Functional alterations in protein kinase C beta II expression in melanoma

Protein kinase C (PKC) is a heterogeneous family of serine/threonine protein kinases that have different biological effects in normal and neoplastic melanocytes (MCs). To explore the mechanism behind their differential response to PKC activation, we analyzed the expression profile of all nine PKC isoforms in normal human MCs, HPV16 E6/E7 immortalized MCs, and a panel of melanoma cell lines. We found reduced PKCβ and increased PKCζ and PKCι expression at both the protein and mRNA levels in immortalized MCs and melanoma lines. We focused on PKCβ as it has been functionally linked to melanin production and oxidative stress response. Re-expression of PKCβ in melanoma cells inhibited colony formation in soft agar, indicating that PKCβ loss in melanoma is important for melanoma growth. PKCβII, but not PKCβI, was localized to the mitochondria, and inhibition of PKCβ significantly reduced UV-induced reactive oxygen species (ROS) in MCs with high PKCβ expression. Thus alterations in PKCβ expression in melanoma contribute to their neoplastic phenotype, possibly by reducing oxidative stress, and may constitute a selective therapeutic target.     

Effects of Calphostin C,Specific PKC Inhibitor on TPA-Induced Normal Human Melanocyte Growth,Morphology and Adhesion

Normal human melanocytes, which rarely undergo mitosis in vivo, require many growth factors and growth-stimulating agents in vitro, such as basic fibroblast growth factor (bFGF) and cyclic adenosine monophosphate-stimulating agents or 12-0-tetrade-canoylphorbol 13-acetate (TPA), to proliferate. TPA, known as a protein kinase C (PKC)-activator, supports normal human melanocyte growth and influences on melanocyte dendrite formation. We have further confirmed the role of the PKC-mediated pathway in the TPA-dependent melanocyte functions—i.e., proliferation, morphology, and adhesion—using Calphostin C (CPC), a highly specific PKC inhibitor. Melanocytes require the continual presence of TPA for growth in culture. Addition of 8 nM TPA to the medium increased melanocyte growth by 198.4 ± 2.3% of that without TPA. The growth induction by TPA was suppressed by the addition of 10 nM CPC at the level comparable to that without TPA without any morphological alterations. Significant levels of PKC were detected in melanocytes chronically exposed to TPA as determined by Western blotting. A long-term exposure to TPA (more than 5 days) resulted in marked reduction of melanocyte adhesion to plastic cell culture dishes, both uncoated and coated with type IV collagen. By the addition of 10 nM CPC in the adhesion assay, the melanocyte adhesion was further inhibited in both conditions. These results indicated the critical involvement of PKC activation in the TPA-dependent melanocyte functions. Continuous activation of PKC by TPA is implicated in melanocyte growth stimulation. TPA also has effects on melanocyte morphology, causing the formation of long extended dendrites with little cytoplasm. However, inhibition of PKC activation by CPC does not affect the melanocyte morphology, and CPC reduces melanocyte adhesion to uncoated or type IV collagen coated plastic cell culture dishes.     

On the role of protein kinases in regulating neutrophil actin association with the cytoskeleton

We have investigated the effect of staurosporine-type protein kinase inhibitors, displaying different enzyme specificity, on the association of actin with the neutrophil cytoskeleton. In resting cells, nanomolar concentrations of staurosporine induced a rapid increase in cytoskeleton-associated actin. Other inhibitors, more specific for protein kinase C (PKC) or kinases dependent on cyclic nucleotides, induced a much smaller response, indicating that inhibition of these enzymes is not involved in the staurosporine-dependent rise. Therefore, inhibition of an unknown staurosporine-sensitive enzyme, not identical with PKC or one of the cyclic nucleotide-dependent kinases, can trigger an increase in cytoskeletal actin. It is well known that chemotactic peptide induces a rapid rise in cytoskeletal actin, followed by a decrease at later times after the onset of activation. Preincubation with CGP 41,251, a relatively specific inhibitor for PKC, did not affect these two events at concentrations of the drug which, in separate experiments, inhibited markedly phorbol ester induced protein phosphorylation in intact neutrophils. Thus the chemotactic peptide-induced changes in the level of cytoskeletal actin appear to be independent of PKC activation.     

