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.     

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.     

Intracellular Trafficking of Presenilin 1 Is Regulated by ��-Amyloid Precursor Protein and Phospholipase D1

Excessive accumulation of β-amyloid peptides in the brain is a major cause for the pathogenesis of Alzheimer disease. β-Amyloid is derived from β-amyloid precursor protein (APP) through sequential cleavages by β- and γ-secretases, whose enzymatic activities are tightly controlled by subcellular localization. Delineation of how intracellular trafficking of these secretases and APP is regulated is important for understanding Alzheimer disease pathogenesis. Although APP trafficking is regulated by multiple factors including presenilin 1 (PS1), a major component of the γ-secretase complex, and phospholipase D1 (PLD1), a phospholipid-modifying enzyme, regulation of intracellular trafficking of PS1/γ-secretase and β-secretase is less clear. Here we demonstrate that APP can reciprocally regulate PS1 trafficking; APP deficiency results in faster transport of PS1 from the trans-Golgi network to the cell surface and increased steady state levels of PS1 at the cell surface, which can be reversed by restoring APP levels. Restoration of APP in APP-deficient cells also reduces steady state levels of other γ-secretase components (nicastrin, APH-1, and PEN-2) and the cleavage of Notch by PS1/γ-secretase that is more highly correlated with cell surface levels of PS1 than with APP overexpression levels, supporting the notion that Notch is mainly cleaved at the cell surface. In contrast, intracellular trafficking of β-secretase (BACE1) is not regulated by APP. Moreover, we find that PLD1 also regulates PS1 trafficking and that PLD1 overexpression promotes cell surface accumulation of PS1 in an APP-independent manner. Our results clearly elucidate a physiological function of APP in regulating protein trafficking and suggest that intracellular trafficking of PS1/γ-secretase is regulated by multiple factors, including APP and PLD1.An important pathological hallmark of Alzheimer disease (AD)⁴ is the formation of senile plaques in the brains of patients. The major components of those plaques are β-amyloid peptides (Aβ), whose accumulation triggers a cascade of neurodegenerative steps ending in formation of senile plaques and intraneuronal fibrillary tangles with subsequent neuronal loss in susceptible brain regions (1, 2). Aβ is proteolytically derived from the β-amyloid precursor protein (APP) through sequential cleavages by β-secretase (BACE1), a novel membrane-bound aspartyl protease (3, 4), and by γ-secretase, a high molecular weight complex consisting of at least four components: presenilin (PS), nicastrin (NCT), anterior pharynx-defective-1 (APH-1), and presenilin enhancer-2 (PEN-2) (5, 6). APP is a type I transmembrane protein belonging to a protein family that includes APP-like protein 1 (APLP1) and 2 (APLP2) in mammals (7, 8). Full-length APP is synthesized in the endoplasmic reticulum (ER) and transported through the Golgi apparatus. Most secreted Aβ peptides are generated within the trans-Golgi network (TGN), also the major site of steady state APP in neurons (9–11). APP can be transported to the cell surface in TGN-derived secretory vesicles if not proteolyzed to Aβ or an intermediate metabolite. At the cell surface APP is either cleaved by α-secretase to produce soluble sAPPα (12) or reinternalized for endosomal/lysosomal degradation (13, 14). Aβ may also be generated in endosomal/lysosomal compartments (15, 16). In contrast to neurotoxic Aβ peptides, sAPPα possesses neuroprotective potential (17, 18). Thus, the subcellular distribution of APP and proteases that process it directly affect the ratio of sAPPα to Aβ, making delineation of the mechanisms responsible for regulating trafficking of all of these proteins relevant to AD pathogenesis.Presenilin (PS) is a critical component of the γ-secretase. Of the two mammalian PS gene homologues, PS1 and PS2, PS1 encodes the major form (PS1) in active γ-secretase (19, 20). Nascent PSs undergo endoproteolytic cleavage to generate an amino-terminal fragment (NTF) and a carboxyl-terminal fragment (CTF) to form a functional PS heterodimer (21). Based on observations that PSs possess two highly conserved aspartate residues indispensable for γ-secretase activity and that specific transition state analogue γ-secretase inhibitors bind to PS1 NTF/CTF heterodimers (5, 22), PSs are believed to be the catalytic component of the γ-secretase complex. PS assembles with three other components, NCT, APH-1, and PEN-2, to form the functional γ-secretase (5, 6). Strong evidence suggests that PS1/γ-secretase resides principally in the ER, early Golgi, TGN, endocytic and intermediate compartments, most of which (except the TGN) are not major subcellular sites for APP (23, 24). In addition to generating Aβ and cleaving APP to release the APP intracellular domain, PS1/γ-secretase cleaves other substrates such as Notch (25), cadherin (26), ErbB4 (27), and CD44 (28), releasing their respective intracellular domains. Interestingly, PS1/γ-secretase cleavage of different substrates seems to occur at different subcellular compartments; APP is mainly cleaved at the TGN and early endosome domains, whereas Notch is predominantly cleaved at the cell surface (9, 11, 29). Thus, perturbing intracellular trafficking of PS1/γ-secretase may alter interactions between PS1/γ-secretase and APP, contributing to either abnormal Aβ generation and AD pathogenesis or decreased access of PS1/γ-secretase to APP such that Aβ production is reduced. However, mechanisms regulating PS1/γ-secretase trafficking warrant further investigation.In addition to participating in γ-secretase activity, PS1 regulates intracellular trafficking of several membrane proteins, including other γ-secretase components (nicastrin, APH-1, and PEN-2) and the substrate APP (reviewed in Ref. 30). Intracellular APP trafficking is highly regulated and requires other factors such as mint family members and SorLA (2). Moreover, we recently found that phospholipase D1 (PLD1), a phospholipid-modifying enzyme that regulates membrane trafficking events, can interact with PS1, and can regulate budding of APP-containing vesicles from the TGN and delivery of APP to the cell surface (31, 32). Interestingly, Kamal et al. (33) identified an axonal membrane compartment that contains APP, BACE1, and PS1 and showed that fast anterograde axonal transport of this compartment is mediated by APP and kinesin-I, implying a traffic-regulating role for APP. Increased APP expression is also shown to decrease retrograde axonal transport of nerve growth factor (34). However, whether APP indeed regulates intracellular trafficking of proteins including BACE1 and PS1/γ-secretase requires further validation. In the present study we demonstrate that intracellular trafficking of PS1, as well as that of other γ-secretase components, but not BACE1, is regulated by APP. APP deficiency promotes cell surface delivery of PS1/γ-secretase complex and facilitates PS1/γ-secretase-mediated Notch cleavage. In addition, we find that PLD1 also regulates intracellular trafficking of PS1 through a different mechanism and more potently than APP.     

