A possible signal transduction pathway for cyclin D2 expression by a pectic polysaccharide from the roots of bupleurum falcatum L. in murine B cell

Bupleuran 2IIc, a pectic polysaccharide isolated from the roots of bupleurum falcatum L., was previously characterized as a T-cell-independent B cell mitogen. This study focuses on elucidating the mechanism by which bupleuran 2IIc induces cyclin D2 production for inducing mitogenesis in murine B cells. Bupleuran 2IIc was digested with endo-alpha-(1-->4)-D-polygalacturonase and the resulting bupleuran 2IIc/PG-1 ("ramified" region) strongly stimulated cyclin D2 expression. When murine B cells were stimulated with bupleuran 2IIc/PG-1, phosphorylation of tyrosine residues of a number of proteins was observed. Cyclin D2 expression by bupleuran 2IIc/PG-1 was inhibited by the tyrosine kinase inhibitors, genistein and herbimycin A, and the Src family tyrosine kinase inhibitor, PP2, suggesting a possible role for tyrosine kinases. The stimulation by bupleuran 2IIc/PG-1 of cyclin D2 expression was significantly decreased by inhibitors, PI 3-kinase (LY294002 and Wortmannin), PLCgamma (U73122), PKC (H-7), receptor-operated calcium entry inhibitor (SK&F 96365), and calcineurin (FK506). Both PD98059 and U0126, highly selective inhibitors of MEK1 and MEK1/2, respectively, did not strongly suppress the expression of cyclin D2 after stimulation by bupleuran 2IIc/PG-1. The results suggest that (1) bupleuran 2IIc/PG-1 is the active site for induction of cyclin D2 by bupleuran 2IIc, (2) the expression of the cyclin D2 gene by bupleuran 2IIc/PG-1 may be mediated via the activation of PI 3-kinase and PLCgamma followed by activation of PKC and calcium mobilization, and (3) the ERK1/2 cascade is not a central signaling pathway for bupleuran 2IIc/PG-1-induced cyclin D2 expression.     

Multiple protein kinases determine the phosphorylated state of the small heat shock protein, HSP27, in SH-SY5Y neuroblastoma cells

In SH-SY5Y human neuroblastoma cells, the cholinergic agonist, carbachol, stimulates phosphorylation of the small heat shock protein 27 (HSP27). Carbachol increases phosphorylation of both Ser-82 and Ser-78 while the phorbol ester, phorbol-12, 13-dibutyrate (PDB) affects only Ser-82. Muscarinic receptor activation by carbachol was confirmed by sensitivity of Ser-82 phosphorylation to hyoscyamine with no effect of nicotine or bradykinin. This response to carbachol is partially reduced by inhibition of protein kinase C (PKC) with GF 109203X and p38 mitogen-activated protein kinase (MAPK) with SB 203580. In contrast, phosphorylation produced by PDB is completely reversed by GF 109203X or CID 755673, an inhibitor of PKD. Inhibition of phosphatidylinositol 3-kinase or Akt with LY 294002 or Akti-1/2 stimulates HSP27 phosphorylation while rapamycin, which inhibits mTORC1, does not. The stimulatory effect of Akti-1/2 is reversed by SB 203580 and correlates with increased p38 MAPK phosphorylation. SH-SY5Y cells differentiated with a low concentration of PDB and basic fibroblast growth factor to a more neuronal phenotype retain carbachol-, PDB- and Akti-1/2-responsive HSP27 phosphorylation. Immunofluorescence microscopy confirms increased HSP27 phosphorylation in response to carbachol or PDB. At cell margins, PDB causes f-actin to reorganize forming lamellipodial structures from which phospho-HSP27 is segregated. The resultant phenotypic change in cell morphology is dependent upon PKC, but not PKD, activity. The major conclusion from this study is that the phosphorylated state of HSP27 in SH-SY5Y cells results from integrated signaling involving PKC, p38 MAPK and Akt.     

