Inhibition of Bradykinin-Induced Phosphoinositide Hydrolysis and Ca Mobilisation by Phorbol Ester in Rat Cultured Vascular Smooth Muscle Cells

Regulation of the increase in inositol phosphate (IP) production and intracellular Ca²⁺ concentration ([Ca²⁺]_i by protein kinase C (PKC) was investigated in cultured rat vascular smooth muscle cells (VSMCs). Pretreatment of VSMCs with phorbol 12-myristate 14-acetate (PMA, 1 μM) for 30 min almost abolished the BK-induced IP formation and Ca²⁺ mobilisation. This inhibition was reduced after incubating the cells with PMA for 4 h, and within 24 h the BK-induced responses were greater than those of control cells. The concentrations of PMA giving a half-maximal (pEC₅₀) and maximal inhibition of BK induced an increase in [Ca²⁺]_i, were 7.8 ± 0.3 M and 1 μM, n = 8, respectively. Prior treatment of VSMCs with staurosporine (1 μM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Paralleling the effect of PMA on the BK-induced IP formation and Ca²⁺ mobilisation, the translocation and downregulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of the cells with PMA for various times, translocation of PKC-, βI, βII, δ, ε, and ζ isozymes from the cytosol to the membrane were seen after 5 min, 30 min, 2 h, and 4 h of treatment. However, 24-h treatment caused a partial downregulation of these PKC isozymes in both fractions. Treatment of VSMCs with 1 μM PMA for either 1 or 24 h did not significantly change the K_D and B_max of the BK receptor for binding (control: K_D = 1.7 ± 0.2 nM; B_max = 47.3 ± 4.4 fmol/mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. In conclusion, these resuts demonstrate that translocation of PKC-, βI, βII, δ, ε, and ζ induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca²⁺ mobilisation in VSMCs.     

Reduction of β-Amyloid Levels by Novel Protein Kinase C? Activators

Isoform-specific protein kinase C (PKC) activators may be useful as therapeutic agents for the treatment of Alzheimer disease. Three new ϵ-specific PKC activators, made by cyclopropanation of polyunsaturated fatty acids, have been developed. These activators, AA-CP4, EPA-CP5, and DHA-CP6, activate PKCϵ in a dose-dependent manner. Unlike PKC activators that bind to the 1,2-diacylglycerol-binding site, such as bryostatin and phorbol esters, which produce prolonged down-regulation, the new activators produced sustained activation of PKC. When applied to cells expressing human APPSwe/PS1δ, which produce large quantities of β-amyloid peptide (Aβ), DCP-LA and DHA-CP6 reduced the intracellular and secreted levels of Aβ by 60–70%. In contrast to the marked activation of α-secretase produced by PKC activators in fibroblasts, the PKC activators produced only a moderate and transient activation of α-secretase in neuronal cells. However, they activated endothelin-converting enzyme to 180% of control levels, suggesting that the Aβ-lowering ability of these PKCϵ activators is caused by increasing the rate of Aβ degradation by endothelin-converting enzyme and not by activating nonamyloidogenic amyloid precursor protein metabolism.     

Differential down-regulation of protein kinase C subspecies in KM3 cells

The down-regulation of protein kinase C (PKC) subspecies in KM3 cells (a pre-B, pre-T cell line) has been examined. The PKC from KM3 cells was resolved into two subspecies, type II (mainly beta II) and type III (alpha), upon hydroxyapatite column chromatography. Biochemical and immunocytochemical analysis revealed that, when these cells were treated with 12-O-tetradecanoylphorbol 13-acetate (TPA), the time course of down-regulation of the PKC subspecies was different; type II PKC was translocated and depleted from the cell more quickly than type III enzyme. The results suggest that each PKC subspecies plays a different role in the cellular response to TPA and probably to other external stimuli.     

The heterogeneity of protein kinase C in various rat tissues

Expression of multiple subspecies of protein kinase C (PKC) was studied in various rat tissues. Three types of the enzyme designated type I, II, and III were analyzed, which have the structures of gamma-, beta- (beta I- and beta II-), and alpha-sequence, respectively. Type I enzyme was found only in the central nervous tissue, whereas type III enzyme appeared to be commonly present in various tissues such as liver, spleen, lung, testis, heart, and kidney. Type II enzyme was also found in these tissues. However, immunoblot and biochemical analysis indicated that type II enzyme of lung and heart was distinct from that of other tissues. The tissue-specific expression of PKC suggests that each subspecies of this enzyme has a defined function in processing and modulating tissue responses to external stimuli.     

