JB Review J. Biochem.118, 1091-1103(1995)

Functional Role of Glycosphingolipids in Recognition and Signaling

Sen-itiroh Hakomori and Yasuyuki Igarashi The Biomembrane Institute, 201 Elliott Ave W, Seattle, WA 98119, USA; and Departments of Pathobiology and Microbiology, University of Washington, Seattle, WA 98195, USA

Received for publication, August 4, 1995

Glycosphingolipids (GSLs), cell type-specific markers which change dramatically during ontogenesis and oncogenesis, have been implicated as playing major roles in cellular interactions and control of cell proliferation in multicellular organisms. These functional roles have been partially clarified through two types of studies: (i) Studies of cell recognition mediated by (a) GSL-GSL interaction, (b) GSL-lectin interaction, and (c) GSL-dependent modulation of receptor function. (ii) Studies on control of trans membrane signaling by GSLs and/or sphingosine (Sph) derivatives, with emphasis on effects of these compounds on: (a) signaling pathways initiated by tyrosine kinase-linked receptors; (b) signaling systems mediated by protein kinase C, MAP kinase, other kinases, or cytosolic Ca2+ concentration, leading to changes in cellular phenotypes such as motility, proliferation, differentiation, and apoptosis.

Key words: glycosphingolipid-glycosphingolipid interaction, integrin receptors, mitogen activated protein kinase, sphingosine/N, N-dimethylsphingosine-dependent kinase, tyro sine kinase receptor.

1. Introduction: Enigma regarding the function of maintenance of cell recognition, this observation does not glycosphingolipids conflict with the above-mentioned concept of GSL function Great technological advances in separation and charac in multicellular systems in vivo. A potentially more serious terization of glycosphingolipids (GSLs) have been achieved conflict with this concept is presented by the observation over the past three decades. Over 200 variations in carbo that treatment of medaka (fish) embryos with D-1-phenyl hydrate structure, in combination with at least 10 common -2-decanoylamino-3-morpholino-l-propanol (D-PDMP) led molecular species of ceramide (Cer), theoretically create to major reduction or total depletion of various GSLs (e. g. over 2,000 possible molecular species of GSLs. Although depletion of GT3 and GQ1c from brain and neural tube), the functional implications of these structural variations and yet the embryos still developed normally (5). How are largely unknown, some of them are obviously involved ever, the function of the depleted GSLs may have been in defining antigenic specificities of cells. GSLs are effective compensated by the 30-40% of GSLs which were not antigens and immunogens, and have been shown to function depleted by PDMP, or by glycoprotein glycosylation. as (a) cell type-specific and developmental stage-specific A long-standing enigma regarding GSL function exists, antigens, and (b) isogeneic or heterophile antigens (i. e. particularly when cell type-specific patterns are compared histo-blood group antigens) (1, 2). Anachronic or ectopic among different isogenic populations (i. e. histo-blood expression of GSLs in category a or b occurs frequently in groups) or different species. One classic problem is that tumors, and such GSLs are regarded as tumor-associated lifespan and development of individuals with very rare antigens (3). Our knowledge of GSLs as cell type-specific histo-blood groups such as group p (i. e. genotype pp) and antigens increased tremendously through application of Bombay group (genotype hh), who do not synthesize major monoclonal antibody techniques in the late 1970s and early globo-series GSLs or H antigen (respectively) in various 1980s, but the real functions of GSLs remained largely tissues and cells, are indistinguishable from those of the unknown. Functional studies of GSLs have been based on majority population with histo-blood group P (P1and P2) or (i) exogenous addition of GSLs or derivatives; H, who do synthesize these carbohydrates. (ii) effect of GSL synthesis inhibitors on cell phenotype; Expression of certain globo-series structures appears to and (iii) use of GSL expression mutants. Such studies have be essential for development of very early-stage embryos revealed that GSLs are involved in two major cellular (6, 7). We assume that these structures are expressed functions: cell recognition and modulation of transmem during embryogenesis of even individuals with genotype brane signaling. These two functions are interrelated, and pp. Similarly, if H, Leg, and Leb are essential in blastocyst essential for development and homeostasis of multicellular formation and/or the implantation process (8-10), even organisms. Bombay individuals must express these epitopes at early In vitro growth of a tumor cell mutant, totally defective developmental stages. in GSL synthesis, was found to be indistinguishable from Perhaps the most extensively studied species-specific that of parent tumor cells (4). Since GSLs are involved in patterns of GSLs in a single cell type are those in eryth

