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Concise Reviews |
, a Member of the Novel Subfamily of Protein Kinase C
a Department of Microbiology and Immunology, Faculty of Health Sciences, and the Cancer Research Center, Ben Gurion University of the Negev, Beer Sheva, Israel;
b Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, California, USA
Key Words. PKC • T cell activation • Cell signaling • Isoenzymes • Protein kinase
Dr. Noah Isakov, Department of Microbiology and Immunology, Faculty of Health Sciences, Ben Gurion University of the Negev, P.O. Box 653, Beer Sheva 84105, Israel.
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Abstract |
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 Top Abstract IntroductionTissue Distribution of...PKC{theta} Expression During...Selective Modulation of...Regulation of IL- 2...Potential Regulation of...Interaction of PKC{theta} with...Function of PKC{theta} in...PKC{theta} and Insulin...Potential Regulation of...Concluding Remarks and Future...References |
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isoenzyme exhibits a relatively restricted expression pattern with high protein levels found predominantly in hematopoietic cells and skeletal muscle. PKC was found to be expressed in T, but not B lymphocytes, and to colocalize with the T-cell antigen receptor (TCR) at the site of contact between the antigen-responding T cell and the antigen-presenting cell (APC). Colocalization of PKC with the TCR was selective for this isoenzyme and occurred only upon antigen-mediated responses leading to T-cell activation and proliferation. PKC was found to be involved in the regulation of transcriptional activation of early-activation genes, predominantly AP-1, and its cellular distribution and activation were found to be regulated by the 14-3-3 protein. Other findings indicated that PKC can associate with the HIV negative factor (Nef) protein, suggesting that altered regulation of PKC by Nef may contribute to the T-cell impairments that are characteristic of infection by HIV. PKC is expressed at relatively high levels in skeletal muscle, where it is suggested to play a role in signal transduction in both the developing and mature neuromuscular junction. In addition, PKC appears to be involved in the insulin-mediated response of intact skeletal muscle, as well as in experimentally induced insulin resistance of skeletal muscle. Further studies suggest that PKC is expressed in endothelial cells and is involved in multiple processes essential for angiogenesis and wound healing, including the regulation of cell cycle progression, formation and maintenance of actin cytoskeleton, and formation of capillary tubes. Here, we review recent progress in the study of PKC and discuss its potential role in various cellular responses.
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Introduction |
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TopAbstractIntroductionTissue Distribution of...PKC{theta} Expression During...Selective Modulation of...Regulation of IL- 2...Potential Regulation of...Interaction of PKC{theta} with...Function of PKC{theta} in...PKC{theta} and Insulin...Potential Regulation of...Concluding Remarks and Future...References |
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, that is expressed predominantly in hematopoietic cells and muscle [1]. Parallel studies by other investigators have led to the cloning of the same gene in the mouse and human [2, 3]. Chromosomal mapping indicated that the human PKC gene is found in the short arm of chromosome 10 (10p15), a region prone to mutations leading to T-cell leukemias or lymphomas and T-cell immunodeficiencies [4].
At present, the PKC family of serine/threonine-specific protein kinases includes eleven known members that are expressed in a variety of tissues and cell types. Based on similarity in primary amino acid sequence and domain structure, the distinct PKC isoenzymes are grouped into three subfamilies. Members of the Ca2+-dependent subfamily are termed conventional, or cPKC, and include PKC
, the two alternatively spliced forms of the ß gene, PKCß1 and ß2, and PKC
[5-16]. These isoenzymes contain three conserved domains, namely, the diacylglycerol-binding C1 domain, which contains two repeats of a cysteine-rich zinc finger-like domain, the phospholipid- and Ca2+-binding C2 domain, and the catalytic C3/C4 domains. Members of the second subfamily, termed novel, or nPKC, are Ca2+-independent for their activity and include PKC
, 
, 
, and µ [1-3, 17-25]. The C2-like N-terminal domain of these isoenzymes can bind acidic phospholipids but not Ca2+ ions. A third PKC subfamily termed atypical, or aPKC, includes only two members, PKC
and 
/
, that possess a single cysteine-rich domain which cannot bind phospholipids or phorbol esters [17, 18, 26-29].
PKC isoenzymes are involved in multiple biochemical processes relevant to cell growth, differentiation, and transformation, and play critical roles in transducing signals from a plethora of extracellular receptors, including those for hormones, neurotransmitters, growth factors, and antigens [30-33].
