Atypical Protein Kinase C-iota Research Articles

Abstract 644: PKC-iota specific inhibitor ICA-1S causes the degradation of mutant p53 in ovarian cancer cell lines

Abstract Ovarian cancer is the most lethal malignant disease of female. Treatment options for ovarian cancer are mostly the use of chemotherapeutics, surgery, or radiotherapy. However, due to resistance to drugs, relapse occurs and thus 5 years survival rate is only 47%. The tumor suppressor protein p53 works as a transcription factor to transcribe several proteins such as p21, p16 and PUMA that lead to halt cell cycle and also induce apoptosis when there is a damage in DNA. Mutation in several regions of p53 (mutp53) is quite common in cancers and can lead to the accumulation of p53 with loss of functions or sometimes gain of oncogenic functions. Thus there is a search for drugs that can degrade the mut53 and can restore the functions of normal p53. In this research, it was observed that the treatment of ovarian cancer cell lines (ES-2, HEY and OVCAR3) with atypical protein kinase C-iota (aPKCɩ) specific inhibitors ICA-1S can cause the degradation of mutp53 (53.6%, N=6 trials). Treatment with ICA-1S also lowered the level of phosphorylated p53 (ser15, 87%, N=3 trials). However, the level of MDM2 protein (murine double minute 2), a known p53 E3 ligase was not significantly (98%, N=2 trials) affected by ICA-1S in ES-2 cell line with mut p53 as well as p21 was also not affected. However level of PUMA was decreased both in ES-2 and HEY cell lines. Immunoprecipitation (IP) with aPKCɩ specific beads showed the aPKCɩ is associated with p53, though reverse IP with p53 beads was not able to show the association. Application of proteasome inhibitor MG-132 at a single dose of 0.5µM could not completely inhibit the degradation of mut53 and WTp53 in ICA-1S treated ES-2 cell line and HEY cell line, respectively, indicating the drug may be affecting the transcription of p53 as well. It is therefore postulated that aPKCɩ can phosphorylate p53, stabilizing it and may be affecting the transcription of p53 as well in those cell lines. Further research (kinase activity assay, immunoprecipitation, immunofluorescence) is going on to reveal the mode of this degradation. Citation Format: Mahfuza Marzan, Nuzhat Nowshin Oishee, Abigail Olatunji, Mildred Acevedo-Duncan. PKC-iota specific inhibitor ICA-1S causes the degradation of mutant p53 in ovarian cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 644.

Abstract 4382: PKC-ι/ζ signaling is crucial for apoptosis/pyroptosis inhibition and invasiveness of glioblastoma cells through upregulation of β-catenin, PDK1/Akt1 and Smad cascades via 14-3-3

Abstract Glioblastoma (GB) is an aggressive form of brain cancer derived from astrocytes. Poor predictability of patient outcomes has limited the development of current therapeutic treatments of GB mainly due to the development of drug resistance. Comparative over-expression of atypical protein kinase C-iota and zeta (aPKC-ι/ζ) in GB cells and tissue samples were previously reported. This study establishes the downstream effects of PKC-ι/ζ attenuation on key cellular signaling which governs apoptosis, pyroptosis, survival, proliferation and epithelial-mesenchymal transition (EMT). Our data suggested that aPKC knockdown of expression induces apoptosis and works as an antecedent of pyroptosis. Data also suggested that involvement of Akt1 and PDK1 in aPKC maturation process weakened due to aPKC attenuation. aPKC attenuation downregulated Ras/Erk1 and canonical β-catenin pathways which resulted in reduced transcriptional activities of Stat3, c-Myc, c-Jun and β-catenin. In addition SNAIL1, SLUG, and PRRX1 were diminished that have been known to stimulate EMT. Proteins 14-3-3 and Smad2/3 acted as molecular adaptors between these pathways. A PKC-ι specific inhibitor 5-amino-1-(2,3-dihydroxy-4-(hydroxymethyl)cyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S) and a PKC-ζ specific inhibitor 8-hydroxy-1,3,6-naphthalenetrisulfonic acid (ζ-Stat) were used to conduct in-vivo experiments using mouse models. Intravenous and oral administration of ICA-1S resulted in the reduction of tumor growth by approximately 30% in athymic nude mice for U-87 xenografts. Taken together, these results suggest that not only do aPKCs play a central role in GB progression, invasiveness, and cell survival, but that effective therapeutics can be developed to specifically target oncogenic aPKCs. Citation Format: Luke Lajmi, Wishrawana S. Ratnayake, Khandker M. Khalid, Sloan Breedy, Aaron Todman, Tracess Smalley, Christopher A. Apostolatos, Robert Hill, Mildred Acevedo-Duncan. PKC-ι/ζ signaling is crucial for apoptosis/pyroptosis inhibition and invasiveness of glioblastoma cells through upregulation of β-catenin, PDK1/Akt1 and Smad cascades via 14-3-3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4382.