vol. 118, No. 6, 1995 1091 1092 S. Hakomori and Y. Igarashi

rocytes (11) and neutrophils (12). Results of such studies glycoproteins (21). Although "galectins" (22) and "selec pose a major, ironic problem in assigning specific functions tins" (23) are capable of binding to GSLs having defined of carbohydrates. For example, sialosyl-Lex (SLex) is carbohydrate structure, the major physiological ligands are claimed to be highly expressed on human neutrophils (12) glycoprotein side chains. However, a few lines of evidence but not on hematopoietic progenitor stem cells (13), and to strongly suggest that GSL-binding lectins do exist. Speci be the binding site for selectins (14). However, neutrophils fi city of such lectins should be analogous to that of bacterial in non-human mammal species so far examined (including "adhesins" which recognize GSL -specific structures such as primates) do not react with any antibodies directed to SLex LacCer and globo-series (17). (12). If this epitope is human neutrophil-specific, it cannot In studies by Schnaar et al. (24, 25), a GTlb-specific be considered as a general epitope interacting with selectins binding molecule (presumably lectin) was detected in rat in non-human neutrophils. The extensive observed varia brain membrane fraction, and was claimed to be essential tion in glycosylation pattern in GSLs and glycoproteins for development of the central nervous system. Fatty acid represents the individuality of a cell type. Although the cell residues of various gangliosides were eliminated, and the biological significance of such individuality remains to be amino group of sphingosine (Sph) residue was coupled to elucidated, one plausible hypothesis is that it represents bovine serum albumin (BSA). Multiple gangliosyl-Sph the "wisdom of a cell" to cope with viral and microbial residues carried by radiolabeled BSA were used as a probe. infections, in which carbohydrates are the target.1 The oligosaccharides without Sph conjugated with BSA In this paper, we will summarize our current concept of were found to be less efficiently bound. Thus, a lectin is the functional role of GSLs in control of two major cell capable of recognizing ganglioside molecules rather than phenotypes: cell recognition2 and transmembrane signal oligosaccharides only. One such molecule was recently control. These two phenomena are, of course, closely identified as an immunoglobulin family receptor present on interrelated. -associated glycoprotein (MAG) which binds sialic acid [Yang, L. J.-S., Zeller, C.B., Shaper, N. L., Kiso, M., 2. Organization of GSLs in membranes, and their role Hasegawa, A., Shapiro, R. E., Schnaar, R. L., unpublished as surface receptors in cell recognition data; presented at the "Sphingo(glyco)lipid Symposium," GSLs have been characterized as cell surface antigens Port Ludlow, WA, USA, August 26-28, 1995]. Amphipa (see preceding section), as binding sites for bacterial toxins thic proteins soluble in chloroform-methanol mixture as (e.g. GM1 for cholera toxin, Gb3Cer for Shigella or ver well as in water were detected in ganglioside fractions otoxin) (15, 16), and as sites for bacterial infection (17). isolated from bovine erythrocyte membrane. One of the Since these established research areas have been well fractions had binding affinity to GM3. Others displayed reviewed as cited above, they will not be discussed here. "Paul -Bunnell" antigenicity which was enhanced by addi Recent studies clearly indicate that GSLs are involved in tion of sialosylparagloboside (26). The physiological role of cell-cell and cell-substratum interaction. The effect of these gangliophilic proteins remains to be elucidated. GSLs in control of cell recognition appears to be exerted Recent studies by Hattori et al. (27) revealed the presence through three different mechanisms: (i) GSLs are recog of GM3-binding lectin at the surface of ovarian immature nized by counterpart lectins (including selectins); (ii) GSLs granulosa cells, which are sensitive to follicle-stimulating (particularly gangliosides) modulate adhesion receptors hormone (FSH) and differentiate into mature follicles. such as integrin; (iii) clusters of specific GSLs interact with These granulosa cells also express GM3, and binding counterpart GSL clusters on the surface of another cell between granulosa cells is thereby mediated by GM3 and (Fig. 1). its specific binding lectin. In order to explain the efficient binding of GSLs to Sulfated GSLs such as sulfatide and sulfated GM2 are exogenous ligands, one must assume that GSLs are (i) claimed to bind to laminin (28), thrombospondin, von present (not necessarily exclusively) at the outer leaflet of Wildebrandt factor (29), and other adhesive proteins (see plasma membrane, and (ii) clustered to form multiple for review Ref. 30). Sulfatide and other sulfated GSLs binding sites. A clustered pattern of GSLs, separated and were more recently claimed to bind to P-selectin (31) and distinct from the pattern of glycoproteins, was evidenced L-selectin (32, 33). Since sulfatide and other sulfated by electron micrography of ferritin-labeled antibodies and GSLs have well-documented tendencies to interact with gold sol-labeled lectins applied to freeze-fractured cell many proteins, the physiological significance of these surfaces and artificial liposome membranes (18-20). A. Lectins that recognize GSLs. GSLs at the cell phenomena is unclear. Sulfated glucuronosylparagloboside surface may not be efficiently recognized by plant lectins. (HNK-1 epitope) is claimed to be an efficient ligand for L-selectin (34). HNK-1 epitope was also recently reported Binding specificity of known plant lectins to GSLs differs to be expressed on lymphokine-activated endothelial cells, from that for binding to carbohydrate side chains of and may play an important role in lymphocyte interaction and recruitment (35). 1 This view is based on the ideas of the late Dr. Salvadore Luria, who B. Ganglioside-dependent modulation of integrin regarded the strain-specific glycosylation patterns of bacteria as the receptors. Before the concept of integrin receptors was "wisdom of bacteria" accumulated duri ng the long evolutionary established, Kleinman et al. (36) observed that polysialo development of strategies for surviving infection by phages and gangliosides block fibronectin-dependent cell adhesion, and plasmids, which utilize bacterial lipopolysaccharides as targets (pers. suggested that polysialogangliosides are fibronectin (FN) comm. ). receptors. Cell adhesion and spreading were found to be 2 We use the term "cell recognition" to include all cellular responses associated with cell-to-cell or cell-to-substratum interaction, e.g. sensitive to gangliosides; this was true for adhesion not adhesion, repulsion, motility change, and transmembrane signaling only to FN-coated plates but also to polylysine-coated change following cell interaction. plates and even non-coated plates (37). Subsequently,

J. Biochem. Glycosphingolipid Function 1093

Fig. 1. Proposed scheme for various cell adhesion mechanisms expressed on counterpart cell (stage C2). Stage Cl (GSL-GSL interac based on GSL recognition. Panel A: E. coli adhesion to host cell tion) is a rapid process, but with weak strength of adhesion. Stage C2 membrane via recognition of host GSL (particularly globo-series occurs more slowly but provides much stronger adhesion. Evidence for structure) by fimbrial protein of bacterium. Panel B: Adhesion occurrence of GSL clusters separately distributed on membrane mediated by interaction of sugar binding protein (SBP) with GSL. proteins or glycoproteins has been provided by electron micrographic Various types of SBP expressed on membrane of both cell A and cell studies (see text). Panel D: Possible interaction of GSLs (gangliosides) B bind specifically to GSLs expressed on the counterpart cell. See text with integrin receptors as exemplified by ƒ¿5/31. The GSL binding for examples. Panel C: Adhesion between GSL clusters on membranes domain of ƒ¿5 or ƒÀ1 has not been identified. It may very well be the of adjoining cells, via GSL-GSL interaction (stage Cl). This initial same as the Ca2+ binding domain. Panel E: Effect of concentration of stage induces activation of coexisting integrin receptor (I), which then GM3 co-incorporated with ƒ¿5ƒÀ1 in liposomes on strength of adhesion. interacts with adhesive protein (AP) or with Ig family receptor

