PKCε Promotes HuD-Mediated Neprilysin mRNA Stability and Enhances Neprilysin-Induced Aβ Degradation in Brain Neurons

Amyloid-beta (Aβ) peptide accumulation in the brain is a pathological hallmark of all forms of Alzheimer’s disease. An imbalance between Aβ production and clearance from the brain may contribute to accumulation of neurotoxic Aβ and subsequent synaptic loss, which is the strongest correlate of the extent of memory loss in AD. The activity of neprilysin (NEP), a potent Aβ-degrading enzyme, is decreased in the AD brain. Expression of HuD, an mRNA-binding protein important for synaptogenesis and neuronal plasticity, is also decreased in the AD brain. HuD is regulated by protein kinase Cε (PKCε), and we previously demonstrated that PKCε activation decreases Aβ levels. We hypothesized that PKCε acts through HuD to stabilize NEP mRNA, modulate its localization, and support NEP activity. Conversely, loss of PKCε-activated HuD in AD leads to decreased NEP activity and accumulation of Aβ. Here we show that HuD is associated with NEP mRNA in cultures of human SK-N-SH cells. Treatment with bryostatin, a PKCε-selective activator, enhanced NEP association with HuD and increased NEP mRNA stability. Activation of PKCε also increased NEP protein levels, increased NEP phosphorylation, and induced cell surface expression. In addition, specific PKCε activation directly stimulated NEP activity, leading to degradation of a monomeric form of Aβ peptide and decreased Aβ neuronal toxicity, as measured by cell viability. Bryostatin treatment also rescued Aβ-mediated inhibition of HuD-NEP mRNA binding, NEP protein expression, and NEP cell membrane translocation. These results suggest that PKCε activation reduces Aβ by up-regulating, via the mRNA-binding protein HuD, Aβ-degrading enzymes such as NEP. Thus, PKCε activation may have therapeutic efficacy for AD by reducing neurotoxic Aβ accumulation as well as having direct anti-apoptotic and synaptogenic effects.     

PKCδ Localization at the Membrane Increases Matrix Traction Force Dependent on PLCγ1/EGFR Signaling

Introduction

During wound healing, fibroblasts initially migrate into the wound bed and later contract the matrix. Relevant mediators of transcellular contractility revealed by systems analyses are protein kinase c delta/myosin light chain-2 (PKCδ/MLC-2). PKCδ is activated by growth factor-driven PLCγ1 hydrolysis of phosphoinositide bisphosphate (PIP₂) hydrolysis when it becomes tranlocated to the membrane. This leads to MLC-2 phosphorylation that regulates myosin for contractility. Furthermore, PKCδ n-terminus mediates PKCδ localization to the membrane in relative proximity to PLCγ1 activity. However, the role this localization and the relationship to its activation and signaling of force is not well understood. Therefore, we investigated whether the membrane localization of PKCδ mediates the transcellular contractility of fibroblasts.

Methods

To determine PKCδ activation in targeted membrane locations in mouse fibroblast cells (NR6-WT), two PKCδ constructs were generated; PKCδ-CaaX with farnesylation moiety targeting PKCδ to the membrane and PKCδ-SaaX a non-targeting control.

Results

Increased mean cell force was observed before and during EGF stimulation in fibroblasts expressing membrane-targeted PKCδ (PKCδ-CaaX) when analyzed with 2D cell traction force and 3D compaction of collagen matrix. This effect was reduced in cells deficient in EGFR/PLCy1 signaling. In cells expressing non-membrane targeted PKCδ (PKCδ-SaaX), the cell force exerted outside the ECM (extracellular matrix) was less, but cell motility/speed/persistence was increased after EGF stimulation. Change in cell motility and increased force exertion was also preceded by change in cell morphology. Organization of actin stress fibers was also decreased as a result of increasing membrane targeting of PKCδ.

Conclusion

From these results membrane tethering of PKCδ leads to increased force exertion on ECM. Furthermore, our data show PLCγ1 regulation of PKCδ, at least in part, drives transcellular contractility in fibroblasts.     

