Deciphering the Role and Signaling Pathways of PKCα in Luminal A Breast Cancer Cells.

Protein kinase C (PKC) comprises a family of highly related serine/threonine protein kinases involved in multiple signaling pathways, which control cell proliferation, survival, and differentiation. The role of PKCα in cancer has been studied for many years. However, it has been impossible to establish whether PKCα acts as an oncogene or a tumor suppressor. Here, we analyzed the importance of PKCα in cellular processes such as proliferation, migration, or apoptosis by inhibiting its gene expression in a luminal A breast cancer cell line (MCF-7). Differential expression analysis and phospho-kinase arrays of PKCα-KD vs. PKCα-WT MCF-7 cells identified an essential set of proteins and oncogenic kinases of the JAK/STAT and PI3K/AKT pathways that were down-regulated, whereas IGF1R, ERK1/2, and p53 were up-regulated. In addition, unexpected genes related to the interferon pathway appeared down-regulated, while PLC, ERBB4, or PDGFA displayed up-regulated. The integration of this information clearly showed us the usefulness of inhibiting a multifunctional kinase-like PKCα in the first step to control the tumor phenotype. Then allowing us to design a possible selection of specific inhibitors for the unexpected up-regulated pathways to further provide a second step of treatment to inhibit the proliferation and migration of MCF-7 cells. The results of this study suggest that PKCα plays an oncogenic role in this type of breast cancer model. In addition, it reveals the signaling mode of PKCα at both gene expression and kinase activation. In this way, a wide range of proteins can implement a new strategy to fine-tune the control of crucial functions in these cells and pave the way for designing targeted cancer therapies.

Identification and Validation of Stage-Associated Serum Biomarkers in Colorectal Cancer Using MS-Based Procedures.

PURPOSE: Successful prevention of colorectal cancer (CRC) would benefit from a rapid serum screening for early detection. Here, a novel strategy for CRC biomarker discovery and validation exclusively based on MS procedures is reported. EXPERIMENTAL DESIGN: Identification of CRC serum biomarkers is initially made using label-free quantification on pooled serum samples from different CRC stages followed by two consecutive steps of targeted parallel reaction monitoring assays in different serum cohorts. Relevance of different protein depletion and peptide fractionation extent is investigated. Absolute quantification of a selected peptide is performed as a proof-of-concept. RESULTS: A total of 945 proteins showed differential abundance in the discovery phase. Based on their statistical significance and relative expression in disease stages, 123 potential biomarkers are selected for a training step. In the final validation step, five peptides belonging to four proteins are consistently quantified in individual CRC serum samples and controls. Different statistical analyses indicate that peptides GWVTDGFSSLK (APOC3) and LCNNPTPQFGGK (THBS1) are candidate biomarkers. Absolute quantification of LCNNPTPQFGGK shows statistical significance for the diagnosis of early respect to late CRC stages. CONCLUSIONS AND CLINICAL RELEVANCE: Two peptides from APOC3 and THBS1 are validated by PRM as potential biomarkers for non-invasive diagnosis of colorectal cancer.

Proteomic Characterization of Transcription and Splicing Factors Associated with a Metastatic Phenotype in Colorectal Cancer.

We investigated new transcription and splicing factors associated with the metastatic phenotype in colorectal cancer. A concatenated tandem array of consensus transcription factor (TF)-response elements was used to pull down nuclear extracts in two different pairs of colorectal cancer cells, KM12SM/KM12C and SW620/480, genetically related but differing in metastatic ability. Proteins were analyzed by label-free LC-MS and quantified with MaxLFQ. We found 240 proteins showing a significant dysregulation in highly metastatic KM12SM cells relative to nonmetastatic KM12C cells and 257 proteins in metastatic SW620 versus SW480. In both cell lines there were similar alterations in genuine TFs and components of the splicing machinery like UPF1, TCF7L2/TCF-4, YBX1, or SRSF3. However, a significant number of alterations were cell-line specific. Functional silencing of MAFG, TFE3, TCF7L2/TCF-4, and SRSF3 in KM12 cells caused alterations in adhesion, survival, proliferation, migration, and liver homing, supporting their role in metastasis. Finally, we investigated the prognostic value of the altered TFs and splicing factors in cancer patients. SRSF3 and SFPQ showed significant prognostic value. We observed that SRSF3 displayed a gradual loss of expression associated with cancer progression. Loss of SRSF3 expression was significantly associated with poor survival and shorter disease-free survival, particularly in early stages, in colorectal cancer.

