Phosphorylation of ��6-Tubulin by Protein Kinase C�� Activates Motility of Human Breast Cells

Engineered overexpression of protein kinase Cα (PKCα) was previously shown to endow nonmotile MCF-10A human breast cells with aggressive motility. A traceable mutant of PKCα (Abeyweera, T. P., and Rotenberg, S. A. (2007) Biochemistry 46, 2364–2370) revealed that α6-tubulin is phosphorylated in cells expressing traceable PKCα and in vitro by wild type PKCα. Gain-of-function, single site mutations (Ser → Asp) were constructed at each PKC consensus site in α6-tubulin (Ser¹⁵⁸, Ser¹⁶⁵, Ser²⁴¹, and Thr³³⁷) to simulate phosphorylation. Following expression of each construct in MCF-10A cells, motility assays identified Ser¹⁶⁵ as the only site in α6-tubulin whose pseudophosphorylation reproduced the motile behavior engendered by PKCα. Expression of a phosphorylation-resistant mutant (S165N-α6-tubulin) resulted in suppression of MCF-10A cell motility stimulated either by expression of PKCα or by treatment with PKCα-selective activator diacylglycerol-lactone. MCF-10A cells treated with diacylglycerol-lactone showed strong phosphorylation of endogenous α-tubulin that could be blocked when S165N-α6-tubulin was expressed. The S165N mutant also inhibited intrinsically motile human breast tumor cells that express high endogenous PKCα levels (MDA-MB-231 cells) or lack PKCα and other conventional isoforms (MDA-MB-468 cells). Comparison of Myc-tagged wild type α6-tubulin and S165N-α6-tubulin expressed in MDA-MB-468 cells demonstrated that Ser¹⁶⁵ is also a major site of phosphorylation for endogenously active, nonconventional PKC isoforms. PKC-stimulated motility of MCF-10A cells was nocodazole-sensitive, thereby implicating microtubule elongation in the mechanism. These findings support a model in which PKC phosphorylates α-tubulin at Ser¹⁶⁵, leading to microtubule elongation and motility.     

GSK-3β Protein Phosphorylates and Stabilizes HLXB9 Protein in Insulinoma Cells to Form a Targetable Mechanism of Controlling Insulinoma Cell Proliferation

Insulinomas (pancreatic islet β cell tumors) are the most common type of functioning pancreatic neuroendocrine tumors that occur sporadically or as a part of the MEN1 syndrome that is caused by germ line mutations in MEN1. Tissue-specific tumor predisposition from germ line mutations in ubiquitously expressed genes such as MEN1 could occur because of functional consequences on tissue-specific factors. We previously reported the proapoptotic β cell differentiation factor HLXB9 as a downstream target of menin (encoded by MEN1). Here we show that GSK-3β inactivates the proapoptotic activity of HLXB9 by phosphorylating HLXB9 at Ser-78/Ser-80 (pHLXB9). Although HLXB9 is found in the nucleus and cytoplasm, pHLXB9 is predominantly nuclear. Both pHLXB9 and active GSK-3β are elevated in β cells with menin knockdown, in MEN1-associated β cell tumors (insulinomas), and also in human sporadic insulinomas. Pharmacologic inhibition of GSK-3β blocked cell proliferation in three different rodent insulinoma cell lines by arresting the cells in G₂/M phase and caused apoptosis. Taken together, these data suggest that the combination of GSK-3β and pHLXB9 forms a therapeutically targetable mechanism of insulinoma pathogenesis. Our results reveal that GSK-3β and pHLXB9 can serve as novel targets for insulinoma treatment and have implications for understanding the pathways associated with β cell proliferation.     

Rho Kinases Regulate the Renewal and Neural Differentiation of Embryonic Stem Cells in a Cell Plating Density–Dependent Manner

Background

Rho kinases (ROCKs) mediate cell contraction, local adhesion, and cell motility, which are considered to be important in cell differentiation. We postulated that ROCKs are involved in controlling embryonic stem (ES) cell renewal and differentiation.

