Regulation of Protein Kinase C function by phosphorylation on conserved and non-conserved sites

Protein Kinase C (PKC) is a family of serine/threonine kinases whose function is influenced by phosphorylation. In particular, three conserved phosphorylation sites known as the activation-loop, the turn-motif and the hydrophobic-motif play important roles in controlling the catalytic activity, stability and intracellular localisation of the enzyme. Prevailing models of PKC phosphorylation suggest that phosphorylation of these sites occurs shortly following synthesis and that these modifications are required for the processing of newly-transcribed PKC to the mature (but still inactive) form; phosphorylation is therefore a priming event that enables catalytic activation in response to lipid second messengers. However, many studies have also demonstrated inducible phosphorylation of PKC isoforms at these sites following stimulation, highlighting that our understanding of PKC phosphorylation and its impact on enzymatic function is incomplete. Furthermore, inducible phosphorylation at these sites is often interpreted as catalytic activation, which could be misleading for some isoforms. Recent studies that include systems-wide phosphoproteomic profiling of cells has revealed a host of additional (and in many cases non-conserved) phosphorylation sites on PKC family members that influence their function. Many of these may in fact be more suitable than previously described sites as surrogate markers of catalytic activation. Here we discuss the role of phosphorylation in controlling PKC function and outline our current understanding of the mechanisms that regulate these phosphorylation sites.     

Effects of PKC activation on the meiotic maturation, fertilization and early embryonic development of mouse oocytes

Protein kinase C (PKC) is a family of Ser/Thr protein kinase widely distributed in eukaryotes. There is evidence that PKC plays key roles in the meiotic maturation and activation of mammalian oocytes. However, the mechanism of PKC's actions and the PKC isoforms responsible for these actions are poorly understood. In this study, we reveal in mouse eggs and early embryos: (1) the effects of PKC on the meiotic and mitotic cell cycle progression during oocyte maturation, egg activation and embryonic cleavages; (2) the functional importance of classical PKC subclasses in these processes; and (3) the subcellular localization of the PKC alpha isoform during development from GV stage oocytes to the blastocyst stage embryos. The results indicate that the PKC activator phorbol 12-myristate 13-acetate (PMA) inhibits the meiotic resumption of cumulus-free mouse oocytes by a mechanism dependent not only on classical PKC activity but also on other PKC isoforms. PKC activation after germinal vesicle breakdown leads to the inhibition of mitogen-activated protein kinase phosphorylation and the arrest of cell cycle at MI stage. The second polar body emission and the cleavages of early embryos are blocked after prolonged PKC activation. The subcellular localization of PKC alpha isoform in mouse oocytes and embryos is developmental-stage associated. All these results suggest that PKC has multiple functional roles in the cell cycle progression of mouse oocytes and embryos.     

蛋白激酶C分子的成熟与活化

蛋白激酶C（Proteinkinase C,PKc）是细胞内一类重要的Ser／Thr激酶,调控多种生命活动的信号转导过程,目前已发现了至少11种亚型,其结构有一定的保守性而又有所差别,导致其功能和调控的多样性。新合成的PKC一般需要经历活化茎环（Acti．vation-loop,A—loop）、转角模体（Tummotif,T1V1）以及疏水模体（hydrophobic motif,HM）的程序性磷酸化过程才能成熟,获得进一步活化的功能。本文综述了近年来PKC的程序性磷酸化成熟以及活化的研究进展情况。     

PKC signaling at fertilization in mammalian eggs

Protein kinase C (PKC) has been proposed to regulate major egg activation events during mammalian fertilization. Most of the evidence supporting this assumption has first been obtained using pharmacological activation and inhibition of the kinase, while egg activation was assessed by checking for exocytosis of the cortical granules, extrusion of the second polar body and formation of pronuclei. However, results have been inconclusive and sometimes contradictory regarding the exact role of PKC in regulating egg activation events. The PKC family is composed of various isotypes, which differ in their modular structures and regulatory properties. Hence the need to re-examine the roles of egg PKCs more specifically. Mammalian eggs express many PKC isotypes, the roles of which have been investigated using immunodetection, isotype-specific inhibition and, more recently, live imaging of fluorescent chimaeras. Here, I review the recent development of PKC research in mammalian fertilization and the evidence for a specific role for certain PKC isotypes in fertilization-induced egg activation.     

