PKCι regulates nuclear YAP1 localization and ovarian cancer tumorigenesis.

Atypical protein kinase Cι (PKCι) is an oncogene in lung and ovarian cancer. The PKCι gene PRKCI is targeted for frequent tumor-specific copy number gain (CNG) in both lung squamous cell carcinoma (LSCC) and ovarian serous carcinoma (OSC). We recently demonstrated that in LSCC cells PRKCI CNG functions to drive transformed growth and tumorigenicity by activating PKCι-dependent cell autonomous Hedgehog (Hh) signaling. Here, we assessed whether OSC cells harboring PRKCI CNG exhibit similar PKCι-dependent Hh signaling. Surprisingly, we find that whereas PKCι is required for the transformed growth of OSC cells harboring PRKCI CNG, these cells do not exhibit PKCι-dependent Hh signaling or Hh-dependent proliferation. Rather, transformed growth of OSC cells is regulated by PKCι-dependent nuclear localization of the oncogenic transcription factor, YAP1. Lentiviral shRNA-mediated knockdown (KD) of PKCι leads to decreased nuclear YAP1 and increased YAP1 binding to angiomotin (AMOT), which sequesters YAP1 in the cytoplasm. Biochemical analysis reveals that PKCι directly phosphorylates AMOT at a unique site, Thr750, whose phosphorylation inhibits YAP1 binding. Pharmacologic inhibition of PKCι decreases YAP1 nuclear localization and blocks OSC tumor growth in vitro and in vivo. Immunohistochemical analysis reveals a strong positive correlation between tumor PKCι expression and nuclear YAP1 in primary OSC tumor samples, indicating the clinical relevance of PKCι-YAP1 signaling. Our results uncover a novel PKCι-AMOT-YAP1 signaling axis that promotes OSC tumor growth, and provide a rationale for therapeutic targeting of this pathway for treatment of OSC.

Protein kinase Cα suppresses Kras-mediated lung tumor formation through activation of a p38 MAPK-TGFß signaling axis.

Protein kinase C alpha (PKCα) can activate both pro- and anti-tumorigenic signaling depending upon cellular context. Here, we investigated the role of PKCα in lung tumorigenesis in vivo. Gene expression data sets revealed that primary human non-small lung cancers (NSCLC) express significantly decreased PKCα levels, indicating that loss of PKCα expression is a recurrent event in NSCLC. We evaluated the functional relevance of PKCα loss during lung tumorigenesis in three murine lung adenocarcinoma models (LSL-Kras, LA2-Kras and urethane exposure). Genetic deletion of PKCα resulted in a significant increase in lung tumor number, size, burden and grade, bypass of oncogene-induced senescence, progression from adenoma to carcinoma and a significant decrease in survival in vivo. The tumor promoting effect of PKCα loss was reflected in enhanced Kras-mediated expansion of bronchio-alveolar stem cells (BASCs), putative tumor-initiating cells, both in vitro and in vivo. LSL-Kras/Prkca(-/-) mice exhibited a decrease in phospho-p38 MAPK in BASCs in vitro and in tumors in vivo, and treatment of LSL-Kras BASCs with a p38 inhibitor resulted in increased colony size indistinguishable from that observed in LSL-Kras/Prkca(-/-) BASCs. In addition, LSL-Kras/Prkca(-/-) BASCs exhibited a modest but reproducible increase in TGFß1 mRNA, and addition of exogenous TGFß1 to LSL-Kras BASCs results in enhanced growth similar to untreated BASCs from LSL-Kras/Prkca(-/-) mice. Conversely, a TGFßR1 inhibitor reversed the effects of PKCα loss in LSL-Kras/Prkca(-/-) BASCs. Finally, we identified the inhibitors of DNA binding (Id) Id1-3 and the Wilm's Tumor 1 as potential downstream targets of PKCα-dependent tumor suppressor activity in vitro and in vivo. We conclude that PKCα suppresses tumor initiation and progression, at least in part, through a PKCα-p38MAPK-TGFß signaling axis that regulates tumor cell proliferation and Kras-induced senescence. Our results provide the first direct evidence that PKCα exhibits tumor suppressor activity in the lung in vivo.

