The regulation of smooth muscle contractility by zipper-interacting protein kinase

Smooth muscle contractility is mainly regulated by phosphorylation of the 20 kDa myosin light chains (LC20), a process that is controlled by the opposing activities of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). Recently, intensive research has revealed that various protein kinase networks including Rho-kinase, integrin-linked kinase, zipper-interacting protein kinase (ZIPK), and protein kinase C (PKC) are involved in the regulation of LC20 phosphorylation and have important roles in modulating smooth muscle contractile responses to Ca2+ (i.e., Ca2+ sensitization and Ca2+ desensitization). Here, we review the general background and structure of ZIPK and summarize our current understanding of its involvement in a number of cell processes including cell death (apoptosis), cell motility, and smooth muscle contraction. ZIPK has been found to induce the diphosphorylation of LC20 at Ser-19 and Thr-18 in a Ca2+-independent manner and to regulate MLCP activity directly through its phosphorylation of the myosin-targeting subunit of MLCP or indirectly through its phosphorylation of the PKC-potentiated inhibitory protein of MLCP. Future investigations of ZIPK function in smooth muscle will undoubtably focus on determining the mechanisms that regulate its cellular activity, including the identification of upstream signaling pathways, the characterization of autoinhibitory domains and regulatory phosphorylation sites, and the development of specific inhibitor compounds.     

Complexities of protein kinase C regulation

A report from the Keystone Protein kinase C: structure, regulation and cellular function meeting, New Mexico, February 5-10, 2000.     

The mechanism of protein kinase C regulation

Protein kinase C (PKC) is a family of serine/threonine protein kinases that plays a central role in transducing extracellular signals into a variety of intracellular responses ranging from cell proliferation to apoptosis. Nine PKC genes have been identified in the human genome, which encode 10 proteins. Each member of this protein kinase family displays distinct biochemical characteristics and is enriched in different cellular and subcellular locations. Activation of PKC has been implicated in the regulation of cell growth and differentiation. This review summarizes works of the past years in the field of PKC biochemistry that covers regulation and activation mechanism of different PKC isoforms.     

Role of protein kinase C in cellular regulation

Protein kinase C (PKC) consists of a family of closely related enzymes ubiquitously present in animal tissues. These enzymes respond to second messengers, Ca2+, diacylglycerol and arachidonic acid, to express their activities at membrane locations. Numerous hormones, neurotransmitters, growth factors and antigens are believed to transmit their signals by activation of a variety of phospholipases to generate these messengers. The various PKC isozymes, which exhibit distinct biochemical characteristics and unique cellular and subcellular localizations, may be differentially stimulated depending on the duration and strength of these messengers. Activation of PKC has been linked to the regulation of cell surface receptors, ion channels, secretion, gene expression, and neuronal plasticity and toxicity. The mechanisms of action of PKC in the regulation of these cellular functions are not entirely clear. Further study to identify the target substrates relevant to the various cellular functions is essential to define the functional diversity of this enzyme family.     

Mechanisms of regulation of phospholipase D1 by protein kinase Calpha

It has been suggested that protein-protein interaction is important for protein kinase C (PKC) alpha to activate phospholipase D1 (PLD1). To determine the one or more sites on PKCalpha that are involved in binding to PLD1, fragments containing the regulatory domain, catalytic domain, and C1-C3 domain of PKCalpha were constructed and shown to be functional, but they all failed to bind and activate PLD1 in vivo and in vitro. A C-terminal 23-amino acid (aa) deletion mutant of PKCalpha was also found to be inactive. To define the binding/activation site(s) in the C terminus of PKCalpha, 1- to 11-aa deletion mutants were made in this terminus. Deletion of up to 9 aa did not alter the ability of PKCalpha to bind and activate PLDl, whereas a 10-aa deletion was inactive. The residue at position 10 was Phe(663). Mutations of this residue (F663D and F663A) caused loss of binding, activation, and phosphorylation of PLD1, indicating that Phe(663) is essential for these activities. Time course experiments showed that the activation of PLD1 by PMA was much faster than its phosphorylation, and its activity decreased as phosphorylation increased with time. Staurosporine, a PKC inhibitor, completely inhibited PLD1 phosphorylation in response to 4beta-phorbol 12-myristate 13-acetate PMA and blocked the later decrease in PLD activity. The same results were found with the D481E mutant of PKCalpha, which is unable to phosphorylate PLD1. These results indicate that neither the regulatory nor catalytic domains of PKCalpha alone can bind to or activate PLD1 and that a residue in the C terminus of PKCalpha (Phe(663)) is required for these effects. The initial activation of PLD1 by PMA is highly correlated with the binding of PKCalpha. Although PKCalpha can phosphorylate PLD1, this is a relatively slow process and is associated with inactivation of the enzyme.     