Modes of inhibition of protein kinase C by triphenylacrylonitrile antiestrogens

Protein kinase C (PKC) I (gamma), II (beta) and III (alpha) subspecies' activities are inhibited by three triphenylacrylonitrile (TPE) antiestrogens at micromolar concentrations. TPE 1 (having a p-hydroxy and a p-diethylaminoethoxy group on the 3-, and 3'- phenyl rings respectively) and TPE 2 (having a p-diethylaminoethoxy group on both the 3-, and 3'- phenyl rings) are competitive with the mechanism of activation by phosphatidylserine (PS). TPE 3 (having p-hydroxy groups on each of the three phenyl rings) is non-competitive with PS and inhibits the Ca2+- and PS-independent phosphorylation of protamine sulfate by PKC subspecies. This evidence suggests that PKC activity can be inhibited by different routes depending on the TPE structure: diethylaminoethoxy side chain-substituted TPEs (TPE 1 and 2) interact with PS as well as with the regulatory domain, whereas the trihydroxylated derivative (TPE 3) inhibits the enzyme by interacting with the catalytically active site.     

Isolation, structural elucidation, and partial synthesis of lutein dehydration products in extracts from human plasma

All-E-(3R,6′R)-3-hydroxy-3′,4′-didehydro-β,γ-carotene (anhydrolutein I) and all-E-(3R,6′R)-3-hydroxy-2′,3′-didehydro-β,ε-carotene (2′,3′-anhydrolutein II) have been isolated and characterized from extracts of human plasma using semipreparative high-performance liquid chromatography (HPLC) on a C₁₈ reversed-phase column. The identification of anhydroluteins was accomplished by comparison of the UV-Vis absorption and mass spectral data as well as HPLC-UV-Vis-mass spectrometry (MS) spiking experiments using fully characterized synthetic compounds. Partial synthesis of anhydroluteins from the reaction of lutein with 2% H₂SO₄ in acetone, in addition to anhydrolutein I (54%) and 2′,3′-anhydrolutein II (19%), also gave (3′R)-3′-hydroxy-3,4-dehydro-β-carotene (3′,4′-anhydrolutein III, 19%). While anhydrolutein I has been shown to be usually accompanied by minute quantities of 2′,3′-anhydrolutein II (ca. 7–10%) in human plasma, 3′,4′-anhydrolutein III has not been detected. The presence of anhydrolutein I and II in human plasma is postulated to be due to acid catalyzed dehydration of the dietary lutein as it passes through the stomach. These anhydroluteins have also been prepared by conversion of lutein diacetate to the corresponding anhydrolutein acetates followed by alkaline hydrolysis. However, under identical acidic conditions, loss of acetic acid from lutein diacetate proceeded at a much slower rate than dehydration of lutein. The structures of the synthetic anhydroluteins, including their absolute configuration at C(3) and C(6′) have been unambiguously established by ¹H NMR and in part by ¹³C NMR, and circular dichroism.     

Protein kinase C inhibitors alter neurotensin receptor binding and function in prostate cancer PC3 cells

Prostate cancer PC3 cells expressed constitutive protein kinase C (PKC) activity that under basal conditions suppressed neurotensin (NT) receptor function. The endogenous PKC activity, assessed using a cell-based PKC substrate phosphorylation assay, was diminished by PKC inhibitors and enhanced by phorbol myristic acid (PMA). Accordingly, PKC inhibitors (staurosporine, Go-6976, Go-6983, Ro-318220, BIS-1, chelerythrine, rottlerin, quercetin) enhanced NT receptor binding and NT-induced inositol phosphate (IP) formation. In contrast, PMA inhibited these functions. The cells expressed conventional PKCs (, βI) and novel PKCs (δ, ε), and the effects of PKC inhibitors on NT binding were blocked by PKC downregulation. The inhibition of NT binding by PMA was enhanced by okadaic acid and blocked by PKC inhibitors. However, when some PKC inhibitors (rottlerin, BIS-1, Ro-318220, Go-69830, quercetin) were used at higher concentrations (> 2 μM), they had a different effect characterized by a dramatic increase in NT binding and an inhibition of NT-induced IP formation. The specificity of the agents implicated novel PKCs in this response and indeed, the inhibition of NT-induced IP formation was reproduced by PKCδ or PKCε knockdown. The inhibition of IP formation appeared to be specific to NT since it was not observed in response to bombesin. Scatchard analyses indicated that the PKC-directed agents modulated NT receptor affinity, not receptor number or receptor internalization. These findings suggest that PKC participates in heterologous regulation of NT receptor function by two mechanisms: a) — conventional PKCs inhibit NT receptor binding and signaling; and b) — novel PKCs maintain the ability of NT to stimulate PLC. Since NT can activate PKC upon binding to its receptor, it is possible that NT receptor is also subject to homologous regulation by PKC.     