Uncoupling of betaIIPKC from its targeting protein RACK1 in response to ethanol in cultured cells and mouse brain.

Protein kinase C (PKC) is involved in many neuroadaptive responses to ethanol in the nervous system. PKC activation results in translocation of the enzyme from one intracellular site to another. Compartmentalization of PKC isozymes is regulated by targeting proteins such as receptors for activated C kinase (RACKs). It is possible, therefore, that ethanol-induced changes in the function and compartmentalization of PKC isozymes could be due to changes in PKC targeting proteins. Here we study the response of the targeting protein RACK1 and its corresponding kinase betaIIPKC to ethanol, and propose a novel mechanism to explain how ethanol modulates signaling cascades. In cultured cells, ethanol induces movement of RACK1 to the nucleus without affecting the compartmentalization of betaIIPKC. Ethanol also inhibits betaIIPKC translocation in response to activation. These results suggest that ethanol inhibition of betaIIPKC translocation is due to miscompartmentalization of the targeting protein RACK1. Similar events occurred in mouse brain. In vivo exposure to ethanol caused RACK1 to localize to nuclei in specific brain regions, but did not affect the compartmentalization of betaIIPKC. Thus, some of the cellular and neuroadaptive responses to ethanol may be related to ethanol-induced movement of RACK1 to the nucleus, thereby preventing the translocation and corresponding function of betaIIPKC.     

Structural requirements for localization and activation of protein kinase C mu (PKC mu) at the Golgi compartment.

We here describe the structural requirements for Golgi localization and a sequential, localization-dependent activation process of protein kinase C (PKC) mu involving auto- and transphosphorylation. The structural basis for Golgi compartment localization was analyzed by confocal microscopy of HeLa cells expressing various PKC mu-green fluorescent protein fusion proteins costained with the Golgi compartment-specific markers p24 and p230. Deletions of either the NH(2)-terminal hydrophobic or the cysteine region, but not of the pleckstrin homology or the acidic domain, of PKC mu completely abrogated Golgi localization of PKC mu. As an NH(2)-terminal PKC mu fragment was colocalized with p24, this region of PKC mu is essential and sufficient to mediate association with Golgi membranes. Fluorescence recovery after photobleaching studies confirmed the constitutive, rapid recruitment of cytosolic PKC mu to, and stable association with, the Golgi compartment independent of activation loop phosphorylation. Kinase activity is not required for Golgi complex targeting, as evident from microscopical and cell fractionation studies with kinase-dead PKC mu found to be exclusively located at intracellular membranes. We propose a sequential activation process of PKC mu, in which Golgi compartment recruitment precedes and is essential for activation loop phosphorylation (serines 738/742) by a transacting kinase, followed by auto- and transphosphorylation of NH(2)-terminal serine(s) in the regulatory domain. PKC mu activation loop phosphorylation is indispensable for substrate phosphorylation and thus PKC mu function at the Golgi compartment.     

Phosphorylated MARCKS: a novel centrosome component that also defines a peripheral subdomain of the cortical actin cap in mouse eggs

MARCKS (myristoylated alanine-rich C-kinase substrate) is a major substrate for protein kinase C (PKC), a kinase that has multiple functions during oocyte maturation and egg activation, for example, spindle function and cytoskeleton reorganization. We examined temporal and spatial changes in p-MARCKS localization during maturation of mouse oocytes and found that p-MARCKS is a novel centrosome component based its co-localization with pericentrin and gamma-tubulin within microtubule organizing centers (MTOCs). Like pericentrin, p-MARCKS staining at the MI spindle poles was asymmetric. Based on this asymmetry, we found that one end of the spindle was preferentially extruded with the first polar body. At MII, however, the spindle poles had symmetrical p-MARCKS staining. p-MARCKS also was enriched in the periphery of the actin cap overlying the MI or MII spindle to form a ring-shaped subdomain. Because phosphorylation of MARCKS modulates its actin crosslinking function, this localization suggests p-MARCKS functions as part of the contractile apparatus during polar body emission. Our finding that an activator of conventional and novel PKC isoforms did not increase the amount of p-MARCKS suggested that an atypical isoform was responsible for MARCKS phosphorylation. Consistent with this idea, immunostaining revealed that the staining patterns of p-MARCKS and the active form of the atypical PKC zeta/lambda isoform(s) were very similar. These results show that p-MARCKS is a novel centrosome component and also defines a previously unrecognized subdomain of the actin cap overlying the spindle.     