The regulation of neurotransmitter secretion by protein kinase C

The effect of protein kinase C (PKC) on the release of neurotransmitters from a number preparations, including sympathetic nerve endings, brain slices, synaptosomes, and neuronally derived cell lines, is considered. A comparison is drawn between effects of activation of PKC on neurotransmitter release from small synaptic vesicles and large dense-cored vesicles. The enhancement of neurotransmitter release is discussed in relation to the effect of PKC on: 1. Rearrangement of the F-actin-based cytoskeleton, including the possible role of MARCKS in this process, to allow access of large dense-cored vesicles to release sites on the plasma membrane. 2. Phosphorylation of key components in the SNAP/SNARE complex associated with the docking and fusion of vesicles at site of secretion. 3. Ion channel activity, particularly Ca2+ channels.     

Feedback regulation of phospholipase C-beta by protein kinase C

Treatment of a variety of cells and tissues with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C (PKC) results in the inhibition of receptor-coupled inositol phospholipid-specific phospholipase C (PLC) activity. To determine whether or not the targets of TPA-activated PKC include one or more isozymes of PLC, studies were carried out with PC12, C6Bu1, and NIH 3T3 cells, which contain at least three PLC isozymes, PLC-beta, PLC-gamma, and PLC-delta. Treatment of the cells with TPA stimulated the phosphorylation of serine residues in PLC-beta, but the phosphorylation state of PLC-gamma and PLC-delta was not changed significantly. Phosphorylation of bovine brain PLC-beta by PKC in vitro resulted in a stoichiometric incorporation of phosphate at serine 887, without any concomitant effect on PLC-beta activity. We propose, therefore, that rather than having a direct effect on enzyme activity, the phosphorylation of PLC-beta by PKC may alter its interaction with a putative guanine nucleotide-binding regulatory protein and thereby prevent its activation.     

In situ regulation of cell-cell communication by the cAMP-dependent protein kinase and protein kinase C

The effects of cAMP-dependent protein kinase A and protein kinase C on cell-cell communication have been examined in primary ovarian granulosa cells microinjected with purified components of these two regulatory cascades. These cells possess connexin43 ( 1)-type gap junctions, and are well-coupled electrotonically and as judged by the cell-to-cell transfer of fluorescent dye. Within 2–3 min after injection of the protein kinase A inhibitor (PKI) communication was sharply reduced or ceased, but resumed in about 3 min with the injection of the protein kinase A catalytic subunit. A similar resumption also occurred in PKI-injected cells after exposure to follicle stimulating hormone. Microinjection of the protein kinase C inhibitor protein caused a transient cessation of communication that spontaneously returned within 15–20 min. Treatment of cells with activators of protein kinase C, TPA or OAG for 60 min caused a significant reduction in communication that could be restored within 2–5 min by the subsequent injection of either the protein kinase C inhibitor or the protein kinase A catalytic subunit. With a longer exposure to either protein kinase C activator communication could not be restored and this appeared to be related to the absence of aggregates of connexin43 in membrane as detected immunologically. In cells injected with alkaline phosphatase communication stopped but returned either spontaneously within 20 min or within 2–3 min of injecting the cell with either the protein kinase A catalytic subunit or with protein kinase C. When untreated cells were injected with protein kinase C communication diminished or ceased within 5 min. Collectively these results demonstrate that cell-cell communication is regulated by both protein kinase A and C, but in a complex interrelated manner, quite likely by multiple phosphorylation of proteins within or regulating connexin-43 containing gap junctions.Abbreviations C catalytic subunit of protein kinase A - CKI protein kinase C inhibitor protein - Cx connexin protein - dbcAMP N⁶,2-O-dibutyryladenosine 3:5-cyclic monophosphate - OAG 1-oleoyl-2-acetyl-sn-glycerol - protein kinase A cAMP-dependent protein kinase - protein kinase C Ca²⁺-sensitive phospholipid-dependent protein kinase - PKI protein kinase A inhibitor protein - R regulatory subunit of protein kinase A - TRA 12-O-tetradecanoylphorbol-13-acetate - 8Br-cAMP 8-bromoadenosine 3:5 cyclic monophosphate     

