Protein kinase C plays an essential role in sildenafil-induced cardioprotection in rabbits

Sildenafil citrate (Viagra) is the most widely used pharmacological drug for treating erectile dysfunction in men. It has potent cardioprotective effects against ischemia-reperfusion injury via nitric oxide and opening of mitochondrial ATP-sensitive K(+) channels. We further investigated the role of protein kinase C (PKC)-dependent signaling pathway in sildenafil-induced cardioprotection. Rabbits were treated (orally) with sildenafil citrate (1.4 mg/kg) 30 min before index ischemia for 30 min and reperfusion for 3 h. The PKC inhibitor chelerythrine (5 mg/kg i.v.) was given 5 min before sildenafil. Infarct size (% of risk area) reduced from 33.65 +/- 2.17 in the vehicle (saline) group to 15.07 +/- 0.63 in sildenafil-treated groups, a 45% reduction compared with vehicle (mean +/- SE, P < 0.05). Chelerythrine abolished sildenafil-induced protection, as demonstrated by increase in infarct size to 31.14 +/- 2.4 (P < 0.05). Chelerythrine alone had an infarct size of 33.5 +/- 2.5, which was not significantly different compared with DMSO-treated group (36.8 +/- 1.7, P > 0.05). Western blot analysis demonstrated translocation of PKC-alpha, -, and -delta isoforms from cytosol to membrane after treatment with sildenafil. However, no change in the PKC-beta and -epsilon isoforms was observed. These data provide direct evidence of an essential role of PKC, and potentially PKC-alpha, -, and -delta, in sildenafil-induced cardioprotection in the rabbit heart.     

Protein kinase C phosphorylates ribosomal protein S6 kinase betaII and regulates its subcellular localization

The ribosomal protein S6 kinase (S6K) belongs to the AGC family of Ser/Thr kinases and is known to be involved in the regulation of protein synthesis and the G(1)/S transition of the cell cycle. There are two forms of S6K, termed S6Kalpha and S6Kbeta, which have cytoplasmic and nuclear splice variants. Nucleocytoplasmic shuttling has been recently proposed for S6Kalpha, based on the use of the nuclear export inhibitor, leptomycin B. However, the molecular mechanisms regulating subcellular localization of S6Ks in response to mitogenic stimuli remain to be elucidated. Here we present data on the in vitro and in vivo phosphorylation of S6Kbeta, but not S6Kalpha, by protein kinase C (PKC). The site of phosphorylation was identified as S486, which is located within the C-terminal nuclear localization signal. Mutational analysis and the use of phosphospecific antibodies provided evidence that PKC-mediated phosphorylation at S486 does not affect S6K activity but eliminates the function of its nuclear localization signal and causes retention of an activated form of the kinase in the cytoplasm. Taken together, this study uncovers a novel mechanism for the regulation of nucleocytoplasmic shuttling of S6KbetaII by PKC-mediated phosphorylation.     

Protein kinase C plays an inhibitory role in interleukin 3- and interleukin 4-mediated mast cell proliferation

Interleukin 3 (IL-3) is required for the survival and proliferation of mouse bone marrow derived mast cells (BMMC). Although interleukin 4 (IL-4) has no direct effect on growth activity, it synergizes with IL-3 in promoting the growth of these cells. The intracellular mechanism by which these ligand-receptor interactions promote mast cell growth are not well documented in the literature. Here we present evidence that both IL-3 and IL-4 have been found to activate protein kinase C (PKC) and phosphatidylinositol turnover in BMMC, in a similar time- and dose-dependent manner, indicating that activation of PKC is not sufficient to induce proliferation in these cells. In this work we addressed the question as to whether the activation of PKC is necessary for mast cell proliferation. Activation of PKC by phorbol myristate acetate causes inhibition of IL-3-mediated growth for the first 72 h of incubation. The inhibition in IL-3-mediated proliferation gradually lessens with the stages of PKC depletion, which is complete after 72 h. The enhancement in phorbol myristate acetate-treated cells grows as PKC is depleted. The inactive phorbol ester, 4-alpha-phorbol, had no effect on proliferation of BMMC. Cells, PKC-depleted by chronical phorbol ester treatment, responded to IL-3 or IL-4 with a significant increase in [3H] thymidine uptake over PKC containing cells stimulated with the same lymphokine. Use of antibodies to these lymphokines showed that the enhanced response of the PKC-depleted BMMC was not due to the additional autocrine production of IL-3 or IL-4 by these cells. The PKC-depleted cells retain the capacity to return to almost normal levels of PKC activity and sensitivity to IL-3 and IL-4, after 72 and 120 h, respectively. These results indicate that PKC plays an important inhibitory role in IL-3- and IL-4-mediated proliferation of BMMC.     