Mammalian TOR signaling to the AGC kinases

The mechanistic (or mammalian) target of rapamycin (mTOR), an evolutionarily conserved protein kinase, orchestrates cellular responses to growth, metabolic and stress signals. mTOR processes various extracellular and intracellular inputs as part of two mTOR protein complexes, mTORC1 or mTORC2. The mTORCs have numerous cellular targets but members of a family of protein kinases, the protein kinase (PK)A/PKG/PKC (AGC) family are the best characterized direct mTOR substrates. The AGC kinases control multiple cellular functions and deregulation of many members of this family underlies numerous pathological conditions. mTOR phosphorylates conserved motifs in these kinases to allosterically augment their activity, influence substrate specificity, and promote protein maturation and stability. Activation of AGC kinases in turn triggers the phosphorylation of diverse, often overlapping, targets that ultimately control cellular response to a wide spectrum of stimuli. This review will highlight recent findings on how mTOR regulates AGC kinases and how mTOR activity is feedback regulated by these kinases. We will discuss how this regulation can modulate downstream targets in the mTOR pathway that could account for the varied cellular functions of mTOR.     

Mammalian TOR signaling to the AGC kinases

The mechanistic (or mammalian) target of rapamycin (mTOR), an evolutionarily conserved protein kinase, orchestrates cellular responses to growth, metabolic and stress signals. mTOR processes various extracellular and intracellular inputs as part of two mTOR protein complexes, mTORC1 or mTORC2. The mTORCs have numerous cellular targets but members of a family of protein kinases, the protein kinase (PK)A/PKG/PKC (AGC) family are the best characterized direct mTOR substrates. The AGC kinases control multiple cellular functions and deregulation of many members of this family underlies numerous pathological conditions. mTOR phosphorylates conserved motifs in these kinases to allosterically augment their activity, influence substrate specificity, and promote protein maturation and stability. Activation of AGC kinases in turn triggers the phosphorylation of diverse, often overlapping, targets that ultimately control cellular response to a wide spectrum of stimuli. This review will highlight recent findings on how mTOR regulates AGC kinases and how mTOR activity is feedback regulated by these kinases. We will discuss how this regulation can modulate downstream targets in the mTOR pathway that could account for the varied cellular functions of mTOR.     

Phosphorylation of membrane-bound acetylcholine receptor by protein kinase C: characterization and subunit specificity

Acetylcholine receptor (AChR) from Torpedo electric organ in its membrane-bound or solubilized form is phosphorylated by the Ca2+/phospholipid-dependent protein kinase (PKC). The subunit specificity for PKC is different from that observed for cAMP-dependent protein kinase (PKA). Whereas PKC phosphorylates predominantly the delta subunit and the phosphorylation of the gamma subunit by this enzyme is very low, PKA phosphorylates both subunits to a similar high extent. We have extended our phosphorylation studies to a synthetic peptide from the gamma subunit, corresponding to residues 346-359, which contains a consensus PKA phosphorylation site. This synthetic peptide is phosphorylated by both PKA and PKC, suggesting that in the intact receptor both kinases may phosphorylate the gamma subunit at a similar site, as has been previously demonstrated by us for the delta subunit [Safran, A., et al. (1987) J. Biol. Chem. 262, 10506-10510]. The diverse pattern of phosphorylation of AChR by PKA and PKC may play a role in the regulation of its function.     