Mechanisms involved in desensitization of particulate guanylyl cyclase in human airway smooth muscle: the role of protein kinase C

We recently showed that cultured human airway smooth muscle cells (HASMC) express both soluble and particulate guanylyl cyclases (GC) and that long term treatment with atrial natriuretic peptide (ANP) causes homologous desensitization of particulate GC. Here we determine if protein kinase C (PKC) activation would desensitize particulate GC and probe the role of PKC in particulate GC desensitization. Pretreatment of HASMC with phorbol 12-myristate 13-acetate (PMA), a PKC activator led to time and concentration-dependent desensitization of ANP-stimulated cGMP accumulation. GF109203X, a selective PKC inhibitor, blocked the PMA-induced desensitization, but did not block ANP-induced desensitization. In addition, desensitization by PMA and ANP showed an additive effect. These results suggest that PKC activation can desensitize particulate GC but that the desensitization induced by ANP is PKC-independent.     

Multiple regulation of proenkephalin gene expression by protein kinase C

In the present study we investigated the role of protein kinase C (Ca2+/phospholipid-dependent enzyme)-mediated processes in the regulation of proenkephalin gene expression in primary cultures of bovine adrenal chromaffin cells. Activators of protein kinase C such as 1-oleoyl-2-acetylglycerol, mezerein, and the phorbol esters phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-didecanoate induced a time-dependent increase in proenkephalin mRNA levels, whereas the inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate had no effect. The increase in phorbol ester-induced proenkephalin mRNA was potentiated by low concentrations of the Ca2+ ionophore A23187, suggesting an interaction between protein kinase- and Ca2+-mediated processes in the regulation of proenkephalin mRNA. The phorbol ester-induced stimulation does not appear to be mediated by an interaction with the cAMP-generating system or increases in Ca2+ uptake. However, when proenkephalin mRNA levels were stimulated by KCl (10 mM) and the dihydropyridine BayK8644, PMA exhibited an inhibitory effect on proenkephalin mRNA, which was detectable at a 10-fold lower concentration of PMA than the stimulatory effect. This inhibitory effect appears to be mediated by an inhibition of Ca2+ entry through voltage-dependent Ca2+ channels, as suggested by 45Ca2+ uptake experiments. Thus, the net effect of PMA depends on and varies with the state of voltage-dependent Ca2+ channel activity. A third mode of action by protein kinase C to modulate proenkephalin gene expression is by interaction with the phosphatidylinositol second messenger system. Stimulation of phosphoinositide hydrolysis and proenkephalin mRNA by histaminic H1-receptor activation was inhibited by low concentrations of PMA. We suggest that protein kinase C may act as a positive and negative regulator of proenkephalin gene expression by interacting with at least three receptor-coupled second messenger systems.     