Vol. 118, No. 6, 1995 1094 S . Hakomori and Y. Igarashi receptor function in "vitronectin"-dependent cell adhesion show surface expression of H (10). Thus, H-Ley interaction was investigated. This process was found to be closely may play an important role in embryo implantation at the associated with expression of GD3 ganglioside. Vitronectin endometrial cell surface. This hypothesis is supported by receptor and GD3 ganglioside showed co-adhesion to a the fact that implantation is inhibitable by H oligosaccha GRGDSP column, and GD3 is therefore assumed to be ride (49) and by anti-Leg antibody AH6 (10). involved in vitronectin receptor function (38). However, GM3, the ubiquitous ganglioside expressed in a large GD3 and other polysialogangliosides which are claimed to variety of extraneural cells, interacts with various types of be functionally involved in cell adhesion are absent in a GSLs having GalNAc at the terminus, and with LacCer. large variety of cells which exhibit FN-, laminin-, vitronec GM3 interacts with Gg3Cer and Gb4Cer (globoside). A tin-, or collagen-dependent cell adhesion. typical example based on GM3-Gg3 interaction is the We have observed that: (i) GM3 ganglioside is a ubiqui strong cell adhesion observed between mouse tous component of essentially all types of cells which show B16 (which expresses high GM3) and mouse lymphoma FN-, laminin-, vitronectin-, or collagen-dependent adhe L5178 (which expresses high Gg3Cer) (50). GM3-Gg3Cer sion; (ii) adhesion sites remained on a glass surface when or GM3-LacCer interaction has been found to play a cells were mechanically eliminated by "rubber policeman" physiological role in adhesion of melanoma cells to mouse followed by trea tment of the surface with detergent. These endothelial SPE-1 cells. This adhesion is inhibitable by detergent-insoluble cell adhesion sites may comprise a part GM3- or Gg3Cer-liposome, or antibodies against these of the "adhesion plaque" or "podosome." They were found GSLs. We suggested that melanoma metastasis is initiated to be enriched in GM3, and GM3 inhibited cell spreading by interaction of GM3 with Gg3Cer or LacCer (expressed and adhesion (39). on endothelial cells) (51). This was confimed by the fact FN-dependent adhesion of liposomal membrane vesicles that administration of GM3- or Gg3Cer-liposome inhibits consisting of purified ƒ¿5, ƒÀ1 integrin receptor, phospha spontaneous B16 melanoma metastasis (52). tidyicholine, and various concentrations of GM3, LacCer, A novel experimental system for testing ganglioside-to or GlcCer showed a clear dependence only on GM3 concen ganglioside interaction upon membrane contact was de tration, i. e. GM3 within a narrow concentration range signed by Brewer and Thomas (53). Lipid monolayer (12-44 nmol %) enhanced adhesion, while concentrations vesicle membranes with and without ganglioside were below or above this range inhibited adhesion (40; see Fig. assembled. Two vesicles were brought into contact, and 1E). We assume that GM3 plays an important role in electrical conductance between them was measured. In maintenance of various types of integrin receptor. Further creased electrical conductance was observed between extensive studies with various types of gangliosides in vesicles containing gangliosides as compared to ones combination with various integrin receptors are in pro without gangliosides, suggesting that the presence of gress. gangliosides creates a new environment in the adhesive C. Cell recognition through GSL-GSL interaction. A junction between two membranes. In order to determine new trend of research in cell recognition systems is based the dipole moment of the surrounding lipids and water at on interaction of cell surface carbohydrates with comple the contact site of membrane, the fluorescent probe 6 mentary carbohydrates expressed at the surface of a - propionyl-2-(dimethylamino)naphthalene (PRODAN) was different (counterpart) cell. Carbohydrate-carbohydrate employed. Altered PRODAN emission was found at the interactions have been known for many years among junctional membrane when gangliosides were present. polysaccharides, based on changes of physical properties PRODAN emission was unchanged when gangliosides were such as optical rotation, viscosity, and 1H-NMR spectra lacking in the membranes (54). These findings suggest that associated with the sol-gel transition (41-44). However, essential changes of membrane organization occur through until around 1989-1990, the concept was not invoked to ganglioside-ganglioside interaction. This does not necessar explain cell-cell interaction. ily cause adhesion, but may cause "recognition" resulting In the compaction process, the first cell adhesion event from changes in membrane organization through interac (at morula stage) of preimplantation embryo in mammals, tion between gangliosides. Lex plays an essential role. Compaction was inhibited by multivalent Lex but not by multivalent Lea or multivalent 3. GSL-dependent modulation of cell growth through H. Inhibition by monovalent Lex was negligible or required modification of signal transduction very high concentrations (45). In subsequent studies using A. Types of signaling systems and their susceptibil teratocarcinoma F9 cell adhesion as a model, two fractions ity to GSLs and . Function of GSLs in cell showing inhibitory effects on Lex-dependent F9 cell aggre recognition depends on the GSL carbohydrate moiety. In gation were isolated (46). The fractions contained Lex, contrast, function of GSLs in signal transduction depends suggesting the possibility of Lex-Lex interaction. In further on the entire GSL structure, including the Cer moiety. studies, Lex-dependent F9 cell autoaggregation was shown Some signal transducer molecules are highly susceptible to to indeed be based on Lex-Lex interaction in the presence of Sph and its derivatives, including Cer. Various signaling bivalent cation (Ca2+) (47). This was best demonstrated by pathways initiated by cell surface receptors and leading to adherence of Lex-liposome to Lex glycolipid-coated plates. activation of transducer molecules have been identified, but Lex-Lex interaction can also be observed with embryogly the interrelationships among them remain unclear. Three can isolated from F9 cells (48). types of receptor system elicited by signaling molecules are H-H or H-Ley interaction was found to be as strong as known: (i) ion-channel linked; (ii) G-protein linked, and Lex-Lex interaction, although Ley-Ley interaction is repul (iii) enzyme-linked (tyrosine kinase-dependent) (see Fig. 2 sive rather than attractive in nature. Ley is highly express and its legend). ed on the blastocyst surface, and uterine endometrial cells Ion-channel-linked receptors include Na+/K+ ATPase