Abstract 164: Oncogenic PKC-ι/PKC-ζ/14-3-3 and PKC-ι/PKC-ζ/Smad2/3 signaling cascades are crucial for increased invasiveness of Neuroblastoma cells

Abstract Neuroblastoma (NB) is a the pediatric cancer of the early nerve cells in which solid tumors originate from the peripheral sympathetic nervous system and as such can occur primarily anywhere along the sympathetic chain with development on the adrenal gland being most common. The expression of the disease is highly variable as in some cases lead to spontaneously regression without any medical intervention whereas in other cases the tumors become highly metastatic and therapeutically resistant. Our results demonstrated that atypical protein kinase C-iota and zeta (aPKC-ι/ζ) levels were overexpressed in NB cells (BE-2C and BE-M17) and tissues. The present study shows the downstream effects PKC-ι specific inhibitor 5-amino-1-(2,3-dihydroxy-4-(hydroxymethyl)cyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S) and a PKC-ζ specific inhibitor 8-hydroxy-1,3,6-naphthalenetrisulfonic acid (ζ-Stat) on aPKCs, 14-3-3, p-14-3-3, Akt1, NF-κB p65, Smad2/3 and p-Smad2/3. Decreased Vimentin and increased E-cadherin levels indicated the downregulation of epithelial-mesenchymal transition (EMT). Result also indicated that SNAIL1 transcriptional activity was downregulated as a result of aPKC diminution. SNAIL1 is known to induce EMT. 14-3-3 proteins have the ability to bind a multitude of functionally diverse signaling proteins, including kinases, phosphatases, and transmembrane receptors. Our immunoprecipitation (IP) data suggested that both aPKCs associate with 14-3-3 and Smad2/3. Our data also indicated that both 14-3-3 and Smad2/3 levels were significantly decreased as a result of aPKC inhibition. In addition, pIκB, NFκB p65, p-14-3-3, pAkt1, levels were significantly decreased. Our data indicates that 14-3-3 acts as a central key regulator in aPKC driven cell cycle progression in BE-2C and BE-M17 cells. In addition, we found that Smad2/3 plays a vital role in upregulation of aPKC driven Vimentin dynamics in NB cell lines in relation to 14-3-3. In-vitro migration/invasion assays, IP, immunofluorescence, real time qPCR and Western blot techniques were used to further analyze role of aPKCs in NB malignancy. Data confirmed that aPKC attenuation triggers multiple pro-apoptotic signaling cascades through downregulation of NFκB, Akt, and Smad pathways through downregulation of 14-3-3. In-vivo experiments on murine models are being conducted using ICA-1S and ζ-Stat. Excised tumors will be analyzed for pathways observed in in-vitro experiments. Overall results suggest that aPKCs are crucial for increased invasiveness in NB. Results suggested that aPKC can be targeted to develop customized, tailored therapies for NB which merit further research. Citation Format: Sloan Breedy. Oncogenic PKC-ι/PKC-ζ/14-3-3 and PKC-ι/PKC-ζ/Smad2/3 signaling cascades are crucial for increased invasiveness of Neuroblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 164.

Abstract 150: Prevalent PKC-ι/ζ signaling is crucial for apoptosis inhibition and invasiveness of glioblastoma through upregulation of Pdk1/Akt1/14-3-3 cascade and Smad2/3

