PKC Function Research Articles

Apigenin Inhibition of Involucrin Gene Expression Is Associated with a Specific Reduction in Phosphorylation of Protein Kinase Cδ Tyr311

Apigenin is a plant-derived flavanoid that has significant promise as a skin cancer chemopreventive agent. In the present study, we examine the mechanism whereby apigenin regulates normal human keratinocyte differentiation. Expression of involucrin (hINV), a marker of keratinocyte differentiation, is increased by differentiating agents via a protein kinase Cdelta (PKCdelta), Ras, MEKK1, MEK3 cascade that increases AP1 factor level and AP1 factor binding to DNA elements in the hINV promoter. We show that apigenin inhibits this response. Apigenin suppresses the 12-O-tetradeconylphorbol-13-acetate-dependent increase in AP1 factor expression and binding to the hINV promoter and the increase in hINV promoter activity. Apigenin also inhibits the increase in promoter activity observed following overexpression of PKCdelta, constitutively active Ras, or MEKK1. The suppression of PKCdelta activity is associated with reduced phosphorylation of PKCdelta-Y311. The physiological importance of this phosphorylation event was confirmed by showing that the PKCdelta phosphorylation-defective mutant, PKCdelta-Y311F, is less able to increase hINV promoter activity. Activation of hINV promoter activity by the green tea polyphenol, (-)-epigellocathecin-3-gallate, is also inhibited by apigenin, suggesting that the two chemopreventive agents can produce opposing actions in keratinocytes. Additional studies show that the apigenin-dependent suppression of differentiation is associated with reduced cell proliferation but that there is no evidence of apoptosis.

Serine (Ser) 302 of Cardiac Myosin Binding Protein C (cMyBPC) Is a Unique PKCɛ Phosphorylation Site Both In Vivo and In Vitro

Phosphorylation of cMyBPC by PKA is known to be essential for normal cardiac function and morphology. PKC has also been reported to phosphorylate cMyBPC in vitro. However, the relevant PKC isoforms and functions of PKC-mediated cMyBPC phosphorylation in vivo are unknown. We recently first reported an increase of cMyBPC Ser phosphorylation in transgenic (TG) mice with cardiac-specific overexpression of PKCɛ, which displayed enhanced phosphorylation of sarcomeric proteins and dilated cardiomyopathy. In this study, we further investigated the potential PKCɛ phosphorylation sites on mouse cMyBPC. Purified cardiac myofibrillar proteins were isolated from the 1-year old PKCɛ TG or wild type (WT) mice. Compared to WT controls, the cMyBPC Ser phosphorylation in TG mice was found significantly increased (∼4-fold) by Western blotting. In addition, in vitro PKCɛ kinase treatment (20 min) dramatically increased (∼4.6-fold) the Ser phosphorylation level of cMyBPC in the WT mice to that in the TG mice; whereas cMyBPC from the TG mice showed minimal (∼15%) increase in Ser phosphorylation in response to PKCɛ treatment. This result indicates that cMyBPC Ser phosphorylation sites in vivo were saturated in PKCɛ mice. The total and Ser phosphorylation of cMyBPC was also studied using 2-D gel electrophoresis followed by Pro-Q Diamond phosphoprotein staining or Western blotting, which confirmed the increase of cMyBPC Ser phosphorylation in PKCɛ mice. Interestingly, the total phosphorylation of cMyBPC was not significantly altered in TG mice, suggesting a switch from PKA to PKC-mediated phosphorylation in these mice. Using Netphos analysis, 3 peptide motifs of cMyBPC were identified as the potential consensus sites for PKCɛ with Ser302 as a 100%-matched motif and Ser811 and Ser1203 as 2 nearly matched motifs. Three 18-amino acid peptides were synthesized corresponding to the sequences of above 3 motifs, and subjected to in vitro PKCɛ assay. Peptide phosphorylation was determined by mass spectrometry and anti-p-Ser ELISA assay, demonstrating that only the Ser302 site, but not the Ser811 or Ser1203 site, was phosphorylated by PKCɛ in vitro. A S302A point mutation in the Ser302 peptide abolished the peptide phosphorylation by PKCɛ. Taken together, our data showed that the Ser302 on mouse cMyBPC is a unique PKCɛ phosphorylation site both in vivo and in vitro, which may contribute to the dilated cardiomyopathy associated with elevated PKCɛ level.