Rab7 Activation by Growth Factor Withdrawal Contributes to the Induction of Apoptosis

The Rab7 GTPase promotes membrane fusion reactions between late endosomes and lysosomes. In previous studies, we demonstrated that Rab7 inactivation blocks growth factor withdrawal-induced cell death. These results led us to hypothesize that growth factor withdrawal activates Rab7. Here, we show that growth factor deprivation increased both the fraction of Rab7 that was associated with cellular membranes and the percentage of Rab7 bound to guanosine triphosphate (GTP). Moreover, expressing a constitutively GTP-bound mutant of Rab7, Rab7-Q67L, was sufficient to trigger cell death even in the presence of growth factors. This activated Rab7 mutant was also able to reverse the growth factor-independent cell survival conferred by protein kinase C (PKC) δ inhibition. PKCδ is one of the most highly induced proteins after growth factor withdrawal and contributes to the induction of apoptosis. To evaluate whether PKCδ regulates Rab7, we first examined lysosomal morphology in cells with reduced PKCδ activity. Consistent with a potential role as a Rab7 activator, blocking PKCδ function caused profound lysosomal fragmentation comparable to that observed when Rab7 was directly inhibited. Interestingly, PKCδ inhibition fragmented the lysosome without decreasing Rab7-GTP levels. Taken together, these results suggest that Rab7 activation by growth factor withdrawal contributes to the induction of apoptosis and that Rab7-dependent fusion reactions may be targeted by signaling pathways that limit growth factor-independent cell survival.     

Hypoxic preconditioning promotes the translocation of protein kinase C ? binding with caveolin-3 at cell membrane not mitochondrial in rat heart

Protein kinase C has been shown to play a central role in the cardioprotection of ischemic preconditioning. However, the mechanism underlying PKC-mediated cardioprotection is not completely understood. Given that caveolae are critical for PKC signaling, we sought to determine whether hypoxic preconditioning promotes translocation and association of PKC isoforms with caveolin-3. A cellular model of hypoxic preconditioning from adult rat cardiac myocytes (ARCM) or H9c2 cells was employed to examine PKC isoforms by molecular, biochemical and cellular imaging analysis. Hypoxia was induced by incubating the cells in an airtight chamber in which O₂ was replaced by N₂ with glucose-free Tyrode''s solution. Cells were subjected to hypoxic preconditioning with 10 minutes of hypoxia followed by 30 minutes of reoxygenation. Western blot data indicated that the band intensity for PKCϵ, PKCδ or PKCα, but not PKCβ and PKCζ was enhanced significantly by hypoxic preconditioning from the caveolin-enriched plasma membrane interactions. Immunoprecipitation experiments from the caveolin-enriched membrane fractions of ARCM showed that the level of PKCϵ, PKCδ and PKCα in the anti-caveolin-3 immunoprecipitates was also increased by hypoxic preconditioning. Further, our FRET analysis in H9c2 cells suggested that there is a minimum FRET signal for caveolin-3 and PKCϵ along cell peripherals, but hypoxic preconditioning enhanced the FRET signal, indicating a potential interaction between caveolin-3 and PKCϵ. And also treatment of the cells with hypoxic preconditioning led to a smaller amount of translocation of PKCϵ to the mitochondria than that to the membrane. We demonstrate that hypoxic preconditioning promotes rapid association of PKCϵ, PKCδ and PKCα with the caveolin-enriched plasma membrane microdomain of cardiac myocytes, and PKCϵ via direct molecular interaction with caveolin-3. This regulatory mechanism may play an important role in cardioprotection.     

The selective inhibition of nuclear PKCζ restores the effectiveness of chemotherapeutic agents in chemoresistant cells

The atypical protein kinase C (PKC) isoform zeta (PKCζ) has been implicated in the intracellular transduction of mitogenic and apoptotic signals by acting on different signaling pathways. The key role of these processes in tumorigenesis suggests a possible involvement of PKCζ in this event. PKCζ is activated by cytotoxic treatments, inhibits apoptotic cell death and reduces the sensitivity of cancer cells to chemotherapeutic agents. Here, using pharmacological and DNA recombinant approaches, we show that oxidative stress triggers nuclear translocation of PKCζ and induces resistance to apoptotic agents. Accordingly, chemoresistant cells show accumulation of PKCζ within the nucleus, and a nuclear-targeted PKCζ transfected in tumor cells decreases sensitivity to apoptosis. We thus developed a novel recombinant protein capable of selectively inhibiting the nuclear fraction of PKCζ that restored the susceptibility to apoptosis in cells in which PKCζ was enriched in the nuclear fraction, including chemoresistant cells. These findings establish the importance of PKCζ as a possible target to increase the effectiveness of anticancer therapies and highlight potential sites of intervention.Key words: protein kinase C, chemoresistance, oxidative stress, nuclear translocation, apoptosis     

