Key Specifications Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|H, B, M, R, Rb||IP, NEUT, RIA, WB||M||Purified||Monoclonal Antibody|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Stable for 1 year at -20ºC from date of receipt.|
|Material Size||100 µg|
|Anti-PKC#945;, clone M4 - DAM1828779||DAM1828779|
|Anti-PKC, clone M4 - 2210398||2210398|
|Anti-PKC, clone M4 - DAM1695851||DAM1695851|
|Anti-PKC, clone M4 - DAM1734743||DAM1734743|
|Anti-PKC, clone M4 - DAM1770256||DAM1770256|
|Anti-PKC, clone M4 - DAM1791288||DAM1791288|
|Anti-PKC, clone M4 - JBC1873434||JBC1873434|
|Anti-PKCa, clone M4 - DAM1530428||DAM1530428|
|Anti-PKCalpha, clone M4 - DAM1632056||DAM1632056|
|Anti-PKCalpha;, clone M4 - 14008||14008|
|Anti-PKCalpha;, clone M4 - 17308||17308|
|Anti-PKCalpha;, clone M4 - 18104||18104|
|Anti-PKCalpha;, clone M4 - 21402||21402|
|Anti-PKCalpha;, clone M4 - 23336||23336|
|Anti-PKCalpha;, clone M4 - 32163||32163|
|Anti-PKCα, clone M4 - 2397121||2397121|
|Anti-PKCα, clone M4 - 2488997||2488997|
References | 33 Available | See All References
|Reference overview||Application||Species||Pub Med ID|
|Detailed expression pattern of aldolase C (Aldoc) in the cerebellum, retina and other areas of the CNS studied in Aldoc-Venus knock-in mice. |
Fujita, H; Aoki, H; Ajioka, I; Yamazaki, M; Abe, M; Oh-Nishi, A; Sakimura, K; Sugihara, I
PloS one 9 e86679 2014
Aldolase C (Aldoc, also known as "zebrin II"), a brain type isozyme of a glycolysis enzyme, is expressed heterogeneously in subpopulations of cerebellar Purkinje cells (PCs) that are arranged longitudinally in a complex striped pattern in the cerebellar cortex, a pattern which is closely related to the topography of input and output axonal projections. Here, we generated knock-in Aldoc-Venus mice in which Aldoc expression is visualized by expression of a fluorescent protein, Venus. Since there was no obvious phenotypes in general brain morphology and in the striped pattern of the cerebellum in mutants, we made detailed observation of Aldoc expression pattern in the nervous system by using Venus expression in Aldoc-Venus heterozygotes. High levels of Venus expression were observed in cerebellar PCs, cartwheel cells in the dorsal cochlear nucleus, sensory epithelium of the inner ear and in all major types of retinal cells, while moderate levels of Venus expression were observed in astrocytes and satellite cells in the dorsal root ganglion. The striped arrangement of PCs that express Venus to different degrees was carefully traced with serial section alignment analysis and mapped on the unfolded scheme of the entire cerebellar cortex to re-identify all individual Aldoc stripes. A longitudinally striped boundary of Aldoc expression was first identified in the mouse flocculus, and was correlated with the climbing fiber projection pattern and expression of another compartmental marker molecule, heat shock protein 25 (HSP25). As in the rat, the cerebellar nuclei were divided into the rostrodorsal negative and the caudoventral positive portions by distinct projections of Aldoc-positive and negative PC axons in the mouse. Identification of the cerebellar Aldoc stripes in this study, as indicated in sample coronal and horizontal sections as well as in sample surface photos of whole-mount preparations, can be referred to in future experiments.