Protein kinase C as a stress sensor

While there are many reviews which examine the group of proteins known as protein kinase C (PKC), the focus of this article is to examine the cellular roles of two PKCs that are important for stress responses in neurological tissues (PKC gamma and epsilon) and in cardiac tissues (PKC epsilon). These two kinases, in particular, seem to have overlapping functions and interact with an identical target, connexin 43 (Cx43), a gap junction protein which is central to proper control of signals in both tissues. While PKC gamma and PKC epsilon both help protect neural tissue from ischemia, PKC epsilon is the primary PKC isoform responsible for responding to decreased oxygen, or ischemia, in the heart. Both do this through Cx43. It is clear that both PKC gamma and PKC epsilon are necessary for protection from ischemia. However, the importance of these kinases has been inferred from preconditioning experiments which demonstrate that brief periods of hypoxia protect neurological and cardiac tissues from future insults, and that this depends on the activation, translocation, or ability for PKC gamma and/or PKC epsilon to interact with distinct cellular targets, especially Cx43. This review summarizes the recent findings which define the roles of PKC gamma and PKC epsilon in cardiac and neurological functions and their relationships to ischemia/reperfusion injury. In addition, a biochemical comparison of PKC gamma and PKC epsilon and a proposed argument for why both forms are present in neurological tissue while only PKC epsilon is present in heart, are discussed. Finally, the biochemistry of PKCs and future directions for the field are discussed, in light of this new information.     

Les protéines phosphatases et protéines kinases des plantes supérieures

The recent gain in knowledge concerning enzymes involved in signal transduction pathways is a direct consequence of the considerable advances made in molecular biology. Protein kinases and protein phosphatases, the two major enzymes implicated in post-translational modifications, have been studied in particular. The number of characterized plant genes and/or cDNAs encoding these enzymes is increasing everyday. Since 1991, 26 genes and cDNAs coding for plant protein phosphatases have been isolated and characterized. The huge number of protein kinases (estimated at several thousands) makes it impossible to give an exhaustive list of the genes already identified, but a classification of these enzymes, based on phylogenetic criteria, allows us to appreciate the range of functions this protein family may play in plants.     

Activation and Function of the MAPKs and Their Substrates, the MAPK-Activated Protein Kinases

Summary: The mitogen-activated protein kinases (MAPKs) regulate diverse cellular programs by relaying extracellular signals to intracellular responses. In mammals, there are more than a dozen MAPK enzymes that coordinately regulate cell proliferation, differentiation, motility, and survival. The best known are the conventional MAPKs, which include the extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun amino-terminal kinases 1 to 3 (JNK1 to -3), p38 (α, β, γ, and δ), and ERK5 families. There are additional, atypical MAPK enzymes, including ERK3/4, ERK7/8, and Nemo-like kinase (NLK), which have distinct regulation and functions. Together, the MAPKs regulate a large number of substrates, including members of a family of protein Ser/Thr kinases termed MAPK-activated protein kinases (MAPKAPKs). The MAPKAPKs are related enzymes that respond to extracellular stimulation through direct MAPK-dependent activation loop phosphorylation and kinase activation. There are five MAPKAPK subfamilies: the p90 ribosomal S6 kinase (RSK), the mitogen- and stress-activated kinase (MSK), the MAPK-interacting kinase (MNK), the MAPK-activated protein kinase 2/3 (MK2/3), and MK5 (also known as p38-regulated/activated protein kinase [PRAK]). These enzymes have diverse biological functions, including regulation of nucleosome and gene expression, mRNA stability and translation, and cell proliferation and survival. Here we review the mechanisms of MAPKAPK activation by the different MAPKs and discuss their physiological roles based on established substrates and recent discoveries.     

蛋白激酶C分子的成熟与活化

蛋白激酶C（Proteinkinase C,PKc）是细胞内一类重要的Ser／Thr激酶,调控多种生命活动的信号转导过程,目前已发现了至少11种亚型,其结构有一定的保守性而又有所差别,导致其功能和调控的多样性。新合成的PKC一般需要经历活化茎环（Acti．vation-loop,A—loop）、转角模体（Tummotif,T1V1）以及疏水模体（hydrophobic motif,HM）的程序性磷酸化过程才能成熟,获得进一步活化的功能。本文综述了近年来PKC的程序性磷酸化成熟以及活化的研究进展情况。     

A protein kinase C isozyme is translocated to cytoskeletal elements on activation.

Protein kinase C (PKC)1 isozymes comprise a family of related cytosolic kinases that translocate to the cell particulate fraction on stimulation. The activated enzyme is thought to be on the plasma membrane. However, phosphorylation of protein substrates occurs throughout the cell and is inconsistent with plasma membrane localization. Using an isozyme-specific monoclonal antibody we found that, on activation, this PKC isozyme translocates to myofibrils in cardiac myocytes and to microfilaments in fibroblasts. Translocation of this activated PKC isozyme to cytoskeletal elements may explain some of the effects of PKC on cell contractility and morphology. In addition, differences in the translocation site of individual isozymes--and, therefore, phosphorylation of different substrates localized at these sites--may explain the diverse biological effects of PKC.     

