Involvement of the ERK mitogen-activated protein kinase in cell resistance to complement-mediated lysis

Sublytic doses of complement desensitize cells and make them resistant to lytic complement doses. This process, named complement-induced protection, requires calcium ion influx, protein kinase C activation and protein synthesis. The involvement of the extracellular signal-regulated kinase, ERK, in cell desensitization by sublytic complement was examined in erythroleukaemia K562 cells and in COS-7 cells. As shown here, ERK is activated in K562 and COS-7 cells within 10 min of sublytic immune attack and then shows a decline and a second peak of activation at 20 min. C7- and C8-deficient human sera have a small effect on ERK activity. However, a significant increase in ERK activation is observed when C7 or C8, respectively, is added back to these sera. Complement-induced ERK activation was blocked in cells treated with GF109203X or Go6976, two selective PKC inhibitors, as well as by treatment with PD098059, an inhibitor of MEK1, the ERK kinase. PD098059 treatment also sensitized K562 cells to complement-mediated lysis and prevented complement-induced protection. COS-7 cells transfected with a dominant-negative MEK plasmid were incapable of undergoing the process of complement-induced protection. In conclusion, cell desensitization by sublytic doses of the complement membrane attack complex involves a signalling cascade that includes PKC-mediated ERK activation.     

The activation of the neutrophil respiratory burst by anti-neutrophil cytoplasm autoantibody (ANCA) from patients with systemic vasculitis requires tyrosine kinases and protein kinase C activation.

The ability of antineutrophil cytoplasm autoantibodies (ANCA) from patients with systemic vasculitis to stimulate protein kinase C (PKC) and tyrosine kinases was examined in human neutrophils. Using the superoxide dismutase-inhibitable reduction of ferricytochrome C, the kinetics of ANCA-induced superoxide (O2-) production were characterized and subsequently manipulated by specific inhibitors of PKC and tyrosine kinases. With this approach, ANCA IgG, but not normal IgG or ANCA F(ab')2 fragments caused a time and dose dependent release of O2- from TNF-alpha primed neutrophils. The kinetics of ANCA-induced O2- production showed an initial 10-15 min lag phase compared to the N-formyl-L-methionyl-L-leucyl-L-phenylalanine response, suggesting differences in the signalling pathways recruited by these two stimuli. Inhibitor studies revealed that ANCA-activation involved members of both the Ca2+-dependent and -independent PKC isoforms and also tyrosine kinases. ANCA IgG resulted in the translocation of the betaII isoform of PKC at a time corresponding to the end of the lag phase of O2- production, suggesting that PKC activity may be instrumental in processes regulating the activity of the NADPH oxidase in response to ANCA. Tyrosine phosphorylation of numerous proteins also peaked 10-15 min after stimulation with ANCA but not normal IgG. These data suggest that PKC and tyrosine kinases regulate O2- production from neutrophils stimulated with autoantibodies from patients with systemic vasculitis.     

Roles of CaMKII, PKA, and PKC in the induction and maintenance of LTP of C-fiber-evoked field potentials in rat spinal dorsal horn

Long-term potentiation (LTP) of C-fiber-evoked field potentials in spinal dorsal horn may be relevant to hyperalgesia, an increased response to noxious stimulation. The mechanism underlying this form of synaptic plasticity is, however, still unclear. Considerable evidence has shown that calcium/calmodulin-dependent protein kinase II (CaMKII), protein kinase A (PKA), and protein kinase C (PKC) are important for LTP in hippocampus. In this study, the roles of these three protein kinases in the induction and maintenance of LTP of C-fiber-evoked field potentials were evaluated by application of specific inhibitors of CaMKII (KN-93 and AIP), PKA (Rp-CPT-cAMPS), and PKC (chelerythrine and G? 6983) at the recording segments before and after LTP induction in urethane-anesthetized Sprague-Dawley rats. We found both KN-93 and AIP, when applied at 30 min prior to tetanic stimulation, completely blocked LTP induction. At 30 min after LTP induction, KN-93 and AIP reversed LTP completely, and at 60 min after LTP induction, they depressed spinal LTP in most rats tested. Three hours after LTP induction, however, KN-93 or AIP did not affect the spinal LTP. Rp-CPT-cAMPS, chelerythrine, and G? 6983 blocked the spinal LTP when applied at 30 min before tetanic stimulation and reversed LTP completely at 15 min after LTP induction. In contrast, at 30 min after LTP induction, the drugs never affected the spinal LTP. These results suggest that activation of CaMKII, PKA, and PKC may be crucial for the induction and the early-phase but not for the late-phase maintenance of the spinal LTP.     