Regulation of protein kinases by lipids

Membranes are sites of intense signaling activity within the cell, serving as dynamic scaffolds for the recruitment of signaling molecules and their substrates. The specific and reversible localization of these signaling molecules to membranes is critical for the appropriate activation of downstream signaling pathways. Phospholipid-binding domains, including C1, C2, PH, and PX domains, play critical roles in the membrane targeting of protein kinases. Recent structural studies have identified a new membrane association domain, the Kinase Associated 1 (KA1) domain, which targets a number of yeast and mammalian protein kinases to membranes containing acidic phospholipids. Despite an abundance of localization studies on lipid-binding proteins and structural studies of the isolated lipid-binding domains, the question of how membrane binding is coupled to the activation of the kinase catalytic domain has been virtually untouched. Recently, structural studies on protein kinase C (PKC) have provided some of the first structural insights into the allosteric regulation of protein kinases by lipid second messengers.     

Regulation of signal transduction by protein targeting: the case for CaMKII.

Protein targeting is increasingly being recognized as a mechanism to ensure speed and specificity of intracellular signal transduction in a variety of biological systems. Conceptually, this is of particular importance for second-messenger-regulated protein kinases with a broad spectrum of substrates, such as the serine/threonine protein kinases PKA, PKC, and CaMKII (cyclic-AMP-dependent protein kinase, Ca(2+)-phospholipid-dependent protein kinase, and Ca(2+)/calmodulin-dependent protein kinase II). The activating second messengers of these enzymes can be produced or released in response to a large variety of "upstream" signals, and they can, in turn, regulate a large variety of "downstream" proteins. Targeting, e.g., via anchoring proteins, can link certain incoming stimuli with specific outgoing signals by restricting the subcellular compartment at which activation and/or action of a signaling molecule can take place. Elegant research on PKA and PKC reinforced the biological importance of such mechanisms. We will focus here on CaMKII, as recent advances in the understanding of its targeting have some significant general implications for signal transduction. The interaction of CaMKII with the NMDA receptor, for instance, shows that a targeting protein can not only specify the subcellular localization of a signaling effector, but can also directly influence its regulation.     

Involvement of the ERK signaling cascade in protein kinase C-mediated cell cycle arrest in intestinal epithelial cells

We have reported previously that protein kinase C (PKC) signaling can mediate a program of cell cycle withdrawal in IEC-18 nontransformed intestinal crypt cells, involving rapid disappearance of cyclin D1, increased expression of Cip/Kip cyclin-dependent kinase inhibitors, and activation of the growth suppressor function of pocket proteins. In the current study, we present evidence to support a requisite role for PKC alpha in mediating these effects. Furthermore, analysis of the signaling events linking PKC/PKC alpha activation to changes in the cell cycle regulatory machinery implicate the Ras/Raf/MEK/ERK cascade. PKC/PKC alpha activity promoted GTP loading of Ras, activation of Raf-1, and phosphorylation/activation of ERK. ERK activation was found to be required for critical downstream effects of PKC/PKC alpha activation, including cyclin D1 down-regulation, p21(Waf1/Cip1) induction, and cell cycle arrest. PKC-induced ERK activation was strong and sustained relative to that produced by proliferative signals, and the growth inhibitory effects of PKC agonists were dominant over proliferative events when these opposing stimuli were administered simultaneously. PKC signaling promoted cytoplasmic and nuclear accumulation of ERK activity, whereas growth factor-induced phospho-ERK was localized only in the cytoplasm. Comparison of the effects of PKC agonists that differ in their ability to sustain PKC alpha activation and growth arrest in IEC-18 cells, together with the use of selective kinase inhibitors, indicated that the length of PKC-mediated cell cycle exit is dictated by the magnitude/duration of input signal (i.e. PKC alpha activity) and of activation of the ERK cascade. The extent/duration of phospho-ERK nuclear localization may also be important determinants of the duration of PKC agonist-induced growth arrest in this system. Taken together, the data point to PKC alpha and the Ras/Raf/MEK/ERK cascade as key regulators of cell cycle withdrawal in intestinal epithelial cells.     