J. Biochem. Glycosphingolipid Function 1095

Fig. 2. Major transmembrane signaling molecules present at binding of adapter proteins having src homology domains (SH2 and cell surface membranes, their lipid/sphingolipid/GSL modula SH3), such as Grb2, phospholipase CƒÁ, GTPase activating protein tors, and their roles in controlling signaling. Three types of (GAP), etc. Binding of Grb2, for example, leads to activation of Sos, receptor are involved: ion channel linked (I), G-protein linked (II), the GDP/GTP exchanger for Ras. This triggers a cascade of phospho and tyrosine kinase linked (III). Function of types I and II is thought rylation through Raf (MAP kinase kinase kinase), MAP kinase kinase, to be modulated by gangliosides, but no extensive studies have been and finally MAP kinase (MAPK), which phosphorylates one of the performed (see text). G-protein ("G"), activated upon GTP binding, in transcription factors (TF) for activation of gene transcription. One of turn activates PLCƒÀ. On the other hand, PLCƒÁ, which has SH domain the activated genes encodes MAPK phosphatase, which feeds back to

(src homology domain 2, 3), is capable of adapting to tyrosine inactivate MAPK. An alternative pathway leading to MAPK activa phosphate of receptor type III. Signaling through PLC is controlled by tion and mediated by PKC and PKA is also present. This pathway, GM3 and other gangliosides. GTP-bound, activated G-protein also regarded as the central event of signal transduction, is closely activates adenylate cyclase (ADC), which converts ATP to cAMP, associated with two other well-established pathways which also which in turn activates PKA. Thus, many PKA-dependent pathways depend on PKC and PKA. It has recently become clear that Raf kinase are opened. ADC is known to be susceptible to gangliosides, but activity is stimulated by PKC (118, 119) and inhibited by PKA (120, further extensive studies are needed. Various gangliosides ("Gg") 121). Some types of receptors (e.g. IL-1, TNF-a) show specific linkage bind to calmodulin and may modulate intracellular Ca2+ level. PC is to sphingomyelinase, which induces hydrolysis of SM to give increased converted to lyso-PC through action of PLA, and to phosphatidic acid level of Cer. This triggers activation of Cer-dependent kinase and by PLD. Activity of PLD is enhanced by Sph-1-P. Lyso-PC, in consequent activation of TF (116, 117). Abbreviations and conven combination with GM3 ("•˜"), strongly inhibits tyrosine kinase linked tions: Shaded and non-shaded compounds are proteins and lipids, receptor (type III). PA is converted to lyso-PA, which strongly respectively. ADC, adenyl cyclase; Cer, ceramide; DAG, di enhances activity of tyrosine kinase associated with podosomes, and acylglycerol; DMS, N, N-dimethylsphingosine; G, G-protein; GAP, also enhances polymerization and cell motility. PLCƒÀ, ƒÁ, GTP phosphatase activating protein; Gg, ganglioside; GM3, II3NeuA activated by G-protein or tyrosine kinase receptor, activates PKC via cLcCer; GSL, glycosphingolipid; GTP, guanidine triphosphate; IP3, DAG formation. This event plays a crucial role in signal transduction inositol triphosphate; MAPK, mitogen activated phosphokinase; PA, and opens many signaling pathways. In contrast to DAG, which phosphatidic acid; PC, phosphatidylcholine; PIP2, phosphatidylino sitol diphosphate; PKA, protein kinase A; PLA, phospholipase A; promotes PKC, Sph and DMS strongly inhibit PKC. PKC activity is also inhibited by various lyso-GSLs and gangliosides. Tyrosine kinase PLC, phospholipase C; PLD, phospholipase D; SM, sphingomyelin; linked receptors (type III) are inhibited by GM3, SPG, and other SMase, sphingomyelinase; SPG, sialosylparagloboside (IV3NeuAcn gangliosides. Activation of type III receptors by binding of various Lc4Cer); Sph, sphingosine; Sph-1-P, sphingosine-1-phosphate; TF, signaling molecules (growth factors) results in tyrosine phosphoryla transcription factor; Zn butterfly, a symmetric, cysteine-rich domain tion at cytoplasmic sites ("P-Y"). This creates "docking sites" for showing lipid-binding affinity.