Abstract Glioblastoma (GB) is an aggressive form of cancer derived from astrocytes and often found in brain or spinal cord. Poor prognosis of GB has limitations in current therapeutics mainly due to the development of resistance. Our results demonstrated that atypical protein kinase C-iota and zeta (aPKC-ι/ζ) levels were over-expressed in GB cells (U-87 and T-98) and tissue samples compared to normal brain cells/tissues which is correlated with increased invasiveness. The present study shows the downstream effects of siRNA knockdown of PKC-ι/ζ on the expression of aPKCs, E-cadherin, Vimentin, p-Vimentin, 14-3-3 and p-14-3-3, BAD, p-BAD, Pdk1, Akt1, c-Raf, Tak1, Smad2/3 and p-Smad2/3. Downregulation of Vimentin, p-Vimentin (S33 and S56) and increased E-cadherin indicated the downregulation of epithelial-mesenchymal transition (EMT). Proteolytic E-cadherin fragment of 80 kDa indicates the reduction of cancer progression. Result also indicated that SNAIL1, SLUG and PRRX1 transcriptional activities were downregulated as a result of aPKC diminution. These transcription factors are known to induce and promote EMT. 14-3-3 is known to bind Pdk1, BAD, Akt1 and c-Raf and perform various anti-apoptotic tasks. Our results indicated that total 14-3-3 levels were significantly increased as a result of aPKC attenuation along with dephosphorylated BAD. In addition, p-14-3-3, p-BAD, pAkt1, Pdk1 levels were significantly decreased. Unphosphorylated 14-3-3 does not bind with their partners: phosphorylation on conserved S259 and S621 through Tak1 which is essential for its activation. Our results indicated that Tak1 levels significantly decreased as a result of aPKC diminution. In-vitro migration/invasion assays, immunoprecipitation (IP), immunofluorescence and immuno-gold transmission electron microscopic techniques, real time qPCR and Western blot techniques are being used in this study to further analyze role of aPKCs in GB malignancy. Our IP and kinase activity assay data confirmed that PKC-ι heavily interacts with Pdk1 and PKC-ζ interacts with Akt1, thereby activating those two kinases via phosphorylation. Data confirmed that aPKC attenuation triggers multiple pro apoptotic signaling cascades through downregulation of Akt1, Pdk1, 14-3-3 and Smad2/3.We have used a novel PKC-ι specific inhibitor 5-amino-1-(2,3-dihydroxy-4-(hydroxymethyl)cyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S) and a PKC-ζ specific inhibitor 8-hydroxy-1,3,6-naphthalenetrisulfonic acid (ζ-Stat) to conduct in-vivo experiments on murine models. Excised tumors will be analyzed for pathways observed in in-vitro experiments. Overall results suggest that aPKCs are crucial for increased invasiveness in GB. Results suggested that aPKC can be targeted to develop customized, tailored therapies for Glioblastoma which merit further research. Citation Format: Wishrawana Sarathi Ratnayake, Khandker M. Khalid, Sloan Breedy, Luke Lajmi, Tracess Smalley, Christopher A. Apostolatos, Robert Hill, James Fackrell, Mildred Acevedo-Duncan. Prevalent PKC-ι/ζ signaling is crucial for apoptosis inhibition and invasiveness of glioblastoma through upregulation of Pdk1/Akt1/14-3-3 cascade and Smad2/3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 150.

Abstract 5216: Atypical PKC inhibitors ICA-1S and ζ-Stat show inhibition of neuroblastoma cell proliferation