Physiological and Non-Redundant Functions of PKC Isotypes in T Lymphocytes

This review is about the physiological and non-redundant functions of the PKC gene products in hematopoietic cells, particularly T cells. In spite of the large amount of information on PKC functions in various cell types and tissues, the characterization of the isotype selective functions of the entire PKC family in lymphoid cell lineages is far from complete. Given the established important role of PKCθ as regulator of T cell fate and knowing that several other PKC isotypes are also expressed in T cells at a high level, we here summarize the physiological and non-redundant functions of PKCα, β, δ, ε,ζ and θ isotypes in T cells (with emphasis on the ongoing mouse genetic studies). Their known and/or suspected cellular regulation, effector pathways as well as physiological functions are discussed. While PKCβ,ε, δ and appear to be dispensable during cellular activation of primary CD3+ T cells, PKCα and PKCθ take critical parts in signaling pathways that are necessary for full antigen receptor mediated T cell activation and T lymphocyte immunity. Keywords: T lymphocyte, antigen receptor, gene ablation, PKC isotypes, cellular functions

Involvement of protein kinase C-related anti-apoptosis signaling in radiation-induced apoptosis in murine thymic lymphoma(3SBH5) cells.

Protein kinase C (PKC; also known as PRKC) is known to be an important participant in radiation-induced apoptosis. However, its role is not fully clarified. Using 3SBH5 cells, which are radiation-sensitive thymic lymphoma cells, the involvement and functions of PKC were assessed in radiation- induced apoptosis. PMA (phorbol 12-myristate 13-acetate), a PKC activator, inhibited the radiation-induced apoptosis in 3SBH5 cells. On the other hand, chelerythrine, a PKC inhibitor, potentiated apoptosis. In addition, Gö6976, a classical PKC (cPKC) inhibitor, which specifically inhibits PKC (alpha and betaI), also promoted apoptosis. Interestingly, post-treatment (20 min after irradiation) with Gö6976 had no effect on the radiation-induced apoptosis. These results suggest that cPKC is activated early after irradiation for anti-apoptosis signaling and contributes to the balance between cell survival and death. Indeed, an increase of cPKC activity involving PKC (alpha, betaI and betaII) was observed in the cytosolic fraction 3 min after irradiation with 0.5 Gy. However, no translocation of cPKC was observed in the cells after irradiation. Our findings indicate that activation of cPKC (alpha or beta) soon after irradiation is critical to the understanding of the regulation of radiation-induced apoptosis in radiation-sensitive cells.

αIIbβ3-mediated outside-in signaling induced by the agonist peptide LSARLAF utilizes ADP and thromboxane A2 receptors to cause α-granule secretion by platelets

The peptide LSARLAF (LSA) causes alphaIIbbeta3-dependent platelet activation that results in alpha-granule secretion and aggregation. LSARLAF-induced, alphaIIbbeta3-mediated outside-in signaling causing alpha-granule secretion and platelet aggregation was studied using washed mouse platelets. ADP receptor antagonists, enzyme inhibitors, normal platelets and platelets from mice that lack either Galphaq or thromboxane (Tx) A2 receptors were used for this investigation. The results demonstrate that LSA-induced alphaIIbbeta3-mediated signaling producing aggregation of washed platelets is mediated through the release of ADP and thromboxane, which cause alpha-granule release by mediating their effects though Galphaq and/or Gi depending on the level of LSA used to activate the platelets. Specifically, alphaIIbbeta3 elicited aggregation of washed platelets in response to a low level of LSA requires signaling through the ADP receptor P2Y1 and Galphaq, and the ADP receptor P2Y12 and Gi as well as TxA2 receptors. However, this aggregation is independent of Galphaq and TxA2 signaling in response to high LSA concentrations, but is dependent on ADP signaling through its receptor P2Y12, and therefore presumably Gi, regardless of the level of LSA used to activate the platelets. PKC function is required for ADP secretion and the subsequent signaling through P2Y12 regardless of the level of LSA used to activate the platelets. The end point of the LSA-induced alphaIIbbeta3-mediated signaling characterized in this study is alpha-granule secretion, which provides the fibrinogen required for aggregation of washed platelets.