Regulation of PKC activity is mediated by defined cofactors that interact with specific regions in its regulatory domain [32] as well as transphosphorylation by serine/threonine kinases [34, 35] and autophosphorylation [33, 36]. The activation is accompanied by removal of the basic pseudosubstrate region from the kinase domain [37, 38] and may involve the association with specific proteins, termed receptors for activated PKC (RACK) [39, 40], that were suggested to function as selective scaffolds for activated PKC at discrete subcellular compartments.
Distinct PKC isoenzymes exhibit differences in tissue distribution, intracellular localization, and cofactor requirements, suggesting that they are independently regulated through response to discrete ligands, and that they may possess distinct protein substrates [30, 31]. It is possible that the specific combination of isoenzymes expressed in a given cell determines the outcome of the PKC-dependent response in that particular cell.
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Tissue Distribution of PKC
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TopAbstractIntroductionTissue Distribution of...PKC{theta} Expression During...Selective Modulation of...Regulation of IL- 2...Potential Regulation of...Interaction of PKC{theta} with...Function of PKC{theta} in...PKC{theta} and Insulin...Potential Regulation of...Concluding Remarks and Future...References |
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distribution in mouse and rat tissues was determined by either Northern or Western blot analysis, and comparison to other isoenzymes demonstrated a relatively restricted pattern of expression [1-3, 41-45]. High levels of PKC protein were detected in skeletal muscle and lymphoid tissues, predominantly in the thymus and lymph nodes, with lower expression levels in the spleen and no detectable expression in the bone marrow ( Table 1). PKC was found to be expressed in endothelial cells and some smooth muscles, but expression levels in other tissues were either very low or completely undetectable. Further dissection revealed high levels of PKC in CD4+ and CD8+ single-positive mature T cells, as well as in CD4+CD8+ double-positive thymocytes, but not in B cells ( Fig. 1, Meller et al., submitted). In addition, PKC was found in platelets, but relatively low or undetectable expression levels were found in other hematopoietic cells, including mononuclear phagocytes, polymorphonuclear cells, and erythrocytes (Meller et al., submitted). These findings were further supported by analysis of PKC expression in cell lines from various histological origins ( Table 3). Data accumulated on PKC expression in different lymphoid and myeloid cell types, as well as a large number of distinct hematopoietic cell lines representing either immature or terminally differentiated cell types, demonstrated a selective expression of PKC in T, but not B lymphocytes, and in platelets but not in erythrocytes, polymorphonuclear neutrophils, monocytes, or macrophages.
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PKC Expression During Hematopoietic Cell Development
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TopAbstractIntroductionTissue Distribution of...PKC{theta} Expression During...Selective Modulation of...Regulation of IL- 2...Potential Regulation of...Interaction of PKC{theta} with...Function of PKC{theta} in...PKC{theta} and Insulin...Potential Regulation of...Concluding Remarks and Future...References |
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expression in hematopoietic precursor cells and during their in vivo differentiation is limited, in part due to the low yield and purity of the cells and their limited in vitro survival. Leukemic cell lines are often used as an alternative model, where the cells are classified according to their tissue of origin, phenotypic markers, and potential to differentiate into distinct hematopoietic lineages. A concise summary of published observations on PKC protein expression in various hematopoietic cell lines is presented in Table 1.
The human megakaryocytic leukemia cell lines Dami and MEG-01 retain many morphological and functional properties of bone-marrow megakaryocytes [46, 47], and serve therefore as an in vitro model for megakaryocytic maturation and differentiation. Both cell lines express significant levels of PKC [48, 49], indicating that the expression of PKC by platelets [3, 50, 51] is not a newly acquired, differentiation-dependent trait, but rather a property inherited from their progenitor cells.
Analysis of additional cell lines indicated PKC expression in K562, HEL, BB88, and F-MEL erythroleukemia cell lines [41, 52, 53]. K562 and HEL cells express both early erythroid and early megakaryoblastoid markers, and can be induced to differentiate in either direction, supporting the concept that erythroid and megakaryoblastic lineages are derived from a common bipotential progenitor [54]. Dimethylsulfoxide (DMSO) induces erythroid differentiation of F-MEL cells to an extent where 60%-70% of the cells express hemoglobin. Under similar conditions, the BB88 cells respond poorly to DMSO, exhibiting only a 10%-20% differentiation level. Interestingly, F-MEL differentiation is associated with downregulation of PKC, while the level of expression of PKC in the poorly differentiating BB88 cells is barely affected [53]. These observations support a model in which PKC is expressed in the bipotential progenitor cell, and throughout the stages of differentiation into megakaryocytes and platelets. In contrast, differentiation of these progenitors into erythrocytes appears to correlate with a reduction in expression levels of PKC.