Vol. 118, No. 6, 1995 1096 S. Hakomori and Y. Igarashi

associated with Na+/K+ pump, synaptic signaling between and exoenzyme C3 were recently found to be inhibited by electrically excitable cells, acetylcholine receptors at the various gangliosides, most notably the novel compound "GQlb neuromuscular junction, etc. The observed susceptibility of a" (IV3NeuAcIII6NeuAcII3NeuAc2•¨8NeuAcGg4 ion transport modulator in kidney cells to GM1 ganglioside Cer) (64). Sph-1-phosphate (Sph-1-P)-dependent activa (55), and susceptibility of Ca2+-dependent kinase to GD1a tion of DNA synthesis in Swiss 3T3 cells was recently (56) and polysialoganglioside-dependent myelin protein reported to be inhibitable by addition of pertussis toxin (57), suggest that ion-channel-linked receptors in general (65). These findings suggest that G-protein is involved in are modulated by gangliosides. Further extensive studies regulation of the mitogenic effect of Sph-1-P, and that along this line are needed. ADP-ribosylation (which activates G-protein) is modulated Signaling via G-protein-linked cell surface receptor is by gangliosides. the most widely-occurring system. Unfortunately, no Activation of adenyl cyclase following G-protein activa extensive studies have been made on the effects of GSLs or tion leads to activation of protein kinase A (PICA). Activa sphingolipids on signaling through this type of receptor. tion of phospholipase CƒÁ leads to protein kinase C (PKC) ƒÀ-Adrenergic receptor, which is typical of this class , was activation via production of diacylglycerol (DAG). Many reported to depend on unknown ganglioside(s) (58, 59). signaling pathways are opened through PKA and PKC. PKC Consequent to binding of signaling ligand to G-protein is highly susceptible to not only DAG but also GSL and Sph linked receptor, activation of GS through binding to GTP derivatives. induces activation of various enzyme systems (e. g. adenyl The signaling system initiated by enzyme-linked (tyro cyclase and phospholipase C) which play crucial roles in a sine kinase-dependent) receptor is the type most intensive large variety of subsequent signaling events. Susceptibility ly studied in terms of susceptibility to gangliosides, their to gangliosides or GSLs of these events subsequent to derivatives, GSLs, and other sphingolipids (see Fig. 2). In G-protein activation, or of G-protein itself, has not been this signaling system, several steps are modulated by these carefully investigated, and obviously requires extensive compounds: (i) All receptor-associated tyrosine kinases so future study. far tested are susceptible to gangliosides (Fig. 3). (ii) Some Pertussis toxin is known to be a functional uncoupler of receptor kinases are inhibited or promoted by primary G-protein from its receptors (60). The toxin ADP-ribosyl. degradation products of gangliosides, or GSL derivatives. ates the ƒ¿-subunit of G-protein (GTP-binding protein) (61, (iii) Sph and N, N-dimethyl-Sph (DMS) inhibit MAPK 62). Similarly, exoenzyme C3 of Clostridium botulinum through either inhibition of PKC, or PKC-independent types C and D ADP-ribosylates the Rho family of GTP pathways. (iv) src, ras, and a few unidentified kinases are binding proteins, which plays an essential role in formation strongly stimulated by Sph and DMS. of actin networks in eukaryotic cells (63). In the following subsections, we will describe specific ADP-ribosyltransferase activities of both pertussis toxin patterns of kinase modulation by specific GSLs.

Fig. 3. Six subfamilies of growth factor/hormone recep tor having tyrosine kinases, and their susceptibility to various types of gangliosides and GSLs. These receptors all have a cytoplasmic tyrosine ki nase domain which is susceptible to GSL or sphingolipid. +, pro motion; -, inhibition; DeNAc GM3, de-N-acetyl-GM3; PSY, plasmalopsychosine; VEGF, vas cular endothelial growth factor.

J. Biochem. Glycosphingolipid Function 1097

B. Modulation of receptor-associated tyrosine ki C. Enhancing effects of novel GSL derivatives on nases by gangliosides. GM3 inhibits EGF receptor kinase receptor kinase activity, cell proliferation, and differ and formation of tyrosine phosphate (66). In this case, entiation. A primary degradation product of GM3 (by GM3 may directly inhibit tyrosine phosphorylation, rather elimination of the N-acetyl group of sialic acid) is de-N than inhibiting receptor-receptor interaction (67). Ex acetyl-GM3 (deNAcGM3), which is detectable in various ogenous addition of GM3 inhibits EGF-dependent growth types of cultured cells by application of its specific antibody of A431 cells and KB cells (66), even though GM3 already DH5. DeNAcGM3 is present naturally in minimal quantity, exists in these cells. Interpretation of the physiological role but its exogenous addition promotes EGF receptor phos

of GM3 in EGF-dependent growth of these cells is difficult. phorylation (of serine as well as tyrosine) and enhances cell Perhaps EGF induces membrane perturbation and "flip proliferation (67, 76). DeNAcGM3 is resistant to sialidase fl op" reorganization of GM3, producing cytoplasm-facing and is not further degraded. It may be re-N-acetylated into GM3, which directly interacts with EGF receptor. How GM3. It is possible that a balance between de and re-N ever, GM3 showed a specific inhibitory effect only in the acetylation is involved in control of cell proliferation. De presence of lyso-PC, not other lyso- (68). N-acetylation may occur not only for GM3 but also other Further evidence that GM3 modulates growth factor gangliosides. Recently, deNAcGD3 was detected in human dependent cell growth was provided by two different melanoma cells. De-N-acetylation of GD3 to deNAcGD3 experiment, one by Weis and Davis (69) using an epimer was inhibited by Genistein, suggesting that formation of ase-less mutant of Chinese Hamster Ovary cells (LAID), deNAcGD3 is controlled by tyrosine kinase (77). Identifi and another by us using GM3 mutant FUA169 derived from cation of target molecules that regulate cell proliferation F28-7 cells (70). LAID was incapable of converting UDP and are controlled by deNAcGD3 has not been achieved. Glc to UDP-Gal. Therefore, in the absence of Gal, the cells A novel GSL termed plasmalopsychosine was isolated could not synthesize LacCer or GM3. On addition of Gal in and characterized from human brain white matter (78). culture medium, the cells became capable of synthesizing This compound is the conjugate of plasmal through 4,6 or LacCer or GM3. This mutant did not have EGF receptor 3,4 cyclic acetal linkage to ƒÀ-Gal residue of psychosine. and did not show EGF-dependent cell growth. After trans Plasmalopsychosine is found exclusively in white matter of fection of EGF receptor gene, LAID-EGFR+ cells were brain, but not in gray matter. In contrast to all other isolated. These cells grew well in the presence of EGF, and complex lipids, plasmalopsychosine has a unique structure showed EGF receptor tyrosine phosphorylation. When the with two aliphatic tails oriented in opposite directions. It LA1D-EGFR+ cells were grown in the prresence of Gal, strongly induces differentiation in neuronal cells leading to whereby GM3 was synthesized, EGF-dependent cell neurite outgrowth, in the same way as nerve growth factor growth and EGF receptor tyrosine phosphorylation were (NGF). Neuritogenic differentiation of most neuronal cells both inhibited. is induced by NGF, but rarely by GSLs alone. However,