Enhanced Missing Proteins Detection in NCI60 Cell Lines Using an Integrative Search Engine Approach.

The Human Proteome Project (HPP) aims deciphering the complete map of the human proteome. In the past few years, significant efforts of the HPP teams have been dedicated to the experimental detection of the missing proteins, which lack reliable mass spectrometry evidence of their existence. In this endeavor, an in depth analysis of shotgun experiments might represent a valuable resource to select a biological matrix in design validation experiments. In this work, we used all the proteomic experiments from the NCI60 cell lines and applied an integrative approach based on the results obtained from Comet, Mascot, OMSSA, and X!Tandem. This workflow benefits from the complementarity of these search engines to increase the proteome coverage. Five missing proteins C-HPP guidelines compliant were identified, although further validation is needed. Moreover, 165 missing proteins were detected with only one unique peptide, and their functional analysis supported their participation in cellular pathways as was also proposed in other studies. Finally, we performed a combined analysis of the gene expression levels and the proteomic identifications from the common cell lines between the NCI60 and the CCLE project to suggest alternatives for further validation of missing protein observations.

Functional Identification of Target by Expression Proteomics (FITExP) reveals protein targets and highlights mechanisms of action of small molecule drugs.

Phenomenological screening of small molecule libraries for anticancer activity yields potentially interesting candidate molecules, with a bottleneck in the determination of drug targets and the mechanism of anticancer action. We have found that, for the protein target of a small-molecule drug, the abundance change in late apoptosis is exceptional compared to the expectations based on the abundances of co-regulated proteins. Based on this finding, a novel method to drug target deconvolution is proposed. In a proof of principle experiment, the method yielded known targets of several common anticancer agents among a few (often, just one) likely candidates identified in an unbiased way from cellular proteome comprising more than 4,000 proteins. A validation experiment with a different set of cells and drugs confirmed the findings. As an additional benefit, mapping most specifically regulated proteins on known protein networks highlighted the mechanism of drug action. The new method, if proven to be general, can significantly shorten drug target identification, and thus facilitate the emergence of novel anticancer treatments.

RRP6/EXOSC10 is required for the repair of DNA double-strand breaks by homologous recombination.

The exosome acts on different RNA substrates and plays important roles in RNA metabolism. The fact that short non-coding RNAs are involved in the DNA damage response led us to investigate whether the exosome factor RRP6 of Drosophila melanogaster and its human ortholog EXOSC10 play a role in DNA repair. Here, we show that RRP6 and EXOSC10 are recruited to DNA double-strand breaks (DSBs) in S2 cells and HeLa cells, respectively. Depletion of RRP6/EXOSC10 does not interfere with the phosphorylation of the histone variant H2Av (Drosophila) or H2AX (humans), but impairs the recruitment of the homologous recombination factor RAD51 to the damaged sites, without affecting RAD51 levels. The recruitment of RAD51 to DSBs in S2 cells is also inhibited by overexpression of RRP6-Y361A-V5, a catalytically inactive RRP6 mutant. Furthermore, cells depleted of RRP6 or EXOSC10 are more sensitive to radiation, which is consistent with RRP6/EXOSC10 playing a role in DNA repair. RRP6/EXOSC10 can be co-immunoprecipitated with RAD51, which links RRP6/EXOSC10 to the homologous recombination pathway. Taken together, our results suggest that the ribonucleolytic activity of RRP6/EXOSC10 is required for the recruitment of RAD51 to DSBs.

