The distribution of PKC isoforms in enteric neurons, muscle and interstitial cells of the human intestine

In many organs, different protein kinase C (PKC) isoforms are expressed in specific cell types, suggesting that the different PKCs have cell-specific roles, and also that drugs acting on a particular PKC may have effects on the whole organ that are distinguishable from drugs that target other isoforms. Previous studies of the guinea-pig and mouse intestine indicate that there are cell-specific expressions of PKC isoforms in neurons, muscle and the interstitial cells of Cajal. In the present study we have investigated the expression of different PKCs in human intestine. Immunohistochemical studies showed that the forms that are prominent in human enteric neurons are PKCs γ and ε and in muscle the dominant form is PKCδ. Neurons were weakly stained for PKCβI. These observations parallel findings in guinea-pig and mouse, except that in human PKCγ-IR was not present in the same types of neurons that express it in the guinea-pig. Enteric glial cells were strongly immunoreactive for PKCα, which is also the major isoform in enteric glial cells of guinea-pig. In human and guinea-pig, glial cells also express PKCβI. Spindle-shaped cells in the mucosa were immunoreactive for PKCα and PKCγ and in the muscle layers similar cells had PKCγ-IR and PKCθ-IR. The spindle-shaped cells were similar in morphology to interstitial cells of Cajal. Western analysis and RT-PCR confirmed the presence of the PKC isoform proteins and mRNA in the tissue. We conclude that there is cell-type specific expression of different PKCs in enteric neurons and intestinal muscle in human tissue, and that there are strong similarities in patterns of expression between laboratory animals and human, but some clear differences are also observed.     

Role of MMP-2 in PKCδ-mediated inhibition of Na+ dependent Ca2+ uptake in microsomes of pulmonary smooth muscle: Involvement of a pertussis toxin sensitive protein

Treatment of bovine pulmonary artery smooth muscle with the O₂^•− generating system hypoxanthine plus xanthine oxidase stimulated MMP-2 activity and PKC activity; and inhibited Na⁺ dependent Ca²⁺ uptake in the microsomes. Pretreatment of the smooth muscle with SOD (the O₂^•− scavenger) and TIMP-2 (MMP-2 inhibitor) prevented the increase in MMP-2 activity and PKC activity, and reversed the inhibition of Na⁺ dependent Ca²⁺ uptake in the microsomes. Pretreatment with calphostin C (a general PKC inhibitor) and rottlerin (a PKCδ inhibitor) prevented the increase in PKC activity and reversed O₂^•− caused inhibition of Na⁺ dependent Ca²⁺ uptake without causing any change in MMP-2 activity in the microsomes of the smooth muscle. Treatment of the smooth muscle with the O₂^•− generating system revealed, respectively, 36 kDa RACK-1 and 78 kDa PKCδ immunoreactive protein profile along with an additional 38 kDa immunoreactive fragment in the microsomes. The 38 kDa band appeared to be the proteolytic fragment of the 78 kDa PKCδ since pretreatment with TIMP-2 abolished the increase in the 38 kDa immunoreactive fragment. Co-immunoprecipitation of PKCδ and RACK-1 demonstrated O₂^•− dependent increase in PKCδ-RACK-1 interaction in the microsomes. Immunoblot assay elicited an immunoreactive band of 41 kDa G_iα in the microsomes. Treatment of the smooth muscle tissue with the O₂^•− generating system causes phosphorylation of G_iα in the microsomes and pretreatment with TIMP-2 and rottlerin prevented the phosphorylation. Pretreatment of the smooth muscle tissue with pertussis toxin reversed O₂^•− caused inhibition of Na⁺ dependent Ca²⁺ uptake without affecting the protease activity and PKC activity in the microsomes. We suggest the existence of a pertussis toxin sensitive G protein mediated mechanism for inhibition of Na⁺ dependent Ca²⁺ uptake in microsomes of bovine pulmonary artery smooth muscle under O₂^•− triggered condition, which is regulated by PKCδ dependent phosphorylation and sensitive to TIMP-2 for its inhibition. (Mol Cell Biochem xxx: 107–117, 2005)     

Protein kinase C isoforms from Giardia duodenalis: identification and functional characterization of a β-like molecule during encystment