GM3 mutant FUA169 was isolated from F28-7 cells. plasmalopsychosine by itself, without NGF, induces neu FUA169 did not show insulin-dependent cell growth. ritogenic differentiation. In other words, it mimics NGF Mouse insulin receptor is highly sensitive to GM3, and activity. When PC12 cells were incubated with plasmalop insulin-dependent growth of F28-7 cells was strongly sychosine, tyrosine kinase associated with NGF receptor inhibited in the presence of GM3 (70). However, in human (p140trk) was strongly activated. High concentrations of cells, insulin receptor is most strongly inhibited by sialosyl GM1 were also reported to activate p140trk in PC12 cells

paragloboside (71). 2•¨3 sialosylparagloboside (2•¨3 SPG; (75), but GM 1 per se did not trigger neurite outgrowth. The IV3NeuAcnLc4Cer) inhibits tyrosine kinase of the 95 kDa effect of plasmalopsychosine is the same as when PC 12 cells subunit associated with insulin receptor. Other gangliosides are incubated with NGF. MAP kinase (MAPK) is also including 2•¨6 SPG did not have this effect. Insulin-depen strongly and immediately activated when PC12 cells are dent growth of K562, HL60, and IM9 cells was inhibited by incubated with plasmalopsychosine, and the activity is exogenous addition of 2•¨3 SPG, whereby phosphorylation sustained for a long period. This enhancement of MAPK of the 95 kDa subunit was also blocked (71). occurred within a few minutes of incubation, similar to the growth factor (FGF) -dependent growth of effect of NGF. The effect of plasmalopsychosine may not BHK cells was observed to be inhibited by exogenous directly involve upon activation of NGF receptor and addition of GM3. GM3-enriched BHK cells became re p140trk, since binding of [125I] NGF to its receptor was not fractive to growth stimulation by FGF, and internalization inhibited by plasmalopsychosine. Yet plasmalopsychosine of FGF was completely blocked (72). Further studies on the enhanced p140trk activity to the same extent as NGF, effect of GM3 on FGF receptor function are needed. It is leading to strong sustained enhancement of MAPK (122). clear that FGF does not interact directly with GM3. Thus, plasmalopsychosine may have an independent re Platelet-derived growth factor (PDGF)-dependent cell ceptor which interacts with Trk receptor in the

growth and receptor tyrosine phosphorylation are inhibited and causes membrane perturbation, activating tyrosine by GM1 (73), GD1a, GDlb, and GT1b (74). These ganglio phosphorylation of p140trk sides do not directly interact with PDGF receptor, and do . D. Effects of gangliosides and sphingolipids on not alter affinity of the receptor for PDGF binding (73). protein kinase C. PKC is a ubiquitous molecule present in A synergistic enhancing effect of GM1 on NGF-depen essentially all living cells, from bacteria through higher dent NGF receptor tyrosine kinase was recently reported in animals and plants. It plays a key role in signal transduc association with the differentiation-inducing effect of GM1 tion. The direct substrate of PKC appears to vary exten in the presence of NGF on PC12 cells (75). GM1 alone has sively from one cell type to another. Polysialogangliosides no effect, in contrast to plasmalopsychosine (see following strongly inhibit PKC in HL60 cells (79), and the effect of section). gangliosides on PKC is probably a general phenomenon.

Vol. 118, No. 6, 1995 1098 S . Hakomori and Y. Igarashi

"Lyso -gangliosides" were claimed to strongly inhibit PKC kDa was greatly increased (96). The physiological signifi in brain (80). In 1986, Hannun et al. observed that Sph cance of this phenomenon remains to be studied. inhibits platelet PKC (81); stereoisomeric Sph specificity GD1c ganglioside was found to be highly expressed in was not observed (82). Sph is converted to DMS by meth CD4+ T cells, which produce abundant IL-2 and very little yltransferase in brain. DMS is a more effective inhibitor of IL-4. GDlc expression may be related to helper T cell PKC compared to Sph, and displays stereospecific effect, function (97). Another study provides insight on involve i. e. D-erythro-DMS was a stronger inhibitor than L-threo, ment of gangliosides in CD4 function. CD4 provides essen L-erythro, or D-threo isomers (83). tial signals for helper T cell stimulation through "T cell Enhancement of signal transduction by PKC is often receptor-CD3 complex." Gangliosides may trigger PKC-in observed in cells, and in activated platelets or dependent signals which cause P56trk dissociation from endothelial cells in association with acute or chronic inflam CD4, which initiates helper T cell activation. It is possible matory response. DMS was effectively applied for inhibi that gangliosides have essential regulatory roles in T cell tion of tumor growth (84). N, N, N-Trimethyl-Sph (TMS), function (98). an an alog of DMS, was synthesized and found to be an even F. Effects of Gb3Cer, LacCer, and GlcCer on specific more effective PKC inhibitor and to give a stable aqueous cellular functions. Gb3Cer (Gala l•¨4Gal/31•¨4GlcƒÀ1•¨ solution, unlike DMS. TMS was applied for inhibition of Cer) is present in high concentration in human B lympho tumor cell metastasis (85) and inflammatory response cytes and Burkitt lymphoma cells. Gb3Cer expression in B (86). TMS and DMS effectively inhibit P-selectin expres lymphocytes in tonsils is closely associated with apoptosis. sion on platelets (87) and endothelial cells (86); this is the Verotoxin subunit B has no cytotoxic effect but is capable of basis of the anti-acute inflammatory effect of these com binding to Gb3Cer (99). Addition of this subunit to Gb3 pounds. expressing cells causes internalization of Gb3-verotoxin E. Effects of gangliosides and GSLs on neuronal and subunit B complex and strongly induces apoptosis (100). hematopoietic cells. Neuronal cell survival and differ The pathogenesis of atherosclerosis involves abnormal entiation require nerve growth factors in general. As proliferation of arterial smooth muscle cells, in which described in the preceding section, plasmalopsychosine LacCer (GalƒÀ1•¨4GlcƒÀ1•¨Cer) is highly expressed and substitutes for NGF and mimics its activity through presumably closely associated with atherosclerotic plaque activation of p140trk and subsequent sustained activity of formation (101). LacCer was recently found to initiate MAPK. GQ1b ganglioside on nanomolar orders was previ signal transduction leading to activation of MAPK [Chat ously found to mimic NGF activity in two types of human terjee, S., Shi, W. Y., Wilson, P., Mazumdar, A., unpub neuroblastoma cells, GOTO and NB-l. GQ1b stimulates lished data; presented at the "Sphingo(glyco)lipid Sympo neurite outgrth of these cells, similarly to NGF (88). sium," Port Ludlow, WA, USA, August 26-28, 1995]. GQ1b was claimed to activate cell surface protein kinase GlcCer has been considered as a mitogen of basal layer