Elevated protein kinase C betaII is an early promotive event in colon carcinogenesis.

Protein kinase C (PKC) has been implicated in colon carcinogenesis in humans and in rodent models. However, little is known about the specific role of individual PKC isozymes in this process. We recently demonstrated that elevated expression of PKC betaII in the colonic epithelium induces hyperproliferation in vivo (N. R. Murray et al., J. Cell Biol., 145: 699-711, 1999). Because hyperproliferation is a major risk factor for colon cancer, we assessed whether specific alterations in PKC betaII expression occur during azoxymethane-induced colon carcinogenesis in mice. An increase in PKC betaII expression was observed in preneoplastic lesions (aberrant crypt foci, 3.7-fold) compared with saline-treated animals, and in colon tumors (7.8-fold; P = 0.011) compared with uninvolved colonic epithelium. In contrast, PKC alpha and PKC betaI (a splicing variant of PKC betaII) expression was slightly decreased in aberrant crypt foci and dramatically reduced in colon tumors. Quantitative reverse transcription-PCR analysis revealed that PKC mRNA levels do not directly correlate with PKC protein levels, indicating that PKC isozyme expression is likely regulated at the posttranscriptional/translational level. Finally, transgenic mice expressing elevated PKC betaII in the colonic epithelium exhibit a trend toward increased colon tumor formation after exposure to azoxymethane. Taken together, our results demonstrate that elevated expression of PKC betaII is an important early, promotive event that plays a role in colon cancer development.

Overexpression of protein kinase C betaII induces colonic hyperproliferation and increased sensitivity to colon carcinogenesis.

Protein kinase C betaII (PKC betaII) has been implicated in proliferation of the intestinal epithelium. To investigate PKC betaII function in vivo, we generated transgenic mice that overexpress PKC betaII in the intestinal epithelium. Transgenic PKC betaII mice exhibit hyperproliferation of the colonic epithelium and an increased susceptibility to azoxymethane-induced aberrant crypt foci, preneoplastic lesions in the colon. Furthermore, transgenic PKC betaII mice exhibit elevated colonic beta-catenin levels and decreased glycogen synthase kinase 3beta activity, indicating that PKC betaII stimulates the Wnt/adenomatous polyposis coli (APC)/beta-catenin proliferative signaling pathway in vivo. These data demonstrate a direct role for PKC betaII in colonic epithelial cell proliferation and colon carcinogenesis, possibly through activation of the APC/beta-catenin signaling pathway.

Interleukin-1-induced nuclear factor-kappaB-IkappaBalpha autoregulatory feedback loop in hepatocytes. A role for protein kinase calpha in post-transcriptional regulation of ikappabalpha resynthesis.

The IkappaB inhibitors regulate the activity of the potent transcription factor nuclear factor-kappaB (NF-kappaB). Following signal-induced IkappaB proteolysis, NF-kappaB translocates into the nucleus to activate transcription of target genes, including IkappaBalpha itself, initiating the "NF-kappaB-IkappaBalpha autoregulatory feedback loop." Upon IkappaBalpha resynthesis, NF-kappaB is subsequently inactivated and redistributed back into the cytoplasm. We have previously reported a robust NF-kappaB-IkappaBalpha autoregulatory feedback loop in HepG2 hepatocytes. Sixty minutes after tumor necrosis factor (TNF-alpha) stimulation, IkappaBalpha is resynthesized to approximately 2-fold greater level than in control cells and completely inhibits NF-kappaB binding. Here we investigate the mechanism for IkappaBalpha resynthesis comparing the effect of stimulation of TNF-alpha with that of interleukin-1 (IL-1alpha). Although either TNF-alpha or IL-1alpha stimulation of protein kinase C (PKC)-down-regulated cells equivalently induces NF-kappaB translocation, the kinetics of IkappaBalpha resynthesis is slowed. Moreover, pretreatment with selective calcium-dependent PKC inhibitors selectively slowed the kinetics of the IL-1alpha-induced overshoot without affecting that produced by TNF-alpha. Down-regulation of PKCalpha by antisense phosphorothioate oligonucleotides and expression vectors selectively blocked the IL-1alpha-induced IkappaBalpha overshoot. In the absence of PKCalpha, although IL-1alpha induced similar amounts of IkappaBalpha transcription and changes in steady-state mRNA, a greater component of IkappaBalpha mRNA was retained in the nucleus. These data indicate a selective role for PKCalpha in IL-1alpha-induced IkappaBalpha resynthesis, which is mediated, at least in part, by post-transcriptional control of mRNA export.