Differential activation of protein kinase C isoforms following chemical ischemia in rat cerebral cortex slices

The aim of the current study was to characterize the effects of chemical ischemia and reperfusion at the transductional level in the brain. Protein kinase C isoforms (, β₁, β₂, γ, δ and ) total levels and their distribution in the particulate and cytosolic compartments were investigated in superfused rat cerebral cortex slices: (i) under control conditions; (ii) immediately after a 5-min treatment with 10 mM NaN₃, combined with 2 mM 2-deoxyglucose (chemical ischemia); (iii) 1 h after chemical ischemia (reperfusion). In control samples, all the PKC isoforms were detected; immediately after chemical ischemia, PKC β₁, δ and isoforms total levels (cytosol + particulate) were increased by 2.9, 2.7 and 9.9 times, respectively, while isoform was slightly reduced and γ isoform was no longer detectable. After reperfusion, the changes displayed by , β₁, γ, δ and were maintained and even potentiated, moreover, an increase in β₂ (by 41 ± 12%) total levels became significant. Chemical ischemia-induced a significant translocation to the particulate compartment of PKC isoform, which following reperfusion was found only in the cytosol. PKC β₁ and δ isoforms particulate levels were significantly higher both in ischemic and in reperfused samples than in the controls. Conversely, following reperfusion, PKC β₂ and isoforms displayed a reduction in their particulate to total level ratios. The intracellular calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, 1 mM, but not the N-methyl-d-asparate receptor antagonist, MK-801, 1 μM, prevented the translocation of β₁ isoform observed during ischemia. Both drugs were effective in counteracting reperfusion-induced changes in β₂ and isoforms, suggesting the involvement of glutamate-induced calcium overload. These findings demonstrate that: (i) PKC isoforms participate differently in neurotoxicity/neuroprotection events; (ii) the changes observed following chemical ischemia are pharmacologically modulable; (iii) the protocol of in vitro chemical ischemia is suitable for drug screening.     

Regulation of the Cell Cycle at the G2/M Boundary in Metastatic Melanoma Cells by 12-O-Tetradecanoyl Phorbol-13-acetate (TPA) by Blocking p34Kinase Activity

12-O-Tetradecanoyl phorbol-13-acetate (TPA) inhibits the growth of most malignant melanoma cells but stimulates the growth of normal human melanocytes. We previously showed that addition of TPA inhibits the growth of the human metastatic melanoma cell line, Demel, by blocking cells at both the G1/S and G2/M cell cycle transitions (D. L. Coppocket al.,1992,Cell Growth Differ.3, 485–494). To examine the G2/M transition, we developed a method to synchronize the cells in early S phase using Lovastatin and mevalonate, followed by treatment with hydroxyurea (HU). TPA (30 nM) was effective in blocking cells from entering mitosis and reentering G1 when added up to the end of G2. These cells arrested in G2. Examination of the levels of cyclins A and B1 demonstrated that the levels of these cyclins were not limiting for entrance into M. However, the addition of TPA blocked the increase in p34^cdc2/cyclin B1 kinase activity. In cells treated with TPA, most p34^cdc2was found in the slowly migrating forms on Western blots, which contained increased levels of phosphotyrosine. In addition, the level of the cyclin-dependent kinase inhibitor p21^Cip1/Waf1, but not of p27^Kip1, was increased. We examined the expression of protein kinase C (PKC) isoforms in Demel cells using Western blots to understand which types were involved in the G2 arrest. Demel cells expressed the PKC α, βI, βII, δ, ε, ι/λ, ζ, and μ isozymes. PKC η and PKC θ were not detected. Addition of TPA did not completely down regulate any PKC isozymes over a 12-h period in these synchronized cells. PKC α, βI, βII, δ, and ε isozymes were translocated to the membrane fraction from the cytosolic fraction when treated with TPA. PKC δ appeared as a doublet and the addition of TPA shifted a majority to the slower migrating form. The level of PKC μ was constant; however, a slow mobility form was observed in TPA-treated cells. This reduced mobility was at least partially due to phosphorylation. Thus, the arrest of growth in G2 appears to be due to the inhibition of the p34^cdc2kinase activity which is associated with the increased expression of p21^Cip1/Waf1and increased phosphorylation on tyrosine of p34^cdc2. This arrest, in turn, is associated with a shift of PKC isozymes PKC α, PKC βI, PKC βII, PKC δ, PKC ε, and PKC μ to the membrane fraction which is induced by addition of TPA.     