Respiratory epithelium-dependent inhibition of protein kinase C of airway smooth muscle cells

We have examined the effect of phorbol myristate acetate (PMA) on airway smooth muscle (ASM) in the presence and absence of respiratory epithelium (RE) and analyzed the dependence of this response on extracellular sodium, Na+/H+ exchange, calcium, and cyclooxygenase products; we determined both the resting membrane potential and isometric force developed by ASM preparations. Removal of RE had no effect on the values of the resting membrane potential of ASM cells. In the presence of RE in the preparation, both electrical and contractile responses to PMA (10(-5) M) were significantly different compared with the response of ASM to PMA without RE. When the RE was present, stimulation of protein kinase C caused only a biphasic response in both membrane potential and isometric force. In either the presence or absence of RE, amiloride (10(-5) M) and a low-sodium solution inhibited both electrical and contractile changes of ASM cells caused by PMA. In the presence or absence of RE, verapamil (10(-5) M) attenuated (P less than 0.05) both electrical and contractile responses of ASM cells as induced by PMA. Verapamil, however, had no effect on the last phase of PMA-induced response. Pretreatment of preparations with indomethacin (10(-6) M) changed the PMA-induced response of ASM with RE to a response usually observed in ASM without RE. Finally, the incubation of tracheal preparations without RE with prostaglandin E2 (10(-8) M) altered the response of these preparations in such a way that their electrical and contractile response to PMA was essentially identical to the PMA response observed in preparations with an intact RE.(ABSTRACT TRUNCATED AT 250 WORDS)     

AKAP79 selectively enhances protein kinase C regulation of GluR1 at a Ca2+-calmodulin-dependent protein kinase II/protein kinase C site

Enhancement of AMPA receptor activity in response to synaptic plasticity inducing stimuli may arise, in part, through phosphorylation of the GluR1 AMPA receptor subunit at Ser-831. This site is a substrate for both Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC). However, neuronal protein levels of CaMKII may exceed those of PKC by an order of magnitude. Thus, it is unclear how PKC could effectively regulate this common target site. The multivalent neuronal scaffold A-kinase-anchoring protein 79 (AKAP79) is known to bind PKC and is linked to GluR1 by synapse-associated protein 97 (SAP97). Here, biochemical studies demonstrate that AKAP79 localizes PKC activity near the receptor, thus accelerating Ser-831 phosphorylation. Complementary electrophysiological studies indicate that AKAP79 selectively shifts the dose-dependence for PKC modulation of GluR1 receptor currents approximately 20-fold, such that low concentrations of PKC are as effective as much higher CaMKII concentrations. By boosting PKC activity near a target substrate, AKAP79 provides a mechanism to overcome limitations in kinase abundance thereby ensuring faithful signal propagation and efficient modification of AMPA receptor-mediated responses.     

Heterogeneity of protein kinase C expression and regulation in T lymphocytes

The purpose of the present study was to examine protein kinase C (PKC) isotype expression in T lymphoblasts derived from peripheral blood and the T leukaemic cell Jurkat. Using antisera reactive with PKC alpha, beta 1, and beta 2 and gamma, it was observed that T cells expressed two PKC isotypes, PKC alpha and beta 1. No PKC gamma was detected in T lymphocytes. In lymphoblasts, high levels of PKC beta compared to PKC alpha were found whereas Jurkat cells expressed high levels of alpha compared to PKC beta. Differences in the calcium sensitivity of phorbol ester-induced phosphorylation were observed in Jurkat and T lymphoblasts which correlated with the relative levels of PKC alpha and beta isotypes expressed by the cells.     

Hormonal regulation of protein kinase C in the mouse mammary gland

The hormonal regulation of protein kinase C (PKC) induction over 3 to 14 days was investigated in the mouse mammary gland in vitro and in vivo. In intact mice, estradiol (1 microgram/mouse injected daily for 2 weeks) stimulated PKC activity 70%, while progesterone (1 mg/mouse injected daily) inhibited it by 30%. Prolactin, whose levels were elevated for 2 weeks by two pituitary isografts, had no effect. When mammary gland explants were cultured in insulin and cortisol, the further addition of estradiol (1 ng/ml), progesterone (1 microgram/ml), or prolactin (1 microgram/ml) did not alter PKC activity after 3 days. These data suggest the following conclusions: although previous studies have implicated prolactin in the transient, calcium-phospholipid activation of PKC, it does not appear to elevate total levels of this kinase over prolonged periods. In contrast, the sex steroids do appear to affect long-term levels of this kinase; furthermore, this latter effect may be indirect.     