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.     

The Carboxyl-terminal Domain of Atypical Protein Kinase C�� Binds to Ceramide and Regulates Junction Formation in Epithelial Cells

Atypical protein kinase Cs (PKCs) (aPKCζ and λ/ι) have emerged as important binding partners for ceramide, a membrane-resident cell signaling lipid that is involved in the regulation of apoptosis as well as cell polarity. Using ceramide overlay assays with proteolytic fragments of PKCζ and vesicle binding assays with ectopically expressed protein, we show that a protein fragment comprising the carboxyl-terminal 20-kDa sequence of PKCζ (C20ζ, amino acids 405–592) bound to C16:0 ceramide. This sequence is not identical to the C1 domain (amino acids 131–180), which has been suggested to serve as a potential ceramide binding domain. Using immunocytochemistry, we found that a C20ζ protein fragment ectopically expressed in two epithelial cell types (neural progenitors and Madin-Darby canine kidney cells) co-distributed with ceramide. Stable expression of C20ζ-EGFP in Madin-Darby canine kidney cells disrupted the formation of adherens and tight junctions and impaired the epithelium integrity by reducing transepithelial electrical resistance. Disruption of cell adhesion and loss of transepithelial electrical resistance was prevented by incubation with C16:0 ceramide. Our results show, for the first time, that there is a novel ceramide binding domain (C20ζ) in the carboxyl terminus of aPKC. Our results also show that the interaction of ceramide with this binding domain is essential for cell-to-cell contacts in epithelia. Therefore, ceramide interaction with the C20ζ binding domain is a potential mechanism by which ceramide and aPKC regulate the formation of junctional complexes in epithelial cells.Epithelial cells play essential roles in multicellular organisms by forming physiological and mechanical barriers and controlling tissue architecture, because they acquire apicobasal and cell-to-cell (planar) polarity (1, 2). Adherens junctions (AJs)² and tight junctions (TJs) are major structures responsible for cell-to-cell adhesion in epithelial cells (3). The regulation of junction formation requires endocytosis, redistribution, and recycling of junctional proteins, such as E-cadherin (4), and ZO-1. Many factors, including EGF, EGFR, Src kinase, Rho family GTPases Cdc42 and Rac1, and atypical PKC (aPKC), have been found to regulate junction formation (5–9). In Madin-Darby canine kidney (MDCK) cells, Cdc42 modulates AJs by regulating E-cadherin ubiquitination and degradation (9), whereas aPKC directly localized at TJs is required for the asymmetric differentiation of the premature junction complex during epithelial cell polarization (1, 10).The protein kinase C (PKC) family comprises serine/threonine kinases, which consist of a carboxyl-terminal catalytic domain and an amino-terminal regulatory domain (Fig. 1A). The regulatory domain includes an inhibitory pseudosubstrate domain and allosteric sites for activation by phosphatidylserine and, depending on the isoform, calcium (C2 domain) and/or diacylglycerol (C1 domain). aPKC is a subfamily of PKC, which consists of the isoforms ζ and λ/ι. The aPKC isoforms contain only half of the C1 domain, and hence, their activity is not affected by calcium or diacylglycerol/phorbol esters (see Fig. 1A and Refs. 11–13).Open in a separate windowFIGURE 1.Binding of ceramide to the COOH terminus of PKCζ. A, primary structure of aPKC, the caspase 3 proteolytic fragment ζCasp II, and the NH₂-terminal deletion mutant C20ζ-EGFP. B, 2 μg of recombinant His-tagged PKCζ was proteolytically