Abstract Neuroblastoma, the pediatric cancer of the early nerve cells, has the tendency to become resistant to traditional therapeutics due to its highly heterogeneous nature. Atypical Protein Kinase C-iota (PKC- ι) has been found to promote cell proliferation in neuroblastoma cells through the PKC- ι /Cdk7/Cdk2 signaling pathway. In this study we utilized various cell lines; I-type (intermediate) CHP-212& BE(2)-C, N-types (neuroblastic); BE(2)-M17& SH-SY5Y and S-types (substrate adherent); SK-N-BE(2) & LAI-5s, representing neuroblastoma heterogeneity to investigate the use of aPKC inhibitors as potential neuroblastoma therapeutics. The aPKC inhibitors, 5-amino-1-2,3-dihydroxy-4-(methylcyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S) (PKC- ι specific) and 8-hydroxy-1,3,6-naphthalenetrisulfonic acid (ζ-Stat) (PKC- ζ specific), both display the ability to reduce neuroblastoma proliferation in a cell type specific manner. ICA-1S was shown to reduce proliferation of BE(2)-C, BE(2)-M17, LAI-5s and SK-N-BE(2) to varying degrees. ζ-Stat was able to consistently reduce proliferation of BE(2)-C, BE(2)-M17, SK-N-BE(2), LAI-5s. The degree of proliferation inhibition varied by drug and cell line. Increasing concentration of ICA-1S displayed an inverse relationship between increasing concentration and a decrease in cell proliferation in the four cell lines previously mentioned. ζ-Stat showed a similar relationship to ICA-1S, however had a lower level inhibition than ICA-1S. WST-1 analysis showed that both drugs displayed varying degrees of toxicity in a cell line dependent manner. The results show that I-type cell lines showed the most linearly proportional toxicity to ICA-1S and ζ-Stat in that increasing concentration increased drug toxicity. N-type cell lines displayed a disproportional relationship between concentration and toxicity. The S-type cell lines followed suit with the N-type in their varying degrees of toxicity. Western blot analysis shows that both inhibitors were effective in reducing the expression of total aPKC levels. We observed the downregulation of various mesenchymal markers using the inhibitors. In particular, ICA-1S appears to have a high percentage of decreased expression of N-cadherin. We also accessed the degree of phosphorylation of Vimentin, S33, S39 and S56 positions which are crucial for its activation. In summary both inhibitors prove to be promising potential neuroblastoma therapeutics. The drugs appear to behave in a more cytostatic manner inhibiting the proliferation of neuroblastoma cells. Further western blots ae still ongoing to determine other pathways through which the drugs act to decrease proliferation. Ongoing experiments include the analysis of tumors collected from mouse models to ascertain the effects of the drugs in vivo as well as the analysis of human tumor samples to ascertain the expression of aPKCs and other markers in patient populations. Experiments also include the use of gene expression knockdowns to compare with the effects from the inhibitors as well as the use of microscopy techniques; fluorescent and electron miscopy to observe the localization of mesenchymal and proliferative markers. Citation Format: Sloan Breedy, Wishrawana S. Ratnayake, Christopher Apostolatos, Avijit Dey, Mildred Acevedo-Duncan. Atypical PKC inhibitors ICA-1S and ζ-Stat show inhibition of neuroblastoma cell proliferation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5216.

Fragment-based Discovery of a Small-Molecule Protein Kinase C-iota Inhibitor Binding Post-kinase Domain Residues.

The atypical protein kinase C-iota (PKC-ι) enzyme is implicated in various cancers and has been put forward as an attractive target for developing anticancer therapy. A high concentration biochemical screen identified pyridine fragment weakly inhibiting PKC-ι with IC50 = 424 μM. Driven by structure-activity relationships and guided by docking hypothesis, the weakly bound fragment was eventually optimized into a potent inhibitor of PKC-ι (IC50= 270 nM). Through the course of the optimization, an intermediate compound was crystallized with the protein, and careful analysis of the X-ray crystal structure revealed a unique binding mode involving the post-kinase domain (C-terminal tail) of PKC-ι.

14-3-3ζ/atypical protein kinase C-ιsynergistically facilitate the invasion and metastasis of cholangiocarcinoma via epithelial-mesenchymaltransition

Objective To illuminate the mechanism of 14-3-3ζ/atypical protein kinase C-iota (aPKC-ι) to promote the invasion and metastasis of cholangiocarcinoma (CCA). Methods The expression of 14-3-3ζ and aPKC-ιwereexamined in the samples of 64 CCA, peritumoural and normal tissues respectively by immunohistochemistry, Western blotting and quantitative real-time polymerase chain reaction. And the independent prognostic factors were analyzed by Kaplan-Meier survival analysis and Multivariate Cox regression analysis. Furthermore, theepithelial-mesenchymaltransition (EMT) model of CCA mediated by Transforming Growth Factor-β1 (TGF-β1) was established to analyze the regulation mechanism of 14-3-3ζand aPKC-ι. Finally, the capacity of invasion, migration, proliferation and metastasis of CCA cells with14-3-3ζdepleted were assessed by transwell cell invasion, wound healing, colony formation assays in vitro. Results Overexpression of 14-3-3ζ and aPKC-ιwas detected in CCA tissue (56.25%, 36/64, and 51.56%, 33/64), there was a significant positive correlation between 14-3-3ζ and aPKC-ι(r=0.406, P=0.001). Low expression of14-3-3ζ or aPKC-ιhad a better overall survival compared with those of overexpression (14-3-3ζ: χ2=10.140, P=0.001; aPKC-ι: χ2=12.575, P=0.000). Moreover, both 14-3-3ζ and aPKC-ιwere the independent prognostic factors of CCA. Interfering with 14-3-3ζ or aPKC-ιsignificantly suppressed the changes in EMT-related markers in CCA cells. In vitro, the inhibition of 14-3-3ζ impaired CCA cell invasion, metastasis and proliferation. Conclusion Our study demonstrated that 14-3-3ζ may combine with aPKC-ιandsynergistically facilitate EMT of CCA to promote CCA invasion and metastasis. Key words: 14-3-3ζ; Atypical protein kinase C-iota; Cholangiocarcinoma; Epithelial-mesenchymal transition; Invasion and metastasis

A Novel Atypical PKC-Iota Inhibitor, Echinochrome A, Enhances Cardiomyocyte Differentiation from Mouse Embryonic Stem Cells.