A pectic polysaccharide isolated from the roots of Bupleurum falcatum L. stimulates the tyrosine phosphorylation of lipid rafts of murine B cells

Bupleuran 2IIc, a pectic polysaccharide isolated from the roots of Bupleurum falcatum L., was previously characterized as a T cell-independent B cell mitogen. The endo-(1-->4)-alpha-D-polygalacturonase-resistant moiety of bupleuran 2IIc (bupleuran 2IIc/PG-1) was the active site for expression of the activity, and expression of the cyclin D2 gene by bupleuran 2IIc/PG-1 may be mediated via activation of Src family tyrosine kinase, phosphatidylinositol 3-kinase (PI 3-K) and phospholipase C (PLC)-gamma followed by activation of protein kinase C (PKC) and calcium mobilization (Matsumoto et al., Int. Immunopharmacol., 5, 1373-1386 (2005)). Plasma membrane microdomains (lipid rafts) are enriched in signaling molecules and suggested to be involved in numerous cell functions, including membrane traffic and signaling. When B cells were stimulated with bupleuran 2IIc/PG-1, clustering of membrane lipid rafts was observed. To consider whether lipid rafts are implicated in bupleuran 2IIc/PG-1-mediated B cell proliferation, we analyzed the phosphorylation of tyrosine residues of proteins in lipid rafts. When murine B cells were stimulated with bupleuran 2IIc/PG-1, tyrosine phosphorylation of proteins in lipid rafts fraction was observed within 5 min. Tyrosine phosphorylation in lipid rafts fraction by bupleuran 2IIc/PG-1 was inhibited by the Src-family tyrosine kinase inhibitor, PP2. Together with previously published data, the results presented in this study suggest that activation of signaling molecules in lipid rafts by stimulation of bupleuran 2IIc/PG-1 contributes to B cell proliferation as the membrane-proximal signaling event.     

TRPC, cGMP-dependent protein kinases and cytosolic Ca2+

Ca2+, nitric oxide (NO), and protein kinase G (PKG) are important signaling molecules that play pivotal roles in many physiological processes such as vascular tone control, platelet activation, and synaptic plasticity. TRPC channels allow Ca2+ influx, thus contributing to the production of NO, which subsequently stimulates PKG. It has been demonstrated that PKG can phosphorylate human TRPC3 at Thr-11 and Ser-263 and that this phosphorylation inactivates TRPC3. These two PKG phosphorylation sites, Thr-11 and Ser-263 in human TRPC3, are conserved in other members of the TRPC3/6/7 subfamily, suggesting that PKG may also phosphorylate TRPC6 and TRPC7. In addition, protein kinase C (PKC) also inactivates TRPC3, partly through activating PKG. The PKG-mediated inhibition of TRPC channels may provide a feedback control for the fine tuning of [Ca2+]i levels and protect the cells from the detrimental effects of excessive [Ca2+]i and/or NO.     

P-glycoprotein-independent decrease in drug accumulation by phorbol ester treatment of tumor cells

The effect of a change in the phosphorylation state of the drug transporter P-glycoprotein (P-gp) on its drug transport activity was studied for the substrates daunorubicin (DNR), etoposide (VP-16), and calcein acetoxymethyl ester (Cal-AM). Phorbol ester (PMA), added to stimulate phosphorylation of P-gp by protein kinase C (PKC), caused a decrease in the cellular accumulation of DNR and VP-16, both in multidrug-resistant (MDR) P-gp-oversxpressing cells and in wild-type cells. Since treatment of cells with kinase inhibitor staurosporine (ST) reversed this effect of PMA and the non-PKC-stimulating phorbol ester 4cn-phorbol, 12,13-didecanoate (4αPDD) did not result in a decreased DNR accumulation, we conclude that this effect is the result of kinase activity. The concentration dependence of the inhibition of P-gp by verapamil (Vp) was not influenced by PMA. Accumulation of the P-gp substrate Cal-AM was not influenced by PMA in wild-type cells. Therefore, Cal-AM was used to study the effect of PMA-induced phosphorylation of P-gp on its transport activity. Activation of PKC with PMA or inhibition of protein phosphatase 1/2A (PP1/PP2A) with okadaic acid (OA) did not affect the accumulation of Cal-AM in the MDR cells or wild-type cells. The kinase inhibitor ST increased the Cal-AM accumulation only in the MDR cells. Neither stimulating PKC with PMA nor inhibiting PP1/PP2A with OA led to a decreased inhibition of P-gp by ST, indicating that ST inhibits P-gp directly. From these experiments, we conclude that PKC and PP1/PP2A activity do not regulate the drug transport activity of P-gp. However, these studies provide evidence that PMA-induced PKC activity decreases cellular drug accumulation in a P-gp-independent manner.     