Mammalian Homologue of the Caenorhabditis elegans UNC-76 Protein Involved in Axonal Outgrowth Is a Protein Kinase C ζ–interacting Protein

By the yeast two-hybrid screening of a rat brain cDNA library with the regulatory domain of protein kinase C ζ (PKCζ) as a bait, we have cloned a gene coding for a novel PKCζ-interacting protein homologous to the Caenorhabditis elegans UNC-76 protein involved in axonal outgrowth and fasciculation. The protein designated FEZ1 (fasciculation and elongation protein zeta-1) consisting of 393 amino acid residues shows a high Asp/Glu content and contains several regions predicted to form amphipathic helices. Northern blot analysis has revealed that FEZ1 mRNA is abundantly expressed in adult rat brain and throughout the developmental stages of mouse embryo. By the yeast two-hybrid assay with various deletion mutants of PKC, FEZ1 was shown to interact with the NH₂-terminal variable region (V1) of PKCζ and weakly with that of PKCε. In the COS-7 cells coexpressing FEZ1 and PKCζ, FEZ1 was present mainly in the plasma membrane, associating with PKCζ and being phosphorylated. These results indicate that FEZ1 is a novel substrate of PKCζ. When the constitutively active mutant of PKCζ was used, FEZ1 was found in the cytoplasm of COS-7 cells. Upon treatment of the cells with a PKC inhibitor, staurosporin, FEZ1 was translocated from the cytoplasm to the plasma membrane, suggesting that the cytoplasmic translocation of FEZ1 is directly regulated by the PKCζ activity. Although expression of FEZ1 alone had no effect on PC12 cells, coexpression of FEZ1 and constitutively active PKCζ stimulated the neuronal differentiation of PC12 cells. Combined with the recent finding that a human FEZ1 protein is able to complement the function of UNC-76 necessary for normal axonal bundling and elongation within axon bundles in the nematode, these results suggest that FEZ1 plays a crucial role in the axon guidance machinery in mammals by interacting with PKCζ.     

Targeted Activation of Conventional and Novel Protein Kinases C through Differential Translocation Patterns

Activation of the two ubiquitous families of protein kinases, protein kinase A (PKA) and protein kinase C (PKC), is thought to be independently coupled to stimulation of Gα_s and Gα_q, respectively. Live-cell confocal imaging of protein kinase C fluorescent protein fusion constructs revealed that simultaneous activation of Gα_s and Gα_q resulted in a differential translocation of the conventional PKCα to the plasma membrane while the novel PKCδ was recruited to the membrane of the endoplasmic reticulum (ER). We demonstrate that the PKCδ translocation was driven by a novel Gα_s-cyclic AMP-EPAC-RAP-PLCε pathway resulting in specific diacylglycerol production at the membrane of the ER. Membrane-specific phosphorylation sensors revealed that directed translocation resulted in phosphorylation activity confined to the target membrane. Specific stimulation of PKCδ caused phosphorylation of the inositol-1,4,5-trisphosphate receptor and dampening of global Ca²⁺ signaling revealed by graded flash photolysis of caged inositol-1,4,5-trisphosphate. Our data demonstrate a novel signaling pathway enabling differential decoding of incoming stimuli into PKC isoform-specific membrane targeting, significantly enhancing the versatility of cyclic AMP signaling, thus demonstrating the possible interconnection between the PKA and PKC pathways traditionally treated independently. We thus provide novel and elementary understanding and insights into intracellular signaling events.     

Genetic Analysis of the Role of Protein Kinase Cθ in Platelet Function and Thrombus Formation

Background

PKCθ is a novel protein kinase C isozyme, predominately expressed in T cells and platelets. PKCθ^−/− T cells exhibit reduced activation and PKCθ^−/− mice are resistant to autoimmune disease, making PKCθ an attractive therapeutic target for immune modulation. Collagen is a major agonist for platelets, operating through an immunoreceptor-like signalling pathway from its receptor GPVI. Although it has recently been shown that PKCθ positively regulates outside-in signalling through integrin α_IIbβ₃ in platelets, the role of PKCθ in GPVI-dependent signalling and functional activation of platelets has not been assessed.