Echinochrome A (EchA) is a marine bioproduct extracted from sea urchins having antioxidant, antimicrobial, anti-inflammatory, and chelating effects, and is the active component of the clinical drug histochrome. We investigated the potential use of Ech A for inducing cardiomyocyte differentiation from mouse embryonic stem cells (mESCs). We also assessed the effects of Ech A on mitochondrial mass, inner membrane potential (Δψm), reactive oxygen species generation, and levels of Ca2+. To identify the direct target of Ech A, we performed in vitro kinase activity and surface plasmon resonance binding assays. Ech A dose-dependently enhanced cardiomyocyte differentiation with higher beating rates. Ech A (50 μM) increased the mitochondrial mass and membrane potential but did not alter the mitochondrial superoxide and Ca2+ levels. The in vitro kinase activity of the atypical protein kinase C-iota (PKCι) was significantly decreased by 50 μM of Ech A with an IC50 for PKCι activity of 107 μM. Computational protein-ligand docking simulation results suggested the direct binding of Ech A to PKCι, and surface plasmon resonance confirmed the direct binding with a low KD of 6.3 nM. Therefore, Ech A is a potential drug for enhancing cardiomyocyte differentiation from mESCs through direct binding to PKCι and inhibition of its activity.

APKC-ι/P-Sp1/Snail signaling induces epithelial-mesenchymal transition and immunosuppression in cholangiocarcinoma.

These results demonstrate that aPKC-ι promotes EMT and induces immunosuppression through the aPKC-ι/P-Sp1/Snail signaling pathway and may be a potential therapeutic target for CCA. (Hepatology 2017;66:1165-1182).

Abstract 7: PKC-ι promotes glioblastoma cell survival by phosphorylating and inhibiting BAD through a phosphotidylinositol 3-kinase pathway

Abstract Robust expression of atypical protein kinase C-iota (PKC-ι) is a hallmark of human glioma, benign and malignant meningiomas [R. Patel, et. al. (2008) Cell Prolif. 41, 122-135]. The phosphoinositide (PI) 3-kinase pathway is frequently found to be over stimulated due to activating mutations in growth factor receptors or loss of function of the tumor suppressor PTEN. Protein kinase C-ι is often activated by the PI 3-kinase pathway and is an essential downstream targeted mediator. We investigated the role of PKC-ι in two human glial tumor derived cell lines T98G and U87MG. PKC-ι directly phosphorylated and inactivated Bad, a pro-apoptotic BH3-only molecule of the Bcl-2 family at three phospho specific residues, Ser-112, Ser-136 and Ser-155. PKC-ι was also observed to co-localize and directly associate with Bad in the cytoplasm of the cells (as shown by Immunofluorescence, Immunoprecipitation, and Western blotting). Furthermore, purified, active PKC-ι induced direct phosphorylation of Bad at all three serine residues subsequently disrupting Bad/Bcl-XL dimerization. Reduction of PKC-ι upon RNA interference exhibited a corresponding reduction in Bad phosphorylation as did Wortmannin and LY294002, known PI3-Kinase inhibitors. Depletion of PKC-ι by siRNA treatment also induced apoptosis with release of cytochrome C, caspase 3 activation and PARP cleavage. These data provide evidence for novel PI3-kinase/PKC-ι/BAD cell survival pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 7. doi:10.1158/1538-7445.AM2011-7

Role of protein kinase C‐iota in transformed non‐malignant RWPE‐1 cells and androgen‐independent prostate carcinoma DU‐145 cells