Molecular modelling of mammalian CYP2B isoforms and their interaction with substrates,inhibitors and redox partners

1. The construction of three-dimensional models of CYP2B isozymes from rat (CYP2B1), rabbit (CYP2B4) and man (CYP2B6), based on a multiple sequence alignment with CYP102,a unique eukaryotic-like bacterial P450 (in terms of possessing an NADPHdependent FAD- and FMN-containing oxidoreductase redox partner) of known crystal structure, is reported. 2. The enzyme models described are shown to be consistent with experimental evidence from site-directed mutagenesis studies, antibody recognitionsites and amino acid residues identified as being associated with redox partner interactions, together with the location of a key serine residue (Ser-128) likely to be involved in protein kinaseA-mediated phosphorylation. 3. A substantial number of known substrates and inhibitors of CYP2B isozymes are shown to fit the putative active sites of the enzyme models inagreement with their reported position of metabolism or mode of inhibition respectively. In particular, there is complementarity between the characteristic non-planar geometries of CYP2B substrates and key groups in the enzymes' active sites. 4. Molecular modelling of CYP2B isozymes appears to rationalize a number of the reported findings from quantitative structure-activity relationship investigations on series of CYP2B substrates and inhibitors.     

Functional regulation of serotonin transporter, a molecule involved in the pathogenesis of anxiety disorder]

The serotonin transporter (SET) is a member of the Na+/Cl(-)-dependent neurotransmitter transporter family and functions as a membrane protein which terminates the serotonergic neuronal transmission by re-uptaking serotonin into the pre-synaptic terminal. SET is thought to be involved in the pathogenesis of affective disorders, drug abuse and anxiety disorder. We have focused on SET regulation by phosphorylation/dephosphorylation since SET has many putative phosphorylation sites in its intracellular region. Our previous studies have revealed that phorbolesters, activators of PKC, decreased in SET uptake activity. Based on a mutagenesis analysis of PKC phosphorylation sites and an in vivo phosphorylation study of SET, we have concluded that PKC regulates SET activity via an indirect mechanism, probably via alternating actin cytoskeleton status. Recent reports and our investigation have demonstrated that the SET C-terminal region interacts with actin binding proteins, suggesting the crucial roles of this region in functional regulation of SET.     

Phosphorylation of Galpha11 protein contributes to agonist-induced desensitization of 5-HT2A receptor signaling