Protein kinase calpha regulates human monocyte O-2 production and low density lipoprotein lipid oxidation.

Our previous studies have shown that human native low density lipoprotein (LDL) can be oxidized by activated human monocytes. In this process, both activation of protein kinase C (PKC) and induction of superoxide anion (O-2) production are required. PKC is a family of isoenzymes, and the functional roles of individual PKC isoenzymes are believed to differ based on subcellular location and distinct responses to regulatory signals. We have shown that the PKC isoenzyme that is required for both monocyte O-2 production and oxidation of LDL is a member of the conventional PKC group of PKC isoenzymes (Li, Q., and Cathcart, M. K. (1994) J. Biol. Chem. 269, 17508-17515). The conventional PKC group includes PKCalpha, PKCbetaI, PKCbetaII, and PKCgamma. With the exception of PKCgamma, each of these isoenzymes was detected in human monocytes. In these studies, we investigated the requirement for select PKC isoenzymes in the process of monocyte-mediated LDL lipid oxidation. Our data indicate that PKC activity was rapidly induced upon monocyte activation with the majority of the activity residing in the membrane/particulate fraction. This enhanced PKC activity was sustained for up to 24 h after activation. PKCalpha, PKCbetaI, and PKCbetaII protein levels were induced upon monocyte activation, and PKCalpha and PKCbetaII substantially shifted their location from the cytosol to the particulate/membrane fraction. To distinguish between these isoenzymes for regulating monocyte O-2 production and LDL oxidation, PKCalpha or PKCbeta isoenzyme-specific antisense oligonucleotides were used to selectively suppress isoenzyme expression. We found that suppression of PKCalpha expression inhibited both monocyte-mediated O-2 production and LDL lipid oxidation by activated human monocytes. In contrast, inhibition of PKCbeta expression (including both PKCbetaI and PKCbetaII) did not affect O-2 production or LDL lipid oxidation. Further studies demonstrated that the respiratory burst oxidase responsible for O-2 production remained functionally intact in monocytes with depressed levels of PKCalpha because O-2 production could be restored by treating the monocytes with arachidonic acid. Taken together, our data reveal that PKCalpha, and not PKCbetaI or PKCbetaII, is the predominant isoenzyme required for O-2 production and maximal oxidation of LDL by activated human monocytes.

Phosphatidylglycerol is a physiologic activator of nuclear protein kinase C.

A major mechanism by which protein kinase C (PKC) function is regulated is through the selective targeting and activation of individual PKC isotypes at distinct subcellular locations. PKC betaII is selectively activated at the nucleus during G2 phase of cell cycle where it is required for entry into mitosis. Selective nuclear activation of PKC betaII is conferred by molecular determinants within the carboxyl-terminal catalytic domain of the kinase (Walker, S. D., Murray, N. R., Burns, D. J., and Fields, A. P. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9156-9160). We previously described a lipid-like PKC activator in nuclear membranes, termed nuclear membrane activation factor (NMAF), that potently stimulates PKC betaII activity through interactions involving this domain (Murray, N. R., Burns, D. J., and Fields, A. P. (1994) J. Biol. Chem. 269, 21385-21390). We have now identified NMAF as phosphatidylglycerol (PG), based on several lines of evidence. First, NMAF cofractionates with PG as a single peak of activity through multiple chromatographic separations and exhibits phospholipase sensitivity identical to that of PG. Second, purified PG, but not other phospholipids, exhibits dose-dependent NMAF activity. Third, defined molecular species of PG exhibit different abilities to stimulate PKC betaII activity. 1,2-Dioleoyl-PG possesses significantly higher activity than other PG species, suggesting that both fatty acid side chain composition and the glycerol head group are important determinants for activity. Fourth, in vitro binding studies demonstrate that PG binds to the carboxyl-terminal region of PKC betaII, the same region we previously implicated in NMAF-mediated activation of PKC betaII. Taken together, our results indicate that specific molecular species of nuclear PG function to physiologically regulate PKC betaII activity at the nucleus.