|Transgenic overexpression of PKCε in the mouse prostate induces preneoplastic lesions. |
Benavides, F; Blando, J; Perez, CJ; Garg, R; Conti, CJ; DiGiovanni, J; Kazanietz, MG
Cell cycle (Georgetown, Tex.) 10 268-77 2011
It is well established that protein kinase C (PKC) isozymes play distinctive roles in mitogenic and survival signaling as well as in cancer progression. PKCε, the product of the PRKCE gene, is up-regulated in various types of cancers including prostate, lung and breast cancer. To address a potential role for PKCs in prostate cancer progression we generated three mouse transgenic lines expressing PKCα, PKCδ, or PKCε in the prostate epithelium under the control of the rat probasin (PB) promoter. Whereas PB-PKCε and PB-PKCδ mice did not show any evident phenotype, PB-PKCε mice developed prostate hyperplasia as well as prostate intraepithelial neoplasia (PIN) that displayed enhanced phospho-Akt, phospho-S6, and phospho-Stat3 levels, as well as enhanced resistance to apoptotic stimuli. PKCε overexpression was insufficient to drive neoplastic changes in the mouse prostate. Notably, overexpression of PKCε by adenoviral means in normal immortalized RWPE-1 prostate cells confers a growth advantage and hyperactivation of Erk and Akt. Our results argue for a causal link between PKCε overexpression and prostate cancer development.Full Text Article
|The Spalt family transcription factor Sall3 regulates the development of cone photoreceptors and retinal horizontal interneurons. |
de Melo, J; Peng, GH; Chen, S; Blackshaw, S
Development (Cambridge, England) 138 2325-36 2011
The mammalian retina is a tractable model system for analyzing transcriptional networks that guide neural development. Spalt family zinc-finger transcription factors play a crucial role in photoreceptor specification in Drosophila, but their role in mammalian retinal development has not been investigated. In this study, we show that that the spalt homolog Sall3 is prominently expressed in developing cone photoreceptors and horizontal interneurons of the mouse retina and in a subset of cone bipolar cells. We find that Sall3 is both necessary and sufficient to activate the expression of multiple cone-specific genes, and that Sall3 protein is selectively bound to the promoter regions of these genes. Notably, Sall3 shows more prominent expression in short wavelength-sensitive cones than in medium wavelength-sensitive cones, and that Sall3 selectively activates expression of the short but not the medium wavelength-sensitive cone opsin gene. We further observe that Sall3 regulates the differentiation of horizontal interneurons, which form direct synaptic contacts with cone photoreceptors. Loss of function of Sall3 eliminates expression of the horizontal cell-specific transcription factor Lhx1, resulting in a radial displacement of horizontal cells that partially phenocopies the loss of function of Lhx1. These findings not only demonstrate that Spalt family transcription factors play a conserved role in regulating photoreceptor development in insects and mammals, but also identify Sall3 as a factor that regulates terminal differentiation of both cone photoreceptors and their postsynaptic partners.Full Text Article
|Different changes in protein and phosphoprotein levels result from serum starvation of high-grade glioma and adenocarcinoma cell lines. |
Levin, VA; Panchabhai, SC; Shen, L; Kornblau, SM; Qiu, Y; Baggerly, KA
Journal of proteome research 9 179-91 2010
Tumor cells undergoing serum starvation in vitro partially mimic metabolically stressed cells trying to adjust to a changed environment in vivo by inducing signal transduction and gene expression so that the tumor continues to grow. Our hypothesis is that the changes in protein and phosphoprotein levels after serum starvation may reflect the adapted phenotype of the tumor, which could be targeted for therapy. We used reverse-phase protein microarrays to interrogate five high-grade glioma cell lines and seven adenocarcinoma cell lines for differences in the level of 81 proteins and 25 phosphoproteins. All cell lines were studied in the well-fed condition of growth with 10% FBS and the starved condition of 0.5% FBS. Protein expression levels were normalized to beta-actin and trichotomized as increased (+1, upper 75th quartile), decreased (-1, lowest 25th quartile), or unchanged (0, others) to focus on the patterns of the biggest (and hopefully most robust) changes in protein and phosphoprotein levels. We examined these trichotomized values to better understand Starved-Fed differences among the cell lines and thereby gain better/clearer insight into the effects of serum starvation on potential cellular responses. In general, the expression of proteins and phosphoproteins 24 h after FBS starvation increased more often in glioma lines than in adenocarcinoma lines, which appeared to have fewer increased protein scores and more decreased scores. Many of the proteins increased in gliomas were downstream targets of the PTEN-PI-3 kinase-AKT, EGFR-MAPK-Stat, and transcription activator-polyamine signaling pathways. In adenocarcinomas, the expression of proteins and phosphoproteins generally increased in apoptosis pathways, while there were minor fluctuations in the other pathways above. Contrawise, gliomas become resistant to apoptosis after 24 h of serum starvation and upregulate transcription activators and polyamines more so than adenocarciomas.