Agonist treatment causes desensitization of many G protein-coupled receptor systems. Recent advances have delineated changes in receptors in the desensitization response; however, the role of G proteins remains unclear. We investigated the role of phosphorylation of Galpha q/11 proteins in agonist-induced desensitization of serotonin 2A (5-HT2A) receptors. In an embryonic rat cortical cell line (A1A1v), 24-h treatment with 100 nM (-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl (DOI), a 5-HT(2A/2C) receptor agonist, decreased DOI-stimulated inositol phosphate accumulation and increased the phosphorylation of Galpha q/11 proteins, as demonstrated by immunoprecipitation of Galpha q/11 and both incorporation of 32P phosphate and labeling with a S/T/Y phosphorylation-dependent antibody. Treatment with DOI for 30 min induced desensitization but did not increase phosphorylation of Galpha q/11 proteins, suggesting that different mechanisms are involved in desensitization after short- and long-term treatments. Mutation of S154A in a protein kinase C (PKC) and calcium/calmodulin dependent kinase (CaMK) consensus site in Galpha11 significantly reduced DOI-stimulated phosphorylation of Galpha11 and DOI-induced desensitization of 5-HT2A receptor signaling. Inhibition of PKC and CaMK attenuated phosphorylation of Galpha q/11 proteins and DOI-induced desensitization of 5-HT2A receptors. Expression of Galpha11 S154D, a phosphorylation mimic, reduced DOI-stimulated inositol phosphate accumulation. DOI treatment for 24 h also produced heterologous desensitization, as indicated by decreased bradykinin-stimulated inositol phosphate accumulation. These data suggest that phosphorylation of Galpha11 protein by PKC and CaMK contributes to agonist-induced homologous desensitization of 5-HT2A receptor signaling as well as heterologous desensitization. The phosphorylation of Galpha protein represents a novel mechanism involved in regulation of receptor signaling and agonist-induced desensitization of G protein-coupled receptors.     

丝氨酸331是蛋白激酶C诱导血栓素α受体磷酰化

目的：检测人类血栓素α受体（TPα）C端丝氨酸／苏氨酸残基的各种丙氨酸诱变体作为PKC底物的能力,以确定被PKC磷酰化和脱敏的特异的丝氨酸／苏氨酸残基。方法：为了易于鉴定被磷酰化的细胞内区段,使用甘胱甘肽S－转移酶（GST）－细胞内区段融合蛋白作纯作的PKC底物,然后突变磷酰化蛋白的cDNA,以找出TPα被PKC磷酰化的主要部位,并将有组氨酸尾野生型或诱变型血栓素受体α稳定地转移到人类胚胎肾（EK）293细胞,以研究该受体的磷酰化和脱敏。结果：仅C－端能充当纯化PKC底物。丝氨酸－331 （mP4)被显示强的磷酰化,丝氨酸－324（mP1)则轻微磷酰化,丝氨酸－329（mP3)则微弱磷酰化,而其它丝氨酸／苏氨酸残基没被发现磷酰化。佛波醇－12－肉豆蔻酸酯－13－乙酸盐（PMA）诱导表达野生型TPα的HEK 293细胞磷酰化。然而不能显示触发表达丝氨酸331丙氨酸诱变受体的HEK 293受体磷酰化。用PMA预处理表达野生型受体的HEK 293细胞能抑制I－BOP诱导Ca^2 释放。然而用PMA预处理表达诱变受体的细胞不能中止I－BOP抑制Ca^2 释放。结论：丝氨酸331是TPα磷酰化和脱敏的主要和关键部位。     

Atypical Protein Kinase Cι as a human oncogene and therapeutic target

Protein kinase inhibitors represent a major class of targeted therapeutics that has made a positive impact on treatment of cancer and other disease indications. Among the promising kinase targets for further therapeutic development are members of the Protein Kinase C (PKC) family. The PKCs are central components of many signaling pathways that regulate diverse cellular functions including proliferation, cell cycle, differentiation, survival, cell migration, and polarity. Genetic manipulation of individual PKC isozymes has demonstrated that they often fulfill distinct, nonredundant cellular functions. Participation of PKC members in different intracellular signaling pathways reflects responses to varying extracellular stimuli, intracellular localization, tissue distribution, phosphorylation status, and intermolecular interactions. PKC activity, localization, phosphorylation, and/or expression are often altered in human tumors, and PKC isozymes have been implicated in various aspects of transformation, including uncontrolled proliferation, migration, invasion, metastasis, angiogenesis, and resistance to apoptosis. Despite the strong relationship between PKC isozymes and cancer, to date only atypical PKCiota has been shown to function as a bona fide oncogene, and as such is a particularly attractive therapeutic target for cancer treatment. In this review, we discuss the role of PKCiota in transformation and describe mechanism-based approaches to therapeutically target oncogenic PKCiota signaling in cancer.     