Specific protein production during melanogenesis in B16/C3 melanoma cells

The mouse melanoma cell line B16/C3 offers an excellent in vitro model for studying melanocyte differentiation. Melanogenesis can be induced by serum, a hormone-supplemented serum-free medium, melanocyte stimulating hormone, and dibutyryl cAMP. The tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate, 5-bromodeoxyuridine, and acidic pH inhibit this process. Using two-dimensional polyacrylamide gel electrophoresis, we have identified four cellular proteins whose production is modulated during melanogenesis, a process which includes concomitant increases in levels of tyrosinase, the rate limiting enzyme for melanin biosynthesis, melanization, and ultimately, cell death. The production of these proteins are coordinately expressed or inhibited in response to the diverse inducers and inhibitors of melanogenesis. We conclude from these studies that these specific proteins are intimately involved in the differentiation of B16/C3 melanoma cells.     

蛋白激酶C分子的成熟与活化

蛋白激酶C(Protein kinase C,PKC)是细胞内一类重要的Ser/Thr激酶,调控多种生命活动的信号转导过程,目前已发现了至少11种亚型,其结构有一定的保守性而又有所差别,导致其功能和调控的多样性。新合成的PKC一般需要经历活化茎环(Activation-loop,A-loop)、转角模体(Turn motif,TM)以及疏水模体(hydrophobic motif,HM)的程序性磷酸化过程才能成熟,获得进一步活化的功能。本文综述了近年来PKC的程序性磷酸化成熟以及活化的研究进展情况。     

Proteome-wide identification of cellular targets affected by bisindolylmaleimide-type protein kinase C inhibitors

Bisindolylmaleimide compounds such as GF109203X are potent inhibitors of protein kinase C (PKC) activity. Although bisindolylmaleimides are not entirely selective for PKC and are known to inhibit a few other protein kinases, these reagents have been extensively used to study the functional roles of PKC family enzymes in cellular signal transduction for more than a decade. Here, we establish a proteomics approach to gain further insights into the cellular effects of this compound class. Functional immobilization of suitable bisindolylmaleimide analogues in combination with the specific purification of cellular binding proteins by affinity chromatography led to the identification of several known and previously unknown enzyme targets. Subsequent in vitro binding and activity assays confirmed the protein kinases Ste20-related kinase and cyclin-dependent kinase 2 (CDK2) and the non-protein kinases adenosine kinase and quinone reductase type 2 as novel targets of bisindolylmaleimide inhibitors. As observed specifically for CDK2, minor chemical variation of the ligand by immobilizing the closely related bisindolylmaleimides III, VIII, and X dramatically affected target binding. These observed changes in affinity correlated with both the measured IC(50) values for in vitro CDK2 inhibition and results from molecular docking into the CDK2 crystal structure. Moreover, the conditions for affinity purification could be adapted in a way that immobilized bisindolylmaleimide III selectively interacted with either PKC alpha or ribosomal S6 protein kinase 1 only after activation of these kinases. Thus, we have established an efficient technique for the rapid identification of cellular bisindolylmaleimide targets and further demonstrate the comparative selectivity profiling of closely related kinase inhibitors within a cellular proteome.     

Protein kinases and the androgen-induced proliferation of accessory sex organ smooth muscle.

