N terminus to increase cell surface expression (24) and with Ca²⁺/calmodulin-dependent protein kinase II to phosphorylate the channel (25). However, whether ASIC1a interacts with additional proteins and with the cytoskeleton remain unknown. Moreover, it is not known whether such interactions alter ASIC1a function.In analyzing the ASIC1a amino acid sequence, we identified cytosolic residues that might bind α-actinins. α-Actinins cluster membrane proteins and signaling molecules into macromolecular complexes and link membrane proteins to the actincytoskeleton (for review, Ref. 26). Four genes encode α-actinin-1, -2, -3, and -4 isoforms. α-Actinins contain an N-terminal head domain that binds F-actin, a C-terminal region containing two EF-hand motifs, and a central rod domain containing four spectrin-like motifs (26–28). The C-terminal portion of the rod segment appears to be crucial for binding to membrane proteins. The α-actinins assemble into antiparallel homodimers through interactions in their rod domain. α-Actinins-1, -2, and -4 are enriched in dendritic spines, concentrating at the postsynaptic membrane (29–35). In the postsynaptic membrane of excitatory synapses, α-actinin connects the NMDA receptor to the actin cytoskeleton, and this interaction is key for Ca²⁺-dependent inhibition of NMDA receptors (36–38). α-Actinins can also regulate the membrane trafficking and function of several cation channels, including L-type Ca²⁺ channels, K⁺ channels, and TRP channels (39–41).To better understand the function of ASIC1a channels in macromolecular complexes, we asked if ASIC1a associates with α-actinins. We were interested in the α-actinins because they and ASIC1a, both, are present in dendritic spines, ASIC1a contains a potential α-actinin binding sequence, and the related epithelial Na⁺ channel (ENaC) interacts with the cytoskeleton (42, 43). Therefore, we hypothesized that α-actinin interacts structurally and functionally with ASIC1a.     

Mechanism of Activation and Functional Role of Protein Kinase C�� in Human Platelets

