Functional interactions of protein kinase A and C in signalling networks: a recapitulation

On the basis of evidence collected from the literature, we propose a general model by which protein kinase (PK) A and the different PKC isoforms can inversely affect cell growth. Molecular switches, which are able to direct the signal towards antiproliferative or mitogenic pathways, are the different isoforms of Raf and PKC. Conflicting data are also reported and discussed in an attempt to reconcile them. Received 10 November 2005; received after revision 28 December 2005; accepted 3 January 2006     

Transforming growth factor-β-inducible early response gene 1 is a novel substrate for atypical protein kinase Cs

The protein kinase C (PKC) family of serine/threonine kinases consists of ten different isoforms grouped into three subfamilies, denoted classical, novel and atypical PKCs (aPKCs). The aPKCs, PKCι/λ and PKCζ serve important roles during development and in processes subverted in cancer such as cell and tissue polarity, cell proliferation, differentiation and apoptosis. In an effort to identify novel interaction partners for aPKCs, we performed a yeast two-hybrid screen with the regulatory domain of PKCι/λ as bait and identified the Krüppel-like factors family protein TIEG1 as a putative interaction partner for PKCι/λ. We confirmed the interaction of both aPKCs with TIEG1 in vitro and in cells, and found that both aPKCs phosphorylate the DNA-binding domain of TIEG1 on two critical residues. Interestingly, the aPKC-mediated phosphorylation of TIEG1 affected its DNA-binding activity, subnuclear localization and transactivation potential.     

Protein kinases mediate nitric oxide-induced apoptosis in the insect cell line IPLB-LdFB

The involvement of protein kinases (PKA, PKC and PKB) in nitric oxide (NO)-induced apoptosis with sodium nitroprusside plus N-acetyl-L-cysteine in the IPLB-LdFB cell line from the insect Lymantria dispar was investigated. The presence of protein kinase-like molecules was demonstrated by western blot analysis. The role of the kinases in programmed cell death was analysed in cytofluorimetric experiments by incubating the insect cells with H-89 (a specific inhibitor of PKA), calphostin C (an inhibitor of PKC) or wortmannin (an inhibitor of phosphatidylinositol 3-kinase). The results show that PKA is correlated with the induction and PKC and PKB with the prevention of NO-induced insect cell death. Moreover, NO-induced apoptosis involves the release of cytochrome c. Received 15 March 2002; accepted 25 March 2002     

Differential water permeability and regulation of three aquaporin 4 isoforms

Aquaporin 4 (AQP4) is expressed in the perivascular glial endfeet and is an important pathway for water during formation and resolution of brain edema. In this study, we examined the functional properties and relative unit water permeability of three functional isoforms of AQP4 expressed in the brain (M1, M23, Mz). The M23 isoform gave rise to square arrays when expressed in Xenopus laevis oocytes. The relative unit water permeability differed significantly between the isoforms in the order of M1 > Mz > M23. None of the three isoforms were permeable to small osmolytes nor were they affected by changes in external K⁺ concentration. Upon protein kinase C (PKC) activation, oocytes expressing the three isoforms demonstrated rapid reduction of water permeability, which correlated with AQP4 internalization. The M23 isoform was more sensitive to PKC regulation than the longer isoforms and was internalized significantly faster. Our results suggest a specific role for square array formation.     

Regulation of membrane trafficking and endocytosis by protein kinase C: emerging role of the pericentrion,a novel protein kinase C-dependent subset of recycling endosomes

The protein kinase C (PKC) family of isoenzymes has been shown to regulate a variety of cellular processes, including receptor desensitization and internalization, and this has sparked interest in further delineation of the roles of specific isoforms of PKC in membrane trafficking and endocytosis. Recent studies have identified a novel translocation of PKC to a juxtanuclear compartment, the pericentrion, which is distinct from the Golgi complex but epicentered on the centrosome. Sustained activation of PKC (longer than 30 min) also results in sequestration of plasma membrane lipids and proteins to the same compartment, demonstrating a global effect on endocytic trafficking. This review summarizes these studies, particularly focusing on the characterization of the pericentrion as a distinct PKC-dependent subset of recycling endosomes. We also discuss emerging insights into a role for PKC as a central hub in regulating vesicular transport pathways throughout the cell, with implications for a wide range of pathobiologic processes, e.g. diabetes and abnormal neurotransmission or receptor desensitization. Received 11 August 2006; received after revision 20 September 2006; accepted 7 November 2006     