Differential regulation of microglial NO production by protein kinase C inhibitors

Nitric oxide (NO) produced by microglia has been implicated in the pathogenesis of various central nervous system diseases; however, the intracellular signal pathways for the production of NO are not well known. Protein kinase C (PKC) plays a key role in a variety of signal transduction processes. To elucidate how PKC regulates microglial NO production, we examined the effects of PKC inhibitors on lipopolysaccharide (LPS)-stimulated NO production by primary cultured rat microglia. Staurosporine, a non-selective PKC inhibitor, increased LPS-induced production of NO at 0.1-10 nM range of concentration. Protein kinase A (PKA) inhibitor, H89, did not affect LPS-induced NO production, suggesting that staurosporine effect is not mediated by inhibition of PKA. However, other two PKC inhibitors, whose specificities for PKC isoforms were different, G?6976 and Ro-32-0432, exhibited different effects on NO production from staurosporine; the former inhibited and the latter showed no effect. Interestingly, an activator of PKC, phorbol 12-myristate 13-acetate (PMA) also increased LPS-induced production of NO at 1-10 nM range of concentration, suggesting that prolonged incubation with PMA caused down-regulation of PKC. These results indicate that the inhibition or down-regulation of some PKC isoforms causes the enhancement of NO production. The different effects of PKC inhibitors on the NO production suggest that the different PKC isoforms play different roles in regulation of NO production in microglia.     

Positive regulation of retinoic acid receptor alpha by protein kinase C and mitogen-activated protein kinase in sertoli cells

Mitogen-activated protein kinase (MAPK) and protein phosphatase 2A (PP2A) regulate oocyte meiosis, yet little is known regarding their mechanisms of action. This study addressed the functional importance of active MAPK and PP2A in regulating oocyte meiosis. Experiments were conducted to identify MAPK activation, PP2A activity, intracellular enzyme trafficking, and ultrastructural associations during meiosis. Questions of requisite kinase and/or phosphatase activity and chromatin condensation, microtubule polymerization, and spindle formation were addressed. At the protein level, MAPK and PP2A were present in constant amounts throughout the first meiotic division. Both MAPK and PP2A were activated following germinal vesicle breakdown (GVBD) in conjunction with metaphase I development. Immunocytochemical studies confirmed the absence of active MAPK in germinal vesicle-intact (GVI) and GVBD oocytes. At metaphase I and during the metaphase I/metaphase II transition, activated MAPK colocalized with microtubules, poles, and plates of meiotic spindles. Protein phosphatase 2A was dispersed evenly throughout the GVI oocyte cytoplasm. Throughout the metaphase I/metaphase II transition, PP2A colocalized with microtubules of meiotic spindles. Both active MAPK and PP2A associated with in vitro-polymerized microtubules, suggesting that active MAPK and PP2A locally regulate spindle formation. Inhibition of MAPK activation resulted in compromised microtubule polymerization, no spindle formation, and loosely condensed chromosomes. Treatment with okadaic acid (OA) or calyculin-A (CL-A), which inhibits oocyte cytoplasmic PP2A, caused an absence of microtubule polymerization and spindles, even though MAPK activity was increased under these treatment conditions. Thus, active MAPK is required, but is not sufficient, for normal meiotic spindle formation and chromosome condensation. In addition, the oocyte OA/CL-A-sensitive PP, presumably PP2A, is essential for microtubule polymerization and meiotic spindle formation.     