PKC has not been detected, however, in the promyelocytic leukemia cell lines HL60 and NB4 [41, 55], which can differentiate in vitro into either monocytes or granulocytes [55, 56] or in the U937 and THP-1 or M1 cell lines [53], which can differentiate in vitro into monocytes or macrophages, respectively [57, 58]. Induction of differentiation of either HL60 or U937 cells into monocytes, and HL60 cells into granulocytes did not result in expression of PKC (Meller et al., submitted), in agreement with the data obtained with isolated peripheral blood monocytes and polymorphonuclear cells showing low levels or complete lack of PKC (Meller et al., submitted).
A putative schematic model for PKC expression during hematopoiesis is presented in Figure 1. While megakaryocytes and their progenitors appear to express PKC and lose it upon differentiation into the erythroid line, it is not yet clear whether the lymphoid progenitor cell expresses PKC and loses it upon differentiation into B cells, or whether it is a PKC- cell that gains expression of PKC upon differentiation into T cells.
The potential role of PKC in hematopoiesis in general, and cell commitment and differentiation into a specific lineage, await further genetic studies in PKC gene transgenic mice or targeted gene disruption models that may provide more conclusive answers.
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Selective Modulation of PKC During T-Cell Activation
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TopAbstractIntroductionTissue Distribution of...PKC{theta} Expression During...Selective Modulation of...Regulation of IL- 2...Potential Regulation of...Interaction of PKC{theta} with...Function of PKC{theta} in...PKC{theta} and Insulin...Potential Regulation of...Concluding Remarks and Future...References |
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in resting T cells demonstrated that a large fraction of the protein resides in the cytosol, and that upon activation with a phorbol ester, PKC translocates to the particulate, membrane-rich fraction, where it apparently becomes active [41]. Pharmacological agents such as phorbol esters stimulate PKC independently of cell surface receptors or their corresponding physiological agonists, and therefore bypass early signaling events that are initiated in T cells following engagement of their TCRs. The effects of such drugs do not necessarily mimic those of physiological agonists, because phorbol esters bind to and activate most of the cellular PKC isoenzymes, regardless of their relevance to signaling via specific cell-surface receptors, such as the TCR [59]. In order to study the effect of TCR-induced activation on individual PKC isoenzymes, Monks et al. have used an experimental system in which antigen-specific T-cell clones were stimulated in vitro by antigen-pulsed antigen-producing cells (APCs) [60]. Under these conditions, the responding T cells physically interact with their APCs to form conjugates, followed by coclustering of the engaged TCRs at the site of contact. As a result, spatially restricted signaling events are initiated in the responding T cells at this site. The effects of such activation on subcellular distribution of individual PKC isoenzymes were evaluated by digital immunofluorescence microscopy.
Immunofluorescent staining by PKC isoenzyme-specific antibodies (Abs) revealed the expression of six different isoenzymes, PKC, -ßI, -, -, -, and -, in different T-cell clones. Following activation with antigen-pulsed APCs, PKC, but none of the other isoenzymes, translocated to the cell contact site at the membrane. Clustering of PKC occurred only upon efficient activation of T cells, by exposure to APCs that are fed with optimal concentrations of the appropriate antigen, under conditions in which coclustering of talin and tubulin resulted in the formation and reorientation of the microtubule organizing center (MOTC). Previous studies have shown that agonist-induced formation and reorientation of MOTC directly correlated with the induction of cell proliferation [61]. TCR engagement-induced translocation of PKC was followed by a selective increase in the activity of PKC, as demonstrated by in vitro kinase assay of PKC immunoprecipitates from the conjugated T cells. These results indicated that PKC translocation to the site of cell contact is succeeded by its catalytic activation and suggested the selective involvement of PKC in early TCR-initiated signaling events that regulate T-cell activation and proliferation.
TCR engagement-induced coclustering of PKC at the site of cell contact also suggests a temporally regulated binding of PKC to one or more proteins at the vicinity of the aggregated TCRs. The identity of these protein(s) is not known; they may represent individual subunits of the engaged TCRs, any of the multiple SH2-containing effector molecules that associate with the phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) found in the cytoplasmic tails of the TCR subunits [62], or PKC-specific membrane-associated RACKs proteins [39]. In an attempt to identify cellular receptors for PKC, Monks et al. have used Abs to various membrane proteins, known to coaggregate with activated TCRs, and tested whether capping of these molecules will result in coclustering of PKC. The results demonstrated that Ab-mediated cross-linking of TCR/CD3, CD4, LFA-1, CD28, CD45, or other cell-surface molecules, failed to induce co-capping of PKC. Although PKC may transiently associate with some other undefined cell-surface molecules, a more attractive hypothesis is that PKC coclustering requires multiple simultaneous or sequential signals that are temporally and spatially coordinated only upon TCR engagement with appropriately presented peptide antigens.