Thiol proteinase inhibitors reverse the increased protein kinase C down-regulation and concanavalin A cap formation in polymorphonuclear leukocytes from Chediak-Higashi syndrome (beige) mouse

Protein kinase C (PKC) plays an essential role in intracellular signal transduction for various cell functions, including concanavalin A (Con A)-induced cap formation. This enzyme is known to be proteolysed by calpain, which is a Ca2(+)-dependent thiol proteinase. As reported previously, in polymorphonuclear leukocytes (PMNs) from beige mouse, the model of Chediak-Higashi syndrome, Con A-induced cap formation significantly increased compared with that in normal mouse. However, after pretreatment of beige PMNs with the thiol proteinase inhibitors leupeptin or E-64, the capping decreased to normal levels. Meanwhile, Con A-induced the translocation of PKC from the cytosolic to membrane fraction within 5 min in both mice, which is essential to the activation of this enzyme. However, after the translocation, an abnormal rapid decline in membrane-bound PKC activity was noted in beige mouse PMNs. Both leupeptin and E-64 also corrected the rapid decline in PKC activity observed in the beige mouse. These findings suggest that the normalization of Con A cap formation in beige mouse PMNs by the thiol proteinase inhibitors is associated with the correction of abnormality in PKC activity.     

Differential recovery of antibody production potential after sublethal, whole-body irradiation of mice

Mice were given single injections of sheep erythrocytes (SE) or polyvinylpyrrolidone (PVP) at various times after sublethal, whole-body irradiation (550 rad 60CO) and direct, antigen-specific, plaque-forming cell (PFC) responses were quantified. Irradiated mice did not respond to SE or PVP when immunized 15 d postirradiation (PI); by day 30 PI, the responses by irradiated mice were 40-126% of normal to SE and 3-38% of normal to PVP. The impaired recovery after irradiation of immune responses to PVP was not due to altered antigen dose requirements or altered time of peak PFC response and occurred after irradiation of mice by doses as low as 200 rad. Both athymic and euthymic mice had impaired responses to PVP after whole-body irradiation. The impaired response of irradiated mice to PVP was repaired by adoptive transfer of normal bone marrow, fetal liver, or spleen cells and also by spleen cell preparations enriched in Ig+ cells but not by spleen cell preparations enriched in Thy.1+ or Ig- cells. With the aid of additional antigens it was observed that by day 30 PI, mice had recovered ability to respond to the T-cell-dependent antigen SE and the T-cell-independent type-1 antigens 2,4,6-trinitrophenyl-Brucella abortus and butanol-extracted bacterial lipopolysaccharide, but at that time they gave impaired responses to the T-cell-independent type-2 antigens PVP, type III pneumococcal polysaccharide, and phenol-extracted bacterial lipopolysaccharide; they had an immune response pattern similar to that of CBA/N mice having an X-linked immunodeficiency.     

吡那地尔预处理提高失血性休克血管的反应性和钙敏感性

目的:观察吡那地尔预处理诱导对失血性休克血管反应性和钙敏感性的保护作用以及与蛋白激酶C(PKC)α、ε的关系。方法:观察不同剂量吡那地尔在休克前不同时间预处理,对失血性休克大鼠去甲肾上腺素(NE)的升压效应[平均动脉压(MAP)增幅]和收缩血管效应[肠系膜上动脉(SMA)管径变化]的影响,以及对SMA一级分支微血管环血管反应性和钙敏感性的影响;观察PKCα、PKCε抑制剂对吡那地尔预处理诱导保护效应的影响,以及吡那地尔预处理对PKCα、PKCε蛋白转位的影响,证实PKCα和PKCε的作用。结果:(1)失血性休克2 h后,NE的升压效应和收缩血管效应、SMA血管反应性和钙敏感性较正常组均显著降低(P0.01),25μg/kgBW吡那地尔休克前30 min预处理可改善休克后的上述变化;(2)PKCα和PKCε的拮抗剂可以消除25μg/kg BW吡那地尔休克前30 min预处理诱导的失血性休克血管反应性和钙敏感性保护,使NE的Emax分别降低42.9%和62.9%(P0.01),使Ca2+的Emax分别降低31.1%和56.1%(P0.01);25μg/kg BW吡那地尔休克前30 min预处理使PKCα和PKCε的胞膜表达较休克2 h增高,胞浆表达较休克2 h降低(P0.01)。结论:吡那地尔预处理可通过诱导PKCα和PKCε的转位和活化,提高大鼠失血性休克后血管的反应性和钙敏感性。     

Cross-linking of surface IgM activates NF-kappa B in B lymphocyte.