Methodology/Principal Findings

CCE, a murine ES cell, was treated with Y-27632 for 48 to 96 hours and colony formation was evaluated. Y-27632 blocked CCE colony formation and induced CCE to grow as individual cells, regardless of the initial seeding cell density either at 10⁴/cm² (“high” seeding density) or 2×10³/cm² (“low” density). However, at high seeding density, Y-27632–treated cells exhibited reduction of alkaline phosphatase (AP) staining and Oct3/4 expression. They expressed SOX-1, nestin, and MAP2c, but not βIII-tubulin or NG-2. They did not express endoderm or mesoderm lineage markers. After removal of Y-27632, the cells failed to form colonies or regain undifferentiated state. Silencing of ROCK-1 or ROCK-2 with selective small interference RNA induced CCE morphological changes similar to Y-27632. Silencing of ROCK-1 or ROCK-2 individually was sufficient to cause reduction of AP and Oct3/4, and expression of SOX-1, nestin, and MAP2c; and combined silencing of both ROCKs did not augment the effects exerted by individual ROCK siRNA. Y-27632–treated CCE cells seeded at 2×10³ or 6.6×10³ cells/cm² did not lose renewal factors or express differentiation markers. Furthermore, they were able to form AP-positive colonies after removal of Y-27632 and reseeding. Similar to ROCK inhibition by Y-27632, silencing of ROCK-1 or ROCK-2 in cells seeded at 2×10³/cm² did not change renewal factors.

Conclusions/Significance

We conclude that ROCKs promote ES cell colony formation, maintain them at undifferentiated state, and prevent them from neural differentiation at high seeding density. ROCK inhibition represents a new strategy for preparing large numbers of neural progenitor cells.     

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.     

C-Nap1, a Novel Centrosomal Coiled-Coil Protein and Candidate Substrate of the Cell Cycle–regulated Protein Kinase Nek2