Further experiments demonstrated that coclustering of PKC occurred only following "productive" stimulation that led to T-cell proliferation [60], and not following a "non-productive" triggering with "altered" peptide antigens [63-65] that, despite their ability to trigger conjugate formation and coclustering of talin, did not promote T-cell proliferation.
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Regulation of IL- 2 Production by PKC
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TopAbstractIntroductionTissue Distribution of...PKC{theta} Expression During...Selective Modulation of...Regulation of IL- 2...Potential Regulation of...Interaction of PKC{theta} with...Function of PKC{theta} in...PKC{theta} and Insulin...Potential Regulation of...Concluding Remarks and Future...References |
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B, NF-AT, and NF-IL-2A, and cooperative interaction between these factors is required for the efficient regulation of gene induction [69]. Among these factors, AP-1, which consists of a dimer of a Jun and a Fos family members [70], appears to be the most critical for IL-2 regulation, apparently due to its ability to interact with two consensus AP-1 regions in the IL-2 promoter [71] and the fact that it serves as an integral component of the NF-AT and NF-IL-2 complexes [72, 73].
PKC has been implicated in the induction of jun and fos genes and in the formation of functional AP-1 complexes in T cells [70, 74-76]. In order to study the role of PKC in the transcriptional regulation of AP-1 and its mode of action in T cells, Baier-Bitterlich et al. have utilized wild-type and mutants of the PKC gene, and the PKC gene for comparison, and tested the effect of their overexpression in T cells on AP-1 activity [77]. Stable overexpression of either PKC or PKC in Jurkat T cells promoted about a sixfold increase in IL-2 production levels following stimulation with phorbol myristate acetate (PMA) and anti-CD3 Abs. In addition, transient overexpression of wild-type PKC in murine EL4 thymoma cells [41, 77] or human Jurkat T cells [78], promoted activation of a chloramphenicol acetyltransferase (CAT) reporter gene driven by the minimal IL-2 promoter. This effect was not isoenzyme-specific, since similar results were obtained following the transient overexpression of PKC [77].
To better define the IL-2 promoter elements that are likely to be differentially regulated by the two PKC isoenzymes, experiments were repeated in EL4 cells with reporter constructs that contain a DNA-binding response element [77]. Overexpression of either PKC or PKC resulted in a similar increase in PMA-induced transcriptional activation of NF-AT. None of the PKC isoenzymes affected the PMA-induced transcriptional activation of NF-IL-2A or NF-B. However, a significant PMA-induced transcriptional activation from an AP-1-containing promoter was obtained upon cotransfection of PKC, but not PKC. Overexpression of a pseudosubstrate mutant constitutively active PKC, but not PKC, resulted in a significant increase of AP-1 CAT expression, even in the absence of PMA stimulation. In contrast, a catalytically inactive "kinase dead" PKC, but not PKC, abrogated PMA-induced AP-1 activation. Cotransfection of a dominant-negative form of Ras, Ha-RasN17, which blocks activation of endogenous Ras, also inhibited PMA-dependent AP-1 activation. In agreement, dominant-negative Ras also inhibited the PMA-independent AP-1 activation induced by constitutively active PKC. These results suggest that PKC is an essential constituent of the signaling pathway leading to AP-1 transcriptional activation and that it operates either upstream or in parallel to Ras. However, it is clear that additional enzymes are involved in the regulation of activation of AP-1, as demonstrated in T cells [77] and other cell types which normally do not express the PKC isoenzyme [79, 80].
Recent work by Werlen et al., (submitted) suggests that the specific effect of PKC on activation of AP-1 is also observed at the level of the complete IL-2 promoter. The contradiction between this study and previous findings [77] may reflect usage by Baier-Bitterlich et al. of EL4 cells that express a constitutively active calcineurin which can provide a complimentary signal to more than one type of PKC isoenzyme [77]. Alternatively, differences in results in the two studies may be due to usage of different expression vectors and/or other experimental conditions.
In an attempt to identify effector molecules that operate downstream of PKC and transduce signals leading to transcription activation of the IL-2 gene, the effect of PKC on distinct members of the mitogen-activated protein kinase (MAPK) cascades was tested. While overexpressed constitutively active PKC induced activation of extracellular receptor kinase (ERK) 1 and ERK 2 enzymes, overexpression of a constitutively active PKC induced a marked activation of the two c-Jun N-terminal kinase (JNK)/stress activated protein kinase (SAPK) isoenzymes, JNK1 and JNK2 ([81], Werlen et al., submitted). The effect on JNK was PKC isoenzyme-specific because other isoenzymes, including PKC, , and /, were ineffective in activation of JNK.