In B lymphocytes, cross-linking of surface IgM activates changes in both the cell cycle and differentiation. In normal B cells and B cell tumors, many stimuli induce the activation of NF-kappa B and its translocation from the cytoplasm to the nucleus. In this study we sought to determine if cross-linking of surface IgM led to the activation of NF-kappa B. Our results show that activation of B cells by cross-linking anti-IgM antibodies activated NF-kappa B in the murine B lymphoid cell lines 70Z/3 and M12, and in the dense fraction of splenic cells. The activation of NF-kappa B required optimal doses of anti-IgM antibodies and took 5 to 10 min to reach maximal levels. Cross-linking of IgM has also been shown to activate protein kinases including protein kinase C (PKC). To test whether PKC activation was required for NF-kappa B translocation, we treated 70Z/3 cells for 18 h with phorbol 12-myristate 13-acetate, a procedure which depletes these cells of functional PKC. This treatment did not abrogate the nuclear translocation of NF-kappa B following anti-IgM cross-linking. These results indicate that the nuclear translocation of NF-kappa B is rapidly induced by surface IgM cross-linking and that this activation appears to use a pathway which does not require PKC.     

Repeat organic dust exposure-induced monocyte inflammation is associated with protein kinase C activity

BACKGROUND: Organic dust exposure results in an inflammatory response that attenuates over time, but repetitive exposures can result in chronic respiratory diseases. Mechanisms underlying this modulated response are not clear. OBJECTIVE: This study investigated the effects of repeat versus single organic dust exposure-induced inflammatory mediators and protein kinase C (PKC) activity in monocytes. METHODS: Settled organic dust was obtained from swine confinement facilities. Promonocytic THP-1 cells and human peripheral blood monocytes were pretreated with or without dust extract and then restimulated. Culture supernatants were evaluated for TNF-alpha, IL-6, CXCL8, and IL-10. Responses were compared with endotoxin-depleted dust, LPS, and peptidoglycan. PKC isoform (alpha, delta, varepsilon, zeta) activation was evaluated by direct kinase activity. PKC isoform inhibitors' effects on TNF-alpha secretion were studied. RESULTS: Single exposure to organic dust stimulated monocyte secretion of TNF-alpha, IL-6, CXCL8, and IL-10 compared with unstimulated cells. TNF-alpha and IL-6 were diminished in pretreated cells restimulated with dust. Secretion of CXCL8 and IL-10 remained persistently elevated. TNF-alpha responses were retained after marked depletion of endotoxin. Dust exposure induced significant PKC alpha, delta, varepsilon, and zeta activation, peaking at 30 to 60 minutes. PKC isoform activation was attenuated in repeat exposed cells. Inhibition of PKCalpha and PKCvarepsilon reduced dust-induced TNF-alpha secretion. CONCLUSION: Repeat organic dust exposure modulated inflammatory mediator production in monocytes independent of endotoxin. The inability of PKC to be reactivated may account for this observation. CLINICAL IMPLICATIONS: Targeting PKC and specific mediators associated with repetitive organic dust exposure may result in novel therapeutic strategies.     

Protein kinase C activation modulates tumour necrosis factor-alpha priming of human neutrophils for zymosan-induced leukotriene B4 release.