The effects of 5-fluorouracil on the proteome of colon cancer cells.

The pyrimidine analogue 5-fluorouracil (5FU) is used as a treatment for solid tumors, but its mechanism of action is not fully understood. We have used mass spectrometry to study the mechanism of action of 5FU, and we have measured the effects of this drug on the composition and on the turnover of the proteome of RKO cancer cells. We have identified novel potential targets of 5FU that are affected after very short exposure times. We have also shown that 5FU has a massive effect on the proteins involved in RNA metabolism. After only 1 h of treatment, 5FU causes a post-transcriptional reduction in the abundance of components of the translation machinery (mostly ribosomal proteins), and this reduction is accompanied by a down-regulation of the translational capacity of the cells. Neither rapamycin nor raltitrexed, two drugs that also block cell proliferation, reduce the abundances of ribosomal proteins as 5FU does, which suggests that the down-regulation of ribosomal proteins is coupled to the mechanism of action of 5FU. Some of our observations conflict with previous reports based on RNA quantification. This shows how important it is to complement RNA profiling studies with analyses of drug toxicity at the protein level.

ATP enhances neuronal differentiation of PC12 cells by activating PKCα interactions with cytoskeletal proteins.

PKCα is a key mediator of the neuronal differentiation controlled by NGF and ATP. However, its downstream signaling pathways remain to be elucidated. To identify the signaling partners of PKCα, we analyzed proteins coimmunoprecipitated with this enzyme in PC12 cells differentiated with NGF and ATP and compared them with those obtained with NGF alone or growing media. Mass spectrometry analysis (LC-MS/MS) identified plectin, peripherin, filamin A, fascin, and ß-actin as potential interacting proteins. The colocalization of PKCα and its interacting proteins increased when PC12 cells were differentiated with NGF and ATP. Peripherin and plectin organization and the cortical remodeling of ß-actin were dramatically affected when PKCα was down-regulated, suggesting that all three proteins might be functional targets of ATP-dependent PKCα signaling. Taken together, these data demonstrate that PKCα is essential for controlling the neuronal development induced by NGF and ATP and interacts with the cytoskeletal components at two levels: assembly of the intermediate filament peripherin and organization of cortical actin.

Are the majority of a(2)-ions cyclic?

Detailed analysis of >18 400 high-mass accuracy tandem mass spectra resulting from higher energy collisional dissociation yields further evidence of the cyclic nature of a(2)-ions.

Structural and mechanistic insights into the association of PKCalpha-C2 domain to PtdIns(4,5)P2.

C2 domains are widely-spread protein signaling motifs that in classical PKCs act as Ca(2+)-binding modules. However, the molecular mechanisms of their targeting process at the plasma membrane remain poorly understood. Here, the crystal structure of PKCalpha-C2 domain in complex with Ca(2+), 1,2-dihexanoyl-sn-glycero-3-[phospho-L-serine] (PtdSer), and 1,2-diayl-sn-glycero-3-[phosphoinositol-4,5-bisphosphate] [PtdIns(4,5)P(2)] shows that PtdSer binds specifically to the calcium-binding region, whereas PtdIns(4,5)P(2) occupies the concave surface of strands beta3 and beta4. Strikingly, the structure reveals a PtdIns(4,5)P(2)-C2 domain-binding mode in which the aromatic residues Tyr-195 and Trp-245 establish direct interactions with the phosphate moieties of the inositol ring. Mutations that abrogate Tyr-195 and Trp-245 recognition of PtdIns(4,5)P(2) severely impaired the ability of PKCalpha to localize to the plasma membrane. Notably, these residues are highly conserved among C2 domains of topology I, and a general mechanism of C2 domain-membrane docking mediated by PtdIns(4,5)P(2) is presented.