Further studies by Werlen et al. indicated that PKC cooperates with calcineurin, and together their signals converge on or upstream to Rac, leading to potent activation of JNK. Similarly, PKC cooperated with calcineurin in the induction of transcription of reporter genes that are driven by the c-jun or IL-2 promoters. The preferential cooperation between PKC and calcineurin was observed in Jurkat T cells, but not in HeLa cells, suggesting the involvement of additional T-cell-specific factors in the integrated PKC- and calcineurin-mediated signals leading to activation of JNK (Werlen et al., submitted). This model of PKC-mediated activation of JNK is not completely resolved, because similar studies by Ghaffari-Tabrizi failed to demonstrate the involvement of Rac in PKC-induced activation of JNK [81].
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Potential Regulation of PKC by 14-3-3 Proteins
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TopAbstractIntroductionTissue Distribution of...PKC{theta} Expression During...Selective Modulation of...Regulation of IL- 2...Potential Regulation of...Interaction of PKC{theta} with...Function of PKC{theta} in...PKC{theta} and Insulin...Potential Regulation of...Concluding Remarks and Future...References |
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Studies which analyzed the effects of 14-3-3 on PKC in vitro reported conflicting results, i.e., inhibition [85-88], activation [89-92], or no effect [93, 94] on PKC activity. This discrepancy may reflect the use of different assay conditions and/or 14-3-3 and PKC preparations. Therefore, we choose to analyze potential interactions between PKC and the 14-3-3
isoform using intact T cells.
In a recent study [78], we detected direct interaction between PKC and the 14-3-3 protein isoform , which is abundantly expressed in T cells, by coimmunoprecipitation of endogenous or transiently overexpressed proteins in Jurkat T cells. This association is likely direct, since recombinant soluble 14-3-3 directly interacted with purified PKC in a Far-Western overlay assay. Transient overexpression of 14-3-3 suppressed stimulation of an IL-2 promoter containing reporter construct, mediated by cotransfected wild-type or constitutively active PKC, as well as by endogenous PKC. In accordance, 14-3-3 also inhibited IL-2 secretion mediated by PKC overexpression. Thus, this interaction may have potential regulatory implications.
Determination of the cellular distribution of PKC under different experimental conditions yielded a possible explanation for the mechanism by which 14-3-3 inhibits PKC activity. Thus, overexpression of wild-type 14-3-3, which is expressed predominantly in the cytosol, inhibited phorbol ester-induced PKC translocation from the cytosol to the membrane. In contrast, a membrane-targeted form of 14-3-3 caused increased localization of PKC in the particulate fraction, even in unstimulated cells. Membrane-targeted 14-3-3 suppressed PKC-dependent IL-2 promoter activity (more effectively than wild-type 14-3-3), suggesting that 14-3-3 inhibits the function of PKC, not only by preventing its translocation to the membrane, but in addition, by associating with it.
Studies of the cellular receptors for activated PKC, the RACK proteins, suggested that the affinity of PKC enzymes for their RACKs increases upon binding of activation cofactors [39]. Thus, RACKs may be involved in determination of the substrate specificity of distinct PKC isoforms by acting as anchoring proteins that target activated PKC enzymes to particular cellular compartments in which their respective substrates are localized. The subcellular distribution of 14-3-3 in T cells and its potential role as a regulator of PKC suggest that 14-3-3 may function as a cytosolic receptor for inactive PKC in resting cells. Upon activation, the relative affinity of PKC for 14-3-3 decreases, resulting in complex dissociation, PKC translocation to the membrane, and its interaction with the appropriate RACK proteins.
Recent observations suggested a similar role for 14-3-3 proteins in other systems. For example, the Cdc25C protein phosphatase promotes cell entry into mitosis, and its phosphorylation on Ser216 during interphase triggers its association with 14-3-3, which prevents uncontrolled cell cycle progression [95]. Thus, the association of 14-3-3 with Cdc25C is likely to block undesired Cdc25 downstream effects. Another 14-3-3-regulated protein is the cell death agonist protein, BAD, that in its unphosphorylated form can heterodimerize with BCL-X(L) at the level of the membrane and promote cell death. In contrast, signals that are provided by the "survival factor" IL-3 induce serine phosphorylation of BAD and its translocation to the cytosol where it associates with 14-3-3 [96]. These and other observations suggest that 14-3-3 may function as a novel type of chaperone that can modulate interactions between distinct components of signal transduction pathways.