digested by 20 ng of recombinant caspase 3. Proteolysis by caspase 3 occurred first after amino acid 239 (4-h incubation) and then after amino acid 459 (10-h incubation, ζCasp II). C, binding to ceramide spotted on nitrocellulose (overlay assay). FL PKCζ and the COOH-terminal proteolytic fragment ζCasp II bound to C16 ceramide. D, C16 ceramide vesicle binding assay (LIMAC). Ectopically expressed C20ζ-EGFP prepared from a cell lysate was bound to ceramide vesicles; EGFP was not. Protein was detected using anti-aPKC and anti-GFP antibodies. Lanes 1–3, loading input for ceramide vesicles; lanes 4–6, eluate of vesicle binding columns (output). Lanes 7 (input) and 8 (output) show that PKCζ-EGFP did not bind to vesicles prepared with sphingomyelin (SM) instead of ceramide. E, subcellular fractionation of cells expressing FL PKCζ-EGFP or C20ζ-EGFP.Apart from its function in apoptosis (13–15) and cell growth (16), aPKC has been found to play a pivotal role in cell polarity, both in neuroepithelial cells (17–20) or other epithelial cell types (1, 10). Consistently, the gene knock-out of aPKC shows loss of cell junction formation and detachment of neural progenitor cells from the neuroepithelium (8, 21–23). We and others have found that the sphingolipid ceramide activates aPKC, recruits it to structured microdomains, and regulates cell polarity and motility (24–28). Using lipid vesicle-mediated affinity chromatography (LIMAC) assays, we showed for the first time that ceramide directly binds to aPKC (25). Yet which domain of aPKC binds to ceramide is not known.Using lipid overlay and LIMAC assays, we show here that a COOH-terminal 20-kDa domain of PKCζ (C20ζ) binds to ceramide. Similar to its full-length counterpart, the C20ζ protein fragment resides in cellular membranes, where it co-distributes with ceramide in both C17.2 (neural progenitor) and MDCK cells. To study the function of this ceramide binding domain, we established a stably transfected MDCK cell line expressing C20ζ-EGFP. In these cells, the protein level of E-cadherin is reduced, and the cellular distribution of E-cadherin, ZO-1, and β-catenin is disrupted when compared with EGFP-transfected cell lines. Further, transepithelial electrical resistance (TER) assays show that the C20ζ-EGFP cell line has reduced impedance when compared with the control cell line expressing EGFP. This finding suggests that the C20ζ protein fragment is a dominant negative mutant of PKCζ. The effects of this dominant negative mutant can be, at least partially, rescued by incubation with C16:0 ceramide, suggesting that ceramide regulates aPKC and aPKC-dependent cell junction formation by interaction with the COOH-terminal domain.     

Protein kinase Cζ and glucose uptake

Protein kinase Cζ (PKCζ) is a member of the PKC family, serving downstream of insulin receptor and phosphatidylinositol (PI) 3-kinase. Many evidences suggest that PKCζ plays a very important role in activating glucose transport response. Not only insulin but also glucose and exercise can activate PKCζ through diverse pathways. PKCζ activation and activity are impaired with insulin resistance in muscle and adipose tissues of type II diabetes individuals, but heightened in liver tissue, wherein it also increases lipid synthesis mediated by SREBP-1c (sterol-regulatory element-binding protein). Many studies have focused on linkage between PKCζ and GLUT4 translocation and activation. Exploring the molecular mechanisms and pathways by which PKCζ mediates glucose transport will highlight the insulin-signaling pathway. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 7, pp. 869–875. Co-first authors.     