The novel class of protein kinase C (nPKC) isoform η is expressed in platelets, but not much is known about its activation and function. In this study, we investigated the mechanism of activation and functional implications of nPKCη using pharmacological and gene knock-out approaches. nPKCη was phosphorylated (at Thr-512) in a time- and concentration-dependent manner by 2MeSADP. Pretreatment of platelets with MRS-2179, a P2Y₁ receptor antagonist, or YM-254890, a G_q blocker, abolished 2MeSADP-induced phosphorylation of nPKCη. Similarly, ADP failed to activate nPKCη in platelets isolated from P2Y₁ and G_q knock-out mice. However, pretreatment of platelets with P2Y₁₂ receptor antagonist, AR-C69331MX did not interfere with ADP-induced nPKCη phosphorylation. In addition, when platelets were activated with 2MeSADP under stirring conditions, although nPKCη was phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by activated integrin α_IIbβ₃ mediated outside-in signaling. Moreover, in the presence of SC-57101, a α_IIbβ₃ receptor antagonist, nPKCη dephosphorylation was inhibited. Furthermore, in murine platelets lacking PP1cγ, a catalytic subunit of serine/threonine phosphatase, α_IIbβ₃ failed to dephosphorylate nPKCη. Thus, we conclude that ADP activates nPKCη via P2Y₁ receptor and is subsequently dephosphorylated by PP1γ phosphatase activated by α_IIbβ₃ integrin. In addition, pretreatment of platelets with η-RACK antagonistic peptides, a specific inhibitor of nPKCη, inhibited ADP-induced thromboxane generation. However, these peptides had no affect on ADP-induced aggregation when thromboxane generation was blocked. In summary, nPKCη positively regulates agonist-induced thromboxane generation with no effects on platelet aggregation.Platelets are the key cellular components in maintaining hemostasis (1). Vascular injury exposes subendothelial collagen that activates platelets to change shape, secrete contents of granules, generate thromboxane, and finally aggregate via activated α_IIbβ₃ integrin, to prevent further bleeding (2, 3). ADP is a physiological agonist of platelets secreted from dense granules and is involved in feedback activation of platelets and hemostatic plug stabilization (4). It activates two distinct G-protein-coupled receptors (GPCRs) on platelets, P2Y₁ and P2Y₁₂, which couple to G_q and G_i, respectively (5–8). G_q activates phospholipase Cβ (PLCβ), which leads to diacyl glycerol (DAG)² generation and calcium mobilization (9, 10). On the other hand, G_i is involved in inhibition of cAMP levels and PI 3-kinase activation (4, 6). Synergistic activation of G_q and G_i proteins leads to the activation of the fibrinogen receptor integrin α_IIbβ₃. Fibrinogen bound to activated integrin α_IIbβ₃ further initiates feed back signaling (outside-in signaling) in platelets that contributes to the formation of a stable platelet plug (11).Protein kinase Cs (PKCs) are serine/threonine kinases known to regulate various platelet functional responses such as dense granule secretion and integrin α_IIbβ₃ activation (12, 13). Based on their structure and cofactor requirements, PKCs are divided in to three classes: classical (cofactors: DAG, Ca²⁺), novel (cofactors: DAG) and atypical (cofactors: PIP₃) PKC isoforms (14). All the members of the novel class of PKC isoforms (nPKC), viz. nPKC isoforms δ, θ, η, and ε, are expressed in platelets (15), and they require DAG for activation. Among all the nPKCs, PKCδ (15, 16) and PKCθ (17–19) are fairly studied in platelets. Whereas nPKCδ is reported to regulate protease-activated receptor (PAR)-mediated dense granule secretion (15, 20), nPKCθ is activated by outside-in signaling and contributes to platelet spreading on fibrinogen (18). On the other hand, the mechanism of activation and functional role of nPKCη is not addressed as yet.PKCs are cytoplasmic enzymes. The enzyme activity of PKCs is modulated via three mechanisms (14, 21): 1) cofactor binding: upon cell stimulus, cytoplasmic PKCs mobilize to membrane, bind cofactors such as DAG, Ca²⁺, or PIP3, release autoinhibition, and attain an active conformation exposing catalytic domain of the enzyme. 2) phosphorylations: 3-phosphoinositide-dependent kinase 1 (PDK1) on the membrane phosphorylates conserved threonine residues on activation loop of catalytic domain; this is followed by autophosphorylations of serine/threonine residues on turn motif and hydrophobic region. These series of phosphorylations maintain an active conformation of the enzyme. 3) RACK binding: PKCs in active conformation bind receptors for activated C kinases (RACKs) and are lead to various subcellular locations to access the substrates (22, 23). Although various leading laboratories have elucidated the activation of PKCs, the mechanism of down-regulation of PKCs is not completely understood.The premise of dynamic cell signaling, which involves protein phosphorylations by kinases and dephosphorylations by phosphatases has gained immense attention over recent years. PP1, PP2A, PP2B, PHLPP are a few of the serine/threonine phosphatases reported to date. Among them PP1 and PP2 phosphatases are known to regulate various platelet functional responses (24, 25). Furthermore, PP1c, is the catalytic unit of PP1 known to constitutively associate with α_IIb and is activated upon integrin engagement with fibrinogen and subsequent outside-in signaling (26). Among various PP1 isoforms, recently PP1γ is shown to positively regulate platelet functional responses (27). Thus, in this study we investigated if the above-mentioned phosphatases are involved in down-regulation of nPKCη. Furthermore, reports from other cell systems suggest that nPKCη regulates ERK/JNK pathways (28). In platelets ERK is known to regulate agonist induced thromboxane generation (29, 30). Thus, we also investigated if nPKCη regulates ERK phosphorylation and thereby agonist-induced platelet functional responses.In this study, we evaluated the activation of nPKCη downstream of ADP receptors and its inactivation by an integrin-associated phosphatase PP1γ. We also studied if nPKCη regulates functional responses in platelets and found that this isoform regulates ADP-induced thromboxane generation, but not fibrinogen receptor activation in platelets.     