Differential regulation of prostaglandin F(2alpha) receptor isoforms by protein kinase C

Prostaglandin F(2alpha) receptors (FP) are G protein-coupled receptors that bind prostaglandin F(2alpha) (PGF(2alpha)), resulting in the activation of an inositol phosphate (IP) second messenger pathway. Alternative mRNA splicing generates two FP receptor isoforms. These isoforms, designated FP(A) and FP(B), are otherwise identical except for their carboxyl termini. FP(B) is essentially a truncated version of FP(A) that lacks the 46 carboxyl-terminal amino acids, including four putative protein kinase C (PKC) phosphorylation sites. Until now, functional differences between these FP receptor isoforms have not been identified. We now report that pretreatment with the PKC inhibitor bisindolylmaleimide I enhanced PGF(2alpha)-stimulated IP accumulation in transfected cells stably expressing the FP(A) isoform but not in cells stably expressing the FP(B) isoform. Whole-cell phosphorylation experiments showed a strong agonist-dependent phosphorylation of the FP(A) isoform but little or no phosphorylation of the FP(B). Pretreatment of cells with bisindolylmaleimide I decreased PGF(2alpha)-stimulated phosphorylation of the FP(A) isoform consistent with a PKC-dependent phosphorylation. In vitro phosphorylation of an FP(A) carboxyl-terminal fusion protein by recombinant PKCalpha showed that the carboxyl terminus of the FP(A) is a substrate for PKC. These results suggest that PKC-dependent phosphorylation is responsible for differential regulation of second messenger signaling by FP prostanoid receptor isoforms.     

Effects of resveratrol on the autophosphorylation of phorbol ester-responsive protein kinases: inhibition of protein kinase D but not protein kinase C isozyme autophosphorylation

The natural product resveratrol is a potent antagonist of phorbol ester-mediated tumor promotion and in vitro cellular responses to phorbol-ester tumor promoters, but it is only weakly inhibitory against the phosphorylation of conventional exogenous substrates by phorbol ester-responsive protein kinase C (PKC) isozymes. In this report, we compare the effects of resveratrol against the autophosphorylation reactions of PKC isozymes versus the novel phorbol ester-responsive kinase, protein kinase D (PKD). We found that resveratrol inhibits PKD autophosphorylation in a concentration-dependent manner, but has only negligible effects against the autophosphorylation reactions of representative members of each PKC isozyme subfamily (cPKC-alpha, -beta(1), and -gamma, nPKC-delta and -epsilon, and aPKC-zeta). Resveratrol was comparably effective against PKD autophosphorylation (IC(50) = 52 microM) and PKD phosphorylation of the exogenous substrate syntide-2 (IC(50) = 36 microM). The inhibitory potency of resveratrol against PKD is in line with the potency of resveratrol observed in cellular systems and with its potency against other purified enzymes and binding proteins that are implicated in the cancer chemopreventive activity of the polyphenol. Thus, PKD inhibition may contribute to the cancer chemopreventive action of resveratrol.     

Evidence for direct protein kinase-C mediated modulation of N-methyl-D-aspartate receptor current

Protein kinase-C (PKC) activation differentially affects currents from N-methyl-D-aspartate (NMDA) type glutamate receptors depending upon their subunit composition. Experiments using chimeras initially indicated that the cytoplasmic C-terminal tails of NR2B (responsive to PKC) and NR2C (unresponsive to PKC) subunits contain the amino acid residues responsible for the observed disparity of PKC effects. However, truncation and point mutation experiments have suggested that PKC action on NMDA receptors may be entirely indirect, working via the phosphorylation of associated proteins. Here we suggest that PKC does, in fact, affect NR2B/NR1-011 NMDA currents by direct phosphorylation of the NR2B tail at residues S1303 and S1323. Replacement of either of these residues with Ala severely reduces PKC potentiation. To verify that S1303 and S1323 are sites of direct phosphorylation by PKC, synthetic peptides from the regions surrounding these sites were used as substrates for in vitro assays with purified rat brain PKC. These results indicate that PKC can directly phosphorylate S1303 and S1323 in the NR2B C terminus, leading to enhanced currents through NMDA receptor channels. The direct action of PKC on certain NMDA receptor subtypes may be important in any physiological or pathological process where PKC and NR2B/NR1 receptors interact.     