Downregulation of protein kinase C-γ is independent of a functional kinase domain

Prolonged activation of protein kinase C (PKC) types and β by tumor-promoting phorbol esters leads to desensitization of the phorbol ester response, downregulation of protein kinase C activity and depletion of the protein kinase C polypeptide. When the γ isoenzyme of PKC is transiently expressed in COS-1 cells and exposed to phorbol esters, PKC-γ is downregulated in COS cells although these cells do not normally express this subtype. A point mutation in the purative ATP-binding site (Lys-380→Met-380) of the protein kinase C γ isoenzyme which results in a kinase-deficient enzyme does not interfere with this downregulation. Our results suggest that autophosphorylation or constitutive signalling through the protein kinase C-γ kinase domain is not a prerequisite for downregulation of PKC activity.     

Chemistry and genetics of withanolides in Withania somnifera hybrids

Hybrid plants of Withania somnifera from cross-pollinations of either chemotypes II or III (Israel) and Indian I (Delhi) have been examined. From both hybrids, 14β-hydroxywithanone (5,14β,17-trihydroxy-6,7-epoxy-1-oxo-22R-witha-2,24-dienolide) has been identified. This compound is the first example of a 14β-substitution among withanolides. From the second hybrid three additional new compounds were characterized: a 2,4,6-trien-1-one (14,2O_F-dihydroxy-i-oxo-22R-witha-2,4,6,24-tetraenolide), a 14-hydroxywithanone (5,14,17-trihydroxy-6,7-epoxy-1-oxo-22R-witha-2,24-dienolide) and a 6β,7β-epoxywithanone (5,14,17-trihydroxy-6β,7β-epoxy-1-oxo-22R-witha-2,24-dienolide). An analysis of the inheritance characteristics of various substituents on the withanolide skeleton was based on the occurrence in per cent of each substituent in relation to the total withanolide content in the hybrid plants and their respective parents.     

Colocalization of prostaglandin F2�� receptor FP and prostaglandin F synthase-I in the spinal cord

Prostaglandin F_2α is synthesized by prostaglandin F synthase, which exists in two types, prostaglandin F synthase I (PGFS I) and prostaglandin F synthase II (PGFS II). Prostaglandin F_2α binds to its specific receptor, FP. Our previous immunohistochemical study showed the distinct localization of prostaglandin F synthases in rat spinal cord. PGFS I exists in neuronal somata and dendrites in the gray substance, and PGFS II exists in ependymal cells and tanycytes surrounding the central canal. Both enzymes are also present in endothelial cells of blood vessels in the white and gray substances of the spinal cord. In this study, we found that FP localizes in neuronal somata and dendrites but not in ependymal cells, tanycytes, or endothelial cells. Immunohistochemical analysis of serial sections showed the colocalization of FP and PGFS I. FP immunoreactivity was intense in spinal laminae I and II of the dorsal horn, a connection site of pain transmission, and was similar to that of PGFS I in neuronal elements. These findings suggest that prostaglandin F_2α synthesized in the neuronal somata and dendrites exert an autocrine action there.—Suzuki-Yamamoto, T., K. Toida, Y. Sugimoto, and K. Ishimura. Colocalization of prostaglandin F_2α receptor FP and prostaglandin F synthase-I in the spinal cord.     

G��q-mediated Activation of GRK2 by Mechanical Stretch in Cardiac Myocytes: THE ROLE OF PROTEIN KINASE C*

G protein-coupled receptor kinase-2 (GRK2) is a critical regulator of β-adrenergic receptor (β-AR) signaling and cardiac function. We studied the effects of mechanical stretch, a potent stimulus for cardiac myocyte hypertrophy, on GRK2 activity and β-AR signaling. To eliminate neurohormonal influences, neonatal rat ventricular myocytes were subjected to cyclical equi-biaxial stretch. A hypertrophic response was confirmed by “fetal” gene up-regulation. GRK2 activity in cardiac myocytes was increased 4.2-fold at 48 h of stretch versus unstretched controls. Adenylyl cyclase activity was blunted in sarcolemmal membranes after stretch, demonstrating β-AR desensitization. The hypertrophic response to mechanical stretch is mediated primarily through the Gα_q-coupled angiotensin II AT₁ receptor leading to activation of protein kinase C (PKC). PKC is known to phosphorylate GRK2 at the N-terminal serine 29 residue, leading to kinase activation. Overexpression of a mini-gene that inhibits receptor-Gα_q coupling blunted stretch-induced hypertrophy and GRK2 activation. Short hairpin RNA-mediated knockdown of PKCα also significantly attenuated stretch-induced GRK2 activation. Overexpression of a GRK2 mutant (S29A) in cardiac myocytes inhibited phosphorylation of GRK2 by PKC, abolished stretch-induced GRK2 activation, and restored adenylyl cyclase activity. Cardiac-specific activation of PKCα in transgenic mice led to impaired β-agonist-stimulated ventricular function, blunted cyclase activity, and increased GRK2 phosphorylation and activity. Phosphorylation of GRK2 by PKC appears to be the primary mechanism of increased GRK2 activity and impaired β-AR signaling after mechanical stretch. Cross-talk between hypertrophic signaling at the level of PKC and β-AR signaling regulated by GRK2 may be an important mechanism in the transition from compensatory ventricular hypertrophy to heart failure.     