Nek2 (for NIMA-related kinase 2) is a mammalian cell cycle–regulated kinase structurally related to the mitotic regulator NIMA of Aspergillus nidulans. In human cells, Nek2 associates with centrosomes, and overexpression of active Nek2 has drastic consequences for centrosome structure. Here, we describe the molecular characterization of a novel human centrosomal protein, C-Nap1 (for centrosomal Nek2-associated protein 1), first identified as a Nek2-interacting protein in a yeast two-hybrid screen. Antibodies raised against recombinant C-Nap1 produced strong labeling of centrosomes by immunofluorescence, and immunoelectron microscopy revealed that C-Nap1 is associated specifically with the proximal ends of both mother and daughter centrioles. On Western blots, anti–C-Nap1 antibodies recognized a large protein (>250 kD) that was highly enriched in centrosome preparations. Sequencing of overlapping cDNAs showed that C-Nap1 has a calculated molecular mass of 281 kD and comprises extended domains of predicted coiled-coil structure. Whereas C-Nap1 was concentrated at centrosomes in all interphase cells, immunoreactivity at mitotic spindle poles was strongly diminished. Finally, the COOH-terminal domain of C-Nap1 could readily be phosphorylated by Nek2 in vitro, as well as after coexpression of the two proteins in vivo. Based on these findings, we propose a model implicating both Nek2 and C-Nap1 in the regulation of centriole–centriole cohesion during the cell cycle.The serine/threonine kinase NIMA of Aspergillus nidulans is considered the founding member of a family of protein kinases with a possible role in cell cycle regulation (for reviews see Fry and Nigg, 1995; Lu and Hunter, 1995a ; Osmani and Ye, 1996). In A. nidulans, NIMA clearly cooperates with the Cdc2 protein kinase to promote progression into mitosis (Osmani et al., 1991), and overexpression of NIMA in a variety of heterologous species promotes a premature onset of chromosome condensation (O''Connell et al., 1994; Lu and Hunter, 1995b ). This has been interpreted to suggest evolutionary conservation of a pathway involving NIMA-related kinases (for review see Lu and Hunter, 1995a ). Indeed, kinases structurally related to NIMA are present in many species (Fry and Nigg, 1997). However, the only bona fide functional homologue of NIMA so far isolated stems from another filamentous fungus, Neurospora crassa (Pu et al., 1995), and the functional relationship between vertebrate NIMA-related kinases and fungal NIMA remains uncertain.The closest known mammalian relative to NIMA is a kinase termed Nek2 (for NIMA-related kinase 2)¹ (Fry and Nigg, 1997). This kinase undergoes cell cycle–dependent changes in abundance and activity, reminiscent of NIMA (Schultz et al., 1994; Fry et al., 1995). It is highly expressed in male germ cells (Rhee and Wolgemuth, 1997; Tanaka et al., 1997), and data have been reported consistent with a role for Nek2 in meiotic chromosome condensation (Rhee and Wolgemuth, 1997). However, overexpression of active Nek2 in somatic cells has no obvious effect on chromosome condensation; instead, it induces striking alterations in the structure of the centrosome, the principal microtubule-organizing center of mammalian cells (Fry et al., 1998). Furthermore, immunofluorescence microscopy and subcellular fractionation concur to demonstrate that endogenous Nek2 associates with centrosomes, strongly suggesting that one physiological function of this kinase may relate to the centrosome cycle (Fry et al., 1998).The mammalian centrosome is an organelle of about 1 μm in diameter. It comprises two barrel-shaped centrioles that are made of nine short triplet microtubules and are surrounded by an amorphous matrix known as the pericentriolar material (PCM) (for review see Brinkley, 1985; Vorobjev and Nadehzdina, 1987; Kimble and Kuriyama, 1992; Kalt and Schliwa, 1993; Kellogg et al., 1994; Lange and Gull, 1996). Major progress has recently been made with the demonstration that microtubules are nucleated from γ-tubulin–containing ring complexes (γ-TuRCs), which are concentrated within the PCM (Moritz et al., 1995; Zheng et al., 1995). γ-Tubulin forms complexes with Spc97/98, two evolutionarily conserved proteins first identified in budding yeast spindle pole bodies (Geissler et al., 1996; Knop et al., 1997; Stearns and Winey, 1997), and there is also evidence for an important role of pericentrin and other coiled-coil proteins in organizing γ-TuRCs into higher order lattice structures (Doxsey et al., 1994; Dictenberg et al., 1998). However, in spite of this recent progress, it is clear that the inventory of centrosome components is far from complete.Centrosome structure and function is regulated in a cell cycle–dependent manner (for reviews see Mazia, 1987; Kellogg et al., 1994; Tournier and Bornens, 1994). Once in every cell cycle, and beginning around the G1/S transition, centrioles are duplicated (e.g., Kuriyama and Borisy, 1981a ; Vorobjev and Chentsov, 1982; Kochanski and Borisy, 1990; Chrétien et al., 1997). Late in G2, centrosomes then grow in size (a process referred to as maturation) through the recruitment of additional PCM proteins (Rieder and Borisy, 1982; Kalt and Schliwa, 1993; Lange and Gull, 1995). At the G2/M transition, the duplicated centrosomes separate and migrate to opposite ends of the nucleus. Concomitantly, their microtubule-nucleating activities increase dramatically in preparation for spindle formation (McGill and Brinkley, 1975; Snyder and McIntosh, 1975; Gould and Borisy, 1977; Kuriyama and Borisy, 1981b ; for reviews see Brinkley, 1985; Vorobjev and Nadehzdina, 1987; Karsenti, 1991). By what mechanisms these events are controlled remains largely unknown, but data obtained using phosphoepitope-specific antibodies strongly suggest that phosphorylation of centrosomal proteins plays a major role (Vandré et al., 1984, 1986; Centonze and Borisy, 1990). More direct support for this view stems from the observation that cyclin-dependent kinases (CDKs) enhance the microtubule-nucleation activity of centrosomes at the G2/M transition (Verde et al., 1990, 1992; Buendia et al., 1992) and are involved in promoting centrosome separation (Blangy et al., 1995; Sawin and Mitchison, 1995). Similarly, polo-like kinase 1, a cell cycle regulatory kinase structurally distinct from CDKs, has recently been implicated in centrosome maturation (Lane and Nigg, 1996).The precise role of Nek2 at the centrosome remains to be determined, but it is intriguing that overexpression of this kinase in human cells causes a pronounced splitting of centrosomes. This led us to propose that Nek2-dependent phosphorylation of previously unidentified proteins may cause a loss of centriole–centriole cohesion, and that this event might represent an early step in centrosome separation at the G2/M transition (Fry et al., 1998). With the aim of identifying potential substrates (or regulators) of Nek2, we have now performed a yeast two-hybrid screen, using full-length Nek2 as a bait. We report here the molecular characterization of a novel coiled-coil protein that we call C-Nap1 (for centrosomal Nek2-associated protein 1). C-Nap1 represents a core component of the mammalian centrosome and the first candidate substrate for a member of the NIMA protein kinase family to be identified.     