Neutrophil (PMN) activation by the yeast component zymosan involves the complement receptor type 3 (CD11b/CD18). Recombinant human tumour necrosis factor-alpha (rhTNF-alpha) augmented the zymosan-stimulated leukotriene B4 (LTB4) release from PMN, reaching a fourfold increase at 10(-9) M. Co-incubation of PMN with 10(-9) M rhTNF-alpha and staurosporine resulted in a further dose-dependent increase, which became significantly greater than a purely additive effect at a staurosporine concentration of 10 nM. This synergy was maintained at all doses of staurosporine tested. In addition, doses of phorbol 12-myristate 13-acetate (PMA) that do not activate protein kinase C (PKC) (below 10(-9) M) also augmented the zymosan-stimulated release of LTB4. However, doses of PMA above 10(-9) M progressively inhibited the response to levels below that of zymosan alone. Staurosporine at 50 nM completely prevented, and 10(-9) M rhTNF-alpha partially but significantly (P less than 0.02 at 10(-8) M PMA, P less than 0.01 at 10(-7) M PMA) reversed, this high-dose PMA inhibition. PKC activation thus opposes the priming effect of rhTNF-alpha on neutrophils, while PKC inhibition may enhance the ability of rhTNF-alpha to prime PMN for zymosan activation. The combined effect of rhTNF-alpha and staurosporine suggests an intracellular synergy rather than simply a direct action due to increased zymosan receptor expression. Thus there appear to be mechanisms whereby the responses of neutrophils may be augmented without activating PKC. Indeed, kinase activation may even exert a degree of feedback control that is antagonized by rhTNF-alpha treatment.     

Early activation defects in T lymphocytes from aged mice

Summary: Aging affects both calcium signals and protein kinase cascades in mouse T lymphocytes. The decline in calcium signal development largely represents differences between naive and memory T cells: the latter are resistant to increases in calcium concentration, and are more common in aged mice. Aging leads to declines in phosphorylation of a wide range of substrates in T cells stimulated by either anti-CD 3 antibodies or by substances, such as phorbol myristate acetate (PMA) or ionomycin, that act at intracellular sites, but some phosphoproteins respond only in old T cells, and others respond regardless of age, Tyrosine phosphorylation of die CD3ζ chain declines with age, both in resting T cells and after activation. but the proportion of Zap-70 that is bound to CD3C increases in T cells from old mice, Zap-70 function and phosphorylation of CD3ζ-associated Zap-70 change only slightly after stimulation of T cells by anti-CD3 and aiHi-CD4, and are at similar levels in activated old and young T cells. Nonetheless, induction of Raf- l, MEK, and ERK kinase activity declines with age in CD4T cells. The effect of aging on T-cell activation is not simply an overall decline in signal intensity, but a set of qualitative changes that differ among subsets and depend at least partly on the nature of the stimulus.     

TNFa-Induced Expression of Endothelial Adhesion Molecules, ICAM-1 and VCAM-1, is Linked to Protein Kinase C Activation

The role of protein kinase C (PKC) in TNFα-induced activation of endothelial adhesion molecules ICAM-1 and VCAM-1 was analysed. Phorbol myristate acetate, which is known to activate PKC, was able lo mimic TNFα-induced up-regulation of ICAM-1 and partly also VCAM-1 expression. Similarly a PKC inhibitor, H7, but not another kinase inhibitor. HA1004, inhibited TNFα-induced enhancement of ICAM-1 expression at both the mRNA and the protein level. Moreover we were able to measure a transient PKC activation peak at 16 min after TNFα induction in endothelial cells analysed by phorbol-dibutyrate binding. These results indicate that the TNFα-induced effect on the regulation of endothelial adhesion molecule expression is at least partly mediated by PKC activation.     

Sevoflurane stimulates MAP kinase signal transduction through the activation of PKC alpha and betaII in fetal rat cerebral cortex cultured neuron

Protein kinase C (PKC) is a key enzyme that participates in various neuronal functions. PKC has also been identified as a target molecule for general anesthetic actions. Raf, mitogen-activated protein kinase (MEK) and extracellular signal-regulated kinase (ERK1/2) have been thought to be target effectors of PKC. In the present study, we attempted to evaluate the effect of sevoflurane on PKC/MAPK cascade signaling in cultured fetal rat cerebral -cortex neurons, prepared from embryonic day 18 fetuses. The effects of sevoflurane on the translocation of 7 PKC isoforms (alpha, betaI, betaII, gamma, delta, varepsilon and zeta) were observed by immunoblotting using isoform-selective antibodies to PKCs. The treatment of neurons with sevoflurane induced the translocation of PKC alpha and PKC betaII species from the cytosol to the membrane fraction, which indicated the activation of these PKC isoforms. In contrast, there was no clear change in the distribution of other PKC isoforms. We next examined whether the specific activation of PKC alpha and betaII by sevoflurane could stimulate the MAP kinase signaling pathway in cultured neurons. Raf phosphorylation was increased by the administration of 0.25 mM sevoflurane. The phosphorylation of Raf proteins reached a maximum at 5-10 min. Subsequently, the phosphorylation of MEK proteins was increased at 10-15 min after sevoflurane treatments. That of ERK proteins was induced at 15-60 min. Moreover, the phosphorylation of ERK induced by sevoflurane was significantly decreased by the treatment of PKC inhibitor (staurosporine) and MEK inhibitor (PD98059). On the other hand, the contents of total Raf, MEK and ERK proteins were relatively constant at all times examined. To examine the -localization of phosphorylated-ERK protein, immunohistochemical staining of sevoflurane-treated cultured neurons was performed. The phosphorylated-ERK proteins were markedly accumulated in both the cytosol of the cell body and the neurites in the neuronal cells with time after 0.25 mM sevoflurane-treatment. These results demonstrated that sevoflurane induced the phosphorylation of the MAP kinase cascade through the activation of the PKC alpha and PKC betaII species.     