Plasmodium falciparum Erythrocyte Membrane Protein 1 Diversity in Seven Genomes – Divide and Conquer

The var gene encoded hyper-variable Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family mediates cytoadhesion of infected erythrocytes to human endothelium. Antibodies blocking cytoadhesion are important mediators of malaria immunity acquired by endemic populations. The development of a PfEMP1 based vaccine mimicking natural acquired immunity depends on a thorough understanding of the evolved PfEMP1 diversity, balancing antigenic variation against conserved receptor binding affinities. This study redefines and reclassifies the domains of PfEMP1 from seven genomes. Analysis of domains in 399 different PfEMP1 sequences allowed identification of several novel domain classes, and a high degree of PfEMP1 domain compositional order, including conserved domain cassettes not always associated with the established group A–E division of PfEMP1. A novel iterative homology block (HB) detection method was applied, allowing identification of 628 conserved minimal PfEMP1 building blocks, describing on average 83% of a PfEMP1 sequence. Using the HBs, similarities between domain classes were determined, and Duffy binding-like (DBL) domain subclasses were found in many cases to be hybrids of major domain classes. Related to this, a recombination hotspot was uncovered between DBL subdomains S2 and S3. The VarDom server is introduced, from which information on domain classes and homology blocks can be retrieved, and new sequences can be classified. Several conserved sequence elements were found, including: (1) residues conserved in all DBL domains predicted to interact and hold together the three DBL subdomains, (2) potential integrin binding sites in DBLα domains, (3) an acylation motif conserved in group A var genes suggesting N-terminal N-myristoylation, (4) PfEMP1 inter-domain regions proposed to be elastic disordered structures, and (5) several conserved predicted phosphorylation sites. Ideally, this comprehensive categorization of PfEMP1 will provide a platform for future studies on var/PfEMP1 expression and function.     

γCOP Is Required for Apical Protein Secretion and Epithelial Morphogenesis in Drosophila melanogaster

Background

There is increasing evidence that tissue-specific modifications of basic cellular functions play an important role in development and disease. To identify the functions of COPI coatomer-mediated membrane trafficking in Drosophila development, we were aiming to create loss-of-function mutations in the γCOP gene, which encodes a subunit of the COPI coatomer complex.

Principal Findings

We found that γCOP is essential for the viability of the Drosophila embryo. In the absence of zygotic γCOP activity, embryos die late in embryogenesis and display pronounced defects in morphogenesis of the embryonic epidermis and of tracheal tubes. The coordinated cell rearrangements and cell shape changes during tracheal tube morphogenesis critically depend on apical secretion of certain proteins. Investigation of tracheal morphogenesis in γCOP loss-of-function mutants revealed that several key proteins required for tracheal morphogenesis are not properly secreted into the apical lumen. As a consequence, γCOP mutants show defects in cell rearrangements during branch elongation, in tube dilation, as well as in tube fusion. We present genetic evidence that a specific subset of the tracheal defects in γCOP mutants is due to the reduced secretion of the Zona Pellucida protein Piopio. Thus, we identified a critical target protein of COPI-dependent secretion in epithelial tube morphogenesis.

Conclusions/Significance

These studies highlight the role of COPI coatomer-mediated vesicle trafficking in both general and tissue-specific secretion in a multicellular organism. Although COPI coatomer is generally required for protein secretion, we show that the phenotypic effect of γCOP mutations is surprisingly specific. Importantly, we attribute a distinct aspect of the γCOP phenotype to the effect on a specific key target protein.     