Methodology/Principal Findings

In the present study we assessed static adhesion, cell spreading, granule secretion, integrin α_IIbβ₃ activation and platelet aggregation in washed mouse platelets lacking PKCθ. Thrombus formation on a collagen-coated surface was assessed in vitro under flow. PKCθ^−/− platelets exhibited reduced static adhesion and filopodia generation on fibrinogen, suggesting that PKCθ positively regulates outside-in signalling, in agreement with a previous report. In contrast, PKCθ^−/− platelets also exhibited markedly enhanced GPVI-dependent α-granule secretion, although dense granule secretion was unaffected, suggesting that PKCθ differentially regulates these two granules. Inside-out regulation of α_IIbβ₃ activation was also enhanced downstream of GPVI stimulation. Although this did not result in increased aggregation, importantly thrombus formation on collagen under high shear (1000 s⁻¹) was enhanced.

Conclusions/Significance

These data suggest that PKCθ is an important negative regulator of thrombus formation on collagen, potentially mediated by α-granule secretion and α_IIbβ₃ activation. PKCθ therefore may act to restrict thrombus growth, a finding that has important implications for the development and safe clinical use of PKCθ inhibitors.     

Regulations of Reversal of Senescence by PKC Isozymes in Response to 12-O-Tetradecanoylphorbol-13-Acetate via Nuclear Translocation of pErk1/2

The mechanism by which 12-O-tetradecanoylphorbol-13-acetate (TPA) bypasses cellular senescence was investigated using human diploid fibroblast (HDF) cell replicative senescence as a model. Upon TPA treatment, protein kinase C (PKC) α and PKCβ1 exerted differential effects on the nuclear translocation of cytoplasmic pErk1/2, a protein which maintains senescence. PKCα accompanied pErk1/2 to the nucleus after freeing it from PEA-15pS¹⁰⁴ via PKCβ1 and then was rapidly ubiquitinated and degraded within the nucleus. Mitogen-activated protein kinase docking motif and kinase activity of PKCα were both required for pErk1/2 transport to the nucleus. Repetitive exposure of mouse skin to TPA downregulated PKCα expression and increased epidermal and hair follicle cell proliferation. Thus, PKCα downregulation is accompanied by in vivo cell proliferation, as evidenced in 7, 12-dimethylbenz(a)anthracene (DMBA)-TPA-mediated carcinogenesis. The ability of TPA to reverse senescence was further demonstrated in old HDF cells using RNA-sequencing analyses in which TPA-induced nuclear PKCα degradation freed nuclear pErk1/2 to induce cell proliferation and facilitated the recovery of mitochondrial energy metabolism. Our data indicate that TPA-induced senescence reversal and carcinogenesis promotion share the same molecular pathway. Loss of PKCα expression following TPA treatment reduces pErk1/2-activated SP1 biding to the p21^WAF1 gene promoter, thus preventing senescence onset and overcoming G1/S cell cycle arrest in senescent cells.     

Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Phosphorylation by Protein Kinase Cδ (PKCδ) Inhibits Mitochondria Elimination by Lysosomal-like Structures following Ischemia and Reoxygenation-induced Injury

After cardiac ischemia and reperfusion or reoxygenation (I/R), damaged mitochondria propagate tissue injury by promoting cell death. One possible mechanism to protect from I/R-induced injury is the elimination of damaged mitochondria by mitophagy. Here we identify new molecular events that lead to mitophagy using a cell culture model and whole hearts subjected to I/R. We found that I/R induces glyceraldehyde-3-phosphate dehydrogenase (GAPDH) association with mitochondria and promotes direct uptake of damaged mitochondria into multiorganellar lysosomal-like (LL) structures for elimination independently of the macroautophagy pathway. We also found that protein kinase C δ (PKCδ) inhibits GAPDH-driven mitophagy by phosphorylating the mitochondrially associated GAPDH at threonine 246 following I/R. Phosphorylated GAPDH promotes the accumulation of mitochondria at the periphery of LL structures, which coincides with increased mitochondrial permeability. Either inhibition of PKCδ or expression of a phosphorylation-defective GAPDH mutant during I/R promotes a reduction in mitochondrial mass and apoptosis, thus indicating rescued mitophagy. Taken together, we identified a GAPDH/PKCδ signaling switch, which is activated during oxidative stress to regulate the balance between cell survival by mitophagy and cell death due to accumulation of damaged mitochondria.     