Delayed sodium pyruvate treatment improves working memory following experimental traumatic brain injury

Protein kinase C (PKC) is a family of serine/threonine-isozymes that are involved in many signaling events in normal and disease states. Previous studies from our lab have demonstrated that ?PKC plays a pivotal role in neuroprotection induced by ischemic preconditioning. However, the role of ?PKC during and after brain ischemia is not clearly defined. Therefore, in the present study, we tested the hypothesis that activation of ?PKC during an ischemic event is neuroprotective. Furthermore, other studies have demonstrated that ?PKC mediates cerebral ischemic tolerance in the rat brain by decreasing vascular tone. Thus, we also tested the effects of ?PKC activation during ischemia on cerebral blood flow (CBF). We found that ψ?-Receptors for Activated C Kinase (RACK), a ?PKC-selective peptide activator, injected intravenously 30 min before induction of global cerebral ischemia conferred neuroprotection in the CA1 region of the rat hippocampus. Moreover, measurements of CBF before, during, and after cerebral ischemia revealed a significant reduction in the reperfusion phase of rats pretreated with ψ?RACK as compared to Tat peptide (vehicle). Our results suggest that ?PKC can protect the rat brain against ischemic damage by regulating CBF. Thus, ?PKC may be one of the treatment modalities against ischemic injury.     

Lysis-sensitive targets stimulate an elevation of cAMP in human natural killer cells.

Natural killer (NK) cells are lymphocytes that are capable of destroying tumour cells and virally infected cells (cytolysis) without prior sensitization. When cAMP is artificially elevated in NK cells, it is a potent inhibitor of their cytolytic function. We investigated whether NK-cell cAMP levels are modulated in response to tumour target cells to determine the potential of cAMP as a physiological regulator of NK cytotoxic function. When NK cells are exposed to a range of lysis-sensitive (LS) tumour-target cells there is an increase in intracellular cAMP levels in the NK cells over a 60-min period. The peak increase in cAMP (200-400% above control) occurs at 30 min for all LS targets tested. There is no increase in NK-cell cAMP in response to lysis-resistant (LR) tumour-target cells. The cAMP elevation may be dependent on both LS-target-stimulated adenylyl cyclase (AC) activation and LS-target-stimulated phosphodiesterase (PDE) inhibition. When the NK cells are pretreated with the protein tyrosine kinase (PTK) inhibitor, genistein (30 micrograms/ml), the AC-activation component of the cAMP elevation is abolished. Thus, the AC-activation component appears to require PTK activation. When NK cells are pretreated with the protein kinase C (PKC) inhibitor, chelerythrine chloride (10 microM) the cAMP elevation in response to LS targets was not diminished. This indicates that neither the AC-activation component nor any PDE-inhibition component require PKC activation.     

Characterization of T-Cell Receptor αβ Repertoire in Synovial Tissue from Different Temporal Phases of Rheumatoid Arthritis

The role of protein kinase C (PKC) in TNF alpha-induced activation of endothelial adhesion molecules ICAM-1 and VCAM-1 was analysed. Phorbol myristate acetate, which is known to activate PKC, was able to mimic TNF alpha-induced up-regulation of ICAM-1 and partly also VCAM-1 expression. Similarly a PKC inhibitor, H7, but not another kinase inhibitor, HA1004, inhibited TNF alpha-induced enhancement of ICAM-1 expression at both the mRNA and the protein level. Moreover we were able to measure a transient PKC activation peak at 16 min after TNF alpha induction in endothelial cells analysed by phorbol-dibutyrate binding. These results indicate that the TNF alpha-induced effect on the regulation of endothelial adhesion molecule expression is at least partly mediated by PKC activation.     

