Click chemistry-enabled CRISPR screening reveals GSK3 as a regulator of PLD signaling.

Enzymes that produce second messengers are highly regulated. Revealing the mechanisms underlying such regulation is critical to understanding both how cells achieve specific signaling outcomes and return to homeostasis following a particular stimulus. Pooled genome-wide CRISPR screens are powerful unbiased approaches to elucidate regulatory networks, their principal limitation being the choice of phenotype selection. Here, we merge advances in bioorthogonal fluorescent labeling and CRISPR screening technologies to discover regulators of phospholipase D (PLD) signaling, which generates the potent lipid second messenger phosphatidic acid. Our results reveal glycogen synthase kinase 3 as a positive regulator of protein kinase C and PLD signaling. More generally, this work demonstrates how bioorthogonal, activity-based fluorescent tagging can expand the power of CRISPR screening to uncover mechanisms regulating specific enzyme-driven signaling pathways in mammalian cells.

PKCs Sweeten Cell Metabolism by Phosphorylation of Glut1.

In this issue, Lee et al. (2015) show that PKC directly phosphorylates the glucose transporter Glut1, in order to promote glucose uptake in response to growth factor signaling.

A Protein Kinase C Phosphorylation Motif in GLUT1 Affects Glucose Transport and is Mutated in GLUT1 Deficiency Syndrome.

Protein kinase C has been implicated in the phosphorylation of the erythrocyte/brain glucose transporter, GLUT1, without a clear understanding of the site(s) of phosphorylation and the possible effects on glucose transport. Through in vitro kinase assays, mass spectrometry, and phosphospecific antibodies, we identify serine 226 in GLUT1 as a PKC phosphorylation site. Phosphorylation of S226 is required for the rapid increase in glucose uptake and enhanced cell surface localization of GLUT1 induced by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Endogenous GLUT1 is phosphorylated on S226 in primary endothelial cells in response to TPA or VEGF. Several naturally occurring, pathogenic mutations that cause GLUT1 deficiency syndrome disrupt this PKC phosphomotif, impair the phosphorylation of S226 in vitro, and block TPA-mediated increases in glucose uptake. We demonstrate that the phosphorylation of GLUT1 on S226 regulates glucose transport and propose that this modification is important in the physiological regulation of glucose transport.

PKCα/ERK/C7ORF41 axis regulates epidermal keratinocyte differentiation through the IKKα nuclear translocation.

Aberrant differentiation of keratinocytes disrupts the skin barrier and causes a series of skin diseases. However, the molecular basis of keratinocyte differentiation is still poorly understood. In the present study, we examined the expression of C7ORF41 using tissue microarrays by immunohistochemistry and found that C7ORF41 is specifically expressed in the basal layers of skin epithelium and its expression is gradually decreased during keratinocytes differentiation. Importantly, we corroborated the pivotal role of C7ORF41 during keratinocyte differentiation by C7ORF41 knockdown or overexpression in TPA-induced Hacat keratinocytes. Mechanismly, we first demonstrated that C7ORF41 inhibited keratinocyte differentiation mainly through formatting a complex with IKKα in the cytoplasm, which thus blocked the nuclear translocation of IKKα. Furthermore, we also demonstrated that inhibiting the PKCα/ERK signaling pathway reversed the reduction in C7ORF41 in TPA-induced keratinocytes, indicating that C7ORF41 expression could be regulated by upstream PKCα/ERK signaling pathway during keratinocyte differentiation. Collectively, our study uncovers a novel regulatory network PKCα/ERK/C7ORF41/IKKα during keratinocyte differentiation, which provides potential therapeutic targets for skin diseases.

The mTORC2/PKC pathway sustains compensatory insulin secretion of pancreatic ß cells in response to metabolic stress.