Deoxygenated Disaccharide Analogs as Specific Inhibitors of ��1�C4-Galactosyltransferase 1 and Selectin-mediated Tumor Metastasis

The disaccharide peracetylated GlcNAcβ1–3Galβ-O-naphthalenemethanol (disaccharide 1) diminishes the formation of the glycan sialyl Lewis X (Neu5Acα2–3Galβ1–4(Fucα1–3) GlcNAc; sLe^X) in tumor cells. Previous studies showed that the mechanism of action of disaccharide 1 involves three steps: (i) deacetylation by carboxyesterases, (ii) action as a biosynthetic intermediate for downstream enzymes involved in sLe^X assembly, and (iii) generation of several glycans related to sLe^X. In this report, we show that GlcNAcβ1–3Galβ-O-naphthalenemethanol binds to the acceptor site of human β1–4-galactosyltransferase much like the acceptor trisaccharide, GlcNAcβ1–2Manβ1–6Man, which is present on N-linked glycans. The 4′-deoxy analog, in which the acceptor hydroxyl group was replaced by -H, did not act as a substrate but instead acted as a competitive inhibitor of the enzyme. The acetylated form of this compound inhibited sLe^X formation in U937 monocytic leukemia cells, suggesting that it had inhibitory activity in vivo as well. A series of synthetic acetylated analogs of 1 containing -H, -F, -N₃, -NH₂, or -OCH₃ instead of the hydroxyl groups at C-3′- and C-4′-positions of the terminal N-acetylglucosamine residue also blocked sLe^X formation in cells. The reduction of sLe^X by the 4′-deoxy analog also diminished experimental tumor metastasis by Lewis lung carcinoma in vivo. These data suggest that nonsubstrate disaccharides have therapeutic potential through their ability to bind to glycosyltransferases in vivo and to alter glycan-dependent pathologic processes.The sialylated, fucosylated tetrasaccharide, sLe^X,³ is a common carbohydrate determinant present in many O-GalNAc-linked mucins and N-linked glycans that act as selectin ligands (see Ref. 1 and references therein). Expression of sLe^X endows tumor cells with the capacity to bind to platelets and endothelial cells in the vasculature via P- and E-selectins, thus facilitating hematogenous metastasis possibly through protection against innate immune cells and by adhesion to the blood vessel wall. Strategies for blocking selectin-carbohydrate interactions include (i) competition by soluble recombinant forms of selectins, glycoprotein ligands, and glycolipids, (ii) peptides based on the primary sequence of the carbohydrate binding site, (iii) anti-selectin antibodies, (iv) oligosaccharides related to Lewis^X, (v) inositol polyanions and sulfated sugars, (vi) heparin, and (vii) molecular mimics of sLe^X, including oligonucleotides (reviewed in Refs. 2 and 3). Analogs of acceptor substrates of the various glycosyltransferases involved in glycan biosynthesis provide another class of potential inhibitors (reviewed in Refs. 4 and 5). Although many of these analogs are effective in vitro, they generally do not exhibit inhibitory activity in cells due to poor membrane permeability. The large number of polar hydroxyl groups and the lack of membrane transporters for oligosaccharides in most cells presumably prevent their uptake (6).In contrast to many of the inhibitors described above, peracetylated disaccharides (e.g. acetylated Galβ1–4GlcNAcβ-O-naphthalenemethanol (NM), acetylated Galβ1–3GalNAcα-O-NM, and acetylated GlcNAcβ1–3Galβ-O-NM) inhibit sLe^X biosynthesis in cells (6–9). These compounds are taken up by cells by passive diffusion and acted on by cytoplasmic or membrane-associated carboxyesterases, which remove the acetyl groups. The compounds gain access to the biosynthetic enzymes located in the Golgi complex, where they serve as substrates, priming oligosaccharide synthesis and generating products related to O-GalNAc-linked mucin oligosaccharides. Priming in this manner diverts the assembly of the O-linked chains from endogenous glycoproteins, resulting in inhibition of expression of terminal Lewis antigens that are recognized by selectins. Inhibition occurs at a much lower dose than for monosaccharide-based agents, such as GalNAcβ-O-benzyl (∼25 μm versus 1–2 mm, respectively) (10, 11). Furthermore, the disaccharides appear to selectively affect sLe^X formation, since sLe^a expression was unaffected (12). By blocking selectin ligand expression, these compounds block both experimental and spontaneous metastasis (12, 13).In this study, we have examined acetylated disaccharide analogs that have been modified so that after deacetylation their activity as substrates would be altered. Characterization of the 4′-deoxy derivative using β1–4-galactosyltransferase 1 as a model showed that it acts by competitively inhibiting the enzyme. Interestingly, the peracetylated form of this analog maintains the capacity to inhibit sLe^X expression in U937 lymphoma cells and Lewis lung carcinoma (LLC) cells and block tumor formation in vivo. Thus, the deoxy analog presumably inhibits one or more galactosyltransferases in vivo, thereby blocking sLe^X formation and experimental tumor cell metastasis without generation of oligosaccharide products.     