Additional studies are required to determine whether the PKC-14-3-3 interaction can be generalized to other PKC and 14-3-3 isoforms and to characterize the biological role of these interactions.
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Interaction of PKC with the HIV Protein Nef
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TopAbstractIntroductionTissue Distribution of...PKC{theta} Expression During...Selective Modulation of...Regulation of IL- 2...Potential Regulation of...Interaction of PKC{theta} with...Function of PKC{theta} in...PKC{theta} and Insulin...Potential Regulation of...Concluding Remarks and Future...References |
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B and/or AP-1 transcription factors [108-110] and to associate with cellular serine/threonine kinases [111-117].
In an attempt to identify the cellular serine/threonine kinases with which Nef may interact, Smith et al. have chosen to concentrate on members of the PKC family that are known to be involved in antigen-mediated activation of T cells [118]. Among the five most prominently expressed PKC isoenzymes in Jurkat T cells, only PKC was found to associate in vitro with a glutathione-S-transferase (GST)-Nef bacterial fusion protein. This association was also demonstrated by coimmunoprecipitation of Nef and PKC from Nef-expressing Jurkat cells. Nef association with PKC was augmented by phosphatidylserine and diacylglycerol but only ~5% of the total cytosolic PKC interacted with Nef. Binding was unaffected by excess of a PKC pseudosubstrate peptide, suggesting that regions other than the substrate-binding domain are involved in the association.
Indirect support for the possible modulation of PKC by its association with Nef came from analysis of PKC distribution and protein levels in Nef-expressing Jurkat cells. Thus, Nef was found to increase the proportion of PKC in the particulate fraction of the cells. This response was selective, because the cellular distribution of other PKC isoenzymes was not affected by Nef. Finally, Nef appeared also to selectively affect the stability of PKC, since short mitogenic stimulation of cells resulted in a rapid loss of immunoreactive PKC, but not PKCß, -, or -, from the particulate fraction of Nef-expressing cells, but not from control cells. The Nef-induced reduction in PKC levels in activated cells may reflect its direct binding to Nef, which could compete for binding of the activated enzyme to its appropriate RACK. This may leave PKC more susceptible to degradation by cellular proteases.
It is possible, therefore, that the Nef-induced reduction in PKC levels and/or its inappropriate binding to cellular target proteins, such as RACK, could account for at least part of the immune impairments observed in HIV-infected T cells. Furthermore, the results provide an additional support for the selective potential critical role of PKC in the regulation of T-cell activation.
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Function of PKC in Skeletal Muscle
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TopAbstractIntroductionTissue Distribution of...PKC{theta} Expression During...Selective Modulation of...Regulation of IL- 2...Potential Regulation of...Interaction of PKC{theta} with...Function of PKC{theta} in...PKC{theta} and Insulin...Potential Regulation of...Concluding Remarks and Future...References |
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expression in mouse skeletal muscle [2] have led to the analysis of its potential role in the development and function of this tissue. Support for the potential role for PKC in the development of skeletal muscle was provided by analysis of the age-dependent expression of PKC protein levels in the rat hind limb skeletal muscle [44]. This study revealed that the PKC protein levels increased during the first three weeks of life, and that the increase was exclusively in the membrane fraction. This time course coincided with the kinetics of development and maturation of neuromuscular junctions in the same tissue. In comparison, only very modest changes in expression levels of PKC in the cytosol and membrane fractions of skeletal muscle were observed during the same time period. The developmentally regulated expression of PKC was specific to the skeletal muscle tissue and did not occur in another PKC-positive tissue, such as the spleen.
Immunocytochemical staining of skeletal muscle demonstrated that a large fraction of the cellular PKC is found in the cell membrane, while the ubiquitously expressed PKC was found in the cytosol [44]. PKC associated predominantly with the sarcolemata of skeletal muscle and localized in the neuromuscular junction. Co-staining of the skeletal muscle with Abs specific for PKC and the nicotinic acetylcholine receptor (nAChR), which serves as a marker for neuromuscular junction, demonstrated that virtually all of the neuromuscular junctions that were identified exhibited enhanced expression of PKC. Staining of PKC did not precisely overlap with the nAChR label but encompassed a broader area of the membrane. In contrast, PKC staining at neuromuscular junctions was weak in comparison with other sites.