Molecular basis of human 3β-hydroxysteroid dehydrogenase deficiency

The enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD) catalyses an essential step in the biosynthesis of all classes of steroid hormones. Classical 3β-HSD deficiency is responsible for CAHII, a severe form of congenital adrenal hyperplasia (CAH) that impairs steroidogenesis in both the adrenals and gonads. Newborns affected by 3β-HSD deficiency exhibit signs and symptoms of adrenal insufficiency of varying degrees associated with pseudohermaphroditism in males, whereas females exhibit normal sexual differentiation or mild virilization. Elevated ratios of 5-ene-to 4-ene-steroids appear as the best biological parameter for the diagnosis of 3β-HSD deficiency. The nonclassical form has been suggested to be related to an allelic variant of the classical form of 3β-HSD as described for steroid 21-hydroxylase deficiency. To elucidate the molecular basis of the classical form of 3β-HSD deficiency, we have analysed the structure of the highly homologous type I and II 3β-HSD genes in 12 male pseudohermaphrodite 3β-HSD deficient patients as well as in four female patients. The 14 different point mutations characterized were all detected in the type II 3β-HSD gene, which is the gene predominantly expressed in the adrenals and gonads, while no mutation was detected in the type I 3β-HSD gene predominantly expressed in the placenta and peripheral tissues. The finding of a normal type I 3β-HSD gene provides the explanation for the intact peripheral intracrine steroidogenesis in these patients and increased androgen manifestations at puberty. The influence of the detected mutations on enzymatic activity was assessed by in vitro expression analysis of mutant enzymes generated by site-directed mutagenesis in COS-1 cells. The mutant type II 3β-HSD enzymes carrying mutations detected in patients affected by the salt-losing form exhibit no detectable activity in intact tranfected cells, whereas those with mutations found in nonsalt-loser index cases have some residual activity ranging from 1–10% compared to the wild-type enzyme. Although in general, our findings provide a molecular explanation for the enzymatic heterogeneity ranging from the severe salt-losing form to the clinically inapparent salt-wasting form of the disease, we have observed that the mutant L108W or P186L enzymes found in a compound heterozygote male presenting the salt-wasting form of the disease, has some residual activity (1%) similar to that observed for the mutant N100S enzyme detected in an homozygous male patient suffering from a nonsalt-losing form of this disorder. Unlike the classical 3β-HSD deficiency, our study in women presenting nonclassical 3β-HSD deficiency strongly suggests that this disorder is not due to a mutant type II 3β-HSD.     

Regulation of expression of the 3β-hydroxysteroid dehydrogenases of human placenta and fetal adrenal

The appropriate expression of 3β-hydroxysteroid dehydrogenase/Δ^5→4-isomerase (3β-HSD) is vital for mammalian reproduction, fetal growth and life maintenance. Several isoforms of 3β-HSD, the products of separate genes, have been identified in various species including man. Current investigations are targeted toward defining the processes that regulate the levels of specific isoforms in various steroidogenic tissues of man. High levels of expression of 3β-HSD were observed in placental tissues. It has been generally considered that the multinucleated syncytiotrophoblastic cells are the principal sites of 3β-HSD expression and, moreover, that 3β-HSD expression is intimately associated with cyclic AMP-promoted formation of syncytia. Herein we report the presence of 3β-HSD immunoreactive and mRNA species in uninucleate cytotrophoblasts in the chorion laeve, similar to that in syncytia but not cytotrophoblast placenta. In vitro, 3β-HSD levels in chorion laeve cytotrophoblasts were not increased with time nor after treatment with adenylate cyclase activators, whereas villous cytotrophoblasts spontaneously demonstrated progressive, increased 3β-HSD expression. Moreover, 3β-HSD synthesis appeared to precede morphologic syncytial formation. Thus high steroidogenic enzyme expression in placenta is not necessarily closely linked to formation of syncytia. Both Western immunoblot and enzymic activity analyses also indicated that the 3β-HSD expressed in these cytotrophoblastic populations was the 3β-HSD type I gene product (M_r, 45K) and not 3β-HSD type II (M_r, 44K) expressed in fetal testis. In cultures of fetal zone and definitive zone cell of human fetal adrenal, 3β-HSD expression was not detected until ACTH was added. ACTH, likely acting in a cyclic AMP-dependent process, induced 3β-HSD type II activity and mRNA expression. The higher level of 3β-HSD mRNA in definitive zone compared with fetal zone cells was associated with parallel increases in cortisol secretion relative to dehydroepiandrosterone sulfate formation.     