Effects of prenyl pyrophosphates on the binding of PKCgamma with RACK1.

Receptors for activated C kinase (RACKs) are a group of PKC binding proteins that have been shown to mediate isoform-selective functions of PKC and to be crucial in the translocation and subsequent functioning of the PKC isoenzymes on activation. RACK1 cDNA from the shrimp Penaeus japonicus was isolated by homology cloning. The hepatopancreas cDNA from this shrimp was found to encode a 318-residue polypeptide whose predicted amino acid sequence shared 91% homology with human G(beta2)-like proteins. Expression of the cDNA of shrimp RACK1 in vitro yielded a 45-kDa polypeptide with positive reactivity toward the monoclonal antibodies against RACK1 of mammals. The shrimp RACK1 was biotinylated and used to compare the effects of geranylgeranyl pyrophosphate and farnesyl pyrophosphate on its binding with PKCgamma in anti-biotin-IgG precipitates. PKCgammas were isolated from shrimp eyes and mouse brains. Both enzyme preparations were able to inhibit taxol-induced tubulin polymerization. Interestingly, when either geranylgeranyl pyrophosphate or farnesyl pyrophosphate was reduced to the submicrogram level, the recruitment activity of RACK1 with purified PKCgamma was found to increase dramatically. The activation is especially significant for RACK1 and PKCgamma from different species. The observation implies that the deprivation of prenyl pyrophosphate might function as a signal for RACK1 to switch the binding from the conventional isoenzymes of PKC (cPKC) to the novel isoenzymes of PKC (nPKC). A hydrophobic binding pocket for geranylgeranyl pyrophosphate in RACK1 is further revealed via prenylation with protein geranylgeranyl transferase I of shrimp P. japonicus.

PKCeta associates with cyclin E/cdk2/p21 complex, phosphorylates p21 and inhibits cdk2 kinase in keratinocytes.

PKC is activated on the cell membrane by phospholipids, thereby transducing signals to intracellular pathways. We provide here another function of PKC, namely, regulating cell cycle by interaction with the cyclin E/cdk2/p21 complex. Among the 10 isoforms of PKC, PKCeta is predominantly expressed in squamous cell epithelia and induces terminal differentiation of keratinocytes. PKCeta that is endogenously expressed or overexpressed was found to associate with the cyclin E/cdk2/p21 complex in keratinocytes of mice and humans. Requirement of a possible adaptor protein to the binding was suggested by the reconstitution of PKCeta and the cyclin E/cdk2/p21 complex which were prepared from human keratinocytes or Sf9 insect cells. Colocalization of PKCeta with cdk2 and cyclin E was observed in the cytoplasm, particularly in the perinuclear region. p21 was phosphorylated in the complex in a PKC-activator dependent manner. Association of PKCeta with cdk2 resulted in marked inhibition of cdk2-kinase activity when measured by phosphorylation of Rb. Dominant negative PKCeta associated with the cyclin E/cdk2/p21 complex, but caused a little inhibition of cdk2 kinase activity. Among the known regulatory mechanisms of cdk2 activity, dephosphorylation of Thr160 was demonstrated. Oncogene (2000) 19, 6334 - 6341.

Bile Acid-induced Activation of Activator Protein-1 Requires Both Extracellular Signal-regulated Kinase and Protein Kinase C Signaling