|The long noncoding RNA RNCR2 directs mouse retinal cell specification. |
Rapicavoli, NA; Poth, EM; Blackshaw, S
BMC developmental biology 10 49 2010
Recent work has identified that many long mRNA-like noncoding RNAs (lncRNAs) are expressed in the developing nervous system. Despite their abundance, the function of these ncRNAs has remained largely unexplored. We have investigated the highly abundant lncRNA RNCR2 in regulation of mouse retinal cell differentiation.We find that the RNCR2 is selectively expressed in a subset of both mitotic progenitors and postmitotic retinal precursor cells. ShRNA-mediated knockdown of RNCR2 results in an increase of both amacrine cells and Müller glia, indicating a role for this lncRNA in regulating retinal cell fate specification. We further report that RNCR2 RNA, which is normally nuclear-retained, can be exported from the nucleus when fused to an IRES-GFP sequence. Overexpression of RNCR2-IRES-GFP phenocopies the effects of shRNA-mediated knockdown of RNCR2, implying that forced mislocalization of RNCR2 induces a dominant-negative phenotype. Finally, we use the IRES-GFP fusion approach to identify specific domains of RNCR2 that are required for repressing both amacrine and Müller glial differentiation.These data demonstrate that the lncRNA RNCR2 plays a critical role in regulating mammalian retinal cell fate specification. Furthermore, we present a novel approach for generating dominant-negative constructs of lncRNAs, which may be generally useful in the functional analysis of this class of molecules.
|Differential distribution of exchange proteins directly activated by cyclic AMP within the adult rat retina. |
C M Whitaker,N G F Cooper
Neuroscience 165 2010
The recently discovered exchange protein directly activated by cAMP (Epac), a guanine exchange factor for the G-protein RAP-1, is directly activated by cAMP independently of protein kinase A (PKA). While cAMP is known to be an important second messenger in the retina, the presence of Epac has not been investigated in this tissue. The goal of the present study was to determine if the Epac1 and Epac2 genes are present and to characterize their location within the retina. Western blot analysis revealed that Epac1 and Epac2 proteins are expressed within the retina, and the presence of mRNA was demonstrated with the aid of reverse transcriptase polymerase chain reaction (RT-PCR). Additionally, we used immunofluorescence and confocal microscopy to demonstrate that Epac1 and Epac2 have overlapping as well as unique distributions within the retina. Both are present within horizontal cells, rod and cone bipolar cells, cholinergic amacrine cells, retrograde labeled retinal ganglion cells, and Müller cells. Uniquely, Epac2 was expressed by cone photoreceptor inner and outer segments, cell bodies, and synaptic terminals. In contrast, Epac1 was expressed in vesicular glutamate transporter 1 (VGlut1) and C-terminal binding protein 2 (CtBP2) positive photoreceptor synaptic terminals. Together, these results provide evidence that Epac1 and Epac2 are differentially expressed within the retina and provide the framework for further functional studies of cAMP pathways within the retina.Full Text Article
|A Technical Assessment of the Utility of Reverse Phase Protein Arrays for the Study of the Functional Proteome in Non-microdissected Human Breast Cancers. |
Hennessy, BT; Lu, Y; Gonzalez-Angulo, AM; Carey, MS; Myhre, S; Ju, Z; Davies, MA; Liu, W; Coombes, K; Meric-Bernstam, F; Bedrosian, I; McGahren, M; Agarwal, R; Zhang, F; Overgaard, J; Alsner, J; Neve, RM; Kuo, WL; Gray, JW; Borresen-Dale, AL; Mills, GB
Clinical proteomics 6 129-51 2010