Increased expression of protein kinase C alpha plays a key role in retinoic acid-induced melanoma differentiation.

Differentiation of B16 mouse melanoma cells induced by retinoic acid (RA) is preceded by a large increase in protein kinase C alpha (PKC alpha) mRNA and protein. To determine the role of PKC alpha in the differentiation program, we stably transfected B16-F1 cells with a plasmid containing the full length PKC alpha cDNA driven by an SV40 promoter. Two out of thirty-two colonies screened were determined to overexpress PKC by 2-4-fold according to Western blot analysis and PKC enzyme activity. When compared to control cells (wild-type cells and cells transfected only with the neomycin resistance gene), PKC alpha overexpressing clones displayed longer doubling times, diminished anchorage-independent growth, and increased melanin production. RA treatment of control cells mimicked these phenotypic characteristics. When injected subcutaneously into syngeneic mice, PKC alpha overexpressing clones produced smaller tumors and had longer latencies than control cells. These findings, combined with the fact that phorbol esters down-regulate PKC and antagonize RA action suggest that PKC alpha plays a key role in the RA-induced melanoma differentiation.     

Protein kinase C betaII regulates Akt phosphorylation on Ser-473 in a cell type- and stimulus-specific fashion

Akt (= protein kinase B), a subfamily of the AGC serine/threonine kinases, plays critical roles in survival, proliferation, glucose metabolism, and other cellular functions. Akt activation requires the recruitment of the enzyme to the plasma membrane by interacting with membrane-bound lipid products of phosphatidylinositol 3-kinase. Membrane-bound Akt is then phosphorylated at two sites for its full activation; Thr-308 in the activation loop of the kinase domain is phosphorylated by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser-473 in the C-terminal hydrophobic motif by a putative kinase PDK2. The identity of PDK2 has been elusive. Here we present evidence that conventional isoforms of protein kinase C (PKC), particularly PKCbetaII, can regulate Akt activity by directly phosphorylating Ser-473 in vitro and in IgE/antigen-stimulated mast cells. By contrast, PKCbeta is not required for Ser-473 phosphorylation in mast cells stimulated with stem cell factor or interleukin-3, in serum-stimulated fibroblasts, or in antigen receptor-stimulated T or B lymphocytes. Therefore, PKCbetaII appears to work as a cell type- and stimulus-specific PDK2.     

Protein kinase B beta/Akt2 plays a specific role in muscle differentiation

Insulin-like growth factors positively regulate muscle differentiation through activation of the phosphatidylinositol 3-kinase/protein kinase B (PKB/Akt) signaling pathway. Here, we compare the role of the two closely related alpha (Akt1) and beta (Akt2) isoforms of PKB in muscle differentiation. During differentiation of C2.7 or L6D2 myoblasts, PKBbeta was up-regulated whereas expression of PKBalpha was unaltered. Although the two isoforms were found active in both myoblasts and myotubes, cell fractionation experiments indicated that they displayed distinct subcellular localizations in differentiated cells with only PKBbeta localized in the nuclei. In a transactivation assay, PKBbeta (either wild-type or constitutively active) was more efficient than PKBalpha in activating muscle-specific gene expression. Moreover, microinjection of specific antibodies to PKBbeta inhibited differentiation of muscle cells, whereas control or anti-PKBalpha antibodies did not. On the other hand, microinjection of the anti-PKBalpha antibodies caused a block in cell cycle progression in both non muscle and muscle cells, whereas anti-PKBbeta antibodies had no effect. Taken together, these results show that PKBbeta plays a crucial role in the commitment of myoblasts to differentiation that cannot be substituted by PKBalpha.     