BACKGROUND: Compensation of the pancreatic ß cell functional mass in response to metabolic stress is key to the pathogenesis of Type 2 Diabetes. The mTORC2 pathway governs fuel metabolism and ß cell functional mass. It is unknown whether mTORC2 is required for regulating metabolic stress-induced ß cell compensation. METHODS: We challenged four-week-old ß-cell-specific Rictor (a key component of mTORC2)-knockout mice with a high fat diet (HFD) for 4weeks and measured metabolic and pancreatic morphological parameters. We performed ex vivo experiments to analyse ß cell insulin secretion and electrophysiology characteristics. Adenoviral-mediated overexpression and lentiviral-ShRNA-mediated knocking down proteins were applied in Min6 cells and cultured primary mouse islets. RESULTS: ßRicKO mice showed a significant glucose intolerance and a reduced plasma insulin level and an unchanged level ß cell mass versus the control mice under HFD. A HFD or palmitate treatment enhanced both glucose-induced insulin secretion (GIIS) and the PMA (phorbol 12-myristate 13-acetate)-induced insulin secretion in the control islets but not in the ßRicKO islets. The KO ß cells showed similar glucose-induced Ca2+ influx but lower membrane capacitance increments versus the control cells. The enhanced mTORC2/PKC proteins levels in the control HFD group were ablated by Rictor deletion. Replenishing PKCα by overexpression of PKCα-T638D restored the defective GIIS in ßRicKO islets. CONCLUSIONS: The mTORC2/Rictor pathway modulates ß cell compensatory GIIS under nutrient overload mediated by its phosphorylation of PKCα. GENERAL SIGNIFICANCE: This study suggests that the mTORC2/PKC pathway in ß cells is involved in the pathogenesis of T2D.

Protein Kinase C Isoforms Differentially Regulate Hypoxia-Inducible Factor-1α Accumulation in Cancer Cells.

Hypoxia-inducible factor-1α (HIF-1α) is one of the key transcription factors that mediate adaptation to hypoxia. Despite increasing evidence implicating the PKC family as potential modulators of HIF-1α, the molecular mechanisms of PKC isoform-dependent HIF-1α activity under hypoxic conditions have not been systematically elucidated in cancer cell lines. Here, we collectively investigated how each isoform of the PKC family contributes to HIF-1α accumulation in the human cervical cancer cell line HeLa. Among the abundant PKC isoforms, blockade of either PKCα or PKCδ was found to substantially reduce HIF-1α accumulation and transcriptional activity in hypoxic cells. Knockdown of PKCδ resulted in a reduction of HIF-1α mRNA levels, whereas the HIF-1α mRNA level was unchanged regardless of PKCα knockdown. Upon searching for the downstream effectors of these kinases, we found that PKCα controls HIF-1α translation via AKT-mTOR under hypoxic conditions. On the other hand, one of the well-known transcriptional regulation pathways of HIF-1α, nuclear factor-κB (NF-κB) is identified as a downstream effector of PKCδ. Taken together, our findings provide insights into the roles of PKC isoforms as additional, discrete modulators of hypoxia-stimulated HIF-1α accumulation through different signaling pathways.

[Roles of PKCα on the biological functions of T cells].

OBJECTIVE: To study the roles of PKCα on the proliferation, apoptosis, differentiation, cytokine production and inducible regulatory T cell (iTreg) induction of T cells. METHODS: T cells from WT (PKCα⁺/⁺) or PKCα knockout (PKCα⁻/⁻) mice were isolated and cultured in vitro. T cell proliferation and apoptosis were determined using ³H thymidine incorporation and CSFE/Annexin V staining. Cytokines production (IL-2, IL-4, IFN-γ and IL-17) was detected using ELISA. CD4⁺T cells were isolated and cultured in vitro via Th17 or iTreg biased condition. Flow cytometry was used to detect the cell differentiation. RESULTS: The production of IL-2 upon TCR stimulation increased, while the contents of IL-4 and IL-17 decreased in the PKCα⁻/⁻ group compared with the PKCα⁺/⁺ group. The differentiation rate of Th17 cells decreased, while the iTreg production increased in the PKCα⁻/⁻ group compared with the PKCα⁺/⁺ group. CONCLUSIONS: PKC-α is proinflammatory.