Prostate cancer is one of the leading causes of death among men in the USA. In this study, we investigated the role of atypical protein kinase C-iota (PKC-iota) in androgen independent prostate DU-145 carcinoma cellscompared to transformed non-malignant prostate RWPE-1 cells. Western blotting and immunoprecipitations demonstrated that PKC-iotaisassociated with cyclin-dependent kinase activating kinase (CAK/Cdk7) in RWPE-1 cells, but not in DU-145 cells. Treatment of prostate RWPE-1 cells with PKC-iota silencing RNA (siRNA) decreased cell viability,cell-cycle accumulation at G2/M phase, and phosphorylation of Cdk7 and Cdk2. In addition, PKC-iota siRNA treatment caused less phosphorylation ofBad at ser-155, ser-136, and greater Bad/Bcl-xL heterodimerization, leading to apoptosis. In DU-145 cells, PKC-iota was anti-apoptotic and was required for cell survival. Treatment with PKC-iota siRNA blocked increase in cell number, and inhibited G1/S transition by accumulation of cells in G0/G1phase. In addition to cell-cycle arrest, both RWPE-1 and DU-145 cells underwent apoptosis due to mitochondrial dysfunction and apoptosis cascades, such as release of cytochrome c,activation of caspase-7, and poly (ADP-ribose)polymerase (PARP) cleavage. Our results suggest that PKC-iota is required for cell survival in both transformed non-malignant prostate RWPE-1 cells and androgen-independent malignant prostate DU-145 cells, whereas suppressing PKC-iota lead to apoptosis in DU-145 prostate cells.

Involvement of PKC‐ι in glioma proliferation

Atypical protein kinase C-iota (PKC-iota) protects cells against apoptosis and may play a role in cell proliferation. However, in vivo, the status and function of PKC-iota in human normal brain tissue, gliomas, benign and malignant meningiomas as well as its in vitro status in proliferating and confluent glioma cells, remains unknown. The objectives of our research were to determine whether expression of PKC-iota is altered either in gliomas or in benign and malignant meningiomas, compared to normal brain. In addition, we wished to establish the expression of PKC-iota in proliferating plus in cell cycle-arrested glioma cell lines, as well as the relationship between PKC-iota siRNA on PKC-iota protein content and cell proliferation. Western blot analyses for PKC-iota were performed on 12 normal brain biopsies, 15 benign meningiomas, three malignant meningiomas and three gliomas. Results demonstrated no (n = 9) or very weak (n = 3) detection of PKC-iota in normal brain tissue. In comparison, PKC-iota was robustly present in the majority of the benign meningiomas. Similarly, PKC-iota was abundant in all malignant meningiomas and gliomas. Western blotting for PKC-iota in confluent or proliferating glioma cell lines depicted substantial quantities of PKC-iota in proliferating T98G and U-138MG glioma cells. In contrast, confluent cells had either 71% (T98G) or 21% (U-138MG) less PKC-iota than proliferating cells. T98 and U-138 MG glioma cells treated with 100 nm PKC-iota siRNA had lower levels of cell proliferation compared to control siRNA-A and complete down-regulation of PKC-iota protein content. These results support the concept that presence of PKC-iota may be required for cell proliferation to take place.

Crystal Structure of the Catalytic Domain of Human Atypical Protein Kinase C-iota Reveals Interaction Mode of Phosphorylation Site in Turn Motif

Atypical protein kinases C (aPKCs) play critical roles in signaling pathways that control cell growth, differentiation and survival. Therefore, they constitute attractive targets for the development of novel therapeutics against cancer. The crystal structure of the catalytic domain of atypical PKCiota in complex with the bis(indolyl)maleimide inhibitor BIM1 has been determined at 3.0A resolution within the frame of the European Structural Proteomics Project SPINE. The overall structure exhibits the classical bilobal kinase fold and is in its fully activated form. Both phosphorylation sites (Thr403 in the activation loop, and Thr555 in the turn motif) are well defined in the structure and form intramolecular ionic contacts that make an important contribution in stabilizing the active conformation of the catalytic subunit. The phosphorylation site in the hydrophobic motif of atypical PKCs is replaced by the phosphorylation mimic glutamate and this is also clearly seen in the structure of PKCiota (residue 574). This structure determination for the first time provides the architecture of the turn motif phosphorylation site, which is characteristic for PKCs and PKB/AKT, and is completely different from that in PKA. The bound BIM1 inhibitor blocks the ATP-binding site and puts the kinase domain into an intermediate open conformation. The PKCiota-BIM1 complex is the first kinase domain crystal structure of any atypical PKC and constitutes the basis for rational drug design for selective PKCiota inhibitors.