Interleukin-25 Expressed by Brain Capillary Endothelial Cells Maintains Blood-Brain Barrier Function in a Protein Kinase C?-dependent Manner

Interleukin (IL)-25, a member of the IL-17 family of cytokines, is expressed in the brains of normal mice. However, the cellular source of IL-25 and its function in the brain remain to be elucidated. Here, we show that IL-25 plays an important role in preventing infiltration of the inflammatory cells into the central nervous system. Brain capillary endothelial cells (BCECs) express IL-25. However, it is down-regulated by inflammatory cytokines, including tumor necrosis factor (TNF)-α, IL-17, interferon-γ, IL-1β, and IL-6 in vitro, and is also reduced in active multiple sclerosis (MS) lesions and in the inflamed spinal cord of experimental autoimmune encephalomyelitis, an animal model of MS. Furthermore, IL-25 restores the reduced expression of tight junction proteins, occludin, junction adhesion molecule, and claudin-5, induced by TNF-α in BCECs and consequently repairs TNF-α-induced blood-brain barrier (BBB) permeability. IL-25 induces protein kinase Cϵ (PKCϵ) phosphorylation, and up-regulation of claudin-5 is suppressed by PKCϵ inhibitor peptide in the IL-25-stimulated BCECs. These results suggest that IL-25 is produced by BCECs and protects against inflammatory cytokine-induced excessive BBB collapse through a PKCϵ-dependent pathway. These novel functions of IL-25 in maintaining BBB integrity may help us understand the pathophysiology of inflammatory brain diseases such as MS.     

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.     

Vascular Smooth Muscle Cell Motility Is Mediated by a Physical and Functional Interaction of Ca2+/Calmodulin-dependent Protein Kinase IIδ2 and Fyn

In vascular smooth muscle (VSM) cells, Ca²⁺/calmodulin-dependent protein kinase IIδ₂ (CaMKIIδ₂) activates non-receptor tyrosine kinases and EGF receptor, with a Src family kinase as a required intermediate. siRNA-mediated suppression of Fyn, a Src family kinase, inhibited VSM cell motility. Simultaneous suppression of both Fyn and CaMKIIδ₂ was non-additive, suggesting coordinated regulation of cell motility. Confocal immunofluorescence microscopy indicated that CaMKIIδ₂ and Fyn selectively (compared with Src) co-localized with the Golgi in quiescent cultured VSM cells. Stimulation with PDGF resulted in a rapid (<5 min) partial redistribution and co-localization of both kinases in peripheral membrane regions. Furthermore, CaMKIIδ₂ and Fyn selectively (compared with Src) co-immunoprecipitated, suggesting a physical interaction in a signaling complex. Stimulation of VSM cells with ionomycin, a calcium ionophore, resulted in activation of CaMKIIδ₂ and Fyn and disruption of the complex. Pretreatment with KN-93, a pharmacological inhibitor of CaMKII, prevented activation-dependent disruption of CaMKIIδ₂ and Fyn, implicating CaMKIIδ₂ as an upstream mediator of Fyn. Overexpression of constitutively active CaMKII resulted in the dephosphorylation of Fyn at Tyr-527, which is required for Fyn activation. Taken together, these data demonstrate a dynamic interaction between CaMKIIδ₂ and Fyn in VSM cells and indicate a mechanism by which CaMKIIδ₂ and Fyn may coordinately regulate VSM cell motility.     