Targeting of Protein Kinase Cα to Caveolae

Previously, we showed caveolae contain a population of protein kinase Cα (PKCα) that appears to regulate membrane invagination. We now report that multiple PKC isoenzymes are enriched in caveolae of unstimulated fibroblasts. To understand the mechanism of PKC targeting, we prepared caveolae lacking PKCα and measured the interaction of recombinant PKCα with these membranes. PKCα bound with high affinity and specificity to caveolae membranes. Binding was calcium dependent, did not require the addition of factors that activate the enzyme, and involved the regulatory domain of the molecule. A 68-kD PKCα-binding protein identified as sdr (serum deprivation response) was isolated by interaction cloning and localized to caveolae. Antibodies against sdr inhibited PKCα binding. A 100–amino acid sequence from the middle of sdr competitively blocked PKCα binding while flanking sequences were inactive. Caveolae appear to be a membrane site where PKC enzymes are organized to carry out essential regulatory functions as well as to modulate signal transduction at the cell surface.     

IFNγ-mediated inhibition of cell proliferation through increased PKCδ-induced overexpression of EC-SOD

Extracellular superoxide dismutase (EC-SOD) overexpression modulates cellular responses such as tumor cell suppression and is induced by IFNγ. Therefore, we examined the role of EC-SOD in IFNγ-mediated tumor cell suppression. We observed that the dominant-negative protein kinase C delta (PKCδ) suppresses IFNγ-induced EC-SOD expression in both keratinocytes and melanoma cells. Our results also showed that PKCδ-induced ECSOD expression was reduced by pretreatment with a PKCspecific inhibitor or a siRNA against PKCδ. PKCδ-induced ECSOD expression suppressed cell proliferations by the up-regulation of p21 and Rb, and the downregulation of cyclin A and D. Finally, we demonstrated that increased expression of EC-SOD drastically suppressed lung melanoma proliferation in an EC-SOD transgenic mouse via p21 expression. In summary, our findings suggest that IFNγ-induced EC-SOD expression occurs via activation of PKCδ. Therefore, the upregulation of EC-SOD may be effective for prevention of various cancers, including melanoma, via cell cycle arrest. [BMB Reports 2012; 45(11): 659-664]     

C-Terminal Domain of ICA69 Interacts with PICK1 and Acts on Trafficking of PICK1-PKCα Complex and Cerebellar Plasticity

Background

PICK1 (protein interacting with C-kinase 1) is a PKC (protein kinase C)-binding protein, which is essential for synaptic plasticity. The trafficking of PKCα-PICK1 complex to plasma membrane is critical for the internalization of GluR2 and induction of long-term depression. ICA69 (islet cell autoantigen 69 kDa) is identified as a major binding partner of PICK1. While heteromeric BAR domain complex is suggested to underlie the interaction between PICK1 and ICA69, the role of C-terminal domain of ICA69 (ICAC) in PICK1-ICA69 complex is unknown.

Methodology/Principal Findings

We found that ICAC interacted with PICK1 and regulated the trafficking of PICK1-PKCα complex. ICAC and ΔICAC (containing BAR domain) might function distinctly in the association of ICA69 with PICK1. While ΔICAC domain inclined to form clusters, the distribution of ICAC was diffuse. The trafficking of PICK1 to plasma membrane mediated by activated PKCα was inhibited by ICA69. This action might ascribe to ICAC, because overexpression of ICAC, but not ΔICAC, interrupted PKCα-mediated PICK1 trafficking. Notably, infusion of maltose binding protein (MBP) fusion protein, MBP-ICA69 or MBP-ICAC, in cerebellar Purkinje cells significantly inhibited the induction of long-term depression at parallel fiber- and climbing fiber-Purkinje cell synapses.

Conclusions

Our experiments showed that ICAC is an important domain for the ICA69-PICK1 interaction and plays essential roles in PICK1-mediated neuronal plasticity.     

Inhibiting Tyrosine Phosphorylation of Protein Kinase Cδ (PKCδ) Protects the Salivary Gland from Radiation Damage

