Design of a PKCδ-specific small peptide as a theragnostic agent for glioblastoma

Glioblastoma is an aggressive malignant brain tumor that starts in the brain or spine and frequently recurs after anticancer treatment. The development of an accurate diagnostic system combined with effective cancer therapy is essential to improve prognosis of glioma patients. Peptides, produced from phage display, are attractive biomolecules for glioma treatment because of their biostability, nontoxicity, and small size. In this study, we employed phage display methodology to screen for peptides that specifically recognize the target PKCδ as a novel biomarker for glioma. The phage library screening yielded four different peptides displayed on phages with a 20- to 200-pM K_d value for the recombinant PKCδ catalytic domain. Among these four phage peptides, we selected one to synthesize and tagged it with fluorescein isothiocyanate (FITC) based on the sequence of the PKCδ-binding phage clone. The synthetic peptide showed a relative binding affinity for antibody and localization in the U373 glioma cell. The kinase activity of PKCδ was inhibited by FITC-labeled peptide with an IC₅₀ of 1.4 μM in vitro. Consequently, the peptide found in this study might be a promising therapeutic agent against malignant brain tumor.     

Protein Kinase C Activity is Necessary for Estrogen-Induced Erk Phosphorylation in Neocortical Explants

Our laboratory showed previously that estrogen activates ERK in neocortical cultures. To further elucidate the precise signaling sequelae that lead to estrogen-induced ERK activity, we evaluated the involvement of protein kinase C (PKC). We found that neocortical explants expressed primarily PKC gamma and PKC epsilon. Consistent with the involvement of PKC in mediating estrogen-induced ERK phosphorylation, we found that estrogen treatment induced translocation of these PKC isoforms to the plasma membrane. Importantly, inhibition of these isoforms abolished the ability of estrogen to phosphorylate ERK. While direct activation of PKC mimicked the effect of estrogen on ERK, both in pattern of activation and resulting intraneuronal distribution of ERK, PKC-induced ERK phosphorylation required the activity of MEK but not B-Raf. Collectively, these data suggest a critical role for PKC in mediating estrogen induction of ERK activation in the developing brain via a MEK-dependent but B-Raf-independent pathway.     

Functional role of PKC in contraction of cultured human prostatic stromal cells

The contractile activity of prostatic stromal cells contributes to symptoms of benign prostatic hyperplasia (BPH). However, the mechanisms for this contraction have not yet been fully elucidated. In this study, we investigated the role of protein kinase C (PKC) in prostatic contraction by measuring the isometric tension development of cultured human prostatic stromal cells (CHPSCs) derived from BPH patients. Fresh human BPH tissue was used only in a Western blot analysis. A ring preparation made of CHPSCs and collagen gel could develop an isometric tension during activation with various agonists. Phorbol 12,13 dibutyrate (PDBu), a PKC activator, induced a relaxation. A Western blot analysis revealed the expression of PKC-potentiated protein phosphatase-1 inhibitory protein (CPI-17) in both CHPSCs and fresh human BPH tissue to be much lower than that in the rabbit aorta. When CPI-17 was over-expressed, PDBu induced a large contraction, but the agonist-induced contraction did not become larger than expected. In alpha-toxin permeabilized preparations, PDBu induced a relaxation in control CHPSCs, while it induced a contraction at a constant [Ca2+]i in CPI-17 over-expressing CHPSCs. These results indicated that the activation of PKC in CHPSCs induces a relaxation probably due to low expression level of CPI-17 and also that the PKC-CPI-17 pathway does not appear to play a major role in the agonist-induced contraction even when CPI-17 was over-expressed.     

Nutrients and the Pkh1/2 and Pkc1 protein kinases control mRNA decay and P-body assembly in yeast

Regulated mRNA decay is essential for eukaryotic survival but the mechanisms for regulating global decay and coordinating it with growth, nutrient, and environmental cues are not known. Here we show that a signal transduction pathway containing the Pkh1/Pkh2 protein kinases and one of their effector kinases, Pkc1, is required for and regulates global mRNA decay at the deadenylation step in Saccharomyces cerevisiae. Additionally, many stresses disrupt protein synthesis and release mRNAs from polysomes for incorporation into P-bodies for degradation or storage. We find that the Pkh1/2-Pkc1 pathway is also required for stress-induced P-body assembly. Control of mRNA decay and P-body assembly by the Pkh-Pkc1 pathway only occurs in nutrient-poor medium, suggesting a novel role for these processes in evolution. Our identification of a signaling pathway for regulating global mRNA decay and P-body assembly provides a means to coordinate mRNA decay with other cellular processes essential for growth and long-term survival. Mammals may use similar regulatory mechanisms because components of the decay apparatus and signaling pathways are conserved.     