Effect of exercise on protein kinase C activity and localization in human skeletal muscle

To investigate the effect of exercise on protein kinase C (PKC) activity and localization in human skeletal muscle, eight healthy men performed cycle ergometer exercise for 40 min at 76 ± 1% the peak pulmonary O₂ uptake     , with muscle samples obtained at rest and after 5 and 40 min of exercise. PKC expression, phosphorylation and activities were examined by immunoblotting and in vitro kinase assays of fractionated and whole tissue preparations. In response to exercise, total PKC activity was slightly higher at 40 min in an enriched membrane fraction, and using a pSer-PKC-substrate motif antibody it was revealed that exercise increased the serine phosphorylation of a ∼50 kDa protein. There were no changes in conventional PKC (cPKC) or PKCθ activities; however, atypical PKC (aPKC) activity was ∼70% higher at 5 and 40 min, and aPKC expression and Thr^410/403 phosphorylation were unaltered by exercise. There were no effects of exercise on the abundance of PKCα, PKCδ, PKCθ and aPKC within cytosolic or enriched membrane fractions of skeletal muscle. These data indicate that aPKC, but not cPKC or PKCθ, are activated by exercise in contracting muscle suggesting a potential role for aPKC in the regulation of skeletal muscle function and metabolism during exercise in humans.     

Defective CD2 T cell pathway activation in common variable immunodeficiency (CVID).

Clonal T cell expansion requires simultaneous activation of the TCR and secondary signals, e.g. CD2, CD4, CD28. Interference of CD2/CD58 interaction with MoAbs abrogates the primary immune response and antibody production. Given this functional importance of CD2/CD58 interaction for the generation of specific immune responses, we demonstrate for the first time a defective CD2 pathway activation in patients with CVID (seven children and four adults). The costimulatory effect of monocytes upon CD2-triggered proliferation was significantly impaired in CVID patients: 4.080 ct/min versus 20.769 ct/min in controls (P < 0.05). Second, IL-1, which is a strong comitogenic factor for activation via CD2 in normal T cells, showed a defective amplifier function of the CD2 pathway in most patients (median 1.714 ct/min in patients versus 17.521 ct/min in controls; P < 0.05). In addition, by using a mitogenic combination of CD2 plus CD45 MoAb, median proliferation of T cells was severely depressed in patients: 10.577 ct/min versus 34.685 ct/min in controls (P = 0.005). In conclusion, the marked dysfunction seen in responsiveness to phytohaemagglutinin (PHA) (median 24.594 ct/min in patients versus 52.229 ct/min in controls; P < 0.001) and after CD2 triggering, together with the unaffected response to TCR-CD3, suggest that the T cell deficiency in CVID is in part due to deficiencies in the CD2 pathway. Since direct activation of protein kinase C(PKC) by phorbol ester restores defective T cell responses to normal, our results suggest that an early signal-transducing defect might exist at a step proximal to PKC activation in patients with CVID.     

Structural basis of protein kinase C isoform function

Protein kinase C (PKC) isoforms comprise a family of lipid-activated enzymes that have been implicated in a wide range of cellular functions. PKCs are modular enzymes comprised of a regulatory domain (that contains the membrane-targeting motifs that respond to lipid cofactors, and in the case of some PKCs calcium) and a relatively conserved catalytic domain that binds ATP and substrates. These enzymes are coexpressed and respond to similar stimulatory agonists in many cell types. However, there is growing evidence that individual PKC isoforms subserve unique (and in some cases opposing) functions in cells, at least in part as a result of isoform-specific subcellular compartmentalization patterns, protein-protein interactions, and posttranslational modifications that influence catalytic function. This review focuses on the structural basis for differences in lipid cofactor responsiveness for individual PKC isoforms, the regulatory phosphorylations that control the normal maturation, activation, signaling function, and downregulation of these enzymes, and the intra-/intermolecular interactions that control PKC isoform activation and subcellular targeting in cells. A detailed understanding of the unique molecular features that underlie isoform-specific posttranslational modification patterns, protein-protein interactions, and subcellular targeting (i.e., that impart functional specificity) should provide the basis for the design of novel PKC isoform-specific activator or inhibitor compounds that can achieve therapeutically useful changes in PKC signaling in cells.