Pharmacology of the receptors for the phorbol ester tumor promoters: multiple receptors with different biochemical properties

The phorbol ester tumor promoters and related analogs are widely used as potent activators of protein kinase C (PKC). The phorbol esters mimic the action of the lipid second messenger diacylglycerol (DAG). The aim of this commentary is to highlight a series of important and controversial concepts in the pharmacology and regulation of phorbol ester receptors. First, phorbol ester analogs have marked differences in their biological properties. This may be related to a differential regulation of PKC isozymes by distinct analogs. Moreover, it seems that marked differences exist in the ligand recognition properties of the C1 domains, the phorbol ester/DAG binding sites in PKC isozymes. Second, an emerging theme that we discuss here is that phorbol esters also target receptors unrelated to PKC isozymes, a concept that has been largely ignored. These novel receptors lacking kinase activity include chimaerins (a family of Rac-GTPase-activating proteins), RasGRP (a Ras exchange factor), and Unc-13/Munc-13 (a family of proteins involved in exocytosis). Unlike the classical and novel PKCs, these "non-kinase" phorbol ester receptors possess a single copy of the C1 domain. Interestingly, each receptor class has unique pharmacological properties and biochemical regulation. Lastly, it is well established that phorbol esters and related analogs can translocate each receptor to different intracellular compartments. The differential pharmacological properties of the phorbol ester receptors can be exploited to generate specific agonists and antagonists that will be helpful tools to dissect their cellular function.     

Vasoactive intestinal peptide (VIP) activation of nuclear protein kinase C in purified nuclei of rat splenocytes

We have examined the actions of vasoactive intestinal peptide (VIP) and certain other known immune modulators on a nuclear pool(s) of protein kinase C (PKC) in isolated rat splenocyte nuclei. Rat splenocyte nuclei pure by enzymatic and electron microscope criteria demonstrated a time- and concentration-dependent activation of nuclear PKC (nPKC) by VIP. A biphasic pattern of three bell-shaped curves was observed with peak phosphorylation at 10⁻¹⁵, 10⁻⁹ and 10⁻⁶M VIP. The phosphorylation of endogenous nuclear substrates was characterized as a PKC-mediated event by use of three known PKC inhibitors, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), sphingosine, and staurosporine, which produced similar phosphate incorporation measurements. Also, this activity was blocked with the addition of a monoclonal antibody to PKC. Inhibitors of the ability of VIP to activate nPKC included somatostatin, 8-bromo-cAMP, peripheral benzodiazepine receptor modulators, and the PKC inhibitors, sphingosine and staurosporine. These data have direct relevance to our knowledge of cell-mediated immunity.     

Pharmacologic modulation of protein kinase C isozymes: the role of RACKs and subcellular localisation.

Protein kinase C (PKC) isozymes are highly homologous kinases and several different isozymes can be present in a cell. Each isozyme is likely to mediate unique functions, but pharmacological tools to explore their isozyme-specific roles have not been available until recently. In this review, we describe the development and application of isozyme-selective inhibitors of PKC. The identification of these inhibitors stems from the observation that PKC isozymes are each localised to unique subcellular locations following activation. Inhibitors of this isozyme-unique localisation have been shown to act as selective inhibitors of the functions of individual isozymes. The identification of isozyme-specific inhibitors should allow the exploration of individual PKC isozyme function in a wide range of cell systems.     