Epidermal growth factor induces fibroblast contractility and motility via a protein kinase C delta-dependent pathway

Myosin-based cell contractile force is considered to be a critical process in cell motility. However, for epidermal growth factor (EGF)-induced fibroblast migration, molecular links between EGF receptor (EGFR) activation and force generation have not been clarified. Herein, we demonstrate that EGF stimulation increases myosin light chain (MLC) phosphorylation, a marker for contractile force, concomitant with protein kinase C (PKC) activity in mouse fibroblasts expressing human EGFR constructs. Interestingly, PKCdelta is the most strongly phosphorylated isoform, and the preferential PKCdelta inhibitor rottlerin largely prevented EGF-induced phosphorylation of PKC substrates and MARCKS. The pathway through which EGFR activates PKCdelta is suggested by the fact that the MEK-1 inhibitor U0126 and the phosphatidylinositol 3-kinase inhibitor LY294002 had no effect on PKCdelta activation, whereas lack of PLCgamma signaling resulted in delayed PKCdelta activation. EGF-enhanced MLC phosphorylation was prevented by a specific MLC kinase inhibitor ML-7 and the PKC inhibitors chelerythrine chloride and rottlerin. Further indicating that PKCdelta is required, a dominant-negative PKCdelta construct or RNAi-mediated PKCdelta depletion also prevented MLC phosphorylation. In the absence of PLC signaling, MLC phosphorylation and cell force generation were delayed similarly to PKCdelta activation. All of the interventions that blocked PKCdelta activation or MLC phosphorylation abrogated EGF-induced cell contractile force generation and motility. Our results suggest that PKCdelta activation is responsible for a major part of EGF-induced fibroblast contractile force generation. Hence, we identify here a new pathway helping to govern cell motility, with PLC signaling playing a role in activation of PKCdelta to promote the acute phase of EGF-induced MLC activation.     

ATP-sensitive potassium channel traffic regulation by adenosine and protein kinase C

ATP-sensitive potassium (K(ATP)) channels activate under metabolic stress to protect neurons and cardiac myocytes. However, excessive channel activation may cause arrhythmia in the heart and silence neurons in the brain. Here, we report that PKC-mediated downregulation of K(ATP) channel number, via dynamin-dependent channel internalization, can act as a brake mechanism to control K(ATP) activation. A dileucine motif in the pore-lining Kir6.2 subunit of K(ATP), but not the site of PKC phosphorylation for channel activation, is essential for PKC downregulation. Whereas K(ATP) activation results in a rapid shortening of the action potential duration (APD) in metabolically inhibited ventricular myocytes, adenosine receptor stimulation and consequent PKC-mediated K(ATP) channel internalization can act as a brake to lessen this APD shortening. Likewise, in hippocampal CA1 neurons under metabolic stress, PKC-mediated, dynamin-dependent K(ATP) channel internalization can also act as a brake to dampen the rapid decline of excitability due to K(ATP) activation.     

Ceramide-dependent regulation of p42/p44 mitogen-activated protein kinase and c-Jun N-terminal-directed protein kinase in cultured airway smooth muscle cells

Previous studies have demonstrated that a number of biochemical actions of ceramide are mediated through protein kinase signalling pathways, such as p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) and c-Jun N-terminal directed protein kinase (JNK). Ceramide-activated protein kinases, such as the kinase suppressor of Ras (KSR) and protein kinase Czeta (PKCzeta), are involved in the regulation of c-Raf, which promotes sequential activation of MEK-1 and p42/p44 MAPK in mammalian cells. However, in cultured airway smooth muscle (ASM) cells, neither KSR nor PKCzeta are involved in the C2-ceramide (C2-Cer)-dependent activation of this kinase cascade. Instead, we found that C2-Cer utilises a novel pathway involving tyrosine kinases, phosphoinositide 3-kinase (PI3K) and conventional PKC isoform(s). We also found that despite its ability to stimulate p42/p44 MAPK, C2-Cer inhibited platelet-derived growth factor (PDGF)-stimulated DNA synthesis. The possibility that growth arrest could be mediated by JNK was discounted on the basis that PDGF, as well as ceramide, stimulated JNK in these cells. Therefore, growth arrest in response to ceramide is mediated by an alternative mechanism.