Phospholipase D regulates myogenic differentiation through the activation of both mTORC1 and mTORC2 complexes

How phospholipase D (PLD) is involved in myogenesis remains unclear. At the onset of myogenic differentiation of L6 cells induced by the PLD agonist vasopressin in the absence of serum, mTORC1 complex was rapidly activated, as reflected by phosphorylation of S6 kinase1 (S6K1). Both the long (p85) and short (p70) S6K1 isoforms were phosphorylated in a PLD1-dependent way. Short rapamycin treatment specifically inhibiting mTORC1 suppressed p70 but not p85 phosphorylation, suggesting that p85 might be directly activated by phosphatidic acid. Vasopressin stimulation also induced phosphorylation of Akt on Ser-473 through PLD1-dependent activation of mTORC2 complex. In this model of myogenesis, mTORC2 had a positive role mostly unrelated to Akt activation, whereas mTORC1 had a negative role, associated with S6K1-induced Rictor phosphorylation. The PLD requirement for differentiation can thus be attributed to its ability to trigger via mTORC2 activation the phosphorylation of an effector that could be PKCα. Moreover, PLD is involved in a counter-regulation loop expected to limit the response. This study thus brings new insights in the intricate way PLD and mTOR cooperate to control myogenesis.     

GSK3 beta mediates acentromeric spindle stabilization by activated PKC zeta

Upon fertilization, the mammalian egg undergoes a precise series of signaling events that orchestrate its conversion into a zygote. Mouse eggs contain acentrosomal spindle poles when arrested at meiotic metaphase II. The meiotic spindle is thought to provide a scaffold that mediates spatial and temporal regulation of the signaling pathways orchestrating post-fertilization events. Many kinases have been found to be enriched at the MII meiotic spindle, such as Protein Kinase C (PKC), and are thought to have an important role in regulating signaling events initiated through fertilization. In this study phosphorylated PKCζ (p-PKCζ) and Glycogen Synthase Kinase 3β (GSK3β) were found to be enriched at both acentrosomal spindle poles and the kinetochore region. Phosphorylated PKCζ (p-PKCζ) was immunopurified from MII eggs and was found to co-localize with known microtubule stabilizing components found in somatic cells, including GSK3β and Partition deficit protein 6 (Par6). Both fluorescence resonance energy transfer (FRET) and immunofluorescence confirmed the existence and close association of these proteins with p-PKCζ at the meiotic spindle. When GSK3β is phosphorylated on ser9 its activity is inhibited and the spindle is stabilized. However, when GSK3β is dephosphorylated (on ser9) it becomes active and the spindle is destabilized. The mechanism by which p-PKCζ maintains spindle organization appears to be through GSK3β and suggests that p-PKCζ phosphorylates GSK3β on the ser9 position inactivating GSK3β and consequently maintaining spindle stability during meiotic metaphase arrest.     

Ca-dependent binding of calcium-binding protein 1 to presynaptic group III metabotropic glutamate receptors and blockage by phosphorylation of the receptors

Presynaptic group III metabotropic glutamate receptors (mGluRs) and Ca²⁺ channels are the main neuronal activity-dependent regulators of synaptic vesicle release, and they use common molecules in their signaling cascades. Among these, calmodulin (CaM) and the related EF-hand Ca²⁺-binding proteins are of particular importance as sensors of presynaptic Ca²⁺, and a multiple of them are indeed utilized in the signaling of Ca²⁺ channels. However, despite its conserved structure, CaM is the only known EF-hand Ca²⁺-binding protein for signaling by presynaptic group III mGluRs. Because the mGluRs and Ca²⁺ channels reciprocally regulate each other and functionally converge on the regulation of synaptic vesicle release, the mGluRs would be expected to utilize more EF-hand Ca²⁺-binding proteins in their signaling. Here I show that calcium-binding protein 1 (CaBP1) bound to presynaptic group III mGluRs competitively with CaM in a Ca²⁺-dependent manner and that this binding was blocked by protein kinase C (PKC)-mediated phosphorylation of these receptors. As previously shown for CaM, these results indicate the importance of CaBP1 in signal cross talk at presynaptic group III mGluRs, which includes many molecules such as cAMP, Ca²⁺, PKC, G protein, and Munc18-1. However, because the functional diversity of EF-hand calcium-binding proteins is extraordinary, as exemplified by the regulation of Ca²⁺ channels, CaBP1 would provide a distinct way by which presynaptic group III mGluRs fine-tune synaptic transmission.     