The lack of large panels of validated antibodies, tissue handling variability, and intratumoral heterogeneity potentially hamper comprehensive study of the functional proteome in non-microdissected solid tumors. The purpose of this study was to address these concerns and to demonstrate clinical utility for the functional analysis of proteins in non-microdissected breast tumors using reverse phase protein arrays (RPPA).Herein, 82 antibodies that recognize kinase and steroid signaling proteins and effectors were validated for RPPA. Intraslide and interslide coefficients of variability were less than 15%. Multiple sites in non-microdissected breast tumors were analyzed using RPPA after intervals of up to 24 h on the benchtop at room temperature following surgical resection.Twenty-one of 82 total and phosphoproteins demonstrated time-dependent instability at room temperature with most variability occurring at later time points between 6 and 24 h. However, the 82-protein functional proteomic "fingerprint" was robust in most tumors even when maintained at room temperature for 24 h before freezing. In repeat samples from each tumor, intratumoral protein levels were markedly less variable than intertumoral levels. Indeed, an independent analysis of prognostic biomarkers in tissue from multiple tumor sites accurately and reproducibly predicted patient outcomes. Significant correlations were observed between RPPA and immunohistochemistry. However, RPPA demonstrated a superior dynamic range. Classification of 128 breast cancers using RPPA identified six subgroups with markedly different patient outcomes that demonstrated a significant correlation with breast cancer subtypes identified by transcriptional profiling.Thus, the robustness of RPPA and stability of the functional proteomic "fingerprint" facilitate the study of the functional proteome in non-microdissected breast tumors.Full Text Article
|Role for protein kinase C-alpha in keratinocyte growth arrest. |
Jerome-Morais, A; Rahn, HR; Tibudan, SS; Denning, MF
The Journal of investigative dermatology 129 2365-75 2009
Multiple protein kinase C (PKC) isoforms have been associated with the epidermal keratinocyte (KC) granular layer differentiation program. Here we show PKCalpha membrane localization and substrate phosphorylation in the first suprabasal KCs of normal human epidermis, suggesting activation in vivo in the lower spinous layers where terminal differentiation-associated growth arrest occurs. To determine if PKCalpha is sufficient for KC growth arrest, we expressed a constitutively active PKCalpha (PKCalpha Delta22-28) in normal human KCs and observed growth arrest and accumulation of cells in G1. PKCalpha Delta22-28 inhibited DNA synthesis through the induction of the cyclin-dependent kinase inhibitor p21. Furthermore, downregulation of PKCalpha in an in vitro organotypic epidermis resulted in increased basal and suprabasal proliferation marker expression, decreased differentiation, and reduced epidermal stratification. Together these results indicate that PKCalpha activation is both necessary and sufficient to trigger irreversible growth arrest during human KC differentiation.
|Developmental sources of conservation and variation in the evolution of the primate eye. |
Michael A Dyer,Rodrigo Martins,Manoel da Silva Filho,José Augusto P C Muniz,Luiz Carlos L Silveira,Constance L Cepko,Barbara L Finlay
Proceedings of the National Academy of Sciences of the United States of America 106 2009
Conserved developmental programs, such as the order of neurogenesis in the mammalian eye, suggest the presence of useful features for evolutionary stability and variability. The owl monkey, Aotus azarae, has developed a fully nocturnal retina in recent evolution. Description and quantification of cell cycle kinetics show that embryonic cytogenesis is extended in Aotus compared with the diurnal New World monkey Cebus apella. Combined with the conserved mammalian pattern of retinal cell specification, this single change in retinal progenitor cell proliferation can produce the multiple alterations of the nocturnal retina, including coordinated reduction in cone and ganglion cell numbers, increase in rod and rod bipolar numbers, and potentially loss of the fovea.Full Text Article
|Phorbol ester-stimulated NF-kappaB-dependent transcription: roles for isoforms of novel protein kinase C. |
Neil S Holden, Paul E Squires, Manminder Kaur, Rosemary Bland, Carol E Jones, Robert Newton
Cellular signalling 20 1338-48 2008