ENaC activity and expression is decreased in the lungs of protein kinase C-α knockout mice.

We used a PKC-α knockout model to investigate the regulation of alveolar epithelial Na(+) channels (ENaC) by PKC. Primary alveolar type II (ATII) cells were subjected to cell-attached patch clamp. In the absence of PKC-α, the open probability (Po) of ENaC was decreased by half compared with wild-type mice. The channel density (N) was also reduced in the knockout mice. Using in vivo biotinylation, membrane localization of all three ENaC subunits (α, ß, and γ) was decreased in the PKC-α knockout lung, compared with the wild-type. Confocal microscopy of lung slices showed elevated levels of reactive oxygen species (ROS) in the lungs of the PKC-α knockout mice vs. the wild-type. High levels of ROS in the knockout lung can be explained by a decrease in both cytosolic and mitochondrial superoxide dismutase activity. Elevated levels of ROS in the knockout lung activates PKC-δ and leads to reduced dephosphorylation of ERK1/2 by MAP kinase phosphatase, which in turn causes increased internalization of ENaC via ubiquitination by the ubiquitin-ligase Nedd4-2. In addition, in the knockout lung, PKC-δ activates ERK, causing a decrease in ENaC density at the apical alveolar membrane. PKC-δ also phosphorylates MARCKS, leading to a decrease in ENaC Po. The effects of ROS and PKC-δ were confirmed with patch-clamp experiments on isolated ATII cells in which the ROS scavenger, Tempol, or a PKC-δ-specific inhibitor added to patches reversed the observed decrease in ENaC apical channel density and Po. These results explain the decrease in ENaC activity in PKC-α knockout lung.

PKC α regulates netrin-1/UNC5B-mediated survival pathway in bladder cancer.

BACKGROUND: Netrin-1 and its receptor UNC5B play important roles in angiogenesis, embryonic development, cancer and inflammation. However, their expression patttern and biological roles in bladder cancer have not been well characterized. The present study aims to investigating the clinical significance of PKC α, netrin-1 and UNC5B in bladder cancer as well as their association with malignant biological behavior of cancer cells. METHODS: Netrin-1 and UNC5B expression was examined in 120 bladder cancer specimens using immunohistochemistry and in 40 fresh cancer tissues by western blot. Immunofluorescence was performed in cancer cell lines. PKC α agonist PMA and PKC siRNA was employed in bladder cancer cells. CCK-8, wound healing assays and flow cytometry analysis were used to examine cell proliferation, migration and cell cycle, respectively. RESULTS: Netrin-1 expression was positively correlated with histological grade, T stage, metastasis and poor prognosis in bladder cancer tissues. Immunofluorescence showed elevated netrin-1 and decreased UNC5B expression in bladder cancer cells compared with normal bladder cell line. Furthermore, cell proliferation, migration and cell cycle progression were promoted with PMA treatment while inhibited by calphostin C. In addition, PMA treatment could induce while calphostin C reduce netrin-1 expression in bladder cancer cells. CONCLUSIONS: The present study identified netrin-1/UNC5B, which could be regulated by PKC signaling, was important mediators of bladder cancer progression.

Endothelial heparanase regulates heart metabolism by stimulating lipoprotein lipase secretion from cardiomyocytes.