Protein kinase C (PKC) is a family of serine/threonine kinases that regulate many different cellular processes such as cell growth and differentiation in eukaryotic cells. Using specific polyclonal antibodies raised against mammalian PKC isoforms, it was demonstrated here for the first time that Giardia duodenalis expresses several PKC isoforms (beta, delta, epsilon, theta and zeta). All PKC isoforms detected showed changes in their expression pattern during encystment induction. In addition, selective PKC inhibitors blocked the encystment in a dose-dependent manner, suggesting that PKC isozymes may play important roles during this differentiation process. We have characterized here the only conventional-type PKC member found so far in Giardia, which showed an increased expression and changes in its intracellular localization pattern during cyst formation. The purified protein obtained by chromatography on DEAE-cellulose followed by size-exclusion chromatography, displayed in vitro kinase activity using histone HI-IIIS as substrate, which was dependent on cofactors required by conventional PKCs, i.e., phospholipids and calcium. An open reading frame in the Giardia Genome Database that encodes a homolog of PKCβ catalytic domain was identified and cloned. The expressed recombinant protein was also recognized by a mammalian anti-PKCβ antibody and was referred as giardial PKCβ on the basis of all these experimental evidence.     

Protein kinase C activity and isoform expression during early postnatal development of rat myocardium

Total protein kinase C (PKC) activity, its isoform expression, and concentration and fatty acid (FA) composition of diacylglycerol (DAG) were determined in the left ventricular myocardium of the rat during early postnatal development (d 2, 3, 5, 7, and 10). PKC activity measured by the incorporation of ³²P into histone IIIS decreased between d 2 and 10 in the homogenate as well as in cytosolic, membrane (100,000g), and nuclear-cytoskeletal-myofilament fractions (1000g). Likewise, the expression of PKC isoforms (α, δ, and ε) determined by immunoblotting generally declined during the period analyzed, although with a variable pattern. In the membrane and nuclear cytoskeletal myofilament fractions, PKCδ and PKCε expression decreased markedly by d 3, returning to or close to the d 2 level immediately on d 5. PKCα expression in the membrane fraction remained almost unchanged by d 7, declining thereafter. PKCδ and PKCε were associated predominantly with particulate fractions, whereas PKCα was more abundant in the cytosolic fraction. DAG concentration exhibited a significant decline by d 5, consistent with the decrease in maximal PKC activity. The unsaturation index of FA in DAG tended to decrease on d 3 owing to the lowered proportion of all polyunsaturated FA of n−6 and n−3 series. These results demonstrate that the developmental decrease in PKC activity and expression in the rat myocardium is not linear and that subcellular localization of the enzyme exhibits isoform-specific day-by-day changes during the early postnatal period. These changes are compatible with the view that PKC signaling may be involved in the control of a rapid switch of myocardial growth pattern during the first week of life.     

Localization of protein kinase C in normal and galactosemic bovine lens epithelial cells in culture

 The localization of the two major isoforms of protein kinase C (PKC), PKCα and PKCγ, present in normal and galactosemic bovine lens epithelial cells in culture, was determined using PKC isoform-specific antisera and visualized with FITC-conjugated secondary antisera. The results indicated that the localization of PKC changed upon exposure to 40 mM galactose after 1day. The subcellular distribution of control cells was cytoplasmic and perinuclear for PKCα, while, in 40 mM galactose-treated cells, PKCα was also localized to nuclei. In contrast, upon exposure to 40 mM galactose the PKCγ of the lens epithelial cells was observed in nucleoli. These results suggest that the subcellular distribution of the PKC isoforms in bovine lens epithelial cells differs and is altered upon exposure to 40 mM galactose. Accepted: 7 January 1998     

Adaptive response is differently induced depending on the sensitivity to radiation-induced cell death in mouse epidermal cells

We investigated the relationship between induction of radio-adaptive response and cell death in mouse normal and neoplastic epidermal cells. Mouse normal primary keratinocytes (PK), cancer-prone cells [v-ras ^Ha-transfected mouse keratinocytes (ras-PK), and line 308 cells (mouse skin papilloma cells which have activatedras ^Ha gene with A-to-T transversion at codon 61) were primed with a low dose of γ-rays (0.01 Gy), and were challenged with a high dose (4 Gy) after a 4 or 7 h interval. The induction of cell death in PK was 2–10 times higher and was also more rapid in PK than in ras-PK or 308 cells. Low-dose pretreatment with a 4 h interval decreased cell death, and this adaptive response was prominent in PK, whereas it was less obvious in the cases of ras-PK and 308 cells. The response of each protein kinase C (PKC) isozymes to high-dose radiation, especially PKCα, PKCδ, PKCε, and PKCη, were different between the normal andras oncogene-activated neoplastic keratinocytes; translocation of these isozymes to membrane occurred more rapidly in normal than in neoplastic cells. Furthermore, low-dose pretreatment did not induce the translocation of PKCδ in PK significantly more than in ras-PK and 308. Thus, the difference in the induction of radio-adaptive responses between mouse normal and neoplastic epidermal cells reflects difference in the rapidity of cell death, and responsiveness of PKC may affect this adaptive response. This revised version was published online in July 2006 with corrections to the Cover Date.     