Protein kinase C zeta plays an essential role for Mycobacterium tuberculosis-induced extracellular signal-regulated kinase 1/2 activation in monocytes/macrophages via Toll-like receptor 2

This study characterized the upstream signalling molecules involved in extracellular signal-regulated kinase (ERK) 1/2 activation and determined their effects on differential tumour necrosis factor (TNF)-alpha expression by monocytes/macrophages infected with virulent or avirulent mycobacteria. The avirulent Mycobacterium tuberculosis (MTB) strain H37Ra (MTBRa) induced higher levels of activation of ERK 1/2 and the upstream MAPK kinase (MEK)1 and, subsequently, higher levels of TNF-alpha expression in human primary monocytes and monocyte-derived macrophages, as compared with MTB strain H37Rv (MTBRv). The MTB-induced activation of ERK 1/2 was not dependent on Ras or Raf. However, inhibition of the activity of atypical protein kinase C (PKC) zeta decreased the in vitro phosphorylation of MEK, ERK 1/2 activation and subsequent TNF-alpha induction caused by MTBRv or MTBRa. Toll-like receptor (TLR) 2 was found to play a major role in MTB-induced TNF-alpha expression and PKCzeta phosphorylation. Co-immunoprecipitation experiments showed that PKCzeta interacts physically with TLR2 after MTB stimulation. Moreover, PKCzeta phosphorylation was increased more in macrophages following MTBRa, versus MTBRv, infection. This is the first demonstration that PKCzeta interacts with TLR2 to play an essential role in MTB-induced ERK 1/2 activation and subsequent TNF-alpha expression in monocytes/macrophages.     

Iron-mediated oxidative stress plays an essential role in ferritin-induced cell death

Previously, we have demonstrated an apoptosis-inducing activity of an acidic, H-chain-rich isoferritin secreted from primary rat hepatocytes in vitro. Because this proapoptotic property may be responsible for the growth-inhibitory and immunosuppressive effects described for certain ferritin species, we aimed to address the mechanism by which ferritin can trigger cell death. Suggesting a pivotal role for iron, iron chelation by desferrioxamine significantly abrogates ferritin-mediated apoptosis and necrosis in primary rat hepatocytes and substantially lowers the extent of protein modification by 4-hydroxynonenal (HNE)—a major lipid peroxidation (LPO) product. Furthermore, supplementing the cultures with the radical-scavenging compound trolox also provided significant protection from ferritin-mediated apoptosis. Moreover, a significant increase in micronucleated cells upon exposure to ferritin indicates that ferritin also introduces damage to DNA. Based on these observations we therefore propose that endocytosis of extracellular ferritin increases the level of free ferrous iron in the lysosomal compartment, promoting Fenton chemistry-based oxidative stress involving LPO and increased lysosomal membrane permeability. Subsequently, the release of reactive lysosomal content leads to cellular damage, in particular modification of protein and DNA induced by HNE and other reactive aldehydic LPO products. Together, these effects will trigger apoptosis and necrosis based on the upregulation of p53, increased mitochondrial membrane permeability, and proapoptotic Fas signaling as described recently. In conclusion, based on their iron-storing ability, secreted acidic isoferritins may act as soluble mediators of oxidative stress under certain physiological and pathophysiological conditions.     