The PtdIns(4,5)P2 ligand itself influences the localization of PKCalpha in the plasma membrane of intact living cells.

Rapamycin-triggered heterodimerization strategy is becoming an excellent tool for rapidly modifying phosphatidylinositol(4,5)-bisphosphate [PtdIns(4,5)P2] levels at the plasma membrane and for studying their influence in different processes. In this work, we studied the effect of modulation of the PtdIns(4,5)P2 concentration on protein kinase C (PKC) alpha membrane localization in intact living cells. We showed that an increase in the PtdIns(4,5)P2 concentration enlarges the permanence of PKCalpha in the plasma membrane when PC12 cells are stimulated with ATP, independently of the diacylglycerol generated. The depletion of this phosphoinositide decreases both the percentage of protein able to translocate to the plasma membrane and its permanence there. Our results demonstrate that the polybasic cluster located in the C2 domain of PKCalpha is responsible for this phosphoinositide-protein interaction. Furthermore, the C2 domain acts as a dominant interfering module in the neural differentiation process of PC12 cells, a fact that was also supported by the inhibitory effect obtained by knocking down PKCalpha with small interfering RNA duplexes. Taken together, these data demonstrate that PtdIns(4,5)P2 itself targets PKCalpha to the plasma membrane through the polybasic cluster located in the C2 domain, with this interaction being critical in the signaling network involved in neural differentiation.

The C2 domains of classical PKCs are specific PtdIns(4,5)P2-sensing domains with different affinities for membrane binding.

C2 domains are conserved protein modules in many eukaryotic signaling proteins, including the protein kinase (PKCs). The C2 domains of classical PKCs bind to membranes in a Ca(2+)-dependent manner and thereby act as cellular Ca(2+) effectors. Recent findings suggest that the C2 domain of PKCalpha interacts specifically with phosphatidylinositols 4,5-bisphosphate (PtdIns(4,5)P(2)) through its lysine rich cluster, for which it shows higher affinity than for POPS. In this work, we compared the three C2 domains of classical PKCs. Isothermal titration calorimetry revealed that the C2 domains of PKCalpha and beta display a greater capacity to bind to PtdIns(4,5)P(2)-containing vesicles than the C2 domain of PKCgamma. Comparative studies using lipid vesicles containing both POPS and PtdIns(4,5)P(2) as ligands revealed that the domains behave as PtdIns(4,5)P(2)-binding modules rather than as POPS-binding modules, suggesting that the presence of the phosphoinositide in membranes increases the affinity of each domain. When the magnitude of PtdIns(4,5)P(2) binding was compared with that of other polyphosphate phosphatidylinositols, it was seen to be greater in both PKCbeta- and PKCgamma-C2 domains. The concentration of Ca(2+) required to bind to membranes was seen to be lower in the presence of PtdIns(4,5)P(2) for all C2 domains, especially PKCalpha. In vivo experiments using differentiated PC12 cells transfected with each C2 domain fused to ECFP and stimulated with ATP demonstrated that, at limiting intracellular concentration of Ca(2+), the three C2 domains translocate to the plasma membrane at very similar rates. However, the plasma membrane dissociation event differed in each case, PKCalpha persisting for the longest time in the plasma membrane, followed by PKCgamma and, finally, PKCbeta, which probably reflects the different levels of Ca(2+) needed by each domain and their different affinities for PtdIns(4,5)P(2).

The C2 domain of PKCalpha is a Ca2+ -dependent PtdIns(4,5)P2 sensing domain: a new insight into an old pathway.