Control of TANK-binding Kinase 1-mediated Signaling by the ��134.5 Protein of Herpes Simplex Virus 1

TANK-binding kinase 1 (TBK1) is a key component of Toll-like receptor-dependent and -independent signaling pathways. In response to microbial components, TBK1 activates interferon regulatory factor 3 (IRF3) and cytokine expression. Here we show that TBK1 is a novel target of the γ₁34.5 protein, a virulence factor whose expression is regulated in a temporal fashion. Remarkably, the γ₁34.5 protein is required to inhibit IRF3 phosphorylation, nuclear translocation, and the induction of antiviral genes in infected cells. When expressed in mammalian cells, the γ₁34.5 protein forms complexes with TBK1 and disrupts the interaction of TBK1 and IRF3, which prevents the induction of interferon and interferon-stimulated gene promoters. Down-regulation of TBK1 requires the amino-terminal domain. In addition, unlike wild type virus, a herpes simplex virus mutant lacking γ₁34.5 replicates efficiently in TBK1^-/- cells but not in TBK1^+/+ cells. Addition of exogenous interferon restores the antiviral activity in both TBK1^-/- and TBK^+/+ cells. Hence, control of TBK1-mediated cell signaling by the γ₁34.5 protein contributes to herpes simplex virus infection. These results reveal that TBK1 plays a pivotal role in limiting replication of a DNA virus.Herpes simplex virus 1 (HSV-1)³ is a large DNA virus that establishes latent or lytic infection, in which the virus triggers innate immune responses. In HSV-infected cells, a number of antiviral mechanisms operate in a cell type- and time-dependent manner (1). In response to double-stranded RNA (dsRNA), Toll-like receptor 3 (TLR3) recruits an adaptor TIR domain-containing adaptor inducing IFN-β and stimulates cytokine expression (2, 3). In the cytoplasm, RNA helicases, RIG-I (retinoid acid-inducible gene-I), and MDA5 (melanoma differentiation associated gene 5) recognize intracellular viral 5′-triphosphate RNA or dsRNA (2, 4). Furthermore, a DNA-dependent activator of IFN-regulatory factor (DAI) senses double-stranded DNA in the cytoplasm and induces cytokine expression (5). There is also evidence that viral entry induces antiviral programs independent of TLR and RIG-I pathways (6). While recognizing distinct viral components, these innate immune pathways relay signals to the two IKK-related kinases, TANK-binding kinase 1 (TBK1) and inducible IκB kinase (IKKi) (2).The IKK-related kinases function as essential components that phosphorylate IRF3 (interferon regulatory factor 3), as well as the closely related IRF7, which translocates to the nucleus and induces antiviral genes, such as interferon-α/β and ISG56 (interferon-stimulated gene 56) (7, 8). TBK1 is constitutively expressed, whereas IKKi is engaged as an inducible gene product of innate immune signaling (9, 10). IRF3 activation is attenuated in TBK1-deficient but not in IKKi-deficient cells (11, 12). Its activation is completely abolished in double-deficient cells (12), suggesting a partially redundant function of TBK1 and IKKi. Indeed, IKKi also negatively regulates the STAT-signaling pathway (13). TBK1/IKKi interacts with several proteins, such as TRAF family member-associated NF-κB activator (TANK), NAP1 (NAK-associated protein 1), similar to NAP1TBK1 adaptor (SINTBAD), DNA-dependent activator of IFN-regulatory factors (DAI), and secretory protein 5 (Sec5) in host cells (5, 14–18). These interactions are thought to regulate TBK1/IKKi, which delineates innate as well as adaptive immune responses.Upon viral infection, expression of HSV proteins interferes with the induction of antiviral immunity. When treated with UV or cycloheximide, HSV induces an array of antiviral genes in human lung fibroblasts (19, 20). Furthermore, an HSV mutant, with deletion in immediate early protein ICP0, induces ISG56 expression (21). Accordingly, expression of ICP0 inhibits the induction of antiviral programs mediated by IRF3 or IRF7 (21–23). However, although ICP0 negatively regulates IFN-β expression, it is not essential for this effect (24). In HSV-infected human macrophages or dendritic cells, an immediate early protein ICP27 is required to suppress cytokine induction involving IRF3 (25). In this context, it is notable that an HSV mutant, lacking a leaky late gene γ₁34.5, replicates efficiently in cells devoid of IFN-α/β genes (26). Additionally, the γ₁34.5 null mutant induces differential cytokine expression as compared with wild type virus (27). Thus, HSV modulation of cytokine expression is a complex process that involves multiple viral components. Currently, the molecular mechanism governing this event is unclear. In this study, we show that HSV γ₁34.5 targets TBK1 and inhibits antiviral signaling. The data herein reveal a previously unrecognized mechanism by which γ₁34.5 facilitates HSV replication.     