Inactivation of DNA–Dependent Protein Kinase Promotes Heat–Induced Apoptosis Independently of Heat–Shock Protein Induction in Human Cancer Cell Lines

The inhibition of DNA damage response pathway seems to be an attractive strategy for cancer therapy. It was previously reported that in rodent cells exposed to heat stress, cell growth was promoted by the activity of DNA-dependent protein kinase (DNA-PK), an enzyme involved in DNA non-homologous end joining (NHEJ) required for double-strand break repair. The absence of a functioning DNA-PK was associated with down regulation of heat shock protein 70 (HSP70). The objective of this study is thus to investigate the role of DNA-PK inhibition in heat-induced apoptosis in human cell lines. The inhibitors of phosphorylation of the DNA-PK catalytic subunit (DNA-PKcs) at Ser2056, such as NU7026 and NU7441, were utilized. Furthermore, knock down of DNA-PKcs was carried out using small interfering RNA (siDNA-PKcs). For heat exposure, cells were placed in water bath at 44°C for 60 min. Apoptosis was evaluated after 24 h incubation flow cytometrically. Proteins were extracted after 24 h and analyzed for HSP70 and HSP40 expression by Western blotting. Total RNA was extracted 6 h after treatment and analyzed using a GeneChip® microarray system to identify and select the up-regulated genes (≥1.5 fold). The results showed an enhancement in heat-induced apoptosis in absence of functioning DNA-PKcs. Interestingly, the expression levels of HSP70 and HSP40 were elevated in the absence of DNA-PKcs under heat stress. The results of genetic network analysis showed that HSPs and JUN genes were up-regulated independently of DNA-PKcs in exposed parent and knock out cells. In the presence of functioning DNA-PKcs, there was an observed up-regulation of anti-apoptotic genes, such as NR1D1, whereas in the absence of DNA-PKcs the pro-apoptotic genes, such as EGR2, were preferentially up-regulated. From these findings, we concluded that in human cells, the inactivation of DNA-PKcs can promote heat-induced apoptosis independently of heat-shock proteins.     

Low Molecular Weight Hyaluronan Activates Cytosolic Phospholipase A2α and Eicosanoid Production in Monocytes and Macrophages

Hyaluronan (HA) is the major glycosaminoglycan in the extracellular matrix. During inflammation, there is an increased breakdown of HA, resulting in the accumulation of low molecular weight (LMW) HA and activation of monocytes and macrophages. Eicosanoids, derived from the cytosolic phospholipase A₂ group IVA (cPLA₂α) activation, are potent lipid mediators also attributed to acute and chronic inflammation. The aim of this study was to determine the effect of LMW HA on cPLA₂α activation, arachidonic acid (AA) release, and subsequent eicosanoid production and to examine the receptors and downstream mechanisms involved in these processes in monocytes and differently polarized macrophages. LMW HA was a potent stimulant of AA release in a time- and dose-dependent manner, induced cPLA₂α, ERK1/2, p38, and JNK phosphorylation, as well as activated COX2 expression and prostaglandin (PG) E₂ production in primary human monocytes, murine RAW 264.7, and wild-type bone marrow-derived macrophages. Specific cPLA₂α inhibitor blocked HA-induced AA release and PGE₂ production in all of these cells. Using CD44, TLR4, TLR2, MYD88, RHAMM or STAB2 siRNA-transfected macrophages and monocytes, we found that AA release, cPLA₂α, ERK1/2, p38, and JNK phosphorylation, COX2 expression, and PGE₂ production were activated by LMW HA through a TLR4/MYD88 pathway. Likewise, PGE₂ production and COX2 expression were blocked in Tlr4^−/− and Myd88^−/− mice, but not in Cd44^−/− mice, after LMW HA stimulation. Moreover, we demonstrated that LMW HA activated the M1 macrophage phenotype with the unique cPLA₂α/COX2^high and COX1/ALOX15/ALOX5/LTA4H^low gene and PGE₂/PGD₂/15-HETE^high and LXA₄^low eicosanoid profile. These findings reveal a novel link between HA-mediated inflammation and lipid metabolism.     