OBJECTIVE: After diabetes mellitus, transfer of lipoprotein lipase (LPL) from cardiomyocytes to the coronary lumen increases, and this requires liberation of LPL from the myocyte surface heparan sulfate proteoglycans with subsequent replenishment of this reservoir. At the lumen, LPL breaks down triglyceride to meet the increased demand of the heart for fatty acid. Here, we examined the contribution of coronary endothelial cells (ECs) toward regulation of cardiomyocyte LPL secretion. APPROACH AND RESULTS: Bovine coronary artery ECs were exposed to high glucose, and the conditioned medium was used to treat cardiomyocytes. EC-conditioned medium liberated LPL from the myocyte surface, in addition to facilitating its replenishment. This effect was attributed to the increased heparanase content in EC-conditioned medium. Of the 2 forms of heparanase secreted from EC in response to high glucose, active heparanase released LPL from the myocyte surface, whereas latent heparanase stimulated reloading of LPL from an intracellular pool via heparan sulfate proteoglycan-mediated RhoA activation. CONCLUSIONS: Endothelial heparanase is a participant in facilitating LPL increase at the coronary lumen. These observations provide an insight into the cross-talk between ECs and cardiomyocytes to regulate cardiac metabolism after diabetes mellitus.

Potential involvement of µ-opioid receptor dysregulation on the reduced antinociception of morphine in the inflammatory pain state in mice.

The antinociceptive effect of morphine in the inflammatory pain state was described in the von Frey filament test using the complete Freund's adjuvant (CFA)-induced mouse inflammatory pain model. After an i.pl. injection of CFA, mechanical allodynia was observed in the ipsilateral paw. The antinociceptive effect of morphine injected s.c. and i.t. against mechanical allodynia was reduced bilaterally at 1 day and 4 days after the CFA pretreatment. The expression level of mRNA for µ-opioid receptors at 1 day after the CFA pretreatment was reduced bilaterally in the lumbar spinal cord and dorsal root ganglion (DRG). In contrast, the protein level of µ-opioid receptors at 1 day after CFA pretreatment was decreased in the ipsilateral side in the DRG but not the lumbar spinal cord. Single or repeated i.t. pretreatment with the protein kinase Cα (PKCα) inhibitor Ro-32-0432 completely restored the reduced morphine antinociception in the contralateral paw but only partially restored it in the ipsilateral paw in the inflammatory pain state. In conclusion, reduced morphine antinociception against mechanical allodynia in the inflammatory pain state is mainly mediated via a decrease in µ-opioid receptors in the ipsilateral side and via the desensitization of µ-opioid receptors in the contralateral side by PKCα-induced phosphorylation.

Calcium-dependent isoforms of protein kinase C mediate glycine-induced synaptic enhancement at the calyx of Held.

Depolarization of presynaptic terminals that arises from activation of presynaptic ionotropic receptors, or somatic depolarization, can enhance neurotransmitter release; however, the molecular mechanisms mediating this plasticity are not known. Here we investigate the mechanism of this enhancement at the calyx of Held synapse, in which presynaptic glycine receptors depolarize presynaptic terminals, elevate resting calcium levels, and potentiate release. Using knock-out mice of the calcium-sensitive PKC isoforms (PKC(Ca)), we find that enhancement of evoked but not spontaneous synaptic transmission by glycine is mediated primarily by PKC(Ca). Measurements of calcium at the calyx of Held indicate that deficits in synaptic modulation in PKC(Ca) knock-out mice occur downstream of presynaptic calcium increases. Glycine enhances synaptic transmission primarily by increasing the effective size of the pool of readily releasable vesicles. Our results reveal that PKC(Ca) can enhance evoked neurotransmitter release in response to calcium increases caused by small presynaptic depolarizations.

SMB myosin heavy chain knockout enhances tonic contraction and reduces the rate of force generation in ileum and stomach antrum.