Oligomeric form of the light-harvesting chlorophyll a + b-protein complex CP II,phosphatidyldiacylglycerol, Δ3-trans-hexadecenoic acid and energy transfer in Chlamydomonas reinhardtii,wild type and mutants

Analyses of chlorophyll-protein complexes and of lipids were performed with the wild type of Chlamydomonas reinhardtii and three non-photosynthetic mutants: Fl 39, which was a ‘classical’ high-fluorescent Photosystem II (PS II)-lacking mutant, and mf 1 and mf 2, which lacked also functional PS II but were low-fluorescent and showed an abnormally predominant energy transfer from the main light-harvesting antenna towards Photosystem I. An oligomeric form of the chlorophyll a + b-protein complex CP II was clearly isolated from the wild type and the mutant Fl 39 but it was not detected in the mutants mf 1 and mf 2. The three mutants showed total lipid contents close to or greater than that of the wild type. Their phosphatidyldiacylglycerol (PG) contents, on a chlorophyll basis, were higher (Fl 39) or 1.4- (mf 1) and 2.0- (mf 2) times lower than that of the wild type. The fatty acid compositions of the wild type and of the mutant Fl 39 were comparable, showing about equal amounts of a C18 series and a C16 series which included the Δ3-trans-hexadecenoic acid (C16:1-trans). This C16:1-trans was not detected in the mutants mf 1 and mf 2 which contained the other fatty acids. These results indicate correlations between lack of C16:1-trans-containing PG, lack of an oligomeric form of CP II and an impaired mechanism of the regulation of excitation energy transfer from the main chlorophyll a + b antenna.     

Identification of a Novel Bacterial Outer Membrane Interleukin-1Β-Binding Protein from Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans is a gram-negative opportunistic oral pathogen. It is frequently associated with subgingival biofilms of both chronic and aggressive periodontitis, and the diseased sites of the periodontium exhibit increased levels of the proinflammatory mediator interleukin (IL)-1β. Some bacterial species can alter their physiological properties as a result of sensing IL-1β. We have recently shown that this cytokine localizes to the cytoplasm of A. actinomycetemcomitans in co-cultures with organotypic gingival mucosa. However, current knowledge about the mechanism underlying bacterial IL-1β sensing is still limited. In this study, we characterized the interaction of A. actinomycetemcomitans total membrane protein with IL-1β through electrophoretic mobility shift assays. The interacting protein, which we have designated bacterial interleukin receptor I (BilRI), was identified through mass spectrometry and was found to be Pasteurellaceae specific. Based on the results obtained using protein function prediction tools, this protein localizes to the outer membrane and contains a typical lipoprotein signal sequence. All six tested biofilm cultures of clinical A. actinomycetemcomitans strains expressed the protein according to phage display-derived antibody detection. Moreover, proteinase K treatment of whole A. actinomycetemcomitans cells eliminated BilRI forms that were outer membrane specific, as determined through immunoblotting. The protein was overexpressed in Escherichia coli in both the outer membrane-associated form and a soluble cytoplasmic form. When assessed using flow cytometry, the BilRI-overexpressing E. coli cells were observed to bind 2.5 times more biotinylated-IL-1β than the control cells, as detected with avidin-FITC. Overexpression of BilRI did not cause binding of a biotinylated negative control protein. In a microplate assay, soluble BilRI bound to IL-1β, but this binding was not specific, as a control protein for IL-1β also interacted with BilRI. Our findings suggest that A. actinomycetemcomitans expresses an IL-1β-binding surface-exposed lipoprotein that may be part of the bacterial IL-1β-sensing system.     