Since protein kinase C (PKC) isoforms are variously implicated in the activation of NF-kappaB, we have investigated the role of PKC in the activation of NF-kappaB-dependent transcription by the diacyl glycerol (DAG) mimetic, phorbol 12-myristate 13-acetate (PMA), and by tumour necrosis factor (TNF) alpha in pulmonary A549 cells. The PKC selective inhibitors, Ro31-8220, Gö6976, GF109203X and Gö6983, revealed no effect on TNFalpha-induced NF-kappaB DNA binding and a similar lack of effect on serine 32/36 phosphorylated IkappaBalpha and the loss of total IkappaBalpha indicates that activation of the core IKK-IkappaBalpha-NF-kappaB cascade by TNFalpha does not involve PKC. In contrast, differential sensitivity of an NF-kappaB-dependent reporter to Ro31-8220, Gö6976, GF109203X and Gö6983 (EC(50)s 0.46 microM, 0.34 microM, >10 microM and >10 microM respectively) suggests a role for protein kinase D in transcriptional activation by TNFalpha. Compared with TNFalpha, PMA weakly induces NF-kappaB DNA binding and this effect was not associated with serine 32/36 phosphorylation of IkappaBalpha. However, PMA-stimulated NF-kappaB DNA binding was inhibited by Ro31-8220 (10 microM), GF109203X (10 microM) and Gö6983 (10 microM), but not by Gö6976 (10 microM), suggesting a role for novel PKC isoforms. Furthermore, a lack of positive effect of calcium mobilising agents on both NF-kappaB DNA binding and on transcriptional activation argues against major roles for classical PKCs. This, combined with the ability of both GF109203X and Gö6983 to prevent enhancement of TNFalpha-induced NF-kappaB-dependent transcription by PMA, further indicates a role for novel PKCs in NF-kappaB transactivation. Finally, siRNA-mediated knockdown of PKCdelta and epsilon expression did not affect TNFalpha-induced NF-kappaB-dependent transcription. However, knockdown of PKCdelta expression significantly inhibited PMA-stimulated luciferase activity, whereas knockdown of PKCepsilon was without effect. Furthermore, combined knockdown of PKCdelta and epsilon revealed an increased inhibitory effect on PMA-stimulated NF-kappaB-dependent transcription suggesting that PMA-induced NF-kappaB-dependent transcription is driven by novel PKC isoforms, particularly PKCdelta and epsilon.
|The last 10 amino acid residues beyond the hydrophobic motif are critical for the catalytic competence and function of protein kinase Calpha. |
Yeong, SS; Zhu, Y; Smith, D; Verma, C; Lim, WG; Tan, BJ; Li, QT; Cheung, NS; Cai, M; Zhu, YZ; Zhou, SF; Tan, SL; Duan, W
The Journal of biological chemistry 281 30768-81 2006
The segment C-terminal to the hydrophobic motif at the V5 domain of protein kinase C (PKC) is the least conserved both in length and in amino acid identity among all PKC isozymes. By generating serial truncation mutants followed by biochemical and functional analyses, we show here that the very C terminus of PKCalpha is critical in conferring the full catalytic competence to the kinase and for transducing signals in cells. Deletion of one C-terminal amino acid residue caused the loss of approximately 60% of the catalytic activity of the mutant PKCalpha, whereas deletion of 10 C-terminal amino acid residues abrogated the catalytic activity of PKCalpha in immune complex kinase assays. The PKCalpha C-terminal truncation mutants were found to lose their ability to activate mitogen-activated protein kinase, to rescue apoptosis induced by the inhibition of endogenous PKC in COS cells, and to augment melatonin-stimulated neurite outgrowth. Furthermore, molecular dynamics simulations revealed that the deletion of 1 or 10 C-terminal residues results in the deformation of the V5 domain and the ATP-binding pocket, respectively. Finally, PKCalpha immunoprecipitated using an antibody against its C terminus had only marginal catalytic activity compared with that of the PKCalpha immunoprecipitated by an antibody against its N terminus. Therefore, the very C-terminal tail of PKCalpha is a novel determinant of the catalytic activity of PKC and a promising target for selective modulation of PKCalpha function. Molecules that bind preferentially to the very C terminus of distinct PKC isozymes and suppress their catalytic activity may constitute a new class of selective inhibitors of PKC.