The APC/C subunit 10 plays an essential role in cell proliferation during leaf development

The largest E3 ubiquitin-ligase complex, known as anaphase-promoting complex/cyclosome (APC/C), regulates the proteolysis of cell cycle regulators such as CYCLIN B and SECURIN that are essential for sister-chromatid separation and exit from mitosis. Despite its importance, the role of APC/C in plant cells and the regulation of its activity during cell division remain poorly understood. Here, the Arabidopsis thaliana APC/C subunit APC10 was characterized and shown to functionally complement an apc10 yeast mutant. The APC10 protein was located in specific nuclear bodies, most probably resulting from its association with the proteasome complex. An apc10 Arabidopsis knockout mutant strongly impaired female gametogenesis. Surprisingly, constitutive overexpression of APC10 enhanced leaf size. Through kinematic analysis, the increased leaf size was found to be due to enhanced rates of cell division during the early stages of leaf development and, at the molecular level, by increased APC/C activity as measured by an amplification of the proteolysis rate of the mitotic cyclin, CYCB1;1.     

The C terminus of RON tyrosine kinase plays an autoinhibitory role

RON is a receptor tyrosine kinase in the MET family. We have expressed and purified active RON using the Sf9/baculovirus system. The constructs used in this study comprise the kinase domain alone and the kinase domain plus the C-terminal region. The construct containing the kinase domain alone has a higher specific activity than the construct containing the kinase and C-terminal domains. Purified RON undergoes autophosphorylation, and the exogenous RON C terminus serves as a substrate. Peptides containing a dityrosine motif derived from the C-terminal tail inhibit RON in vitro or when delivered into intact cells, consistent with an autoinhibitory mechanism. Phenylalanine substitutions within these peptides increase the inhibitory potency. Moreover, introduction of these Phe residues into the dityrosine motif of the RON kinase leads to a decrease in kinase activity. Taken together, our data suggest a model in which the C-terminal tail of RON regulates kinase activity via an interaction with the kinase catalytic domain.     

Protein kinase C: properties and possible role in cellular division and differentiation

Protein kinase C was first described some eight years ago. Recent results indicate that this kinase may have a crucial role in signal transduction for substances involved in cellular differentiation and division. Protein kinase C is activated by attachment to plasma membranes, in the presence of calcium and diacylglycerol. The activator is produced in the membrane following the signal-induced breakdown of phosphoinositides. Tumor promoters, such as phorbol ester, can substitute for diacylglycerol. The recent findings that: tyrosine kinases might be involved in the phosphoinositide turnover and, phosphorylation of growth factor receptors by protein kinase C regulates some of their functions, indicate more and more clearly that this kinase is involved in the control of cell growth division and differentiation. Purification procedures, properties and mechanisms of regulation will be summarized and discussed.     

Protein kinase C and its role in normal and transformed cells

Data are reviewed on protein kinase C (PK-C) and its function in phosphorylation and regulation of different cellular proteins--enzymes, receptors, contractile and cytoskeletal proteins, as well as expression of cellular oncogenes. Besides, interactions between PK-C and cAMP-dependent PK are discussed. The evidence provided suggests that PK-C-phosphorylation may be one of the mechanisms of transformation of extracellular transmembrane signals (hormones, neuromediators, growth factors) into responsive biochemical cell reactions.     

Protein kinase Calpha plays a critical role in mannosylerythritol lipid-induced differentiation of melanoma B16 cells

Mannosylerythritol lipid (MEL), a novel extracellular glycolipid from yeast, was found to inhibit the proliferation of mouse melanoma B16 cells in a dose-dependent manner and to induce the apoptosis of B16 cells at concentrations higher than 10 microm (Zhao, X., Wakamatsu, Y., Shibahara, M., Nomura, N., Geltinger, C., Nakahara, T., Murata, T., and Yokoyama, K. K. (1999) Cancer Res. 59, 482-486). We show here that exposure of B16 cells to MEL (5 microm) for 2 days resulted in an increase of the levels of differentiation-associated markers of melanoma cells such as melanogenesis and tyrosinase activity, which were accompanied by morphological changes. The MEL-induced differentiation of B16 cells at this concentration was closely associated with arrest of the cell cycle at G(1) phase, but no significant population of apoptotic cells was identified. Expression of protein kinase Calpha (PKCalpha) was enhanced after exposure of B16 cells to MEL for 48 h. Antisense oligodeoxynucleotides against the mouse gene for PKCalpha prevented MEL-induced melanogenesis in B16 cells. Conversely, the effects of the expression of a constitutively active form of PKCalpha mimicked the effects of MEL on B16 cells. These data suggest that MEL, a yeast-derived glycolipid, triggers the differentiation of B16 melanoma cells through a signaling pathway that involves PKCalpha.     