Characterization of the Differential Roles of the Twin C1a and C1b Domains of Protein Kinase C��

Classic and novel protein kinase C (PKC) isozymes contain two zinc finger motifs, designated “C1a” and “C1b” domains, which constitute the recognition modules for the second messenger diacylglycerol (DAG) or the phorbol esters. However, the individual contributions of these tandem C1 domains to PKC function and, reciprocally, the influence of protein context on their function remain uncertain. In the present study, we prepared PKCδ constructs in which the individual C1a and C1b domains were deleted, swapped, or substituted for one another to explore these issues. As isolated fragments, both the δC1a and δC1b domains potently bound phorbol esters, but the binding of [³H]phorbol 12,13-dibutyrate ([³H]PDBu) by the δC1a domain depended much more on the presence of phosphatidylserine than did that of the δC1b domain. In intact PKCδ, the δC1b domain played the dominant role in [³H]PDBu binding, membrane translocation, and down-regulation. A contribution from the δC1a domain was nonetheless evident, as shown by retention of [³H]PDBu binding at reduced affinity, by increased [³H]PDBu affinity upon expression of a second δC1a domain substituting for the δC1b domain, and by loss of persistent plasma membrane translocation for PKCδ expressing only the δC1b domain, but its contribution was less than predicted from the activity of the isolated domain. Switching the position of the δC1b domain to the normal position of the δC1a domain (or vice versa) had no apparent effect on the response to phorbol esters, suggesting that the specific position of the C1 domain within PKCδ was not the primary determinant of its activity.One of the essential steps for protein kinase C (PKC)² activation is its translocation from the cytosol to the membranes. For conventional (α, βI, βII, and γ) and novel (δ, ε, η, and θ) PKCs, this translocation is driven by interaction with the lipophilic second messenger sn-1,2-diacylglycerol (DAG), generated from phosphatidylinositol 4,5-bisphosphate upon the activation of receptor-coupled phospholipase C or indirectly from phosphatidylcholine via phospholipase D (1). A pair of zinc finger structures in the regulatory domain of the PKCs, the “C1” domains, are responsible for the recognition of the DAG signal. The DAG-C1 domain-membrane interaction is coupled to a conformational change in PKC, both causing the release of the pseudosubstrate domain from the catalytic site to activate the enzyme and triggering the translocation to the membrane (2). By regulating access to substrates, PKC translocation complements the intrinsic enzymatic specificity of PKC to determine its substrate profile.The C1 domain is a highly conserved cysteine-rich motif (∼50 amino acids), which was first identified in PKC as the interaction site for DAG or phorbol esters (3). It possesses a globular structure with a hydrophilic binding cleft at one end surrounded by hydrophobic residues. Binding of DAG or phorbol esters to the C1 domain caps the hydrophilic cleft and forms a continuous hydrophobic surface favoring the interaction or penetration of the C1 domain into the membrane (4). In addition to the novel and classic PKCs, six other families of proteins have also been identified, some of whose members possess DAG/phorbol ester-responsive C1 domains. These are the protein kinase D (5), the chimaerin (6), the munc-13 (7), the RasGRP (guanyl nucleotide exchange factors for Ras and Rap1) (8), the DAG kinase (9), and the recently characterized MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) families (10). Of these C1 domain-containing proteins, the PKCs have been studied most extensively and are important therapeutic targets (11). Among the drug candidates in clinical trials that target PKC, a number such as bryostatin 1 and PEP005 are directed at the C1 domains of PKC rather than at its catalytic site.Both the classic and novel PKCs contain in their N-terminal regulatory region tandem C1 domains, C1a and C1b, which bind DAG/phorbol ester (12). Multiple studies have sought to define the respective roles of these two C1 domains in PKC regulation, but the issue remains unclear. Initial in vitro binding measurements with conventional PKCs suggested that 1 mol of phorbol ester bound per mole of PKC (13-15). On the other hand, Stubbs et al., using a fluorescent phorbol ester analog, reported that PKCα bound two ligands per PKC (16). Further, site-directed mutagenesis of the C1a and C1b domains of intact PKCα indicated that the C1a and C1b domains played equivalent roles for membrane translocation in response to phorbol 12-myristate 13-acetate (PMA) and (-)octylindolactam V (17). Likewise, deletion studies indicated that the C1a and C1b domains of PKCγ bound PDBu equally with high potency (3, 18). Using a functional assay with PKCα expression in yeast, Shieh et al. (19) deleted individual C1 