The C2 domain is a targeting domain that responds to intracellular Ca2+ signals in classical protein kinases (PKCs) and mediates the translocation of its host protein to membranes. Recent studies have revealed a new motif in the C2 domain, named the lysine-rich cluster, that interacts with acidic phospholipids. The purpose of this work was to characterize the molecular mechanism by which PtdIns(4,5)P2 specifically interacts with this motif. Using a combination of isothermal titration calorimetry, fluorescence resonance energy transfer and time-lapse confocal microscopy, we show here that Ca2+ specifically binds to the Ca2+ -binding region, facilitating PtdIns(4,5)P2 access to the lysine-rich cluster. The magnitude of PtdIns(4,5)P2 binding is greater than in the case of other polyphosphate phosphatidylinositols. Very importantly, the residues involved in PtdIns(4,5)P2 binding are essential for the plasma membrane localization of PKCalpha when RBL-2H3 cells are stimulated through their IgE receptors. Additionally, CFP-PH and CFP-C1 domains were used as bioprobes to demonstrate the co-existence of PtdIns(4,5)P2 and diacylglycerol in the plasma membrane, and it was shown that although a fraction of PtdIns(4,5)P2 is hydrolyzed to generate diacylglycerol and IP3, an important amount still remains in the membrane where it is available to activate PKCalpha. These findings entail revision of the currently accepted model of PKCalpha recruitment to the membrane and its activation.

Molecular mechanisms of PKCalpha localization and activation by arachidonic acid. The C2 domain also plays a role.

Arachidonic acid, one of the major unsaturated fatty acids released during cell stimulation, participates in the signaling necessary for activation of different enzymes, including protein kinase C (PKC). Here, we demonstrate that arachidonic acid is a direct activator of PKCalpha, but needs the cooperation of Ca(2+) to exert its function. By using several mutants of the C2 and C1 domains, we were able to determine the molecular mechanism of this activation. More specifically, site-directed mutagenesis in key residues found in the C2 domain showed that the Ca(2+)-binding region was essential for the arachidonic acid-dependent localization and activation of PKCalpha. However, the lysine-rich cluster, also located in the C2 domain, played no relevant role in either the membrane localization or activation of the enzyme. Moreover, site-directed mutagenesis in key residues placed in the C1A and C1B subdomains, which are responsible for the diacylglycerol/phorbil ester interaction, demonstrated that the C1A subdomain was involved in the membrane localization and activation mechanism. Taken together, these data suggest a very precise mechanism for PKCalpha activation by arachidonic acid, involving a sequential model of activation in which an increase in intracytosolic Ca(2+) leads to the interaction of arachidonic acid with the Ca(2+)-binding region; only after this step, does the C1A subdomain interact with arachidonic acid, leading to full activation of the enzyme.

The ATP-dependent membrane localization of protein kinase Calpha is regulated by Ca2+ influx and phosphatidylinositol 4,5-bisphosphate in differentiated PC12 cells.

Signal transduction through protein kinase Cs (PKCs) strongly depends on their subcellular localization. Here, we investigate the molecular determinants of PKCalpha localization by using a model system of neural growth factor (NGF)-differentiated pheochromocytoma (PC12) cells and extracellular stimulation with ATP. Strikingly, the Ca2+ influx, initiated by the ATP stimulation of P2X receptors, rather than the Ca2+ released from the intracellular stores, was the driving force behind the translocation of PKCalpha to the plasma membrane. Furthermore, the localization process depended on two regions of the C2 domain: the Ca2+-binding region and the lysine-rich cluster, which bind Ca2+ and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], respectively. It was demonstrated that diacylglycerol was not involved in the localization of PKCalpha through its C1 domain, and in lieu, the presence of PtdIns(4,5)P2 increased the permanence of PKCalpha in the plasma membrane. Finally, it also was shown that ATP cooperated with NGF during the differentiation process of PC12 cells by increasing the length of the neurites, an effect that was inhibited when the cells were incubated in the presence of a specific inhibitor of PKCalpha, suggesting a possible role for this isoenzyme in the neural differentiation process. Overall, these results show a novel mechanism of PKCalpha activation in differentiated PC12 cells, where Ca2+ influx, together with the endogenous PtdIns(4,5)P2, anchor PKCalpha to the plasma membrane through two distinct motifs of its C2 domain, leading to enzyme activation.