Protein kinase Cα and Src kinase support human prostate-distributed dihydrotestosterone-metabolizing UDP-glucuronosyltransferase 2B15 activity

Because human prostate-distributed UDP-glucuronosyltransferase (UGT) 2B15 metabolizes 5α-dihydrotestosterone (DHT) and 3α-androstane-5α,17β-diol metabolite, we sought to determine whether 2B15 requires regulated phosphorylation similar to UGTs already analyzed. Reversible down-regulation of 2B15-transfected COS-1 cells following curcumin treatment and irreversible inhibition by calphostin C, bisindolylmaleimide, or röttlerin treatment versus activation by phorbol 12-myristate 13-acetate indicated that 2B15 undergoes PKC phosphorylation. Mutation of three predicted PKC and two tyrosine kinase sites in 2B15 caused 70–100 and 80–90% inactivation, respectively. Anti-UGT-1168 antibody trapped 2B15-His-containing co-immunoprecipitates of PKCα in 130–140- and >150-kDa complexes by gradient SDS-PAGE analysis. Complexes bound to WT 2B15-His remained intact during electrophoresis, whereas 2B15-His mutants at phosphorylation sites differentially dissociated. PKCα siRNA treatment inactivated >50% of COS-1 cell-expressed 2B15. In contrast, treatment of 2B15-transfected COS-1 cells with the Src-specific activator 1,25-dihydroxyvitamin D₃ enhanced activity; treatment with the Src-specific PP2 inhibitor or Src siRNA inhibited >50% of the activity. Solubilized 2B15-His-transfected Src-free fibroblasts subjected to in vitro [γ-³³P]ATP-dependent phosphorylation by PKCα and/or Src, affinity purification, and SDS gel analysis revealed 2-fold more radiolabeling of 55–58-kDa 2B15-His by PKCα than by Src; labeling was additive for combined kinases. Collectively, the evidence indicates that 2B15 requires regulated phosphorylation by both PKCα and Src, which is consistent with the complexity of synthesis and metabolism of its major substrate, DHT. Whether basal cells import or synthesize testosterone for transport to luminal cells for reduction to DHT by 5α-steroid reductase 2, comparatively low-activity luminal cell 2B15 undergoes a complex pattern of regulated phosphorylation necessary to maintain homeostatic DHT levels to support occupation of the androgen receptor for prostate-specific functions.     

Human T cell activation by phorbol esters and diacylglycerol analogues

Activation of protein kinase C (PKC), by the phorbol ester PMA, or the membrane-permeable diacylglycerol 1-oleoyl 2-acetylglycerol (OAG), had different effects on the proliferation-associated responses of a more than 99% pure population of human T cells. Treatment with PMA or OAG caused down-regulation of the TCR-CD3 complex, but only PMA, in combination with ionomycin, was capable of stimulating IL-2R expression and proliferation. Immunocytochemical staining with antisera specific for the PKC subspecies alpha, beta I, beta II, and gamma showed that untreated resting T cells normally coexpress alpha, beta I, and beta II PKC subspecies, which are distributed diffusely throughout the cell, with some localization around the periphery of the nucleus. There was no difference between the responses of these PKC subspecies to OAG and PMA, redistributing, after 10 min of treatment, to a discrete focal area within the cell. Treatment with OAG resulted in transient redistribution of PKC, maximal at 10 min, while in PMA-stimulated cells, the PKC redistribution was prolonged, persisting for at least 24 h. The results suggest that the difference in cellular response to treatment with PMA and OAG is not a consequence of differential activation of various PKC subspecies.     

Insights into diterpene cyclization from structure of bifunctional abietadiene synthase from Abies grandis

Abietadiene synthase from Abies grandis (AgAS) is a model system for diterpene synthase activity, catalyzing class I (ionization-initiated) and class II (protonation-initiated) cyclization reactions. Reported here is the crystal structure of AgAS at 2.3 Å resolution and molecular dynamics simulations of that structure with and without active site ligands. AgAS has three domains (α, β, and γ). The class I active site is within the C-terminal α domain, and the class II active site is between the N-terminal γ and β domains. The domain organization resembles that of monofunctional diterpene synthases and is consistent with proposed evolutionary origins of terpene synthases. Molecular dynamics simulations were carried out to determine the effect of substrate binding on enzymatic structure. Although such studies of the class I active site do lead to an enclosed substrate-Mg²⁺ complex similar to that observed in crystal structures of related plant enzymes, it does not enforce a single substrate conformation consistent with the known product stereochemistry. Simulations of the class II active site were more informative, with observation of a well ordered external loop migration. This “loop-in” conformation not only limits solvent access but also greatly increases the number of conformational states accessible to the substrate while destabilizing the nonproductive substrate conformation present in the “loop-out” conformation. Moreover, these conformational changes at the class II active site drive the substrate toward the proposed transition state. Docked substrate complexes were further assessed with regard to the effects of site-directed mutations on class I and II activities.     