domains and reported that C1a and C1b were both functional and equivalent upon stimulation by PMA, with either deletion causing a similar reduction in potency of response, whereas for mezerein the response depended essentially on the C1a domain, with much weaker response if only the C1b domain was present. Using isolated C1 domains, Irie et al. (20) suggested that the C1a domain of PKCα but not those of PKCβ or PKCγ bound [³H]PDBu preferentially; different ligands showed a generally similar pattern but with different extents of selectivity. Using synthesized dimeric bisphorbols, Newton''s group reported (21) that, although both C1 domains of PKCβII are oriented for potential membrane interaction, only one C1 domain bound ligand in a physiological context.In the case of novel PKCs, many studies have been performed on PKCδ to study the equivalency of the twin C1 domains. The P11G point mutation of the C1a domain, which caused a 300-fold loss of binding potency in the isolated domain (22), had little effect on the phorbol ester-dependent translocation of PKCδ in NIH3T3 cells, whereas the same mutation of the C1b caused a 20-fold shift in phorbol ester potency for inducing translocation, suggesting a major role of the C1b domain for phorbol ester binding (23). A secondary role for the C1a domain was suggested, however, because mutation in the C1a domain as well as the C1b domain caused a further 7-fold shift in potency. Using the same mutations in the C1a and C1b domains, Bögi et al. (24) found that the binding selectivity for the C1a and C1b domains of PKCδ appeared to be ligand-dependent. Whereas PMA and the indole alkaloids indolactam and octylindolactam were selectively dependent on the C1b domain, selectivity was not observed for mezerein, the 12-deoxyphorbol 13-monoesters prostratin and 12-deoxyphorbol 13-phenylacetate, and the macrocyclic lactone bryostatin 1 (24). In in vitro studies using isolated C1a and C1b domains of PKCδ, Cho''s group (25) described that the two C1 domains had opposite affinities for DAG and phorbol ester; i.e. the C1a domain showed high affinity for DAG and the C1b domain showed high affinity for phorbol ester. No such difference in selectivity was observed by Irie et al. (20).PKC has emerged as a promising therapeutic target both for cancer and for other conditions, such as diabetic retinopathy or macular degeneration (26-30). Kinase inhibitors represent one promising approach for targeting PKC, and enzastaurin, an inhibitor with moderate selectivity for PKCβ relative to other PKC isoforms (but still with activity on some other non-PKC kinases) is currently in multiple clinical trials. An alternative strategy for drug development has been to target the regulatory C1 domains of PKC. Strong proof of principle for this approach is provided by multiple natural products, e.g. bryostatin 1 and PEP005, which are likewise in clinical trials and which are directed at the C1 domains. A potential advantage of this approach is the lesser number of homologous targets, <30 DAG-sensitive C1 domains compared with over 500 kinases, as well as further opportunities for specificity provided by the diversity of lipid environments, which form a half-site for ligand binding to the C1 domain. Because different PKC isoforms may induce antagonistic activities, inhibition of one isoform may be functionally equivalent to activation of an antagonistic isoform (31).Along with the benzolactams (20, 32), the DAG lactones have provided a powerful synthetic platform for manipulating ligand: C1 domain interactions (31). For example, the DAG lactone derivative 130C037 displayed marked selectivity among the recombinant C1a and C1b domains of PKCα and PKCδ as well as substantial selectivity for RasGRP relative to PKCα (33). Likewise, we have shown that a modified DAG lactone (dioxolanones) can afford an additional point of contact in ligand binding to the C1b domain of PKCδ (34). Such studies provide clear examples that ligand-C1 domain interactions can be manipulated to yield novel patterns of recognition. Further selectivity might be gained with bivalent compounds, exploiting the spacing and individual characteristics of the C1a and C1b domains (35). A better understanding of the differential roles of the two C1 domains in PKC regulation is critical for the rational development of such compounds. In this study, by molecularly manipulating the C1a or C1b domains in intact PKCδ, we find that both the C1a and C1b domains play important roles in PKCδ regulation. The C1b domain is predominant for ligand binding and for membrane translocation of the whole PKCδ molecule. The C1a domain of intact PKCδ plays only a secondary role in ligand binding but stabilizes the PKCδ molecule at the plasma membrane for downstream signaling. In addition, we show that the effect of the individual C1 domains of PKCδ does not critically depend on their position within the regulatory domain.     