An investigation into signal transduction mechanisms involved in insulin-induced long-term depression in the CA1 region of the hippocampus

Recent work has demonstrated that brief application of insulin to hippocampal slices can induce a novel form of long-term depression (insulin-LTD) in the CA1 region of the hippocampus; however, the molecular details of how insulin triggers LTD remain unclear. Using electrophysiological and biochemical approaches in the hippocampal slices, we show here that insulin-LTD (i) is specific to 3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor- but not NMDA receptor-mediated synaptic transmission; (ii) is induced and expressed postsynaptically but does not require the activation of ionotropic and metabotropic glutamate receptors; (iii) requires a concomitant Ca(2+) influx through l-type voltage-activated Ca(2+) channels (VACCs) and the release of Ca(2+) from intracellular stores; (iv) requires the series of protein kinases, including protein tyrosine kinase (PTK), phosphatidylinositol 3-kinase (PI3K), and protein kinase C (PKC); (v) is mechanistically distinct from low-frequency stimulation-induced LTD (LFS-LTD) and independent on protein phosphatase 1/2 A (PP1/2 A) and PP2B activation; (vi) is dependent on a rapamycin-sensitive local translation of dendritic mRNA, and (vii) is associated with a persistent decrease in the surface expression of GluR2 subunit. These results suggest that a PI3K/PKC-dependent insulin signaling, which controls postsynaptic surface AMPA receptor numbers through PP-independent endocytosis, may be a major expression mechanism of insulin-LTD in hippocampal CA1 neurons.     

Designing of Protein Kinase C β-II Inhibitors against Diabetic complications: Structure Based Drug Design, Induced Fit docking and analysis of active site conformational changes

Protein Kinase C β-II (PKC β-II) is an important enzyme in the development of diabetic complications like cardiomyopathy, retinopathy, neuropathy, nephropathy and angiopathy. PKC β-II is activated in vascular tissues during diabetic vascular abnormalities. Thus, PKC β-II is considered as a potent drug target and the crystal structure of the kinase domain of PKC β-II (PDB id: 2I0E) was used to design inhibitors using Structure-Based Drug Design (SBDD) approach. Sixty inhibitors structurally similar to Staurosporine were retrieved from PubChem Compound database and High Throughput Virtual screening (HTVs) was carried out with PKC β-II. Based on the HTVs results and the nature of active site residues of PKC β-II, Staurosporine inhibitors were designed using SBDD. Induced Fit Docking (IFD) studies were carried out between kinase domain of PKC β-II and the designed inhibitors. These IFD complexes showed favorable docking score, glide energy, glide emodel and hydrogen bond and hydrophobic interactions with the active site of PKC β-II. Binding free energy was calculated for IFD complexes using Prime MM-GBSA method. The conformational changes induced by the inhibitor at the active site of PKC β-II were observed for the back bone Cα atoms and side-chain chi angles. PASS prediction tool was used to analyze the biological activities for the designed inhibitors. The various physicochemical properties were calculated for the compounds. One of the designed inhibitors successively satisfied all the in silico parameters among the others and seems to be a potent inhibitor against PKC β-II.     

Hallmarks for senescence in carcinogenesis: novel signaling players

Cellular senescence is a potent anti-cancer mechanism controlled by tumor suppressor genes, particularly p53 and pRb, which is characterized by the irreversible loss of proliferation. Senescence induced by DNA damage, oncogenic stimulation, or excessive mitogenic input, serves as a barrier that counteracts cancer progression. Emerging evidence in cellular and in in vivo models revealed the involvement of additional signaling players in senescence, including PML, CK2, Bcl-2, PI3K effectors such as Rheb, Rho small GTPases, and cytokines. Recent studies have also implicated protein kinase C (PKC) isozymes as modulators of senescence phenotypes and showed that phorbol esters, widely used PKC activators, can induce senescence in a number of cancer cells. These novel findings suggest a complex array of cross-talks between senescence pathways and may have significant implications in cancer therapy.