ERKs are the point of divergence of PKA and PKC activation by PTHrP in human skin fibroblasts

Parathyroid hormone-related peptide (PTHrP) receptors, coupled to trimeric G proteins, operate in most target cells through at least three different transduction routes: Gαs-mediated stimulation of adenylylcyclase (AC), Gαq-mediated activation of phospholipase Cβ (PLC) and mitogen-activated protein kinase (MAPK) activation. In this study we investigated the relative role of different pathways in human skin fibroblast prolifera-tion. Using chemical inhibitors and activators of signal transduction, we demonstrated that: (i) AC/cAMP and PLC/1,4,5 inositol triphosphate/diacylglycerol second-messenger systems are simultaneously activated following PTHrP binding to its receptors; (ii) the mitogenic response to PTHrP derives from a balance between two counteracting pathways – an activating route mediated by protein kinase C (PKC) and an inhibitory route mediated by protein kinase A (PKA); (iii) PTHrP mitogenic effects are largely dependent on MAPKs, whose activity can be modulate d by both PKA and PKC. Our results indicate that MAPKs are common targets of both transduction routes and, at the same time, their point of divergence in mediating PTHrP dual and opposite mitogenic effects. Received 2 August 2002; received after revision 10 September 2002; accepted 18 October 2002 RID="*" ID="*"Corresponding author.     

The ubiquitin-specific protease USP25 interacts with three sarcomeric proteins

The biological functions of the more than one hundred genes coding for deubiquitinating enzymes in the human genome remain mostly unknown. The USP25 gene, located at 21q11.2, encodes three protein isoforms produced by alternative splicing. While two of the isoforms are expressed nearly ubiquituously, the expression of the longer USP25 isoform (USP25m) is restricted to muscular tissues and is upregulated during myogenesis. USP25m interacts with three sarcomeric proteins: actin alpha-1 (ACTA1), filamin C (FLNC), and myosin binding protein C1 (MyBPC1), which are critically involved in muscle differentiation and maintenance, and have been implicated in the pathogenesis of severe myopathies. Biochemical analyses demonstrated that MyBPC1 is a short-lived proteasomal substrate, and its degradation is prevented by over-expression of USP25m but not by other USP25 isoforms. In contrast, ACTA1 and FLNC appear to be stable proteins, indicating that their interaction with USP25m is not related to their turnover rate. Received 7 November 2005; received after revision 7 January 2006; accepted 13 January 2006     

The effect of isoforms of the cell polarity protein, human ASIP, on the cell cycle and Fas/FasL-mediated apoptosis in human hepatoma cells

Human ASIP (hASIP) is expressed as numerous alternative splicing isoforms and there is an atypical protein kinease C (aPKC) phosphorylation site in exon 17b of the encoded sequence. We have identified an important role for exon 17b in cancer cells. Our results showed that hASIP-sa and sb had different effects on cell growth and Fas/FasL-mediated apoptosis in BEL-7404 human hepatoma cells. Human ASIP-sa modified the S phase of the cell cycle and might stimulate cell proliferation. Growth inhibition by hASIP-a antisense oligonucleotide-confirmed the positive action of hASIP-sa. Compared with hASIP-sa, hASIP-sb accelerated Fas/FasL-induced apoptosis, examined by sub-G1 accumulation, chromatin condensation, nuclear fragmentation, PARP cleavage, caspase-8 degradation and mitochondria- regulated cell death. Treatment with aPKC inhibitor could enhance Fas/FasL-mediated apoptosis in hASIP-sa-overexpressing cells, suggesting that hASIP-sa and its interaction with aPKC might contribute to the malignant growth and the blocking of Fas/FasL-mediated apoptosis, while hASIP-sb might function as an antagonist of hASIP-sa.Received 24 March 2005; received after revision 31 May 2005; accepted 21 June 2005     

Crosstalk of protein kinase C ε with Smad2/3 promotes tumor cell proliferation in prostate cancer cells by enhancing aerobic glycolysis