The Chaperones Hsp90 and Cdc37 Mediate the Maturation and Stabilization of Protein Kinase C through a Conserved PXXP Motif in the C-terminal Tail

The life cycle of protein kinase C (PKC) is tightly controlled by mechanisms that mature the enzyme, sustain the activation-competent enzyme, and degrade the enzyme. Here we show that a conserved PXXP motif (Kannan, N., Haste, N., Taylor, S. S., and Neuwald, A. F. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 1272–1277), in the C-terminal tail of AGC (c-AMP-dependent protein kinase/protein kinase G/protein kinase C) kinases, controls the processing phosphorylation of conventional and novel PKC isozymes, a required step in the maturation of the enzyme into a signaling-competent species. Mutation of both Pro-616 and Pro-619 to Ala in the conventional PKC βII abolishes the phosphorylation and activity of the kinase. Co-immunoprecipitation studies reveal that conventional and novel, but not atypical, PKC isozymes bind the chaperones Hsp90 and Cdc37 through a PXXP-dependent mechanism. Inhibitors of Hsp90 and Cdc37 significantly reduce the rate of processing phosphorylation of PKC. Of the two C-terminal sites processed by phosphorylation, the hydrophobic motif, but not the turn motif, is regulated by Hsp90. Overlay of purified Hsp90 onto a peptide array containing peptides covering the catalytic domain of PKC βII identified regions surrounding the PXXP segment, but not the PXXP motif itself, as major binding determinants for Hsp90. These Hsp90-binding regions, however, are tethered to the C-terminal tail via a “molecular clamp” formed between the PXXP motif and a conserved Tyr (Tyr-446) in the αE-helix. Disruption of the clamp by mutation of the Tyr to Ala recapitulates the phosphorylation defect of mutating the PXXP motif. These data are consistent with a model in which a molecular clamp created by the PXXP motif in the C-terminal tail and determinants in the αE-helix of the catalytic domain allows the chaperones Hsp90 and Cdc37 to bind newly synthesized PKC, a required event in the processing of PKC by phosphorylation.Protein kinases, which comprise ∼2% of the human genome, are key signal transducers that regulate a wide variety of cellular processes, such as growth, proliferation, and metabolism, through catalysis of specific phosphorylation events (1). By integrating signals from extracellular stimuli and transmitting them to targeted downstream substrates, protein kinases serve as a pivotal point of regulation within the cell. Deregulation and mutation of protein kinases play a causal role in human pathology, notably cancer, poising kinases as important targets for the design of therapeutics (2–5). Therefore, understanding the mechanisms that regulate protein kinases, such as those important for maturation and processing, would be critical for designing therapeutics that would maintain the correct functioning of signal transduction pathways.Heat shock proteins (Hsp),³ such as Hsp90, are ubiquitously expressed molecular chaperones that facilitate protein folding, regulate quality control, and guide protein turnover in an effort to maintain cellular homeostasis (6–8). Unlike other chaperones such as Hsp70, which non-specifically assists in folding of nascent polypeptide chains (7), Hsp90 works with a specific and discrete set of client proteins, particularly protein kinases (9). Many of the known client kinases of Hsp90, Src (10), Akt (11, 12), phosphoinositide dependent kinase-1 (PDK-1) (13), and ErbB2-/HER2 (14, 15), require the activity of Hsp90 to reach an activation-competent and mature state. Hsp90 is recruited to its kinase clients through interactions with cochaperones, such as Cdc37, which bridge the interaction between Hsp90 and the kinase client (16, 17); this mechanism is revealed in a structural analysis of the Cdc37-Cdk4-Hsp90 complex (18). Cdc37, originally identified in yeast (19), is a cochaperone specific to the kinome that not only assists Hsp90 function but can also recognize and stabilize clients independently of Hsp90 (20). By binding specific regions of the catalytic domains of these kinases, the Hsp90-Cdc37 complex utilizes ATP to promote and stabilize functional conformations of its clients (8, 16, 17, 21). Pharmacological inhibition of Hsp90 by ansamycin antibiotics such as 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) leads to the destabilization and subsequent proteasomal degradation of its clients (6, 22). Recent studies have identified Hsp90 as a promising therapeutic target in cancer as levels of chaperones and activity of client kinases are frequently up-regulated (23–25).The protein kinase C (PKC) family of Ser/Thr kinases serves as a paradigm of how conformation and processing by phosphorylation regulate activity, localization, and inter- and intramolecular interactions (26). The mammalian PKC family consists of 10 isozymes divided into three subclasses (conventional, novel, and atypical) based on their primary structure and second messenger mode of regulation. In the case of conventional PKC isozymes, newly synthesized enzyme is loosely engaged on the membrane in a conformation that exposes the activation loop for phosphorylation by the upstream kinase, PDK-1 (28). This phosphorylation triggers two sequential phosphorylations on the C terminus, one on the turn motif and one on the hydrophobic motif. Phosphorylation of the turn motif is required for phosphorylation of the hydrophobic motif and has recently been shown to depend on the mammalian target of rapamycin complex 2 (mTORC2), a complex consisting of the mammalian target of rapamycin (mTOR), Rictor, Sin1, and mLST8 (29, 30). Turn motif phosphorylation is rapidly followed by phosphorylation at the hydrophobic motif, a reaction that occurs by an intramolecular mechanism in vitro (31). Fully stey phosphorylated PKC is released into the cytosol in a closed conformation in which an autoinhibitory pseudosubstrate sequence occupies the substrate-binding cavity. Upon generation of the lipid second messenger diacylglycerol and elevation of intracellular Ca²⁺, conventional PKC isozymes (α, βI, βII, γ) translocate to membranes via their membrane-targeting C1 and C2 domains, where they adopt an open conformation in which the pseudosubstrate is expelled from the substrate-binding cavity, permitting phosphorylation of downstream substrates (32). Novel PKC isozymes (δ, ε, θ, and η) only respond to diacylglycerol, and their processing phosphorylations can occur through additional mechanisms (33, 34). Atypical PKC isozymes (ζ and ι/λ) do not respond to either Ca²⁺ or diacylglycerol but can also undergo regulation of their processing phosphorylations by external stimuli (35). In fact, atypical PKC isozymes contain a Glu at their hydrophobic motif site. Thus, although these three sites are conserved among PKC family members, additional layers of regulation generate specificity in how these kinases become signaling-competent enzymes.The mechanisms of regulation of the activity and signaling properties of PKC by lipid second messengers and phosphorylation have been well characterized; however, mounting evidence suggests that there are many other regulatory inputs for PKC function. Recent analysis of the evolutionary constraints acting on AGC kinase sequences have underscored the importance of the C-terminal tail as a critical regulatory module (36). Deletion mutants of the C-terminal tail have been shown to abrogate PKC activity (37). In addition to containing the key regulatory phosphorylation sites and docking the upstream kinase PDK-1, the C-terminal tail contains key conserved motifs, found within all AGC kinases, that facilitate ATP binding, promote substrate binding, and structure the catalytic core (36). One such motif comprises the segment PXXP; this motif makes key contacts with the catalytic core, where it is important for modulating movement of the catalytic domain (36). Although this PXXP motif is conserved in all the PKC isozymes, its functional role is unknown.In this study, we address the role of the conserved Pro residues in the PXXP motif of the C-terminal tail of PKC βII. We show that mutation of these two Pro to Ala (P616A and P619A) results in a kinase that is not processed by phosphorylation in cells and is thus inactive. Further analysis reveals that this mutant is not able to bind the chaperones Hsp90 and Cdc37, an event that is required for the processing of PKC by phosphorylation. Our peptide array data indicate that Hsp90 binds to regions of the catalytic core such as the αC-β4 loop and the αD-helix that serve as hinge points for C-helix movement (36). Structural analysis delineates that these hinge points are tethered to the C-terminal tail through a molecular clamp formed between the PXXP segment and AGC conserved residues in the αE-helix. Mutation of one of the “clamping” residues, a conserved Tyr (Tyr-446), recapitulates the defect resulting from mutation of the PXXP motif. Our data support a model in which the PXXP motif participates in an intramolecular clamp with determinants in the αE helix of the kinase core, by providing a recognition surface for Hsp90 to bind and facilitate the maturation of PKC, a required step in the processing of the enzyme.     

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (α, βI, βII and γ), novel type I PKCs (ε and η), novel type II PKCs (δ and θ), and atypical PKCs (ζ and ι). The classical PKCs are activated by Ca²⁺ and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca²⁺-independent. The atypical PKCs are activated by neither Ca²⁺ nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation ^1-2. Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail ³. In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article ⁴, expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.Download video file.^{(60M, mov)}