Characterization of protein kinase C-delta in mouse oocytes throughout meiotic maturation and following egg activation

Changes in protein kinase C (PKC) activity influence the progression of meiosis; however, the specific function of the various PKC isoforms in female gametes is not known. In the current study, the protein expression and subcellular distribution profile of PKC-delta (PKC-delta), a novel isoform of the PKC family, was determined in mouse oocytes undergoing meiotic maturation and following egg activation. The full-length protein was observed as a doublet (76 and 78 kDa) on Western blot analysis. A smaller (47 kDa) carboxyl-terminal fragment, presumably the truncated catalytic domain of PKC-delta, was also strongly expressed. Both the full-length protein and the catalytic fragment became phosphorylated coincident with the resumption of meiosis and remained phosphorylated throughout metaphase II (MII) arrest. Immunofluorescence staining showed PKC-delta distributed diffusely throughout the cytoplasm of oocytes during maturation and associated with the spindle apparatus during the first meiotic division. Discrete foci of the protein also localized with the chromosomes in some mature eggs. Following the completion of meiosis, PKC-delta became dephosphorylated within 2 h of in vitro fertilization or parthenogenetic activation. The protein also accumulated in the nuclei of early embryos and was phosphorylated during M-phase of the initial mitotic cleavage division. By the two-cell stage, expression of the truncated catalytic fragment was minimal. These data demonstrate that the subcellular distribution and posttranslational modification of PKC-delta is cell cycle dependent, suggesting that its activity and/or function likely vary with the progression of meiosis and egg activation.     

Functional independency between catalytic activity and subcellular targeting of Polo-like kinase-1: Phenotypes of ectopic overexpression of various mutants

The mammalian polo-like kinase-1 (Plk1) plays key role in the progression of the M-phase. As cells enter the M-phase, Plk1 is phosphorylated by an upstream kinase on Thr-210 in N-terminal region, which is essential for Plk1 activation. In addition to catalytic activation, the subcellular localization is also an important task for Plk1, and the C-terminal polo-boxes are responsible for Plk1 targeting. Although both catalytic activation and targeting might be essential and work concurrently, it is still unclear which process precedes the other in mitotic progression, and which is more essential for the function of Plk1 through the M-phase. In order to investigate this mechanism, this study introduced several point mutations in the catalytic and polo-box domains, and overexpressed these constructs in HeLa cells. Although the cellular effects by ectopic expression were different in the cells containing various Plk1 constructs, phosphorylation itself, not the catalytic activity, was found to be important for the progression of the M-phase. Plk1 phosphorylation was also responsible for targeting by polo-box, and compensated for the polo-box mutation. These results suggested that Plk1 is phosphorylated in the starting M-phase, and this phosphorylation induces polo-box targeting through a possible conformational change. In this situation, catalytic activation does not appear to be a critical factor for the function of Plk1.     

Protein kinase C regulates integrin-induced activation of the extracellular regulated kinase pathway upstream of Shc