The role of SMA and SMB smooth muscle myosin heavy chain (MHC) isoforms in tonic and phasic contractions was studied in phasic (longitudinal ileum and stomach circular antrum) and tonic (stomach circular fundus) smooth muscle tissues of SMB knockout mice. Knocking out the SMB MHC gene eliminated SMB MHC protein expression and resulted in upregulation of the SMA MHC protein without altering the total MHC protein level. Switching from SMB to SMA MHC protein expression decreased the rate of the force transient and increased the sustained tonic force in SMB((-/-)) ileum and antrum with high potassium (KPSS) but not with carbachol (CCh) stimulation. The increased tonic contraction under the depolarized condition was not through changes in second messenger signaling pathways (PKC/CPI-17 or Rho/ROCK signaling pathway) or LC(20) phosphorylation. Biochemical analyses showed that the expression of contractile regulatory proteins (MLCK, MLCP, PKCδ, and CPI-17) did not change significantly in tissues tested except for PKCα protein expression being significantly decreased in the SMB((-/-)) antrum. However, specifically activating PKCα with phorbol dibutyrate (PDBu) was not significantly different in knockout and wild-type tissues, with total force being a fraction of the force generation with KPSS or CCh stimulation in SMB((-/-)) ileum and antrum. Taken together, these data show removing the SMB MHC protein expression with a compensatory increase in the SMA MHC protein results in enhanced sustained KPSS-induced tonic contraction with a reduced rate of force generation in these phasic tissues.

Desmosomal adhesiveness is developmentally regulated in the mouse embryo and modulated during trophectoderm migration.

During embryonic development tissues remain malleable to participate in morphogenetic movements but on completion of morphogenesis they must acquire the toughness essential for independent adult life. Desmosomes are cell-cell junctions that maintain tissue integrity especially where resistance to mechanical stress is required. Desmosomes in adult tissues are termed hyper-adhesive because they adhere strongly and are experimentally resistant to extracellular calcium chelation. Wounding results in weakening of desmosomal adhesion to a calcium-dependent state, presumably to facilitate cell migration and wound closure. Since desmosomes appear early in mouse tissue development we hypothesised that initial weak adhesion would be followed by acquisition of hyper-adhesion, the opposite of what happens on wounding. We show that epidermal desmosomes are calcium-dependent until embryonic day 12 (E12) and become hyper-adhesive by E14. Similarly, trophectodermal desmosomes change from calcium-dependence to hyper-adhesiveness as blastocyst development proceeds from E3 to E4.5. In both, development of hyper-adhesion is accompanied by the appearance of a midline between the plasma membranes supporting previous evidence that hyper-adhesiveness depends on the organised arrangement of desmosomal cadherins. By contrast, adherens junctions remain calcium-dependent throughout but tight junctions become calcium-independent as desmosomes mature. Using protein kinase C (PKC) activation and PKCα-/- mice, we provide evidence suggesting that conventional PKC isoforms are involved in developmental progression to hyper-adhesiveness. We demonstrate that modulation of desmosomal adhesion by PKC can regulate migration of trophectoderm. It appears that tissue stabilisation is one of several roles played by desmosomes in animal development.

The faces and friends of RhoGDI2.

RhoGDI2 is a guanine nucleotide dissociation inhibitor (GDI) specific for the Rho family of small GTPases that plays dual opposite roles in tumor progression, being both a promoter in tissues such as breast and a metastasis suppressor in tissues such as the bladder. Despite a clear role for this protein in modulating the invasive and metastatic process, the mechanisms through which RhoGDI2 executes these functions remain unclear. This review will highlight the current state of our knowledge regarding how RhoGDI2 functions in metastasis with a focus on bladder cancer and will also seek to highlight other potential underappreciated avenues through which this protein may affect cancer cell behavior.

Conventional protein kinase C-α (PKC-α) and PKC-ß negatively regulate RIG-I antiviral signal transduction.