Independent ��-Arrestin2 and Gq/Protein Kinase C�� Pathways for ERK Stimulated by Angiotensin Type 1A Receptors in Vascular Smooth Muscle Cells Converge on Transactivation of the Epidermal Growth Factor Receptor

Recent studies in receptor-transfected cell lines have demonstrated that extracellular signal-regulated kinase (ERK) activation by angiotensin type 1A receptor and other G protein-coupled receptors can be mediated by both G protein-dependent and β-arrestin-dependent mechanisms. However, few studies have explored these mechanisms in primary cultured cells expressing endogenous levels of receptors. Accordingly, here we utilized the β-arrestin biased agonist for the angiotensin type 1A receptor, SII-angiotensin (SII), and RNA interference techniques to investigate angiotensin II (ANG)-activated β-arrestin-mediated mitogenic signaling pathways in rat vascular smooth muscle cells. Both ANG and SII induced DNA synthesis via the ERK activation cascade. Even though SII cannot induce calcium influx (G protein activation) after receptor stimulation, it does cause ERK activation, although less robustly than ANG. Activation by both ligands is diminished by depletion of β-arrestin2 by small interfering RNA, although the effect is more complete with SII. ERK activation at early time points but not later time points is strongly inhibited by those protein kinase C inhibitors that can block protein kinase Cζ. Moreover, ANG- and SII-mediated ERK activation require transactivation of the epidermal growth factor receptor via metalloprotease 2/9 and Src kinase. β-Arrestin2 facilitates ANG and SII stimulation of Src-mediated phosphorylation of Tyr-845 on the EGFR, a known site for Src phosphorylation. These studies delineate a convergent mechanism by which G protein-dependent and β-arrestin-dependent pathways can independently mediate ERK-dependent transactivation of the EGFR in vascular smooth muscle cells thus controlling cellular proliferative responses.G protein-coupled receptors, also known as seven transmembrane (7TM)² receptors, control virtually all known physiological processes in mammals (1). The various functions of these receptors are mediated and modulated by three families of proteins, which share the property that they interact virtually universally with the receptors in a strictly stimulus-dependent way (1). These three families of proteins are the heterotrimeric G proteins, the G protein-coupled receptor kinases (GRKs), and the β-arrestins. Activation of the receptors stimulates classical G protein-dependent signaling, often involving regulation of levels of second messengers such as cAMP and diacyglycerol. However, as has been known for many years, interaction of activated receptors with GRKs leading to their phosphorylation, and subsequent interaction with β-arrestins leads to desensitization of G protein signaling.In recent years, however, it has become increasingly clear that the β-arrestin-GRK system is in fact bifunctional (2). Thus, even as it desensitizes G protein signaling by the receptors, it also serves as a signal transduction system in its own right, activating a growing list of signaling pathways. These positive signaling functions are often mediated by the ability of β-arrestin to serve as an adaptor or scaffold molecule, bringing elements of diverse signaling pathways into proximity with one another and the receptors and thereby facilitating their activation. This new paradigm for understanding the previously unrecognized signaling properties of the β-arrestin-GRK system has been explored in a wide variety of transfected cultured cell systems.However, to date, relatively little investigation of these novel signaling pathways has been carried out in primary cell culture systems expressing endogenous levels of 7TM receptors. In seeking such a system in which to characterize and compare β-arrestin and G protein-mediated signaling pathways from a typical 7TM receptor, our attention was drawn to cultured rat vascular smooth muscle cells (VSMCs). Several features of rat VSMCs suggest this to be a relevant system for these purposes. Rat VSMCs express a variety of physiologically important 7TM receptors including the angiotensin II type 1A receptor (AT1R) (3). This receptor has been the focus of extensive study in transfected cell systems with respect to its β-arrestin-mediated signaling to a variety of pathways, most particularly extracellular signal-regulated kinase (ERK). Moreover, the AT1R mediates the physiologically important effects of angiotensin II (ANG) on vascular tone as well as on proliferation and chemotaxis (4, 5). Pathophysiologically, ANG stimulation of this receptor has been implicated in VSMC proliferation and chemotaxis, which are thought to play an important role in such important disease processes as atherosclerosis and restenosis after angioplasty (6, 7). Moreover, a ligand has been characterized [Sar¹,Ile⁴,Ile⁸](SII)-angiotensin (SII), a triply mutated angiotensin octapeptide that, in transfected cell systems, acts as a specific agonist for β-arrestin-mediated signaling, although not activating G protein-mediated signaling (8).Accordingly, in the studies described here, we set out to investigate the characteristics of activation of ERK in rat VSMCs that might be mediated through G protein as well as β-arrestin signaling. The results not only demonstrate the importance of β-arrestin-mediated signaling in ERK-mediated proliferative responses of these cells, but also shed new light on the molecular mechanisms and interrelationships between the β-arrestin and classical G protein-mediated activation of these pathways.     