Adhesion of fibroblasts to extracellular matrices via integrin receptors is accompanied by extensive cytoskeletal rearrangements and intracellular signaling events. The protein kinase C (PKC) family of serine/threonine kinases has been implicated in several integrin-mediated events including focal adhesion formation, cell spreading, cell migration, and cytoskeletal rearrangements. However, the mechanism by which PKC regulates integrin function is not known. To characterize the role of PKC family kinases in mediating integrin-induced signaling, we monitored the effects of PKC inhibition on fibronectin-induced signaling events in Cos7 cells using pharmacological and genetic approaches. We found that inhibition of classical and novel isoforms of PKC by down-regulation with 12-0-tetradeconoyl-phorbol-13-acetate or overexpression of dominant-negative mutants of PKC significantly reduced extracellular regulated kinase 2 (Erk2) activation by fibronectin receptors in Cos7 cells. Furthermore, overexpression of constitutively active PKCalpha, PKCdelta, or PKCepsilon was sufficient to rescue 12-0-tetradeconoyl-phorbol-13-acetate-mediated down-regulation of Erk2 activation, and all three of these PKC isoforms were activated following adhesion. PKC was required for maximal activation of mitogen-activated kinase kinase 1, Raf-1, and Ras, tyrosine phosphorylation of Shc, and Shc association with Grb2. PKC inhibition does not appear to have a generalized effect on integrin signaling, because it does not block integrin-induced focal adhesion kinase or paxillin tyrosine phosphorylation. These results indicate that PKC activity enhances Erk2 activation in response to fibronectin by stimulating the Erk/mitogen-activated protein kinase pathway at an early step upstream of Shc.     

Importance of protein kinase C targeting for the phosphorylation of its substrate, myristoylated alanine-rich C-kinase substrate

We visualized the translocation of myristoylated alanine-rich protein kinase C substrate (MARCKS) in living Chinese hamster ovary-K1 cells using MARCKS tagged to green fluorescent protein (MARCKS-GFP). MARCKS-GFP was rapidly translocated from the plasma membrane to the cytoplasm after the treatment with phorbol ester, which translocates protein kinase C (PKC) to the plasma membrane. In contrast, PKC activation by hydrogen peroxide, which was not accompanied by PKC translocation, did not alter the intracellular localization of MARCKS-GFP. Non-myristoylated mutant of MARCKS-GFP was distributed throughout the cytoplasm, including the nucleoplasm, and was not translocated by phorbol ester or by hydrogen peroxide. Phosphorylation of wild-type MARCKS-GFP was observed in cells treated with phorbol ester but not with hydrogen peroxide, whereas non-myristoylated mutant of MARCKS-GFP was phosphorylated in cells treated with hydrogen peroxide but not with phorbol ester. Phosphorylation of both MARCKS-GFPs reduced the amount of F-actin. These findings revealed that PKC targeting to the plasma membrane is required for the phosphorylation of membrane-associated MARCKS and that a mutant MARCKS existing in the cytoplasm can be phosphorylated by PKC activated in the cytoplasm without translocation but not by PKC targeted to the membrane.     

Molecular analysis reveals localization of Saccharomyces cerevisiae protein kinase C to sites of polarized growth and Pkc1p targeting to the nucleus and mitotic spindle

The catalytic activity and intracellular localization of protein kinase C (PKC) are both highly regulated in vivo. This family of kinases contains conserved regulatory motifs, i.e., the C1, C2, and HR1 domains, which target PKC isoforms to specific subcellular compartments and restrict their activity spatially. Saccharomyces cerevisiae contains a single PKC isozyme, Pkc1p, which contains all of the regulatory motifs found in mammalian PKCs. Pkc1p localizes to sites of polarized growth, consistent with its main function in maintaining cell integrity. We dissected the molecular basis of Pkc1p localization by expressing each of its domains individually and in combinations as green fluorescent protein fusions. We find that the Rho1p-binding domains, HR1 and C1, are responsible for targeting Pkc1p to the bud tip and cell periphery, respectively. We demonstrate that Pkc1p activity is required for its normal localization to the bud neck, which also depends on the integrity of the septin ring. In addition, we show for the first time that yeast protein kinase C can accumulate in the nucleus, and we identify a nuclear exit signal as well as nuclear localization signals within the Pkc1p sequence. Thus, we propose that Pkc1p shuttles in and out of the nucleus and consequently has access to nuclear substrates. Surprisingly, we find that deletion of the HR1 domain results in Pkc1p localization to the mitotic spindle and that the C2 domain is responsible for this targeting. This novel nuclear and spindle localization of Pkc1p may provide a molecular explanation for previous observations that suggest a role for Pkc1p in regulating microtubule function.     