Fulvestrant-Induced Cell Death and Proteasomal Degradation of Estrogen Receptor α Protein in MCF-7 Cells Require the CSK c-Src Tyrosine Kinase

Fulvestrant is a representative pure antiestrogen and a Selective Estrogen Receptor Down-regulator (SERD). In contrast to the Selective Estrogen Receptor Modulators (SERMs) such as 4-hydroxytamoxifen that bind to estrogen receptor α (ERα) as antagonists or partial agonists, fulvestrant causes proteasomal degradation of ERα protein, shutting down the estrogen signaling to induce proliferation arrest and apoptosis of estrogen-dependent breast cancer cells. We performed genome-wide RNAi knockdown screenings for protein kinases required for fulvestrant-induced apoptosis of the MCF-7 estrogen-dependent human breast caner cells and identified the c-Src tyrosine kinase (CSK), a negative regulator of the oncoprotein c-Src and related protein tyrosine kinases, as one of the necessary molecules. Whereas RNAi knockdown of CSK in MCF-7 cells by shRNA-expressing lentiviruses strongly suppressed fulvestrant-induced cell death, CSK knockdown did not affect cytocidal actions of 4-hydroxytamoxifen or paclitaxel, a chemotherapeutic agent. In the absence of CSK, fulvestrant-induced proteasomal degradation of ERα protein was suppressed in both MCF-7 and T47D estrogen-dependent breast cancer cells whereas the TP53-mutated T47D cells were resistant to the cytocidal action of fulvestrant in the presence or absence of CSK. MCF-7 cell sensitivities to fulvestrant-induced cell death or ERα protein degradation was not affected by small-molecular-weight inhibitors of the tyrosine kinase activity of c-Src, suggesting possible involvement of other signaling molecules in CSK-dependent MCF-7 cell death induced by fulvestrant. Our observations suggest the importance of CSK in the determination of cellular sensitivity to the cytocidal action of fulvestrant.     

Staphylococcus aureus α-Hemolysin Activates the NLRP3-Inflammasome in Human and Mouse Monocytic Cells

Community Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA) causes severe necrotizing infections of the skin, soft tissues, and lungs. Staphylococcal α-hemolysin is an essential virulence factor in mouse models of CA-MRSA necrotizing pneumonia. S. aureus α-hemolysin has long been known to induce inflammatory signaling and cell death in host organisms, however the mechanism underlying these signaling events were not well understood. Using highly purified recombinant α-hemolysin, we now demonstrate that α-hemolysin activates the Nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain containing 3 protein (NLRP3)-inflammasome, a host inflammatory signaling complex involved in responses to pathogens and endogenous danger signals. Non-cytolytic mutant α-hemolysin molecules fail to elicit NLRP3-inflammasome signaling, demonstrating that the responses are not due to non-specific activation of this innate immune signaling system by bacterially derived proteins. In monocyte-derived cells from humans and mice, inflammasome assembly in response to α-hemolysin results in activation of the cysteine proteinase, caspase-1. We also show that inflammasome activation by α-hemolysin works in conjunction with signaling by other CA-MRSA-derived Pathogen Associated Molecular Patterns (PAMPs) to induce secretion of pro-inflammatory cytokines IL-1β and IL-18. Additionally, α-hemolysin induces cell death in these cells through an NLRP3-dependent program of cellular necrosis, resulting in the release of endogenous pro-inflammatory molecules, like the chromatin-associated protein, High-mobility group box 1 (HMGB1). These studies link the activity of a major S. aureus virulence factor to a specific host signaling pathway. The cellular events linked to inflammasome activity have clear relevance to the disease processes associated with CA-MRSA including tissue necrosis and inflammation.     

Protein Kinase Cα Isoform Regulates the Activation of the MAP Kinase ERK1/2 in Human Glioma Cells: Involvement in Cell Survival and Gene Expression

Protein kinase C is a family of serine/threonine protein kinases involved in many cellular responses, including cell survival and apoptosis. We have recently found that specific inhibition of the PKCα isoform by nucleic acid enzymes induced apoptosis in sensitive cells. Here we show that in PKCα DNA enzyme-treated glioma cells the activation of MAP kinases ERK1/2 is inhibited, whereas their total level was not significantly affected by the treatment. Similar results were obtained when the overall activity of the PKC was inhibited by calphostin, a specific inhibitor for PKC. These results would indicate that the ERK1/2 signaling pathway plays an important role in glioma cell survival and that the PKCα isoform is the main modulator of this pathway. Furthermore, we show that the ERK1/2 signaling pathway is required for the constitutive expression of the basic fibroblast growth factor, a potent mitogen for glioma cell growth.     