Atypical protein kinase C iota protects human leukemia cells against drug-induced apoptosis.

Protein kinase C (PKC) isozymes play distinct roles in cellular function. In human K562 leukemia cells, PKC alpha is important for cellular differentiation and PKC betaII is required for proliferation. In this report, we assess the role of the atypical PKC isoform PKC iota in K562 leukemia cell physiology. K562 cells were stably transfected with expression plasmids containing the cDNA for human PKC iota in sense or antisense orientation to increase or decrease cellular PKC iota levels, respectively. Overexpression or inhibition of expression of PKC iota had no significant effect on the proliferative capacity of K562 cells nor their sensitivity to phorbol myristate acetate-induced cytostasis and megakaryocytic differentiation, suggesting that PKC iota does not play a critical role in these processes. Rather, PKC iota serves to protect K562 cells against drug-induced apoptosis. K562 cells, which are resistant to most apoptotic agents, undergo apoptosis when treated with the protein phosphatase inhibitor okadaic acid (OA). Overexpression of PKC iota leads to increased resistance to OA-induced apoptosis whereas inhibition of PKC iota expression sensitizes cells to OA-induced apoptosis. Overexpression of the related atypical PKC zeta has no protective effect, demonstrating that the effect is isotype-specific. PKC iota also protects K562 cells against taxol-induced apoptosis, indicating that it plays a general protective role against apoptotic stimuli. These data support a role for PKC iota in leukemia cell survival.

A role for nuclear phosphatidylinositol-specific phospholipase C in the G2/M phase transition.

Protein kinase C (PKC) is activated at the nucleus during the G2 phase of cell cycle, where it is required for mitosis. However, the mechanisms controlling cell cycle-dependent activation of nuclear PKC are not known. We now report that nuclear levels of the major physiologic PKC activator diacylglycerol (DAG) fluctuate during cell cycle. Specifically, nuclear DAG levels in G2/M phase cells are 2. 5-3-fold higher than in G1 phase cells. In synchronized cells, nuclear DAG levels rise to a peak coincident with the G2/M phase transition and return to basal levels in G1 phase cells. This increase in DAG level is sufficient to stimulate betaII PKC-mediated phosphorylation of its mitotic nuclear envelope substrate lamin B in vitro. Isolated nuclei from G2 phase cells contain an active phospholipase activity capable of generating DAG in vitro. Nuclear phospholipase activity is inhibited by the selective phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitor 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine and neomycin sulfate, but not by the phosphatidylcholine-PLC selective inhibitor D609 or inhibitors of phospholipase D-mediated DAG generation. Treatment of synchronized cells with 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine leads to decreased nuclear PI-PLC activity and cell cycle blockade in the G2 phase, suggesting a role for nuclear PI-PLC in the G2/M phase transition. Our data are consistent with the hypothesis that nuclear PI-PLC generates DAG to activate nuclear betaII PKC, whose activity is required for mitosis.

Hypophosphorylation of topoisomerase II in etoposide (VP-16)-resistant human leukemia K562 cells associated with reduced levels of beta II protein kinase C.