Isolation and physiological and biochemical characterization of the Chlamydomonas reinhardtii C-41 mutant,a superproducer of ζ-carotene

The composition of the carotenes and xanthophylls of Chlamydomonas reinhardtii Dang. C-41, a mutant of a unicellular green alga and a superproducer of ζ-carotene, was studied. The light-harvesting complexes and a complex of the PS-II reaction center were established to be disrupted in the C-41 mutant. However, the mutant retained a high (up to 46%) photosynthetic activity and the capacity to accumulate chlorophylls and carotenoids (up to 50%). The composition of carotenes was studied, and it was shown that, in contrast to wild-type K(+) cells, which accumulate up to 95% of β-carotene and 5% α-carotene, cells of the C-41 mutant contained 43% β-carotene, 19% β-zeacarotene, and 38% ζ-carotene. The high level of C-41 mutant biomass accumulation made it possible to recommend the mutant as a superproducer of ζ-carotene in phytobiotechnology.     

Phosphorylation of the Consensus Sites of Protein Kinase A on ��1D L-type Calcium Channel

The novel α_1D L-type Ca²⁺ channel is expressed in supraventricular tissue and has been implicated in the pacemaker activity of the heart and in atrial fibrillation. We recently demonstrated that PKA activation led to increased α_1D Ca²⁺ channel activity in tsA201 cells by phosphorylation of the channel protein. Here we sought to identify the phosphorylated PKA consensus sites on the α₁ subunit of the α_1D Ca²⁺ channel by generating GST fusion proteins of the intracellular loops, N terminus, proximal and distal C termini of the α₁ subunit of α_1D Ca²⁺ channel. An in vitro PKA kinase assay was performed for the GST fusion proteins, and their phosphorylation was assessed by Western blotting using either anti-PKA substrate or anti-phosphoserine antibodies. Western blotting showed that the N terminus and C terminus were phosphorylated. Serines 1743 and 1816, two PKA consensus sites, were phosphorylated by PKA and identified by mass spectrometry. Site directed mutagenesis and patch clamp studies revealed that serines 1743 and 1816 were major functional PKA consensus sites. Altogether, biochemical and functional data revealed that serines 1743 and 1816 are major functional PKA consensus sites on the α₁ subunit of α_1D Ca²⁺ channel. These novel findings provide new insights into the autonomic regulation of the α_1D Ca²⁺ channel in the heart.L-type Ca²⁺ channels are essential for the generation of normal cardiac rhythm, for induction of rhythm propagation through the atrioventricular node and for the contraction of the atrial and ventricular muscles (1–5). L-type Ca²⁺ channel is a multisubunit complex including α₁, β and α₂/δ subunits (5–7). The α₁ subunit contains the voltage sensor, the selectivity filter, the ion conduction pore, and the binding sites for all known Ca²⁺ channel blockers (6–9). While α_1C Ca²⁺ channel is expressed in the atria and ventricles of the heart (10–13), expression of α_1D Ca²⁺ channel is restricted to the sinoatrial (SA)² and atrioventricular (AV) nodes, as well as in the atria, but not in the adult ventricles (2, 3, 10).Only recently it has been realized that α_1D along with α_1C Ca²⁺ channels contribute to L-type Ca²⁺ current (I_Ca-L) and they both play important but unique roles in the physiology/pathophysiology of the heart (6–9). Compared with α_1C, α_1D L-type Ca²⁺ channel activates at a more negative voltage range and shows slower current inactivation during depolarization (14, 15). These properties may allow α_1D Ca²⁺ channel to play critical roles in SA and AV nodes function. Indeed, α_1D Ca²⁺ channel knock-out mice exhibit significant SA dysfunction and various degrees of AV block (12, 16–19).The modulation of α_1C Ca²⁺ channel by cAMP-dependent PKA phosphorylation has been extensively studied, and the C terminus of α₁ was identified as the site of the modulation (20–22). Our group was the first to report that 8-bromo-cAMP (8-Br-cAMP), a membrane-permeable cAMP analog, increased α_1D Ca²⁺ channel activity using patch clamp studies (2). However, very little is known about potential PKA phosphorylation consensus motifs on the α_1D Ca²⁺ channel. We therefore hypothesized that the C terminus of the α₁ subunit of the α_1D Ca²⁺ channel mediates its modulation by cAMP-dependent PKA pathway.     