To test whether a nerve may regulate the subcellular location of PKC and its extent of expression in the muscle, a diaphragm muscle has been tested before and at various time points following denervation [44, 119]. The presynaptic nerve terminal at the neuromuscular junction tends to rapidly degenerate following denervation, while the postsynaptic motor endplate remains intact for weeks. In this study, PKC persisted in the neuromuscular junction for long time periods following denervation, indicating that it is distributed postsynaptically. Protein levels of PKC decreased by 65% within two weeks post-operation, while the level of expression of PKC increased by 80%. Furthermore, the levels of expression of PKC in cultured myoblasts was relatively low but increased following coculture with a neuroblastoma x glioma hybrid cell line (NG108-15) that is capable of forming functional synapses. In contrast, levels of expression of PKC in cultured myoblasts increased during differentiation and exceeded those of adult skeletal muscle but were unaffected by coculture with the NG108-15 hybrid cells. Addition of tetradotoxin to block spontaneous muscle contraction had little effect on basal PKC expression levels, or on expression levels of PKC in NG108-15-stimulated myotubes. The results indicate, therefore, that the expression level of PKC, but not of PKC, in skeletal muscle is regulated by trans-synaptic interaction with the nerve, either by nerve-derived soluble factors or as a result of the direct cell-to-cell contact, and is independent of the contractile activity of the muscle. The results also suggest that PKC may play a critical role in the development and formation of the neuromuscular junction, and possibly, in its physiological function.
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PKC and Insulin Resistance
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TopAbstractIntroductionTissue Distribution of...PKC{theta} Expression During...Selective Modulation of...Regulation of IL- 2...Potential Regulation of...Interaction of PKC{theta} with...Function of PKC{theta} in...PKC{theta} and Insulin...Potential Regulation of...Concluding Remarks and Future...References |
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in white fibers were 2.5-fold higher than in red fibers [42], and insulin was found to stimulate the translocation of PKC to the membrane fraction [122]. The inverse relationship between insulin sensitivity of red versus white muscle and their levels of expression of PKC suggest that PKC, but not other PKC isoenzymes, may contribute to the negative regulation of insulin action.
The effect of insulin on skeletal muscle PKC was not always selective for PKC, and in vivo administration of insulin to rats or in vitro stimulation of skeletal muscle with insulin augmented mRNA expression levels and cytosol-to-membrane translocation of PKC, ß, and , in addition to PKC [122, 125].
Induction of insulin resistance in rats by a fructose-enriched diet induced a selective diacylglycerol-mediated increase in PKC levels in the membrane fraction of the white, but not the red muscle, without affecting expression levels or activity of conventional PKCs [42]. Insulin resistance induced by high-fat diet resulted in decreased PKC expression levels in red, but not white muscle, predominantly in the cytosol fraction [126]. In agreement with the data on PKC expression levels in red versus white muscle, these experiments support the assumption that diet-induced changes in PKC contribute to the development of insulin resistance. Previous studies have shown that PKC-mediated phosphorylation of the insulin receptor reduces the catalytic activity of its integral cytoplasmic tyrosine kinase [127, 128]. This study and the above-mentioned observations by Donnelly et al. [42], may suggest a potential role for PKC in phosphorylation and downregulation of the insulin receptor. Alternatively, PKC may increase resistance to insulin by phosphorylating other substrates, such as insulin receptor downstream effector molecules (e.g., IRS-1 and Shc [129]), or components of the glucose transport or metabolism (e.g., glycogen synthase [130, 131]).
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Potential Regulation of Endothelial Cell Cycle Progression and Cytoskeleton Reorganization by PKC
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TopAbstractIntroductionTissue Distribution of...PKC{theta} Expression During...Selective Modulation of...Regulation of IL- 2...Potential Regulation of...Interaction of PKC{theta} with...Function of PKC{theta} in...PKC{theta} and Insulin...Potential Regulation of...Concluding Remarks and Future...References |
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Cultured quiescent RCE cells responded to serum stimulation by increased expression of PKC, both at the protein and mRNA levels. In contrast, expression levels of PKC, , , and , that were also detectable in RCE cells, were unaltered. Localization of PKC in quiescent nonconfluent RCE cells predominated in the cytoplasm and nucleus, but following serum stimulation or contact triggering (in confluent cultures), PKC translocated to the outer cell membrane.
To further analyze the potential role of PKC in serum-stimulated endothelial cells, stable RCE transfectants were prepared that express either constitutively active PKC or kinase-negative PKC under the control of a glucocorticoid-inducible promoter. Serum stimulation of kinase-negative PKC expressing RCE cells resulted in a significant inhibition of cell replication and closure of endothelial wound, suggesting the involvement of PKC in the regulation of both mitogenesis and motility of endothelial cells. Furthermore, kinase-negative PKC but not constitutively active PKC, inhibited the formation of capillary tube and ring-like structures in a gel containing either collagen or a basement membrane matrix, representing a three-dimensional in vitro model for angiogenesis.