Elevated concentrations of fecal bile aids are known to promote colon cancer and increasing evidence suggests that alterations in cellular signaling and gene expression may play an important role in this process. In this study, we examined the molecular mechanisms underlying bile acid-mediated gene regulation using GADD153 as our model gene. Promoter deletion analyses revealed that the activator protein-1 (AP-1) transcription factor was crucial for deoxycholic acid (DCA)-mediated GADD153 gene transcription. Electrophoretic mobility shift assays and transient transfection analyses demonstrated that both DNA binding and transactivation activities of AP-1 were induced by DCA in a dose-dependent manner. The AP-1 complex induced by DCA consisted of JunD, Fra-1, and c-Fos. Examination of the signaling pathways stimulated by DCA showed that extracellular signal-regulated kinases (ERKs) were required for AP-1 activation. Inhibition of ERK by the mitogen-activated protein kinase/ERK kinase inhibitor PD 98059 or by expression of a dominant negative mutant ERK suppressed AP-1 activation. Notably, the PKC inhibitor, calphostin C, also abolished DCA-induced AP-1 activation but did not affect DCA-mediated ERK activation, suggesting that ERK and PKC function in separate signaling pathways that cooperatively mediate DCA-induced AP-1 activation. Hence, bile acid-stimulated signaling appears to converge on the AP-1 protooncogene.

The Npc1 mutation causes an altered expression of caveolin-1, annexin II and protein kinases and phosphorylation of caveolin-1 and annexin II in murine livers

We have previously demonstrated (1) an increased expression of caveolin-1 in murine heterozygous and homozygous Niemann-Pick type C (NPC) livers, and (2) an increased concentration of unesterified cholesterol in a detergent insoluble caveolae-enriched fraction from homozygous livers. To define further the relationship between caveolin-1 function and the cholesterol trafficking defect in NPC, we examined the expression and distribution of additional caveolar and signal transduction proteins. The expression of annexin II was significantly increased in homozygous liver homogenates and the Triton X-100 insoluble floating fraction (TIFF). Phosphoamino acid analysis of caveolin-1 and annexin II from the homozygous TIFF demonstrated an increase in serine and tyrosine phosphorylation, respectively. To determine the basis for increased phosphorylation of these proteins, the expression and distribution of several protein kinases was examined. The expression of PKCα, PKCζ and pp60-src (protein kinases) were significantly increased in both heterozygous and homozygous liver homogenates, while PKCδ was increased only in homozygous livers. Of the protein kinases analyzed, only CK IIα was significantly enriched in the heterozygous TIFF. Finally, the concentration of diacylglycerol in the homozygous TIFF was significantly increased and this elevation may modulate PKC distribution and function. These results provide additional evidence for involvement of a caveolin-1 containing cellular fraction in the pathophysiology of NPC and also suggest that the Npc1 gene product may directly or indirectly, regulate the expression and distribution of signaling molecules.

Sphingosylphosphorylcholine is a Potent Inducer of Intercellular Adhesion Molecule-1 Expression in Human Keratinocytes

We recently reported that the epidermis of patients with atopic dermatitis contains an abnormally expressed sphingomyelin deacylase that yields a large amount of sphingosylphosphorylcholine (SPC) rather than ceramide. In this study, we characterize inflammatory roles of newly discovered chemicals in the epidermis by elucidating biologic effects of SPC on intercellular adhesion molecules-1 (ICAM-I) expression by human keratinocytes in culture in comparison with other sphingolipids. Using fluorescence-activated cell sorter analysis, we found that SPC treatment at concentrations of 10-20 microM significantly enhanced the expression of ICAM-I by cultured human keratinocytes in a dose-dependent manner after incubation for 15-24 h, and, using northern blot analysis, that this was accompanied by increased expression of ICAM-1 mRNA within 4 h of incubation. Transforming necrosis factor-alpha (TNF-alpha) levels in the medium of keratinocytes treated at a 10 microM concentration of SPC were significantly increased by 200%. Furthermore, the SPC-induced ICAM-1 expression was partially abolished by the concomitant addition of anti-TNF-alpha, suggesting a partial autocrine involvement of TNF-alpha in ICAM-1 expression. Assay of mitogen-activated protein kinase revealed that 10 microM SPC induced a rapid activation of mitogen-activated protein kinase in human keratinocytes, including an increase in its phosphorylation within 5 min, which then declined to the baseline control level after 30 min. In contrast, sphingomyelin or sphingosine had no significant potential to activate mitogen-activated protein kinase at the same concentration. These findings suggest that SPC plays an important role in the inflammatory process of epidermis in skin diseases, such as atopic dermatitis, with high expression of sphingomyelin deacylase.