Retinoic acid-inducible gene I (RIG-I) is a key sensor for viral RNA in the cytosol, and it initiates a signaling cascade that leads to the establishment of an interferon (IFN)-mediated antiviral state. Because of its integral role in immune signaling, RIG-I activity must be precisely controlled. Recent studies have shown that RIG-I CARD-dependent signaling function is regulated by the dynamic balance between phosphorylation and TRIM25-induced K63-linked ubiquitination. While ubiquitination of RIG-I is critical for RIG-I's ability to induce an antiviral IFN response, phosphorylation of RIG-I at S8 or T170 suppresses RIG-I signal-transducing activity under normal conditions. Here, we not only further define the roles of S8 and T170 phosphorylation for controlling RIG-I activity but also identify conventional protein kinase C-α (PKC-α) and PKC-ß as important negative regulators of the RIG-I signaling pathway. Mutational analysis indicated that while the phosphorylation of S8 or T170 potently inhibits RIG-I downstream signaling, the dephosphorylation of RIG-I at both residues is necessary for optimal TRIM25 binding and ubiquitination-mediated RIG-I activation. Furthermore, exogenous expression, gene silencing, and specific inhibitor treatment demonstrated that PKC-α/ß are the primary kinases responsible for RIG-I S8 and T170 phosphorylation. Coimmunoprecipitation showed that PKC-α/ß interact with RIG-I under normal conditions, leading to its phosphorylation, which suppresses TRIM25 binding, RIG-I CARD ubiquitination, and thereby RIG-I-mediated IFN induction. PKC-α/ß double-knockdown cells exhibited markedly decreased S8/T170 phosphorylation levels of RIG-I and resistance to infection by vesicular stomatitis virus. Thus, these findings demonstrate that PKC-α/ß-induced RIG-I phosphorylation is a critical regulatory mechanism for controlling RIG-I antiviral signal transduction under normal conditions.

Adenosine A1 receptors link to smooth muscle contraction via CYP4a, protein kinase C-α, and ERK1/2.

Adenosine A1 receptor (A1AR) activation contracts smooth muscle, although signaling mechanisms are not thoroughly understood. Activation of A1AR leads to metabolism of arachidonic acid, including the production of 20-hydroxyeicosatetraenoic acid (20-HETE) by cytochrome P4504a (CYP4a). The 20-HETE can activate protein kinase C-α (PKC-α), which crosstalks with extracellular signal-regulated kinase (ERK1/2) pathway. Both these pathways can regulate smooth muscle contraction, we tested the hypothesis that A1AR contracts smooth muscle through a pathway involving CYP4a, PKC-α, and ERK1/2. Experiments included isometric tension recordings of aortic contraction and Western blots of signaling molecules in wild type (WT) and A1AR knockout (A1KO) mice. Contraction to the A1-selective agonist 2-chloro-N cyclopentyladenosine (CCPA) was absent in A1KO mice aortae, indicating the contractile role of A1AR. Inhibition of CYP4a (HET0016) abolished 2-chloro-N cyclopentyladenosine-induced contraction in WT aortae, indicating a critical role for 20-HETE. Both WT and A1KO mice aortae contracted in response to exogenous 20-HETE. Inhibition of PKC-α (Gö6976) or ERK1/2 (PD98059) attenuated 20-HETE-induced contraction equally, suggesting that ERK1/2 is downstream of PKC-α. Contractions to exogenous 20-HETE were significantly less in A1KO mice; reduced protein levels of PKC-α, p-ERK1/2, and total ERK1/2 supported this observation. Our data indicate that A1AR mediates smooth muscle contraction via CYP4a and a PKC-α-ERK1/2 pathway.

Generation of reactive oxygen species (ROS) is a key factor for stimulation of macrophage proliferation by ceramide 1-phosphate.