Cell cycle analysis of RCE cells revealed that the kinase-negative PKC delays the cell cycle at the G2/M phase but does not affect the progression through G1 and entry of the cells into the S phase. In addition, the kinase-negative PKC induced specific morphological changes in subconfluent cultures of RCE cells that tended to grow in clusters or colonies and exhibited an overall abnormal round shape. This is in contrast to the wild-type cells and the constitutively-active PKC expressing RCE cells that were spread and had a polar migratory shape, typical of capillary endothelial cells.
Proliferation, migration, and ring or tubule formation by endothelial cells require tightly regulated changes in actin cytoskeleton. Upon cell contact, proliferating endothelial cells become quiescent and develop a static actin cytoskeleton with a prominent peripheral actin band. When contact inhibition is released, endothelial cells lose their peripheral actin band, reorganize the actin filaments, and re-enter the cell cycle. Wild-type and constitutively active PKC-expressing RCE cells were found to develop prominent stress fibers, filopodia, and lamellipodia that are characteristics of migrating cells, as demonstrated by immunohistochemical examination. In contrast, the kinase-negative PKC-expressing RCE cells demonstrated a very prominent dense peripheral band of actin but had relatively few stress fibers or filopodia. No impairment of adhesion of the endothelial cells to vitronectin was noted, suggesting no involvement of PKC in signaling events that regulate integrin binding to the extracellular solid matrix.
A support for the potential role for PKC in cytoskeletal rearrangement has recently been obtained by studies on moesin, a member of the ezrin-radixin-moesin (ERM) family of proteins that link cytoskeletal elements to the cell's membrane [135]. Pietromonaco et al., found that PKC specifically phosphorylates moesin on Thr558 [136] within the conserved carboxy-terminal actin-binding domain. This site has also been shown to undergo phosphorylation in vivo upon thrombin stimulation of platelets [137], and the flanking sequences on both sides of Thr558 were found to have a high degree of homology to that of the PKC pseudosubstrate region. Pietromonaco et al. suggested that one function of PKC may be to regulate the interaction of ERM proteins with the actin cytoskeleton. Because moesin was found to undergo phosphorylation in RAW macrophages [136] (in contrast to other normal macrophages or macrophage cell lines; Table 1 and Fig. 1), it is possible that either RAW cells express PKC or that an enzyme besides PKC can phosphorylate moesin on the same threonine residue. It is interesting to note that the PKC-induced phosphorylation of moesin was dependent on Mn2+ and phosphoglycerol, with less efficient activation by phosphatidylinositol and phosphatidylserine plus diacylglycerol.
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Concluding Remarks and Future Directions |
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TopAbstractIntroductionTissue Distribution of...PKC{theta} Expression During...Selective Modulation of...Regulation of IL- 2...Potential Regulation of...Interaction of PKC{theta} with...Function of PKC{theta} in...PKC{theta} and Insulin...Potential Regulation of...Concluding Remarks and Future...References |
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suggests its involvement in cellular functions that are unique to PKC-expressing cell types. However, in spite of a large body of information on PKC, its mode of regulation within cells and the cellular functions which it may regulate are yet unknown. T and B lymphocytes are derived from the same cell lineage and are likely to share more than 99% of their expressed genes. The fact that PKC is expressed at high levels in T, but not in B, lymphocytes, in analogy to other T-cell-specific proteins, such as ZAP-70 [138] and LAT [139], makes it likely that PKC plays a role in signaling pathways unique to the TCR. This notion is supported by the exclusive translocation of PKC in antigen-stimulated T cells to the site of contact with the APC and the selective association of PKC with the immunoregulatory protein, Nef. The observed changes in PKC expression levels and distribution during skeletal muscle development and its localization at neuromuscular junctions indicate that PKC is involved in regulation of nerve-muscle interactions. While the general homology between the functions of PKC in T cells and muscle is not obvious, the PKC-regulated signaling pathways in the two cell types may possess similar effectors and/or share similar downstream signaling elements. Determination of the specific signaling pathways in normal cells in which PKC is involved is difficult, predominantly because of the presence of several distinct PKC isoenzymes within any cell. More conclusive information about the exact role of PKC in activation processes awaits studies in PKC-negative cells or in other experimental models in which PKC-dependent phenotypes will be obvious and easy to follow.
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Acknowledgments |
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References |
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TopAbstractIntroductionTissue Distribution of...PKC{theta} Expression During...Selective Modulation of...Regulation of IL- 2...Potential Regulation of...Interaction of PKC{theta} with...Function of PKC{theta} in...PKC{theta} and Insulin...Potential Regulation of...Concluding Remarks and Future...References |
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