("ectoprotein kinase") in GOTO cells (89). Mouse neuro epidermal cells. Topical application of ƒÀ-glucosidase in blastoma Neuro2a shows strong neuritogenic response to hibitor, which enhances G1cCer synthesis in epidermis, exogenous addition of various gangliosides and synthetic stimulates mitogenesis of basal cells. D-PDMP, which sialosyl compounds in the absence of NGF. Gangliosides inhibits G1cCer synthesis, suppresses mitogenesis of these and NGF function synergistically to affect neuritogenesis in cells (102). These interesting observations provide strong these cells (90). It is possible that a signaling pathway evidence that LacCer and GlcCer, the basic constituents of initiated by ectoprotein kinase may trigger activation of GSLs, play important roles in proliferation of certain types pl40trk and consequent MAPK activation. of cells, although the signal transduction mechanism and Endogenous de novo synthesis of GD3 ganglioside by enzymes involved in the process remain to be studied. transfection of the gene for GD3 synthetase (CMP-sialic G. Effects of GSLs and Sph-1-phosphate on cytosolic acid: GD3-ƒ¿2-8 sialyltransferase) (91, 92) in Neuro2a Ca2+ and cell motility. Cytosolic Ca2+ concentration is on cells induces expression of not only GD3 but also b-series the order of 10-7 M, whereas endoplasmic, mitochondrial,

gangliosides. These transfectants were characterized as and extracellular concentrations are on the order of 10-3 M. showing cholinergic differentiation and neurite formation Cells use Ca2+ as an intracellular signaling molecule, and (93). Here again, the signal pathway triggered by de novo resting cells must keep cytosolic Ca2+ level low. Upon cell synthesis of b-series gangliosides needs to be identified. stimulation, Ca2+ level increases within seconds. This Ca2+ Promyelocytic leukemia HL60 cells were found to differ influx is mediated by various types of GSLs and Sph entiate into granulocytes or monocytes/ after derivatives, in analogy to the well-established inositol exogenous addition of neolacto-series gangliosides or GM3 triphosphate (IP3) effect (see Fig. 2). Antibody directed to (94, 95). How signaling mechanisms in HL60 cells are GalCer induces a strong, sustained increase in Ca2+ level in affected by granulocytes remains to be elucidated. the majority of primary culture of mouse oligodendroglia Hematopoietic cells, in general, are highly glycosylated (103). Sph-1-P stimulates DNA synthesis in Swiss 3T3 and have high content of gangliosides. Our knowledge of fi broblasts via induction of Ca2+ mobilization (104). Sph-1 activation mechanisms, interaction between B and T cells, - P also induces strong Ca2+ influx in platelets, triggering recognition of antigen by Ig receptor and T cell receptor, platelet activation (105). In most tumor cells, motility is etc. has increased greatly in recent years. Nevertheless, our stopped in the presence of nanomolar order concentrations understanding of control mechanisms in immunocyte func of Sph-1-P, whereas motility of normal and tion affected by gangliosides and GSLs is highly fragmen endothelial cells is unaffected by Sph-1-P (106). Motility of tary. IL-2-dependent CTLL cell growth was strongly human arterial smooth muscle cells stimulated by platelet inhibited by DMS when CTLL cells were incubated with derived growth factor was inhibited by Sph-1-P because of D-PDMP, whereby tyrosine phosphorylation of 90-100 blocking of actin nucleation (107). Calmodulin, a ubiqui