In Vitro Characterization of a Recombinant Blh Protein from an Uncultured Marine Bacterium as a ��-Carotene 15,15��-Dioxygenase

Codon optimization was used to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli. The expressed enzyme cleaved β-carotene at its central double bond (15,15′) to yield two molecules of all-trans-retinal. The molecular mass of the native purified enzyme was ∼64 kDa as a dimer of 32-kDa subunits. The K_m, k_cat, and k_cat/K_m values for β-carotene as substrate were 37 μm, 3.6 min⁻¹, and 97 mm⁻¹ min⁻¹, respectively. The enzyme exhibited the highest activity for β-carotene, followed by β-cryptoxanthin, β-apo-4′-carotenal, α-carotene, and γ-carotene in decreasing order, but not for β-apo-8′-carotenal, β-apo-12′-carotenal, lutein, zeaxanthin, or lycopene, suggesting that the presence of one unsubstituted β-ionone ring in a substrate with a molecular weight greater than C₃₅ seems to be essential for enzyme activity. The oxygen atom of retinal originated not from water but from molecular oxygen, suggesting that the enzyme was a β-carotene 15,15′-dioxygenase. Although the Blh protein and β-carotene 15,15′-monooxygenases catalyzed the same biochemical reaction, the Blh protein was unrelated to the mammalian β-carotene 15,15′-monooxygenases as assessed by their different properties, including DNA and amino acid sequences, molecular weight, form of association, reaction mechanism, kinetic properties, and substrate specificity. This is the first report of in vitro characterization of a bacterial β-carotene-cleaving enzyme.Vitamin A (retinol) is a fat-soluble vitamin and important for human health. In vivo, the cleavage of β-carotene to retinal is an important step of vitamin A synthesis. The cleavage can proceed via two different biochemical pathways (1, 2). The major pathway is a central cleavage catalyzed by mammalian β-carotene 15,15′-monooxygenases (EC 1.14.99.36). β-Carotene is cleaved by the enzyme symmetrically into two molecules of all-trans-retinal, and retinal is then converted to vitamin A in vivo (3–5). The second pathway is an eccentric cleavage that occurs at double bonds other than the central 15,15′-double bond of β-carotene to produce β-apo-carotenals with different chain lengths, which are catalyzed by carotenoid oxygenases from mammals, plants, and cyanobacteria (6). These β-apo-carotenals are degraded to one molecule of retinal, which is subsequently converted to vitamin A in vivo (2).β-Carotene 15,15′-monooxygenase was first isolated as a cytosolic enzyme by identifying the product of β-carotene cleavage as retinal (7). The characterization of the enzyme and the reaction pathway from β-carotene to retinal were also investigated (4, 8). The enzyme activity has been found in mammalian intestinal mucosa, jejunum enterocytes, liver, lung, kidney, and brain (5, 9, 10). Molecular cloning, expression, and characterization of β-carotene 15,15′-monooxygenase have been reported from various species, including chickens (11), fruit flies (12), humans (13), mice (14), and zebra fishes (15).Other proteins thought to convert β-carotene to retinal include bacterioopsin-related protein (Brp) and bacteriorhodopsin-related protein-like homolog protein (Blh) (16). Brp protein is expressed from the bop gene cluster, which encodes the structural protein bacterioopsin, consisting of at least three genes as follows: bop (bacterioopsin), brp (bacteriorhodopsin-related protein), and bat (bacterioopsin activator) (17). brp genes were reported in Haloarcula marismortui (18), Halobacterium sp. NRC-1 (19), Halobacterium halobium (17), Haloquadratum walsbyi, and Salinibacter ruber (20). Blh protein is expressed from the proteorhodopsin gene cluster, which contains proteorhodopsin, crtE (geranylgeranyl-diphosphate synthase), crtI (phytoene dehydrogenase), crtB (phytoene synthase), crtY (lycopene cyclase), idi (isopentenyl diphosphate isomerase), and blh gene (21). Sources of blh genes were previously reported in Halobacterium sp. NRC-1 (19), Haloarcula marismortui (18), Halobacterium salinarum (22), uncultured marine bacterium 66A03 (16), and uncultured marine bacterium HF10 49E08 (21). β-Carotene biosynthetic genes crtE, crtB, crtI, crtY, ispA, and idi encode the enzymes necessary for the synthesis of β-carotene from isopentenyl diphosphate, and the Idi, IspA, CrtE, CrtB, CrtI, and CrtY proteins have been characterized in vitro (23–28). Blh protein has been proposed to catalyze or regulate the conversion of β-carotene to retinal (29, 30), but there is no direct proof of the enzymatic activity.In this study, we used codon optimization to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli, and we performed a detailed biochemical and enzymological characterization of the expressed Blh protein. In addition, the properties of the enzyme were compared with those of mammalian β-carotene 15,15′-monooxygenases.     