The possible role of second messenger systems in androgen-dependent smooth muscle proliferation was investigated. Focusing on the hormone-sensitive guinea pig seminal vesicle, we analyzed changes in protein kinase C (PKC) and cAMP-dependent type I and II protein kinases during the androgen-dependent smooth muscle proliferation of puberty, as well as in the transition to the nonproliferative state of the adult. The androgenic sensitivity of the cAMP-dependent type I and II protein kinases and the cAMP-dependent phosphorylations of soluble muscle proteins did not correlate with the qualitative change in the androgenic sensitivity of the prepubertal vs. adult animals. In contrast to the cAMP-dependent protein kinases, regulation of the soluble and particulate forms of PKC corresponded to the androgen-induced smooth muscle proliferation. That is, in the seminal vesicle muscle of prepubertal castrated animals, androgen treatment reduced both the soluble and particulate forms of PKC during the increase in smooth muscle DNA synthesis, and in adult seminal vesicle smooth muscle, which was resistant to androgen-induced proliferation, both forms of the enzyme were resistant to androgenic stimulation. It is concluded that PKC may be a component of an autocrine mitogenic mechanism involved in the coupling and uncoupling of androgen-induced smooth muscle proliferation.     

Structural and functional diversity of the microbial kinome

The eukaryotic protein kinase (ePK) domain mediates the majority of signaling and coordination of complex events in eukaryotes. By contrast, most bacterial signaling is thought to occur through structurally unrelated histidine kinases, though some ePK-like kinases (ELKs) and small molecule kinases are known in bacteria. Our analysis of the Global Ocean Sampling (GOS) dataset reveals that ELKs are as prevalent as histidine kinases and may play an equally important role in prokaryotic behavior. By combining GOS and public databases, we show that the ePK is just one subset of a diverse superfamily of enzymes built on a common protein kinase-like (PKL) fold. We explored this huge phylogenetic and functional space to cast light on the ancient evolution of this superfamily, its mechanistic core, and the structural basis for its observed diversity. We cataloged 27,677 ePKs and 18,699 ELKs, and classified them into 20 highly distinct families whose known members suggest regulatory functions. GOS data more than tripled the count of ELK sequences and enabled the discovery of novel families and classification and analysis of all ELKs. Comparison between and within families revealed ten key residues that are highly conserved across families. However, all but one of the ten residues has been eliminated in one family or another, indicating great functional plasticity. We show that loss of a catalytic lysine in two families is compensated by distinct mechanisms both involving other key motifs. This diverse superfamily serves as a model for further structural and functional analysis of enzyme evolution.     

Diacylglycerol Kinase Inhibition and Vascular Function

Diacylglycerol kinases (DGKs), a family of lipid kinases, convert diacylglycerol (DG) to phosphatidic acid (PA). Acting as a second messenger, DG activates protein kinase C (PKC). PA, a signaling lipid, regulates diverse functions involved in physiological responses. Since DGK modulates two lipid second messengers, DG and PA, regulation of DGK could induce related cellular responses. Currently, there are 10 mammalian isoforms of DGK that are categorized into five groups based on their structural features. These diverse isoforms of DGK are considered to activate distinct cellular functions according to extracellular stimuli. Each DGK isoform is thought to play various roles inside the cell, depending on its subcellular localization (nuclear, ER, Golgi complex or cytoplasm). In vascular smooth muscle, vasoconstrictors such as angiotensin II, endothelin-1 and norepinephrine stimulate contraction by increasing inositol trisphosphate (IP(3)), calcium, DG and PKC activity. Inhibition of DGK could increase DG availability and decrease PA levels, as well as alter intracellular responses, including calcium-mediated and PKC-mediated vascular contraction. The purpose of this review is to demonstrate a role of DGK in vascular function. Selective inhibition of DGK isoforms may represent a novel therapeutic approach in vascular dysfunction.     

Conserved spatial patterns across the protein kinase family

Protein kinases are a large family of enzymes heavily involved in signal transduction, regulation of metabolism, and control of cell growth and differentiation. These functions require precise recognition of widely diverse signals and substrates, and very detailed control of protein kinase activity. Large molecules interact primarily through recognition of surface features. Comparison of surfaces is complicated by both sequence diversity and conformational variability, including multiple possible rotameric states of side chains. We used a recently developed method of protein surface comparison to compare different serine/threonine and tyrosine kinases. As we have shown, two hydrophobic cores inside a protein kinase molecule are connected by a unique formation, called the "spine". It exists only in the active conformation of protein kinases and is dynamically disassembled during the inactivation process. Detection of such structures by any other method was not possible as the residues which comprise the spine do not form any sequence or 3D motifs in a traditional sense.