Radiation therapy for head and neck cancer can result in extensive damage to normal adjacent tissues such as the salivary gland and oral mucosa. We have shown previously that tyrosine phosphorylation at Tyr-64 and Tyr-155 activates PKCδ in response to apoptotic stimuli by facilitating its nuclear import. Here we have identified the tyrosine kinases that mediate activation of PKCδ in apoptotic cells and have explored the use of tyrosine kinase inhibitors for suppression of irradiation-induced apoptosis. We identify the damage-inducible kinase, c-Abl, as the PKCδ Tyr-155 kinase and c-Src as the Tyr-64 kinase. Depletion of c-Abl or c-Src with shRNA decreased irradiation- and etoposide-induced apoptosis, suggesting that inhibitors of these kinases may be useful therapeutically. Pretreatment with dasatinib, a broad spectrum tyrosine kinase inhibitor, blocked phosphorylation of PKCδ at both Tyr-64 and Tyr-155. Expression of “gate-keeper” mutants of c-Abl or c-Src that are active in the presence of dasatinib restored phosphorylation of PKCδ at Tyr-155 and Tyr-64, respectively. Imatinib, a c-Abl-selective inhibitor, also specifically blocked PKCδ Tyr-155 phosphorylation. Dasatinib and imatinib both blocked binding of PKCδ to importin-α and nuclear import, demonstrating that tyrosine kinase inhibitors can inhibit nuclear accumulation of PKCδ. Likewise, pretreatment with dasatinib also suppressed etoposide and radiation induced apoptosis in vitro. In vivo, pre-treatment of mice with dasatinib blocked radiation-induced apoptosis in the salivary gland by >60%. These data suggest that tyrosine kinase inhibitors may be useful prophylactically for protection of nontumor tissues in patients undergoing radiotherapy of the head and neck.     

Regulation of Basal Lateral Membrane Mobility and Permeability to Divalent Cations by Membrane Associated-Protein Kinase C

Biological membrane stabilization is essential for maintenance of cellular homeostasis, functionality and appropriate response to various stimuli. Previous studies have showed that accumulation of PKCs in the cell membrane significantly downregulates the membrane fluidity and Ca²⁺ influxes through the membranes in activated cells. In addition, membrane-inserted form of PKCs has been found in a variety of resting mammalian cells and tissues. This study is aimed to investigate possible role of the endogenous membrane-associated PKCs in the modulation of basal membrane fluidity. Here, we showed that interfering PKC expression by chronic activation of PKC with phorbol myristate acetate (PMA) or shRNA targeting at PKCα lowered the levels of PKCα in cytosol, peripheral membrane and integral membrane pools, while short-term activation of PKC with PMA induced accumulation of PKCα in the membrane pool accompanied by a dramatic decrease in the cytosol fraction. The lateral membrane mobility increased or decreased in accordance with the abundance alterations in the membrane-associated PKCα by these treatments. In addition, membrane permeability to divalent cations including Ca²⁺, Mn²⁺ and Ba²⁺ were also potentiated or abrogated along with the changes in PKC expression on the plasma membrane. Membrane stabilizer ursodeoxycholate abolished both of the enhanced lateral membrane mobility and permeability to divalent cations due to PKCα deficiency, whereas Gö6983, a PKC antagonist, or Gd³⁺ and 2-aminoethyoxydipheyl borne, two Ca²⁺ channels blockers, showed no effect, suggesting that this PKC-related regulation is independent of PKC activation or a modulation of specific divalent cation channel. Thus, these data demonstrate that the native membrane-associated PKCα is involved in the maintenance of basal membrane stabilization in resting cells.     

Widespread Expression of PICOT in Mouse and Human Tissues With Predominant Localization to Epithelium

The protein kinase C-interacting cousin of thioredoxin (PICOT; also termed glutaredoxin 3) protein was discovered a decade ago as a protein kinase C θ (PKCθ)-binding protein in human T lymphocytes. PICOT possesses an amino-terminal monothiol thioredoxin-like domain and a carboxy-terminal tandem repeat of a monothiol glutaredoxin-like domain. Nevertheless, the enzymatic activities of PICOT and its potential substrates have not yet been characterized and its biological importance is unknown. Earlier studies reported the presence of PICOT in several different cell lines and tissues, but its expression pattern has not been thoroughly investigated. We performed Northern blot analysis of 19 different human organs and tissues and found the expression of PICOT mRNA in all organs and tissues tested. Western blot analysis confirmed the expression of PICOT at the protein level in all organs and tissues tested and showed, in addition, that PICOT and PKCθ expression in different tissues only partially overlap. These findings support the involvement of PICOT in biological functions that are independent of PKCθ. To analyze the in vivo expression pattern of PICOT within cells of different human organs, we performed immunohistochemical staining using PICOT-specific antibodies. Analysis of breast, pituitary, adrenal, pancreas, and kidney sections demonstrated a differential expression of PICOT in various cell types, with a predominant cytosolic staining of epithelial cells and low or undetectable levels of PICOT in the stroma. (J Histochem Cytochem 58:799–806, 2010)