We previously demonstrated that ceramide 1-phosphate (C1P) is mitogenic for fibroblasts and macrophages. However, the mechanisms involved in this action were only partially described. Here, we demonstrate that C1P stimulates reactive oxygen species (ROS) formation in primary bone marrow-derived macrophages, and that ROS are required for the mitogenic effect of C1P. ROS production was dependent upon prior activation of NADPH oxidase by C1P, which was determined by measuring phosphorylation of the p40phox subunit and translocation of p47phox from the cytosol to the plasma membrane. In addition, C1P activated cytosolic calcium-dependent phospholipase A(2) and protein kinase C-α, and NADPH oxidase activation was blocked by selective inhibitors of these enzymes. These inhibitors, and inhibitors of ROS production, blocked the mitogenic effect of C1P. By using BHNB-C1P (a photolabile caged-C1P analog), we demonstrate that all of these C1P actions are caused by intracellular C1P. It can be concluded that the enzyme responsible for C1P-stimulated ROS generation in bone marrow-derived macrophages is NADPH oxidase, and that this enzyme is downstream of PKC-α and cPLA(2)-α in this pathway.

Relief of Mg²âº-dependent inhibition of TRPM1 by PKCα at the rod bipolar cell synapse.

In the retina, light onset hyperpolarizes photoreceptors and depolarizes ON bipolar cells at the sign inverting photoreceptor-ON bipolar cell synapse. Transmission at this synapse is mediated by a signaling cascade comprised of mGluR6, a G-protein containing G(αo), and the cation channel TRP melastatin 1 (TRPM1). This system is thought to be common to both the rod- and ON-cone-driven pathways, which control vision under scotopic and photopic conditions, respectively. In this study, we present evidence that the rod pathway is uniquely susceptible to modulation by PKCα at the rod-rod bipolar cell synapse. Decreased production of DAG (an activator of PKC) by inhibition of PIP2 (phosphatidylinositol-4,5-bisphosphate) hydrolysis caused depression of the TRPM1 current. Conversely, addition of a DAG analog, 2-acetyl-1-oleoyl-sn-glycerol (OAG), potentiated the current in rod bipolar cells but not in ON-cone bipolar cells. The potentiating effects of OAG were absent both in mutant mice that lack PKCα expression and in wild-type mice in which enzymatic activity of PKCα was pharmacologically inhibited. In addition, we found that, like other members of the TRPM subfamily, TRPM1 current is susceptible to voltage-independent inhibition by intracellular magnesium, and that modulation by PKCα relieves this inhibition, as the potentiating effects of OAG are absent in low intracellular magnesium. We conclude that activation of PKCα initiates a modulatory mechanism at the rod-rod bipolar cell synapse whose function is to reduce inhibition of the TRPM1 current by magnesium, thereby increasing the gain of transmission at this synapse.

Protein kinase C α regulates nuclear pri-microRNA 15a release as part of endothelin signaling.

Endothelin-1 induced signaling is characterized by an early induction of a nuclear factor-kappa B p65/mitogen-activated phosphokinase p38 transcription complex via its A-receptor versus a late induction via diacylglycerol, and protein kinase C. A possible interaction between these two pathways and a potential function for protein kinase C in this context has not previously been elucidated. Here we report that in Caki-1 tumor cells, protein kinase C α is a part of the transcription complex. With importin α4 and α5 as chaperones, the transcription complex transmigrates into the nucleus. Protein kinase C α blocks the nuclear release of pri-microRNA 15a by direct binding shown by electrophoretic mobility shift assay and Duolink immune histology. The expression levels of miRNA 15a can be further manipulated by transfection of si-protein kinase C α, or an expression vector containing protein kinase C α or miRNA 15. The miRNA 15a regulation by protein kinase C α is detectable in different malignant human tumor cell lines (renal cell carcinoma, breast carcinoma, and melanoma). Furthermore, all three cell lines harbor both endothelin receptors (ETAR/ETBR). Specific blockage of each receptor leads to major reduction of miRNA 15a expression due to increased nuclear protein kinase C α translocation. We conclude that the nuclear binding of pri-microRNA 15a is a novel function of protein kinase C α, which plays an important role in endothelin-1 mediated signaling. Since several endothelin-sensitive, malignant tumor cell lines harbor this regulation, it could indicate a more general role in tumor biology.