We selected and characterized a 30-fold etoposide (VP-16)-resistant subline of K562 human leukemia cells (K/VP.5) that exhibits quantitative and qualitative changes in topoisomerase II, including hypophosphorylation of this drug target. The initial rate of topoisomerase II phosphorylation was reduced 3-fold in K/VP.5 compared with K562 cells, but the rate of dephosphorylation was similar. Analysis of potential topoisomerase II protein kinases revealed a 3-fold reduction in the level of the beta II protein kinase C (PKC) in K/VP.5 cells, whereas levels of alpha- and epsilon PKC, casein kinase II, p42map kinase, and p34cdc2 kinase were comparable for both cell lines. The PKC activator, bryostatin 1, together with K562 nuclear extracts potentiated VP-16-induced topoisomerase II/DNA covalent complex formation in nuclei isolated from K/VP.5 cells but not from K562 cells. Bryostatin 1 effects were blocked by the PKC inhibitor 7-O-methyl-hydroxy-staurosporine. Bryostatin 1 also up-regulated topoisomerase II phosphorylation and potentiated VP-16 activity in intact K/VP.5 cells but had no enhancing effect in K562 cells. 4 beta-Phorbol-12,13-dibutyrate and 12-O-tetradecanoylphorbol-13-acetate did not potentiate VP-16-induced topoisomerase II/DNA complex formation in intact cells or in isolated K/VP.5 nuclei. Together, our results indicate that beta II PKC plays a role in modulating the VP-16-induced DNA binding activity of topoisomerase II in resistant K/VP.5 cells through a mechanism linked to phosphorylation of topoisomerase II.

Protein kinase C chimeras: catalytic domains of alpha and beta II protein kinase C contain determinants for isotype-specific function.

Protein kinase C (PKC) is involved in the proliferation and differentiation of many cell types. In human erythroleukemia (K-562) cells, the PKC isoforms alpha and beta II play distinct functional roles. alpha PKC is involved in phorbol 12-myristate 13-acetate-induced cytostasis and megakaryocytic differentiation, whereas beta II PKC is required for proliferation. To identify regions within alpha and beta II PKC that allow participation in these divergent pathways, we constructed chimeras in which the regulatory and catalytic domains of alpha and beta II PKC were exchanged. These PKC chimeras can be stably expressed, exhibit enzymatic properties similar to native alpha and beta II PKC in vitro, and participate in alpha and beta II PKC isotype-specific pathways in K-562 cells. Expression of the beta/alpha PKC chimera induces cytostasis in the same manner as overexpression of wild-type alpha PKC. In contrast, the alpha/beta II PKC chimera, like wild-type beta II PKC, selectively translocates to the nucleus and leads to increased phosphorylation of the nuclear envelope polypeptide lamin B in response to bryostatin-1. Therefore, the catalytic domains of alpha and beta II PKC contain determinants important for alpha and beta II PKC isotype function. These results suggest that the catalytic domain represents a potential target for modulating PKC isotype activity in vivo.

Human parainfluenza virus type 3 phosphoprotein: identification of serine 333 as the major site for PKC zeta phosphorylation.

The human parainfluenza virus type 3 P protein is an RNA polymerase subunit involved in both transcription and replication during the life cycle of the virus. Our laboratory has recently shown that the P protein is phosphorylated both in vitro and in vivo by the cellular protein kinase C (PKC) isoform zeta and that this phosphorylation is essential for viral replication. To identify the site(s) of phosphorylation, we have used CNBr cleavage, phosphoamino acid analysis, and two-dimensional tryptic peptide mapping of the in vitro and in vivo phosphorylated P protein. We demonstrate that when bacterially expressed unphosphorylated P is labeled in vitro with either commercial PKC or purified recombinant PKC zeta P protein has one major phosphorylation site. By site-directed mutagenesis of PKC consensus sites in the P protein, the primary phosphorylation site is found to be Ser 333. The same site appeared to be modified when viral P protein was phosphorylated in vitro by the PKC packaged within the virion and in the P protein of progeny virion labeled in vivo.