|CXCR2 ligands and G-CSF mediate PKCalpha-induced intraepidermal inflammation. |
Cataisson, C; Pearson, AJ; Tsien, MZ; Mascia, F; Gao, JL; Pastore, S; Yuspa, SH
The Journal of clinical investigation 116 2757-66 2006
Transgenic mice overexpressing PKCalpha in the epidermis (K5-PKCalpha mice) exhibit an inducible severe intraepidermal neutrophilic inflammation and systemic neutrophilia when PKCalpha is activated by topical 12-O-tetradecanoylphorbol-13-acetate (TPA). This inducible model of cutaneous inflammation was used to define mediators of skin inflammation that may have clinical relevance. Activation of cutaneous PKCalpha increased the production of the chemotactic factors cytokine-induced neutrophil chemoattractant (KC) and macrophage inflammatory protein 2 (MIP-2) in murine plasma. TPA treatment of cultured K5-PKCalpha keratinocytes also released KC and MIP-2 into culture supernatants through an NF-kappaB-dependent pathway. MIP-2 and KC mediated the infiltration of neutrophils into the epidermis, since this was prevented by ablating CXCR2 in K5-PKCalpha mice or administering neutralizing antibodies against KC or MIP-2. The neutrophilia resulted from PKCalpha-mediated upregulation of cutaneous G-CSF released into the plasma independent of CXCR2. These responses could be inhibited by topical treatment with a PKCalpha-selective inhibitor. Inhibiting PKCalpha also reduced the basal and TNF-alpha- or TPA-induced expression of CXCL8 in cultured psoriatic keratinocytes, suggesting that PKCalpha activity may contribute to psoriatic inflammation. Thus, skin can be the source of circulating factors that have both local and systemic consequences, and these factors, their receptors, and possibly PKCalpha could be therapeutic targets for inhibition of cutaneous inflammation.Full Text Article
|Involvement of the ERK signaling cascade in protein kinase C-mediated cell cycle arrest in intestinal epithelial cells. |
Clark, Jennifer A, et al.
J. Biol. Chem., 279: 9233-47 (2004) 2004
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.
|Protein Kinase C Promotes Apoptosis in LNCaP Prostate Cancer Cells through Activation of p38 MAPK and Inhibition of the Akt Survival Pathway |
Tanaka, Y., et al
J Biol Chem, 278:33753-62 (2003) 2003
|Protein kinase calpha targeting is regulated by temporal and spatial changes in intracellular free calcium concentration [Ca(2+)](i) |
Maasch, C., et al
Faseb J, 14:1653-63 (2000) 2000
|Immunocytochemistry, Immunoblotting (Western)||10929000|
|Protein kinase C signaling mediates a program of cell cycle withdrawal in the intestinal epithelium |
Frey, M. R., et al
J Cell Biol, 151:763-78 (2000) 2000
|Up-regulation of the Pit-2 phosphate transporter/retrovirus receptor by protein kinase C epsilon |
Jobbagy, Z., et al
J Biol Chem, 274:7067-71 (1999) 1999
|Evidence for multiple protein kinase C isoforms in the leukocytes of a marine teleost, Sciaenops ocellatus |
Mericko, P. A. and Burnett, K. G.