Immunolocalization of Lacrimal Gland PKC Isoforms. Effect of Phorbol Esters and Cholinergic Agonists on Their Cellular Distribution

In previous studies, we showed that lacrimal gland acini express three isoforms of protein kinase C (PKC): PKCα,-δ, and -ε. In the present study, we report the identification of two other PKC isoforms, namely PKCμ and -ι/λ. Using immunofluorescence techniques, we showed that these isoforms are differentially located. PKCα and -μ showed the most prominent membrane localization, whereas PKCδ, -ε and -ι/λ were mainly cytosolic. Using cell fractionation and western blotting techniques, we showed that the phorbol ester, phorbol 12, 13-dibutyrate (PdBu, 10⁻⁶ m), translocated all PKC isoforms, except PKCι/λ, from the soluble fraction into the particulate fraction. The effect was maximum at 5 min and persisted at 10 min. PKCε was the most responsive to PdBu reaching almost maximal translocation at a PdBu concentration as low as 10⁻⁹ m. The cholinergic agonist, carbachol (10⁻⁵ and 10⁻³ m), induced translocation which was transient for PKCδ, and -μ, but persisted for 10 min for PKCε. Carbachol did not translocate PKCα and, like PdBu, did not translocate PKCι/λ. We concluded that lacrimal gland PKC isoforms are differentially localized and that they translocate differentially in response to phorbol esters and cholinergic agonists. Received: 25 June 1996/Revised: 24 December 1996     

Involvement of PKCζ and GSK3β in the stability of the metaphase spindle

In the somatic cell, the mitotic spindle apparatus is centrosomal, and several isoforms of protein kinase C (PKC) have been associated with the mitotic spindle, but their role in stabilizing the mitotic spindle is still unclear. Other protein kinases such as, glycogen synthase kinase 3β (GSK3β) have also been shown to be associated with the mitotic spindle apparatus. In this study, we show the enrichment of active (phosphorylated) PKCζ at the centrosomal region of the spindle apparatus in metaphase stage of 3T3 cells. In order to understand whether the two kinases PKC and GSK3β are associated with the mitotic spindle, first, the co-localization of phosphorylated PKC isoforms with GSK3β was studied at the poles in metaphase cells. Fluorescence resonance energy transfer (FRET) analysis was used to demonstrate close molecular proximity of phospho-PKCζ with phospho(ser9)GSK3β. Second, the involvement of inactive GSK3β in maintaining an intact mitotic spindle in 3T3 cells was shown. Third, this study also showed that addition of a phospho-PKCζ specific inhibitor to cells can disrupt the mitotic spindle microtubules and some of the proteins associated with it. The mitotic spindle at metaphase in mouse fibroblasts appears to be maintained by PKCζ acting through GSK3β. Phospho-PKCζ is in close molecular proximity to GSK3β, whereas the other isoforms of PKC such as pPKCβII, pPKCγ, pPKCμ, and pPKCθ are not close enough to have significant FRET readings. The close molecular proximity supports the idea that GSK3β may be a substrate of PKCζ.     

Phosphorylation is Involved in the Regulation of the Taurine Influx Via the β-system in Ehrlich Ascites Tumor Cells

The role of 3′,5′-cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), protein kinase C (PKC) and phosphatases in the regulation of the taurine influx via the β-system in Ehrlich ascites tumor cells has been investigated. The taurine uptake by the β-system in Ehrlich cells is inhibited when PKC is activated by phorbol 12-myristate 13-acetate (PMA) and when protein phosphatases are inhibited by calyculin A (CLA). On the other hand, taurine uptake by the β-system is stimulated by an increased level of cAMP or following addition of N⁶,2′-O-dibutyryl-3′,5′-cyclic adenosine monophosphate (dbcAMP). The effect of dbcAMP is partially blocked by addition of the protein kinase inhibitor H-89, and suppressed in the presence of CLA. It is proposed that the β-system in the Ehrlich cells exists in three states of activity: State I, where a PKC phosphorylation site on the transporter or on a regulator is phosphorylated and transport activity is low. State II, where the PKC phosphorylation site is dephosphorylated and transport activity is normal. State III, representing a state with high transport activity, induced by an elevated cellular cAMP level. Apparently, cAMP preferentially stimulates taurine transport when the β-system is in State II. Received: 8 September/Revised: 9 November 1995     