Annexins — Scaffolds modulating PKC localization and signaling

Spatial and temporal organization of signal transduction is critical to link different extracellular stimuli with distinct cellular responses. A classical example of hormones and growth factors creating functional diversity is illustrated by the multiple signaling pathways activated by the protein kinase C (PKC) family of serine/threonine protein kinases. The molecular requirements for diacylglycerol (DAG) and calcium (Ca^2 +) to promote PKC membrane translocation, the hallmark of PKC activation, have been clarified. However, the underlying mechanisms that establish selectivity of individual PKC family members to facilitate differential substrate phosphorylation and varied signal output are still not fully understood. It is now well believed that the coordinated control and functional diversity of PKC signaling involves the formation of PKC isozyme-specific protein complexes in certain subcellular sites. In particular, interaction of PKC isozymes with compartment and signal-organizing scaffolds, including receptors for activated C-kinase (RACKs), A-kinase-anchoring proteins (AKAPs), 14-3-3, heat shock proteins (HSP), and importins target PKC isozymes to specific cellular locations, thereby delivering PKC isozymes into close proximity of their substrates. In addition, several annexins (Anx), including AnxA1, A2, A5 and A6, display specific and distinct abilities to interact and promote membrane targeting of different PKC isozymes. Together with the ability of annexins to create specific membrane microenvironments, this is likely to enable PKCs to phosphorylate certain substrates and regulate their downstream effector pathways in specific cellular sites. This review aims to summarize the capacity of annexins to modulate the localization and activity of PKC family members and participate in the spatiotemporal regulation of PKC signaling in health and disease.     

NOM1 targets protein phosphatase I to the nucleolus

Protein phosphatase I (PP1) is an essential eukaryotic serine/threonine phosphatase required for many cellular processes, including cell division, signaling, and metabolism. In mammalian cells there are three major isoforms of the PP1 catalytic subunit (PP1alpha, PP1beta, and PP1gamma) that are over 90% identical. Despite this high degree of identity, the PP1 catalytic subunits show distinct localization patterns in interphase cells; PP1alpha is primarily nuclear and largely excluded from nucleoli, whereas PP1gamma and to a lesser extent PP1beta concentrate in the nucleoli. The subcellular localization and the substrate specificity of PP1 catalytic subunits are determined by their interaction with targeting subunits, most of which bind PP1 through a so-called "RVXF" sequence. Although PP1 targeting subunits have been identified that direct PP1 to a number of subcellular locations and/or substrates, no targeting subunit has been identified that localizes PP1 to the nucleolus. Identification of nucleolar PP1 targeting subunit(s) is important because all three PP1 isoforms are included in the nucleolar proteome, enzymatically active PP1 is present in nucleoli, and PP1gamma is highly concentrated in nucleoli of interphase cells. In this study, we identify NOM1 (nucleolar protein with MIF4G domain 1) as a PP1-interacting protein and further identify the NOM1 RVXF motif required for its binding to PP1. We also define the NOM1 nucleolar localization sequence. Finally, we demonstrate that NOM1 can target PP1 to the nucleolus and show that a specific NOM1 RVXF motif and the NOM1 nucleolar localization sequence are required for this targeting activity. We therefore conclude that NOM1 is a PP1 nucleolar targeting subunit, the first identified in eukaryotic cells.     

Critical role of novel Thr-219 autophosphorylation for the cellular function of PKCtheta in T lymphocytes

Phosphopeptide mapping identified a major autophosphorylation site, phospho (p)Thr-219, between the tandem C1 domains of the regulatory fragment in protein kinase C (PKC)theta. Confirmation of this identification was derived using (p)Thr-219 antisera that reacted with endogenous PKCtheta in primary CD3+ T cells after stimulation with phorbol ester, anti-CD3 or vanadate. The T219A mutation abrogated the capacity of PKCtheta to mediate NF-kappaB, NF-AT and interleukin-2 promoter transactivation, and reduced PKCtheta's ability in Jurkat T cells to phosphorylate endogenous cellular substrates. In particular, the T219A mutation impaired crosstalk of PKCtheta with Akt/PKBalpha in NF-kappaB activation. Yet, this novel (p)Thr-219 site did not affect catalytic activity or second-messenger lipid-binding activity in vitro. Instead, the T219A mutation prevented proper recruitment of PKCtheta in activated T cells. The PKCthetaT219A mutant defects were largely rescued by addition of a myristoylation signal to force its proper membrane localization. We conclude that autophosphorylation of PKCtheta at Thr-219 plays an important role in the correct targeting and cellular function of PKCtheta upon antigen receptor ligation.     