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.     

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.     

Activation of Protein Kinase C�� Increases OAT1 (SLC22A6)- and OAT3 (SLC22A8)-mediated Transport

Organic anion transporters (OATs) play a pivotal role in the clearance of small organic anions by the kidney, yet little is known about how their activity is regulated. A yeast two-hybrid assay was used to identify putative OAT3-associated proteins in the kidney. Atypical protein kinase Cζ (PKCζ) was shown to bind to OAT3. Binding was confirmed in immunoprecipitation assays. The OAT3/PKCζ interaction was investigated in rodent renal cortical slices from fasted animals. Insulin, an upstream activator of PKCζ, increased both OAT3-mediated uptake of estrone sulfate (ES) and PKCζ activity. Both effects were abolished by a PKCζ-specific pseudosubstrate inhibitor. Increased ES transport was not observed in renal slices from OAT3-null mice. Transport of the shared OAT1/OAT3 substrate, ρ-aminohippurate, behaved similarly, except that stimulation was reduced, not abolished, in the OAT3-null mice. This suggested that OAT1 activity was also modified by PKCζ, subsequently confirmed using an OAT1-specific substrate, adefovir. Inhibition of PKCζ also blocked the increase in ES uptake seen in response to epidermal growth factor and to activation of protein kinase A. Thus, PKCζ acted downstream of the epidermal growth factor to protein kinase A signaling pathway. Activation of transport was accompanied by an increase in V_max and was blocked by microtubule disruption, indicating that activation may result from trafficking of OAT3 into the plasma membrane. These data demonstrate that PKCζ activation up-regulates OAT1 and OAT3 function, and that protein-protein interactions play a central role controlling these two important renal drug transporters.Organic anion transporters (OATs)⁷ are members of the solute carrier 22A family and play a pivotal role in the renal clearance of small (<500 Dalton) anionic drugs, xenobiotics, and their metabolites. OAT substrates include a variety of drugs such as β-lactam antibiotics, non-steroidal anti-inflammatory drugs, diuretics, and chemotherapeutics (1). OATs are predominantly expressed in renal proximal tubule, with OATs 1–3 localized to the basolateral membrane and OAT4 and URAT1 on the apical membrane. OATs 1 and 3 are dicarboxylate exchangers, and are indirectly coupled to the sodium gradient maintained by Na,K-ATPase through sodium/dicarboxylate co-transport to drive the uphill basolateral step in renal organic anion secretion (2).Although the ionic gradients, electrophysiology, and underlying kinetics that drive transport by OATs 1 and 3 are well characterized, physiologically important interactions of these basolateral OATs with membrane or cytosolic proteins have yet to be identified (1). Nevertheless, there is clear evidence that other plasma membrane transporters do interact with protein partners, influencing a diverse array of functions including transport itself, cytoskeletal structure, vesicle formation, and trafficking, as well as signaling (3). Among the transporters with activity modulated by protein-protein interactions, particularly by the PDZ proteins, PDZK1 and NHERFs 1 and 2, are apical drug transporters of the SLC22A family, including OCTN1, OCTN2, OAT4, and URAT1 (4–6).In the present study, we have used a yeast two-hybrid assay to identify putative protein partners that interact directly with OAT3. The C-terminal 81 amino acids of OAT3 were used as bait to screen a human cDNA kidney library. Among the 23 positive clones (putative binding partners) was a clone encoding the C-terminal 141 amino acids of atypical protein kinase Cζ (PKCζ). Functional consequences of the putative OAT3/PKCζ interaction were investigated in rodent renal slices. The resulting data indicate that activation of PKCζ by insulin or epidermal growth factor (EGF) increased OAT3- and OAT1-mediated transport. Thus, PKCζ controls function of both major secretory organic anion transporters expressed at the basolateral face of the renal proximal tubule, positioning it to regulate the efficacy of renal drug elimination.     

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.