Protein kinase C ε (PKCε) has emerged as an oncogenic protein kinase and plays important roles in cancer cell survival, proliferation, and invasion. It is, however, still unknown whether PKCε affects cell proliferation via glucose metabolism in cancer cells. Here we report a novel function of PKCε that provides growth advantages for cancer cells by enhancing tumor cells glycolysis. We found that either PKCε or Smad2/3 promoted aerobic glycolysis, expression of the glycolytic genes encoding HIF-1α, HKII, PFKP and MCT4, and tumor cell proliferation, while overexpression of PKCε or Smad3 enhanced aerobic glycolysis and cell proliferation in a protein kinase D- or TGF-β-independent manner in PC-3M and DU145 prostate cancer cells. The effects of PKCε silencing were reversed by ectopic expression of Smad3. PKCε or Smad3 ectopic expression-induced increase in cell growth was antagonized by inhibition of lactate transportation. Furthermore, interaction of endogenous PKCε with Smad2/3 was primarily responsible for phosphorylation of Ser213 in the Samd3 linker region, and resulted in Smad3 binding to the promoter of the glycolytic genes, thereby promoting cell proliferation. Forced expression of mutant Smad3 (S213A) attenuated PKCε-stimulated protein overexpression of the glycolytic genes. Thus, our results demonstrate a novel PKCε function that promotes cell growth in prostate cancer cells by increasing aerobic glycolysis through crosstalk between PKCε and Smad2/3.     

Regulation of muscle creatine kinase by phosphorylation in normal and diabetic hearts

Protein kinase C (PKC) is an important signaling molecule in the heart, but its targets remain unclear. Using a PKC substrate antibody, we detected a 40-kDa phosphorylated cardiac protein that was subsequently identified by tandem mass spectroscopy as muscle creatine kinase (M-CK) with phosphorylation at serine 128. The forward reaction using ATP to generate phosphocreatine was reduced, while the reverse reaction using phosphocreatine to generate ATP was increased following dephosphorylation of immunoprecipitated M-CK with protein phosphatase 2A (PP2A) or PP2C. Despite higher PKC levels in diabetic hearts, decreased phosphorylation of M-CK was more prominent than the reduction in its expression. Changes in CK activity in diabetic hearts were similar to those found following dephosphorylation of M-CK from control hearts. The decrease in phosphorylation may act as a compensatory mechanism to maintain CK activity at an appropriate level for cytosolic ATP regeneration in the diabetic heart. Received 15 September 2008; received after revision 30 September 2008; accepted 13 October 2008     

Atypical protein kinase C in cell motility

Cell motility is defined as cell movement in the three-dimensional space leading to repositioning of the cell. Atypical protein kinase C (aPKC, including ζ and λ/ι) are a subfamily of PKC. Different from classic PKC and novel PKC, the activation of atypical PKC is not dependent on diacylglycerol or calcium. PKCζ can be activated by lipid components, such as phosphatidylinositols, phosphatidic acid, arachidonic acid, and ceramide. Both phosphatidylinositol (3,4,5)-trisphosphate and PDK1 are necessary for the complete and stable activation of PKCζ. Atypical PKC is involved in the regulation of cell polarization, directional sensing, formation of filopodia, and cell motility. It is essential for migration and invasion of multiple cancer cell types. Particularly, atypical PKC has been found in the regulation of the motility of hematopoietic cells. It also participates in the regulation of proteolytic activity of podosomes and invadopodia. It has been found that atypical PKC can work coordinately with other PKC subfamily members and other signaling pathways. Research on the roles of atypical PKC in cell motility may lead to new therapeutic strategies for cancer and other diseases.     

Diacylglycerol kinases in nuclear lipid-dependent signal transduction pathways

Several independent groups have shown that lipid-dependent signal transduction systems operate in the nucleus and that they are regulated independently from their membrane and cytosolic counterparts. A sizable body of evidence suggests that nuclear lipid signaling controls critical biological functions such as cell proliferation and differentiation. Diacylglycerol is a fundamental lipid second messenger which is produced in the nucleus. The levels of nuclear diacylglycerol fluctuate during the cell cycle progression, suggesting that such a molecule has important regulatory roles. Most likely, nuclear diacylglycerol serves as a chemoattractant for some isoforms of protein kinase C that migrate to the nucleus in response to a variety of agonists. The nucleus also contains diacylglycerol kinases, i.e. the enzymes that, by converting diacylglycerol into phosphatidic acid, terminate diacylglycerol-dependent events. A number of diacylglycerol kinases encoded by separate genes are present in the mammalian genome. This review aims at highlighting the different isotypes of diacylglycerol kinases identified at the nuclear level as well as at discussing their potential function and regulation. Received 4 December 2001; received after revision 28 January 2002; accepted 31 January 2002     