Protein kinase catalytic subunit (PKAcat) from bovine lens: purification, characterization and phosphorylation of lens crystallins

The purification and functional characterization of protein kinase A catalytic subunit (PKAcat) from bovine lens cytosol has been described. Purification to homogeneity has been achieved by using 100 kDa cut-off membrane filtration followed by Sephacryl S-300 chromatography and finally fractionating on High Q anion exchange column. The purified protein migrates as a single band of molecular mass ∼41 kDa on 12.5% SDS-PAGE. Proteomic data from ion trap LC-MS when analyzed through NCBI blast program reveals significant homology (52%) with bovine zeta-crystallin and also some homology with pig casein kinase I alpha chain (38%) and SLA-DR1 beta 1 domain (38%). The search does not indicate homology with any known catalytic subunit of PKA. Inspite of the significant homology with the zeta-crystallin, our protein is different from it in terms of molecular mass. pI value of the kinase (5.3) obtained from 2D analysis is also different from zeta-crystallin (8.5). The protein is found to contain 17% α-helix, 26.5% β-sheet, 21.4% turn and 34.7% random coil. The active catalytic subunit of the bovine lens cAMP-dependent kinase belongs to Type I Cα subtype. The enzyme shows maximum activity at 30 min incubation in presence of 5 mM MgCl₂ and 50 μM ATP. The kinase shows broad substrate specificity. It prefers Ser over Thr as phosphorylating residue. Phosphorylation of crystallin proteins, major protein fraction of bovine lens and phosphorylation of chaperone protein α crystallin by the kinase suggests that the kinase plays some crucial role in regulation of chaperone function within lens.     

Protein kinase Cα inhibitor enhances the sensitivity of human pancreatic cancer HPAC cells to <Emphasis Type="Italic">Clostridium perfringens</Emphasis> enterotoxin via claudin-4

Protein kinase C (PKC) is overexpressed in cancer, including pancreatic cancer, compared with normal tissue. Moreover, PKCα is considered one of the biomarkers for the diagnosis of cancers. In several human cancers, the claudin tight junction molecules are abnormally regulated and are thus promising molecular targets for diagnosis and therapy with Clostridium perfringens enterotoxin (CPE). In order to investigate the changes of tight junction functions of claudins via PKCα activation in pancreatic cancer cells, the well-differentiated human pancreatic cancer cell line HPAC, with its highly expressed tight junction molecules and well-developed barrier function, was treated with the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA). Treatment with TPA modified the activity of phosphoPKCα and caused an increase of the Snail family members Snail, Slug and Smad-interacting protein 1 and a decrease of E-cadherin. In HPAC cells treated with TPA, downregulation of claudin-1 and mislocalization of claudin-4 and occludin around the nuclei were observed, together with a decrease in the numbers of tight junction strands and an increase in phosphorylation of claudin-4. The barrier function and the cytotoxicity of CPE were significantly decreased on TPA treatment. All such changes after TPA treatment were prevented by inhibitors of panPKC and PKCα. These findings suggest that, in human pancreatic cancer cells, PKCα activation downregulates tight junction functions as a barrier and as a receptor of CPE via the modification of claudin-1 and −4 during epithelial to mesenchymal transition-like changes. PKCα inhibitors might represent potential therapeutic agents against human pancreatic cancer cells by use of CPE cytotoxicity via claudin-4.     