Uptake of HCO3? and CO2 in Cells and Chloroplasts from the Microalgae Chlamydomonas reinhardtii and Dunaliella tertiolecta

Mass-spectrometric disequilibrium analysis was applied to investigate CO₂ uptake and HCO₃⁻ transport in cells and chloroplasts of the microalgae Dunaliella tertiolecta and Chlamydomonas reinhardtii, which were grown in air enriched with 5% (v/v) CO₂ (high-Ci cells) or in ambient air (low-Ci cells). High- and low-Ci cells of both species had the capacity to transport CO₂ and HCO₃⁻, with maximum rates being largely unaffected by the growth conditions. In high- and low-Ci cells of D. tertiolecta, HCO₃⁻ was the dominant inorganic C species taken up, whereas HCO₃⁻ and CO₂ were used at similar rates by C. reinhardtii. The apparent affinities of HCO₃⁻ transport and CO₂ uptake increased 3- to 9-fold in both species upon acclimation to air. Photosynthetically active chloroplasts isolated from both species were able to transport CO₂ and HCO₃⁻. For chloroplasts from C. reinhardtii, the concentrations of HCO₃⁻ and CO₂ required for half-maximal activity declined from 446 to 33 μm and 6.8 to 0.6 μm, respectively, after acclimation of the parent cells to air; the corresponding values for chloroplasts from D. tertiolecta decreased from 203 to 58 μm and 5.8 to 0.5 μm, respectively. These results indicate the presence of inducible high-affinity HCO₃⁻ and CO₂ transporters at the chloroplast envelope membrane.