Role of the α1 and α2 Integrin Cytoplasmic Domains in Cell Morphology,Motility and Responsiveness to Stimulation by the Protein Kinase C Pathway

The α₁β₁ and α₂β₁ integrins, extracellular matrix receptors for collagens and/or laminins, have similarities in structure and ligand binding. Recent studies suggest that the two receptors mediate distinct post-ligand binding events and are not simply redundant receptors. To discern the mechanisms by which the two receptors differ, we focused on the roles of the cytoplasmic domains of the α subunits. We expressed either full-length α₁ integrin subunit cDNA (XICI), full-length α₂ integrin subunit cDNA (X2C2), chimeric cDNA composed of the extracellular and transmembrane domains of Q₂ subunit and the cytoplasmic domain of α₁ (X2C1), chimeric cDNA composed of the extracellular and transmembrane domains of α₁ subunit and the, cytoplasmic domain of α₂ (X1C2), α₁ cDNA truncated after the GFFKR sequence (X1C0) or α₂ cDNA truncated after the GFFKR sequence (X2C0) in K562 cells. Although the cytoplasmic domains of the a_x and α₂ subunits were not required for adhesion, the extent of adhesion at low substrate density was enhanced by the presence of either the α₁ or α₂ cytoplasmic tail. Spreading was also influenced by the presence of an α subunit cytoplasmic tail. Activation of the protein kinase C pathway with phorbol dibutyrate-stimulated motility that was dependent upon the presence of the α₂ cytoplasmic tail. Both the phosphatidylinosotide-3-OH kinase and the mitogen-activated protein kinase pathways were required for phorbol-activated, α₂-cytoplasmic tail-dependent migration.     

Identification of Regulatory Phosphorylation Sites in a Cell Volume– and Ste20 Kinase–dependent ClC Anion Channel

Changes in phosphorylation regulate the activity of various ClC anion transport proteins. However, the physiological context under which such regulation occurs and the signaling cascades that mediate phosphorylation are poorly understood. We have exploited the genetic model organism Caenorhabditis elegans to characterize ClC regulatory mechanisms and signaling networks. CLH-3b is a ClC anion channel that is expressed in the worm oocyte and excretory cell. Channel activation occurs in response to oocyte meiotic maturation and swelling via serine/threonine dephosphorylation mediated by the type I phosphatases GLC-7α and GLC-7β. A Ste20 kinase, germinal center kinase (GCK)-3, binds to the cytoplasmic C terminus of CLH-3b and inhibits channel activity in a phosphorylation-dependent manner. Analysis of hyperpolarization-induced activation kinetics suggests that phosphorylation may inhibit the ClC fast gating mechanism. GCK-3 is an ortholog of mammalian SPAK and OSR1, kinases that bind to, phosphorylate, and regulate the cell volume–dependent activity of mammalian cation-Cl⁻ cotransporters. Using mass spectrometry and patch clamp electrophysiology, we demonstrate here that CLH-3b is a target of regulatory phosphorylation. Concomitant phosphorylation of S742 and S747, which are located 70 and 75 amino acids downstream from the GCK-3 binding site, are required for kinase-mediated channel inhibition. In contrast, swelling-induced channel activation occurs with dephosphorylation of S747 alone. Replacement of both S742 and S747 with glutamate gives rise to kinase- and swelling-insensitive channels that exhibit activity and biophysical properties similar to those of wild-type CLH-3b inhibited by GCK-3. Our studies provide novel insights into ClC regulation and mechanisms of cell volume signaling, and provide the foundation for studies aimed at defining how conformational changes in the cytoplasmic C terminus alter ClC gating and function in response to intracellular signaling events.     

Cell Cycle Dependent Regulation of Deoxycytidine Kinase,Deoxyguanosine Kinase,and Cytosolic 5′-Nucleotidase I Activity in MOLT-4 Cells

Activation of nucleoside analogues is dependent on kinases and 5′-nucleotidases and the balance between the activity of these enzymes. The purpose of this study was to analyze deoxycytidine kinase, deoxyguanosine kinase, and 4 different 5′-nucleotidases during cell cycle progression in MOLT-4 cells. The activity of both kinases was cell cycle dependent and increased during proliferation while the activity of cytosolic 5′-nucleotidase I decreased. We could show that the kinase activity was higher than the total nucleotidase activity, which was unchanged or decreased during cell cycle progression. These data may be important in designing modern combination therapy with nucleoside analogues.