Comp Biochem Physiol B Biochem Mol Biol, 120:217-22 (1998) 1998
|Immunoblotting (Western), Immunoprecipitation||9787791|
|Protein kinase Cdelta is activated by caspase-dependent proteolysis during ultraviolet radiation-induced apoptosis of human keratinocytes. |
M F Denning, Y Wang, B J Nickoloff, T Wrone-Smith, M F Denning, Y Wang, B J Nickoloff, T Wrone-Smith
The Journal of biological chemistry 273 29995-30002 1998
The elimination of ultraviolet (UV) radiation-damaged keratinocytes via apoptosis is an important mechanism for the protection of the skin from sunlight, an ubiquitous environmental carcinogen. Due to the pleiotropic nature of UV radiation, the molecular mechanisms of UV-induced apoptosis are poorly understood. Protein kinase C (PKC) is a family of enzymes critically involved in the regulation of differentiation in the epidermis, and is associated with the induction of apoptosis by ionizing radiation in other cell types. In normal human keratinocytes, the induction of apoptosis by UV exposure correlated with generation of the catalytic domain of PKCdelta in the soluble fraction. In contrast, phorbol ester 12-O-tetradecanoylphorbol-13-acetate caused translocation of PKCdelta from the soluble to the particulate fraction without inducing apoptosis. The effect of UV radiation on PKCdelta was isoform specific, as UV exposure did not stimulate the cleavage, or effect the subcellular distribution of any other PKC isoform. The soluble, catalytic domain of PKCdelta induced by UV exposure was associated with an increase in soluble PKCdelta activity. Proteases of the caspase family are activated during UV-induced apoptosis. Inhibition of caspases blocked the UV-induced cleavage of PKCdelta and apoptosis. In addition, inhibition of PKC activity specifically inhibited UV-induced apoptosis of keratinocytes, without affecting the G0/G1 cell cycle block induced by UV exposure. These results indicate that PKC activation is involved in the UV-induced death effector pathway of keratinocytes undergoing apoptosis, and defines a novel role for this enzyme in epidermal homeostasis.
|The catalytic domain of protein kinase C-delta in reciprocal delta and epsilon chimeras mediates phorbol ester-induced macrophage differentiation of mouse promyelocytes |
Wang, Q. J., et al
J Biol Chem, 272:76-82 (1997) 1997
|Hypoxia alters the subcellular distribution of protein kinase C isoforms in neonatal rat ventricular myocytes |
Goldberg, M., et al
J Clin Invest, 99:55-61 (1997) 1997
|Protein kinase C isozyme-mediated cell cycle arrest involves induction of p21(waf1/cip1) and p27(kip1) and hypophosphorylation of the retinoblastoma protein in intestinal epithelial cells |
Frey, M. R., et al
J Biol Chem, 272:9424-35 (1997) 1997
|Protein kinase C-alpha activity modulates transepithelial permeability and cell junctions in the LLC-PK1 epithelial cell line |
Rosson, D., et al
J Biol Chem, 272:14950-3 (1997) 1997
|T cell proliferation in response to interleukins 2 and 7 requires p38MAP kinase activation. |
Crawley, J B, et al.
J. Biol. Chem., 272: 15023-7 (1997) 1997
Interleukin-2 (IL-2) is a potent T cell mitogen. However, the signaling pathways by which IL-2 mediates its mitogenic effect are not fully understood. One of the members of the mitogen-activated protein kinase (MAPK) family, p42/44MAPK (ERK2/1), is known to be activated by IL-2. We have now investigated the response to IL-2 of two other members of the MAP kinase family, p54MAP kinase (stress-activated protein kinase (SAPK)/Jun-N-terminal kinase (JNK)) and p38MAP kinase (p38/Mpk2/CSBP/RK), which respond primarily to stressful and inflammatory stimuli (e.g. tumor necrosis factor-alpha, IL-1, and lipopolysaccharide). Here we show that IL-2, and another T cell growth factor, IL-7, activate both SAPK/JNK and p38MAP kinase. Furthermore, inhibition of p38MAP kinase activity with a specific pyrinidyl imidazole inhibitor SB203580 that prevents activation of its downstream effector, MAPK-activating protein kinase-2, correlated with suppression of IL-2- and IL-7-driven T cell proliferation. These data indicate that in T cells p38MAP kinase has a role in transducing the mitogenic signal.