Yeast holoenzyme of protein kinase CK2 requires both β and β′ regulatory subunits for its activity

Protein kinase CK2 is a highly conserved Ser/Thr protein kinase that is ubiquitous among eucaryotic organisms and appears to play an important role in many cellular functions. This enzyme in yeast has a tetrameric structure composed of two catalytic (α and/or α′) subunits and two regulatory β and β′ subunits. Previously, we have reported isolation from yeast cells four active forms of CK2, composed of αα′ββ′, α₂ββ′, α′₂ββ′ and a free α′-catalytic subunit. Now, we report that in Saccharomyces cerevisiae CK2 holoenzyme regulatory β subunit cannot substitute other β′ subunit and only both of them can form fully active enzymatic unit. We have examined the subunit composition of tetrameric complexes of yeast CK2 by transformation of yeast strains containing single deletion of the β or β′ regulatory subunits with vectors carrying lacking CKB1 or CKB2 genes. CK2 holoenzyme activity was restored only in cases when both of them were present in the cell. Additional, co-immunoprecypitation experiments show that polyadenylation factor Fip1 interacts with catalytic α subunits of CK2 and interaction with beta subunits in the holoenzyme decreases CK2 activity towards this protein substrate. These data may help to elucidate the role of yeast protein kinase CK2β/β′ subunits in the regulation of holoenzyme assembly and phosphotransferase activity.     

Upregulation of cAMP-specific PDE-4 activity following ligation of the TCR complex on thymocytes is blocked by selective inhibitors of protein kinase C and tyrosyl kinases

We have previously shown that the major cAMP phosphodiesterase (PDE) isoforms present in murine thymocytes are the cGMP-stimulated PDE activity (PDE-2) and the cAMP-specific PDE activity (PDE-4), and that these isoforms are differentially regulated following ligation of the TCR (Michie, A. M., Lobban, M. D., Mueller, T., Harnett, M. M., and Houslay, M. D. [1996]Cell. Signalling 8, 97–110). We show here that the anti-CD3-stimulated elevation in PDE-4 activity in murine thymocytes is dependent on protein tyrosine kinase and protein kinase C (PKC)-mediated signals as the TCR-coupled increase in PDE-4 activity can be abrogated by both the tyrosine kinase inhibitor, genistein, and the PKC selective inhibitors chelerythrine and staurosporine. Moreover, the PKC-activating phorobol ester, phorbol-12-myristate, 13-acetate (PMA) caused an increase in PDE-4 activity, similar to that observed in cells challenged with anti-CD3 monoclonal antibodies and which was not additive with cochallenge using anti-CD3 antibodies. Both the PMA-and the anti-CD3 antibody-mediated increases in PDE-4 activity were blocked by treatment with either cycloheximide or actinomycin D. Despite the upregulation of PDE-4 activity consequent to TCR ligation, intracellular cAMP levels increased on challenge of thymocytes with anti-CD3 antibody, indicating that adenylate cyclase activity was also increased by TCR ligation. It is suggested that the anti-CD3-mediated increase in PDE-4 activity was owing to a rapid PKC-dependent induction of PDE-4 activity following crosslinking of the TCR complex. This identifies “crosstalk” occurring between the PKA and PKC signaling pathways initiated by ligation of the antigen receptor in murine thymocytes. That both adenylate cyclase and PDE-4 activities were increased may indicate the presence of compartmentalized cAMP responses present in these cells.     

Chelerythrine is a potent and specific inhibitor of protein kinase C

The benzophenanthridine alkaloid chelerythrine is a potent, selective antagonist of the Ca++/phospholopid-dependent protein kinase (Protein kinase C: PKC) from the rat brain. Half-maximal inhibition of the kinase occurs at 0.66 microM. Chelerythrine interacted with the catalytic domain of PKC, was a competitive inhibitor with respect to the phosphate acceptor (histone IIIS) (Ki = 0.7 microM) and a non-competitive inhibitor with respect to ATP. This effect was further evidenced by the fact that chelerythrine inhibited native PKC and its catalytic fragment identically and did not affect [3H]- phorbol 12,13 dibutyrate binding to PKC. Chelerythrine selectively inhibited PKC compared to tyrosine protein kinase, cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase. The potent antitumoral activity of celerythrine measured in vitro might be due at least in part to inhibition of PKC and thus suggests that PKC may be a model for rational design of antitumor drugs.     