Presence of a beta II protein kinase C-selective nuclear membrane activation factor in human leukemia cells.

In human promyelocytic (HL60) leukemia cells beta II protein kinase C (PKC) is selectively translocated to the nucleus in response to proliferative stimuli. At the nucleus, beta II PKC directly phosphorylates the nuclear envelope polypeptide lamin B at two consensus PKC phosphorylation sites, Ser395 and Ser405. Phosphorylation of these sites by beta II PKC leads to solubilization of lamin B indicative of mitotic nuclear envelope breakdown in vitro (Hocevar, B.A., Burns, D.J., and Fields, A.P. (1993) J. Biol. Chem. 268, 7545-7552). We have now investigated the molecular basis for beta II PKC-selective nuclear translocation and lamin B phosphorylation using an in vitro reconstitution system. We find that beta II PKC phosphorylates nuclear envelope lamin B at 10-20 times the rate of alpha PKC, whereas both kinases phosphorylate soluble lamin B at similar rates. Comparative tryptic phosphopeptide analysis demonstrates that alpha PKC and beta II PKC phosphorylate identical sites, Ser395 and Ser405, on soluble lamin B. These data suggest that a component(s) of the nuclear envelope confers beta II PKC-selective nuclear activation and lamin B phosphorylation. Extraction of nuclear envelopes with either non-ionic detergent (2% n-octyl glucoside) or organic solvent (CHCl3/CH3OH/H2O; 10:10:3) abolishes beta II PKC-selective phosphorylation of nuclear lamin B. Nuclear membrane extracts reconstitute beta II PKC-selective phosphorylation, indicating the presence of a beta II PKC-selective nuclear membrane activation factor (NMAF). NMAF selectively activates beta II PKC histone H1 kinase activity 3-4-fold above the level achieved with optimal concentrations of Ca2+, diacylglycerol, and phosphatidylserine. Finally, NMAF activity is not affected by exhaustive protease treatment, suggesting that it is a nuclear membrane lipid(s) or lipid metabolite. These data suggest that NMAF plays a physiologic role in the nuclear activation of beta II PKC.

Protein kinase C isotypes in human erythroleukemia (K562) cell proliferation and differentiation. Evidence that beta II protein kinase C is required for proliferation.

The human erythroleukemia (K562) cell line undergoes megakaryocytic differentiation and cessation of proliferation when treated with phorbol myristate acetate (PMA). To investigate the role of individual protein kinase C (PKC) isotypes in these events, we have assessed PKC isotype expression during leukemic proliferation and PMA-induced differentiation. Immunoblot analysis using isotype-specific antibodies demonstrates that proliferating K562 cells express the alpha, beta II, and zeta PKC isotypes. PMA-induced differentiation and cytostasis lead to a decrease in beta II PKC and increases in alpha and zeta PKC levels. The role of the alpha and beta II PKC isotypes was further assessed in cells overexpressing these isotypes. K562 cells overexpressing human alpha PKC grew more slowly and were more sensitive to the cytostatic effects of PMA than control cells, whereas cells overexpressing beta II PKC were less sensitive to PMA. PMA-induced cytostasis is reversed upon removal of PMA. Resumption of proliferation is accompanied by reexpression of beta II PKC to near control levels, whereas alpha and zeta PKC levels remain elevated for several days after removal of PMA. Proliferation of PMA-withdrawn cells can be partially inhibited by antisense beta II PKC oligodeoxyribonucleotide. Growth inhibition is dose-dependent, specific for beta II PKC-directed antisense oligonucleotide, and associated with significant inhibition of beta II PKC levels indicating that beta II PKC is essential for K562 cell proliferation. Sodium butyrate, which unlike PMA induces megakaryocytic differentiation without cytostasis, causes increases in both alpha and beta II PKC levels. These data demonstrate that beta II PKC is required for K562 cell proliferation, whereas alpha PKC is involved in megakaryocytic differentiation.