|Phosphatidic acid-mediated phosphorylation of the NADPH oxidase component p47-phox. Evidence that phosphatidic acid may activate a novel protein kinase |
Waite, K. A., et al
J Biol Chem, 272:15569-78 (1997) 1997
|Beta2-chimaerin is a high affinity receptor for the phorbol ester tumor promoters |
Caloca, M. J., et al
J Biol Chem, 272:26488-96 (1997) 1997
|Both the catalytic and regulatory domains of protein kinase C chimeras modulate the proliferative properties of NIH 3T3 cells |
Acs, P., et al
J Biol Chem, 272:28793-9 (1997) 1997
|Identification and regulation of protein kinase C-delta in human neutrophils |
Kent, J. D., et al
J Immunol, 157:4641-7 (1996) 1996
|Partial reversal of multidrug resistance in human breast cancer cells by an N-myristoylated protein kinase C-alpha pseudosubstrate peptide |
Gupta, K. P., et al
J Biol Chem, 271:2102-11 (1996) 1996
|Distribution of protein kinase C alpha and accumulation of extracellular Ca2+ during early dentin and enamel formation. |
Bawden, J W, et al.
J. Dent. Res., 73: 1429-36 (1994) 1994
|Protein kinase C is localized in focal contacts of normal but not transformed fibroblasts. |
Hyatt, S L, et al.
Mol. Carcinog., 3: 45-53 (1990) 1990
|Association of type 3 protein kinase C with focal contacts in rat embryo fibroblasts. |
Jaken, S, et al.
J. Cell Biol., 109: 697-704 (1989) 1989
We have used immunocytofluorescence techniques to determine the subcellular distribution of the Ca2+, phospholipid-dependent protein kinase, protein kinase C (PKC). Using monoclonal antibodies that are specific for Type 3 (alpha) PKC, we have determined that there are least two pools of PKC in normal rat embryo fibroblasts (REF52 cells): diffuse cytoplasmic and fiber-associated. Extraction with chelators and detergent before fixing and staining removes the cytoplasmic PKC. The fiber-associated staining remains in these cytoskeleton preparations. The cytoskeleton Type 3 PKC staining closely resembles that of the focal contact protein vinculin and colocalizes with another focal contact protein, talin. Cytochalasin, but not colchicine, coordinately disrupts the staining pattern of vinculin and PKC. Activation of PKC by treatment with phorbol esters causes depolymerization of microfilaments and reorganization of vinculin staining. We propose that Type 3 PKC is a modulatory component of the focal contact and has a primary role in regulation of the association of microfilament bundles with the plasma membrane.
|Monoclonal antibodies specific for type 3 protein kinase C recognize distinct domains of protein kinase C and inhibit in vitro functional activity. |
Leach, K L, et al.
J. Biol. Chem., 263: 13223-30 (1988) 1988
Monoclonal antibodies (mAbs) which distinguish Type 3 protein kinase C (PKC) from Types 1 and 2 have been obtained from mice immunized with purified Type 3 PKC from rabbit brain cytosol. Most of these mAbs (seven out of eight) selectively recognize Type 3 versus Types 1 and 2 PKC in both enzyme-linked immunosorbent and immunoblot assays. Trypsin treatment of Type 3 PKC reduced the immunoreactivity with 82-kDa PKC and generated immunoreactive fragments of 45 and 35 kDa. The mAbs can be divided into two classes based on their ability to recognize the 45-kDa catalytic fragment (5/8) or the 35 kDa regulatory domain fragment (3/8). Each of the mAbs inhibits phosphorylation of histone or lipocortin by PKC, although the extent of the inhibition varied. Only those mAbs that recognize the 35-kDa regulatory domain inhibited phorbol ester binding. The inhibition of both kinase and binding activities by this group of mAbs was sensitive to the concentration of phospholipid used in the assay. This functional inhibition suggests that these mAbs may be useful for defining the phospholipid binding domain(s) of Type 3 PKC. The mAbs recognized 82-kDa PKC in a variety of cell types; the presence of smaller molecular weight fragments was not consistently found. Distinct immunofluorescence staining patterns were observed with mAbs directed toward different epitopes, suggesting that there may be heterogeneity in the subcellular localization of PKC. The type specificity of these mAbs will make them valuable tools for studying activation and regulation of Type 3 PKC in cell culture model systems.