Low temperature-stimulated phosphorylation regulates the binding of nuclear factors to the promoter of Wcs120 , a cold-specific gene in wheat

The Wcs120 gene encodes a highly abundant protein which appears to play an important role during cold acclimation of wheat. To understand the regulatory mechanism controlling its expression at low temperature, the promoter region has been characterized. Electrophoretic mobility shift assays using short promoter fragments revealed the presence in nuclear extracts from non-acclimated (NA) plants of multiple DNA-binding proteins which interact with several elements. In contrast, no DNA-binding activity was observed in the nuclear extracts from cold-acclimated (CA) plants. In vitro dephosphorylation of these CA nuclear extracts with alkaline phosphatase restored the binding activity. Moreover, okadaic acid (a potent phosphatase inhibitor) markedly stimulated the in vivo accumulation of the WCS120 family of proteins. This suggests that protein phosphatases PP1 and/or PP2A negatively regulate the expression of the Wcs120 gene. In addition, both Ca²⁺-dependent and Ca²⁺-independent kinase activities were found to be significantly higher in the CA nuclear extracts. Western analysis using antibodies directed against protein kinase C (PKC) isoforms showed that a PKCγ homolog (84 kDa) is selectively translocated into the nucleus in response to low temperature. Taken together, our results suggest that, in vivo, the expression of the Wcs120 gene may be regulated by nuclear factors whose binding activity is modulated by a phosphorylation/dephosphorylation mechanism. Received: 9 June 1997 / Accepted: 18 August 1997     

PKCα translocation from mitochondria to nucleus is closely related to induction of apoptosis in gastric cancer cells

PKCs have been implicated in the regulation of cellular differentiation, proliferation, apoptosis and signal transduction. It was demonstrated in this study that PKCα was located both at mitochondria and in cytosol in gastric cancer cell line BGC-823. Treatment of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in the translocation of PKCα from both mitochondria and cytosol to nucleus as clearly shown by laserscanningconfocal microscopy, while the protein level of PKCα was not changed by TPA treatment as detected by Western blot. The results also revealed that TPA-induced translocation of PKCα was in close association with apoptosis induction, and such association was further affirmed by other experiments where various apoptotic stimuli and specific inhibitors of PKC were used. Taken together, these findings indicate that translocation of PKCα from both mitochondria and cytosol to nucleus in gastric cancer cell is accompanied by induction of apoptosis, and may imply a new mechanism of the potential linking between cell apoptosis and PKCα translocation.     

Protein kinase Cζ regulates phospholipase D activity in rat-1 fibroblasts expressing the α<Subscript>1A</Subscript> adrenergic receptor

Background  

Phenylephrine (PHE), an α₁ adrenergic receptor agonist, increases phospholipase D (PLD) activity, independent of classical and novel protein kinase C (PKC) isoforms, in rat-1 fibroblasts expressing α_1A adrenergic receptors. The aim of this study was to determine the contribution of atypical PKCζ to PLD activation in response to PHE in these cells.     

The use of time-resolved fluorescence imaging in the study of protein kinase C localisation in cells

Background  

Two-photon-excitation fluorescence lifetime imaging (2P-FLIM) was used to investigate the association of protein kinase C alpha (PKCα) with caveolin in CHO cells. PKCα is found widely in the cytoplasm and nucleus in most cells. Upon activation, as a result of increased intracellular Ca²⁺ and production of DAG, through G-protein coupled-phospholipase C signalling, PKC translocates to a variety of regions in the cell where it phosphorylates and interacts with many signalling pathways. Due to its wide distribution, discerning a particular interaction from others within the cell is extremely difficult     

Isoflurane Inhibits Protein Kinase Cγ and Calcium/Calmodulin Dependent Protein Kinase II-α Translocation to Synaptic Membranes in Ischemic Mice Brains

Volatile anesthetics isoflurane possibly improves the ischemic brain injury. However, its molecular actions are still unclear. In ischemia, protein kinase C (PKC)γ and calcium/calmodulin dependent protein kinase II (CaMKII)-α are persistently translocated from cytosol to cell membranes, and diminish these translocation suggested to be neuroprotective. We thus tested a hypothesis that isoflurane inhibits PKCγ and CaMKII-α translocation after ischemic brain insults. C57Bl/6J male mice were made to inhale 1 or 2 MAC isoflurane, after which 3 or 5 min cerebral ischemia was induced by decapitation. The sampled cerebrum cortex was then homogenized and centrifuged into crude synaptosomal fractions (P2), cytosolic fractions (S3), and particulate fractions (P3). CaMKII-α and PKCγ levels of these fractions were analyzed by immunoblotting. PKCγ and CaMKII-α are translocated to synaptic membrane from cytosol by cerebral ischemia, although isoflurane significantly inhibited such translocation. These results may explain in part the cellular and molecular mechanisms of neuroprotective effects of isoflurane.