Identification of glycoinositol phospholipids in rat liver by reductive radiomethylation of amines but not in H4IIE hepatoma cells or isolated hepatocytes by biosynthetic labeling with glucosamine.

The identification of free glycoinositol phospholipids (GPIs) following biosynthetic labeling with [3H]glucosamine in cultured cells has been reported by several laboratories. We applied this procedure to two of the cell types used in these studies, H4IIE hepatoma cells and isolated hepatocytes, but were unable to detect a [3H]glucosamine-containing lipid that met any of the criteria for GPIs, including sensitivity to phosphatidylinositol-specific phospholipase C (PIPLC) or GPI-specific phospholipase D. Part of the difficulty in radiolabeling a GPI by this procedure was the rapid metabolic conversion of [3H]glucosamine to galactosamine and neutral or anionic derivatives. A PIPLC-sensitive radiolabeled lipid was detected only after 16 h of labeling. The water-soluble fragments released from this lipid by PIPLC corresponded largely to myo-inositol 1,2-cyclic phosphate and myo-inositol 1-phosphate, products expected from PIPLC cleavage of phosphatidylinositol or lyso-phosphatidylinositol. In an alternative approach that we introduce here, free GPIs in lipid extracts from rat liver plasma membranes were labeled by reductive radiomethylation. This procedure, which radiomethylates primary and secondary amines, has been shown to label a glucosamine residue adjacent to inositol in all GPIs characterized to date. The labeled extracts were fractionated by two-dimensional thin-layer chromatography, and a cluster of polar labeled lipids were assigned as GPIs based upon the following observations. 1) They were cleaved by PIPLC, 2) after hydrolysis in 6 N HCl, both radiomethylated glucosamine and a glucosamine-inositol conjugate were identified by cation exchange chromatography, and 3) hydrolysis in 4 M trifluoroacetic acid generated a fragment consistent with glucosamine-inositol-phosphate. These results illustrate new criteria for the identification of GPIs. The labeled GPIs also contained radiomethylated ethanolamine, another component found in GPI anchors of proteins and in mature lipid precursors of GPI anchors, suggesting that the liver plasma membrane GPIs retained considerable structural homology to GPI anchors.

Conversion of human erythrocyte acetylcholinesterase from an amphiphilic to a hydrophilic form by phosphatidylinositol-specific phospholipase C and serum phospholipase D.

Each catalytic subunit in the amphiphilic dimer of human erythrocyte acetylcholinesterase (AChE) is anchored in the plasma membrane exclusively by a glycoinositol phospholipid. In contrast to erythrocyte AChEs in other mammalian species, the human enzyme is resistant to direct cleavage by phosphatidylinositol-specific phospholipase C (PtdIns-specific PLC). The resistance is due to the existence of an additional fatty acyl chain on the inositol ring which blocks the action of PtdIns-specific PLC [Roberts et al. (1988) J. Biol. Chem. 263, 18766-18775]. In this report, nondenaturing polyacrylamide gel electrophoresis was applied to permit rapid and unambiguous distinction between amphiphilic AChE, in which each catalytic subunit binds one nonionic detergent micelle, and hydrophilic AChE, which does not interact with detergent. Deacylation of human erythrocyte AChE by an alkaline treatment with hydroxylamine rendered the amphiphilic AChE susceptible to PtdIns-specific PLC with the consequent release of hydrophilic AChE. Although serum anchor-specific phospholipase D (PLD) cleaves the intact human erythrocyte AChE anchor, this treatment, as judged by nondenaturing electrophoresis, did not release hydrophilic AChE. Hydroxylamine treatment before or after PLD digestion was necessary to achieve the conversion. These observations indicate that binding of a single detergent micelle was maintained when any of the three fatty acyl or alkyl groups in the human erythrocyte AChE anchor phospholipid were retained. For proteins that can be identified following nondenaturing gel electrophoresis, these procedures provide methods both for detecting glycoinositol phospholipid anchors resistant to PtdIns-specific PLC and for indicating fatty acyl and/or alkyl chains in these anchors.