Evaluation of signal transduction pathways mediating the nuclear exclusion of the androgen receptor by melatonin

The intracellular signaling pathways mediating the nuclear exclusion of the androgen receptor (AR) by melatonin were evaluated in PC3 cells stably transfected with the AR. The melatonin-induced nuclear exclusion of the AR by melatonin (100 nM, 3 h) was blocked by LY 83583 (an inhibitor of guanylyl cyclases). 8-Bromo-cGMP (a cell-permeable cGMP analog), mimicked the effect of melatonin, as did ionomycin (a calcium ionophore) and PMA [an activator of protein kinase C (PKC)], and their effects were blocked by GF-109203X (a selective PKC inhibitor). BAPTA (an intracellular calcium chelator) blocked the effects of melatonin and 8-bromo-cGMP but not of PMA. Inhibition or activation of the protein kinase A pathway did not affect basal or melatonin-mediated AR localization. We conclude that the melatonin-mediated rise in cGMP elicits AR nuclear exclusion via a pathway involving increased intracellular calcium and PKC activation. These results define a novel signaling pathway that regulates AR localization and androgen responses in target cells. Received 31 July 2001; received after revision 18 September 2001; accepted 30 October 2001     

Protein kinases: which one is the memory molecule?

Encoding of new experiences is likely to induce activity-dependent modifications in the brain. Studies in organisms far apart on the phylogenetic scale have shown that similar, sometimes identical, signal transduction pathways subserve plasticity in neuronal systems, and they may play pivotal roles in the formation of long-term memories. It has become evident that phosphorylation/dephosphorylation reactions are critical for the initiation of cellular mechanisms that embody, retain and modify information in neural circuits. Although physiological investigations on synaptic plasticity have had a major impact, we have concentrated our review on behavioural studies that provide direct or indirect evidence for a role of kinases in mechanisms underlying memory formation. From these, it appears that the learning event induces activation of a variety of kinases with specific time courses. For instance, the calcium/calmodulin-dependent protein kinase II seems to participate in an early phase of memory formation. Apparently, activation of both protein tyrosine kinases and mitogen-activated protein kinases is required for much longer and may thus have a particular function during transformation from short-term into long-term memory. Quite different time courses appear for protein kinase C (PKC) and protein kinase A (PKA), which may function at two different time points, shortly after training and again much later. This suggests that PKC and PKA might play a role at early and late stages of memory formation. However, we have considered some examples showing that these signalling pathways do not function in isolation but rather interact in an intricate intracellular network. This is indicative of a more complex contribution of each kinase to the fine tuning of encoding and information processing. To decipher this complexity, pharmacological, biochemical and genetic investigations are more than ever necessary to unravel the role of each kinase in the syntax of learning and memory formation.     

Adducin: structure, function and regulation

Adducin is a ubiquitously expressed membrane-skeletal protein localized at spectrin-actin junctions that binds calmodulin and is an in vivo substrate for protein kinase C (PKC) and Rho-associated kinase. Adducin is a tetramer comprised of either alpha/beta or alpha/gamma heterodimers. Adducin subunits are related in sequence and all contain an N-terminal globular head domain, a neck domain and a C-terminal protease-sensitive tail domain. The tail domains of all adducin subunits end with a highly conserved 22-residue myristoylated alanine-rich C kinase substrate (MARCKS)-related domain that has homology to MARCKS protein. Adducin caps the fast-growing ends of actin filaments and also preferentially recruits spectrin to the ends of filaments. Both the neck and the MARCKS-related domains are required for these activities. The neck domain self-associates to form oligomers. The MARCKS-related domain binds calmodulin and contains the major phosphorylation site for PKC. Calmodulin, gelsolin and phosphorylation by the kinase inhibit in vitro activities of adducin involving actin and spectrin. Recent observations suggest a role for adducin in cell motility, and as a target for regulation by Rho-dependent and Ca2+-dependent pathways. Prominent physiological sites of regulation of adducin include dendritic spines of hippocampal neurons, platelets and growth cones of axons.