Lithium treatment inhibits protein kinase C translocation in rat brain cortex

RATIONALE: Lithium, an effective psychotropic agent, affects membrane phospholipid metabolism, interferes with phosphoinositide signal transduction, and antagonizes the biological activity of calcium, all major factors of protein kinase C (PKC) activation. Consequently, lithium may interfere with cellular functions requiring PKC. Supporting this hypothesis, lithium was found to inhibit increased neurotransmitter release upon PKC activation and to prevent phorbol ester-mediated PKC translocation. OBJECTIVES: The present study was undertaken to determine whether the frontal cortex of rats treated with lithium exhibits altered PKC activity and translocation in response to phorbol ester, K+, or serotonin (5-HT) receptor stimulation and to determine whether specific PKC isozymes are disproportionately affected. METHODS: Rats were fed either a normal diet or one enriched with LiCl. In cerebrocortical slices or synaptosomes, cytosolic and membranous PKC activity and translocation in response to stimuli were determined after partial purification with anion exchange chromatography. RESULTS: In brain slices, lithium treatment inhibited phorbol 12-myristate, 13-acetate (PMA)-, 5-HT-, or K+-induced PKC translocation from cytosol to membrane without affecting basal membrane or cytosolic PKC activity. In synaptosomes, lithium also attenuated PMA- or K+-evoked translocation of PKC. Immunoblotting with isozyme-specific PKC antibodies revealed that chronic lithium treatment reduced basal cytosolic alphaPKC and deltaPKC but increased membrane-associated zetaPKC immunoreactivities. Stimulation with PMA, 5-HT or K+ elicited translocation of alpha, beta and gammaPKC isozymes and PMA induced translocation of delta and epsilonPKC isozymes. Stimulus-mediated translocation of PKC isozymes was attenuated in cortical tissue obtained from animals that received lithium for 6 weeks. In synaptosomes, PMA- or K+-induced PKC translocation was attenuated by in vitro lithium or chronic lithium treatment. Neither rubidium nor cesium affected PKC activities or PMA-induced translocation. Suppression of PMA-elicited translocation by lithium was partially antagonized by Ca2+. CONCLUSIONS: Lithium treatment reduces PKC translocation induced by either stimulation of a cell surface receptor or by direct enzyme stimulation with phorbol ester. This effect leads to reduced PKC-mediated phosphorylation of cellular proteins and may be responsible for the pharmacotherapeutic action of lithium.     

Regulatory B Subunits of Protein Phosphatase 2A Are Involved in Site-specific Regulation of Tau Protein Phosphorylation

Overexpression of amyloid precursor protein with the Swedish mutation causes abnormal hyperphosphorylation of the microtubule-associated protein tau. Hyperphosphorylated isoforms of tau are major components of neurofibrillary tangles, which are histopathological hallmarks of Alzheimer''s disease. Protein phosphatase 2A (PP2A), a major tau protein phosphatase, consists of a structural A subunit, catalytic C subunit, and a variety of regulatory B subunits. The B subunits have been reported to modulate function of the PP2A holoenzyme by regulating substrate binding, enzyme activity, and subcellular localization. In the current study, we characterized regulatory B subunit-specific regulation of tau protein phosphorylation. We showed that the PP2A B subunit PPP2R2A mediated dephosphorylation of tau protein at Ser-199, Ser-202/Thr-205, Thr-231, Ser-262, and Ser-422. Down-regulation of PPP2R5D expression decreased tau phosphorylation at Ser-202/Thr-205, Thr-231, and Ser-422, which indicates activation of the tau kinase glycogen synthase kinase 3 beta (GSK3β) by PP2A with PPP2R5D subunit. The level of activating phosphorylation of the GSK3β kinase Akt at Thr-308 and Ser-473 were both increased by PPP2R5D knockdown. We also characterized B subunit-specific phosphorylation sites in tau using mass spectrometric analysis. Liquid chromatography-mass spectrometry revealed that the phosphorylation status of the tau protein may be affected by PP2A, depending on the specific B subunits. These studies further our understanding of the function of various B subunits in mediating site-specific regulation of tau protein phosphorylation.