PKC Regulation of Microfilament Network Organization in Keratinocytes Defined by a Pharmacological Study with PKC Activators and Inhibitors

The modulation by PKC activators and inhibitors of adhesion, spreading, migration, actin cytoskeleton organization, and focal complex formation in keratinocytes attaching to type I collagen was studied. Two actin microfilament networks, stress fibers and cortical actin, could be distinguished on the basis of cellular distribution and opposite regulation by growth factors, tyrosine kinase inhibitors, and PKC activators. Stress fiber formation was stimulated by growth factors and by PMA (100 ng/ml) and these stimulations were blocked by tyrosine kinase inhibitors (0.3 mMgenistein and 1 μMherbimycin A). By contrast, the cortical network occurred in quiescent cells, was unaffected by tyrosine kinase inhibitors, and was broken down after PKC activation by PMA. Spreading, migration, and actin polymerization were completely blocked while adhesion efficacy was significantly decreased by three specific PKC inhibitors. Half-inhibition of migration was obtained with 0.025, 1, and 3 μMconcentrations of calphostin C, chelerytrine chloride, andd-erythro-sphingosine, respectively, which are concentrations close to those known to inhibit the PKC kinase functionin vitro.Paxillin clustering, which was observed even in the presence of tyrosine kinase inhibitors, disappeared only when actin polymerization was completely impaired, i.e., in cells treated with PKC inhibitors or with both tyrosine kinase inhibitors and PMA, which indicated that focal complex formation was highly dependent on microfilament reorganization. The analysis of these data underscores a major regulation function of PKC in the molecular events involved in growth factor and adhesion-dependent regulation of microfilament dynamics.

Protein kinase C activity, translocation, and conventional isoforms in aging rat brain

Protein kinase C was studied in various brain areas in aging Wistar rats. Histone-directed kinase activity from the cortex, hippocampus and cerebellum did not change with aging. Using purified protein B-50 as a substrate, between 3 and 8 months a decrease in in vitro phosphorylation was detected in the membrane fraction of the cortex but after this age values remained stable. In hippocampal membranes, B-50 phosphorylation was increased in aged rats. PKC translocation was impaired in aged rats in both the cortex and the hippocampus. PKC α and β mRNA decreased in the cortex between 3 and 8 months with no further decline in aged animals. Hippocampal mRNA for calcium-dependent PKC isoforms was not modified during aging, as assessed by Northern and in situ hybridization. Western blot analysis revealed a change in PKC γ protein only, which was increased in hippocampal membranes from aged rats. The data indicate that the key PKC function that is impaired in aged rats is enzyme translocation irrespective of the brain area investigated.

Zeta PKC induces phosphorylation and inactivation of I kappa B-alpha in vitro.

The zeta isotype of protein kinase C (zeta PKC), a distinct PKC unable to bind phorbol esters, is required during NF-kappa B activation as well as in mitogenic signalling in Xenopus oocytes and mammalian cells. To investigate the mechanism(s) for control of cellular functions by zeta PKC, this enzyme was expressed in Escherichia coli as a fusion protein with maltose binding protein (MBP), to allow immobilization on amylose beads to study signalling proteins in cell extracts that might form complex(es) with zeta PKC. The following evidence for interaction with the NF-kappa B/I kappa B pathway was obtained. MBP-zeta PKC, but not MBP, bound and activated a potentially novel I kappa B kinase of approximately 50 kDa molecular weight able to regulate I kappa B-alpha function. Activation of the I kappa B kinase was dependent on zeta PKC enzymatic activity and ATP, suggesting that zeta PKC controls, directly or indirectly, the activity of a functionally significant I kappa B kinase. Importantly, zeta PKC immunoprecipitates from TNF-alpha-stimulated NIH-3T3 fibroblasts displayed a higher I kappa B phosphorylating activity than untreated controls, indicating the in vivo relevance of these findings. We also show here that zeta PKC associates with and activates MKK-MAPK in vitro, suggesting that one of the mechanisms whereby overexpression of zeta PKC leads to deregulation of cell growth may be accounted for at least in part by activation of the MKK-MAPK complex. However, neither MKK nor MAPK is responsible for the putative I kappa B phosphorylating activity. These data provide a decisive step towards understanding the functions of zeta PKC.