Protein kinase C betaII and TGFbetaRII in omega-3 fatty acid-mediated inhibition of colon carcinogenesis

Increasing evidence demonstrates that protein kinase C betaII (PKCbetaII) promotes colon carcinogenesis. We previously reported that colonic PKCbetaII is induced during colon carcinogenesis in rodents and humans, and that elevated expression of PKCbetaII in the colon of transgenic mice enhances colon carcinogenesis. Here, we demonstrate that PKCbetaII represses transforming growth factor beta receptor type II (TGFbetaRII) expression and reduces sensitivity to TGF-beta-mediated growth inhibition in intestinal epithelial cells. Transgenic PKCbetaII mice exhibit hyperproliferation, enhanced colon carcinogenesis, and marked repression of TGFbetaRII expression. Chemopreventive dietary omega-3 fatty acids inhibit colonic PKCbetaII activity in vivo and block PKCbetaII-mediated hyperproliferation, enhanced carcinogenesis, and repression of TGFbetaRII expression in the colonic epithelium of transgenic PKCbetaII mice. These data indicate that dietary omega-3 fatty acids prevent colon cancer, at least in part, through inhibition of colonic PKCbetaII signaling and restoration of TGF-beta responsiveness.     

miRNAs在干细胞自我更新和分化中的调控作用

干细胞与microRNAs(miRNAs)均为近年来研究的热点问题。干细胞是一类具有自我更新与多项分化潜能的细胞, 因与生物发育和癌症发生的密切联系而越来越受到人们的重视。miRNAs是一类长约22nt的小分子非编码RNA, 具有高度的种间保守性和时空特异性, 在转录后水平调节靶基因的表达, 是细胞内基因表达的基本调控机制之一。最近的一些研究表明, miRNAs在干细胞的自我更新和分化过程中具有重要的调控作用。这些研究主要采用两种策略: (1)缺失/突变干细胞中miRNAs合成途径必需酶(包括Dicer1、Loqs、DGCR8、Argnaute蛋白等), 通过细胞特性变化来研究其功能; (2)直接筛选干细胞中的特异性miRNAs并研究其功能。针对干细胞中miRNAs的研究对深入了解干细胞自我更新和分化的机制以及干细胞的鉴定具有重要的意义。文章基于近年来的研究对干细胞相关的miRNAs进行了综述。     

Palmitoylcarnitine modulates interaction between protein kinase C betaII and its receptor RACK1

Palmitoylcarnitine, known to promote differentiation of neuroblastoma NB-2a cells as well as to inhibit protein kinase C (PKC) activity and to decrease phorbol ester binding, was shown previously to diminish the amount of complex formed between PKCdelta and its substrate GAP-43. In the present work we studied the effect of palmitoylcarnitine on the interaction between PKCbetaII and its receptor RACK1. Palmitoylcarnitine was found to decrease autophosphorylation of PKCbetaII on serine in a concentration-dependent manner and to decrease the amount of PKCbetaII/RACK1 complex. The effect of palmitoylcarnitine on cellular localization was found to be dependent on the presence of ATP; palmitoylcarnitine lowered the amount of PKCbetaII in cytosol and decreased the amount of PKCbetaII-RACK1 complex in membrane in the absence of ATP. Palmitoylcarnitine also reversed the effect of phorbol ester on the increase in the amount of PKCbetaII in membrane. Palmitoylcarnitine binds to PKCbetaII through hydrophobic interactions, although acylation of PKCbetaII by the palmitate moiety has been excluded. The presence of palmitoylcarnitine did not have any additive effect on the diminution of PKCbetaII-RACK1 complex formation in the presence of a RACK1-binding peptide from within the C2 region of PKCbetaII. These results rather exclude a possibility of interaction of palmitoylcarnitine with the C2 domain and suggest a possible interaction with the V5 domain and a conformational change affecting the C1 region.