Protein kinase C modulates tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by targeting the apical events of death receptor signaling

We have further examined the mechanism by which phorbol ester-mediated protein kinase C (PKC) activation protects against tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced cytotoxicity. We now report that activation of PKC targets death receptor signaling complex formation. Pre-treatment with 12-O-tetradecanoylphorbol-13-acetate (PMA) led to inhibition of TRAIL-induced apoptosis in HeLa cells, which was characterized by a reduction in phosphatidylserine (PS) externalization, decreased caspase-8 processing, and incomplete maturation and activation of caspase-3. These effects of PMA were completely abrogated by the PKC inhibitor, bisindolylmaleimide I (Bis I), clearly implicating PKC in the protective effect of PMA. TRAIL-induced mitochondrial release of the apoptosis mediators cytochrome c and Smac was blocked by PMA. This, together with the observed decrease in Bid cleavage, suggested that PKC activation modulates apical events in TRAIL signaling upstream of mitochondria. This was confirmed by analysis of TRAIL death-inducing signaling complex formation, which was disrupted in PMA-treated cells as evidenced by a marked reduction in Fas-associated death domain protein (FADD) recruitment, an effect that could not be explained by any change in FADD phosphorylation state. In an in vitro binding assay, the intracellular domains of both TRAIL-R1 and TRAIL-R2 bound FADD: activation of PKC significantly inhibited this interaction suggesting that PKC may be targeting key apical components of death receptor signaling. Significantly, this effect was not confined to TRAIL, because isolation of the native TNF receptor signaling complex revealed that PKC activation also inhibited TNF receptor-associated death domain protein recruitment to TNF-R1 and TNF-induced phosphorylation of IkappaB-alpha. Taken together, these results show that PKC activation specifically inhibits the recruitment of key obligatory death domain-containing adaptor proteins to their respective membrane-associated signaling complexes, thereby modulating TRAIL-induced apoptosis and TNF-induced NF-kappaB activation, respectively.     

Effect of age, GV transfer and modified nucleocytoplasmic ratio on PKCα in mouse oocytes and early embryos

Protein kinase C (PKC) is a family of Ser/Thr protein kinases that can be activated by Ca2+, phospholipid and diacylglycerol. There is evidence that PKC plays key roles in the meiotic maturation and activation of mammalian oocytes. The present study aimed to monitor the effect of age, germinal vesicle (GV) transfer and modified nucleoplasmic ratio on the subcellular distribution profile of PKCα, an important isozyme of PKC, in mouse oocytes undergoing meiotic maturation and following egg activation. Germinal vesicle oocytes were collected from 6-8-week-old and 12-month-old mice. Germinal vesicle-reconstructed oocytes and GV oocytes with one-half or one-third of the original oocyte volume were created using micromanipulation and electrofusion. The subcellular localization of PKCα was detected by immunocytochemistry and laser confocal microscopy. Our study showed that PKCα had a similar location pattern in oocytes and early embryos from young and old mice. PKCα was localized evenly in ooplasm, with weak staining in GV at the GV stage, and present in the entire meiosis II (MII) spindle at the MII stage. In pronuclear and 2-cell embryos, PKCα was concentrated in the nucleus except for the nucleolus. After the GV oocytes were reconstructed, the resultant MII oocytes and embryos showed a similar distribution of PKCα between reconstructed and unreconstructed controls. After one-half or two-thirds of the cytoplasm was removed from the GV oocytes, PKCα still had a similar location pattern in MII oocytes and early embryos from the GV oocytes with modified nucleoplasmic ratio. Our study showed that age, GV transfer and modified nucleocytoplasmic ratio does not affect distribution of PKCα during mouse oocyte maturation, activation, and early embryonic mitosis.