J. Biochem. Glycosphingolipid Function 1099 tous Ca2+ receptor/modulator, binds to various ganglio lular localization, functional predisposition, etc. This prob sides (108), and calmodulin-dependent phosphodiesterase lem also applies to various other GSLs which are claimed to is inhibited by gangliosides (109). modulate transmembrane signaling with cells. H. Sph, DMS, Sph-1-P, and Cer as second messen gers. The concept that these sphingolipids may act as 4. Conclusions second messengers was based primarily on observed (i) Dramatic changes in GSL composition, structure, and inhibitory or inducing effects of these compounds on known organization in membrane, associated with ontogenesis and or unknown protein kinases, and (ii) increase of these oncogenesis, suggest a functional role of GSLs in cell compounds upon physiological stimulation of cells by recognition and signaling. GSLs are involved directly in cell growth factors or lymphokines, followed by changes in recognition through binding with counterpart GSLs (GSL DNA synthesis or degradation (apoptosis). We originally GSL interaction), or through recognition by GSL-binding described the inhibitory effect of Sph and DMS on PKC (83, proteins (lectins). In addition, some GSLs (e. g. GM3 84), and their enhancing effects on tyrosine kinases as ganglioside) modulate integrin receptor function. In trans sociated with EGF receptor (81) and on src kinase (110). membrane signaling, specific gangliosides directly modu More recently, we found that (i) Sph production is en late receptor tyrosine kinases and PKC activity. Some hanced in TNF-ƒ¿-treated human neutrophils and TPA GSLs strongly promote receptor kinase activity, leading to treated HL60 cells, resulting in apoptosis (111, 112), and enhancement of MAPK. Our knowledge of control of (ii) exogenously-added Sph or DMS induce apoptosis in transmembrane signaling by GSLs is still highly fragmen various solid tumor cells (but not in normal cells) through tary. Fundamental core structures within GSLs (e. g. GM3, inhibition of MAPK (Sakakura, C., Sweeney, E. A., Shira Gb3, LacCer, G1cCer, Cer, Sph) and their derivatives hama, T., Solca, F., Kohno, M., Hakomori, S., Fischer, display a general modulatory effect on various signaling E. H., and Igarashi, Y., manuscript submitted). We have systems, which are highly characteristic of specific types of also identified several phosphorylated bands as a target cells. substrate of Sph- or DMS-dependent kinases in BALB/c 3T3(A31) cells (113). One such protein was identified as We thank Stephen Anderson, Ph. D., for scientific editing and "14 -3-3 protein ," known as a modulator of various signaling preparation of the manuscript. molecules. The phosphorylated form of 14-3-3 inhibits PKC, and the non-phosphorylated form is associated close ly with and enhances Raf kinase. Thus, Sph and DMS affect REFERENCES cell signaling through modulatiof a signal-modulating 1. Yamakawa, T. and Nagai, Y. (1978) Glycolipids at the cell protein, 14-3-3. surface and their biological functions. Trends Biochem. Sci. 3, The identification of Sph-1-P as a second messenger was 128-131 2. Feizi, T. (1985) Demonstration by monoclonal antibodies that based on two observations: (i) triggering by exogenously carbohydrate structures of glycoproteins and glycolipids are added Sph-1-P of a number of signal transduction pathways oncodevelopmental antigens. Nature 314, 53-57 to induce proliferation of a specific type of Swiss 3T3 cell; 3. Hakomori, S. (1989) Aberrant glycosylation in tumors and (ii) PDGF or fetal calf serum-treated, but not EGF-treat tumor-associated carbohydrate antigens. Adv. Cancer Res. 52, ed cells, showed a transient increase of Sph-1-P prior to 257-331 DNA synthesis (104, 114). Our experience indicates that 4. Ichikawa, S., Nakajo, N., Sakiyama, H., and Hirabayashi, Y. proliferative cell response to Sph-1-P is highly limited, (1994) A mouse B16 melanoma mutant deficient in glycolipids. Proc. Natl. Acad. Sci. USA 91, 2703-2707 although motility of many transformed cells is inhibited 5. Fenderson, B. A., Ostrander, G. K., Hausken, Z., Radin, N. S., and strongly by this catabolite (106) (see preceding section). Hakomori, S. (1992) A ceramide analogue (PDMP) inhibits Exogenous addition of Sph-1-P strongly activates platelets glycolipid synthesis in fish embryos. Exp. Cell Res. 198, 362-366 in terms of shape change and Ca2+ influx, suggesting that 6. Kannagi, R., Levery, S. B., Ishigami, F., Hakomori, S., Shevins Sph-1-P acts as an intercellular messenger as well as ky, L. H., Knowles, B. B., and Solter, D. (1983) New globoseries intracellular signaling molecule (105). glycosphingolipids in human teratocarcinoma reactive with the monoclonal antibody directed to a developmentally regulated The concept of Cer as a second messenger is based on the antigen, stage-specific embryonic antigen 3. J. Biol. Chem. 258, fact that Cer increase due to sphingomyelin hydrolysis is 8934-8942 observed in HL60 cells stimulated by vitamin D3 (115). In 7. Kannagi, R., Cochran, N. A., Ishigami, F., Hakomori, S., An cell lines susceptible to stimulation by TNF-ƒ¿,ƒÁ-inter drews, P. W., Knowles, B. B., and Solter, D. (1983) Stage-specific feron, dexamethasone, or vitamin D3 (e. g. various myeloid embryonic antigens (SSEA-3 and -4) are epitopes of a unique cell lines, fibroblasts, and pre-adipocytes [3T3-L1]), such globo-series ganglioside isolated from human teratocarcinoma cells. EMBO J. 2, 2355-2361 stimulation leads to increased sphingomyelinase level and 8. Fenderson, B. A., Holmes, E. H., Fukushi, Y., and Hakomori, S. hydrolysis of sphingomyelin to Cer. The increased Cer level (1986) Coordinate expression of X and Y haptens during murine induces activation of Cer-dependent kinases and conse embryogenesis. Dev. Biol. 114, 12-21 quent characteristic cell response, possibly through activa 9. Fenderson, B. A., Andrews, P. W., Nudelman, E. D., Clausen, H., tion of transcription factor. Numerous studies along this and Hakomori, S. (1987) Glycolipid core structure switching line have been performed by Y. Hannun's and R. Kole from globo- to lacto- and ganglio-series during retinoic acid-in snick's groups, and reviewed by them (116, 117). duced differentiation of TERA-2-derived human embryonal carcinoma cells. Dev. Biol. 122, 21-34 Cer is present in significant quantity in all cell types. 10. Zhu, Z., Kojima, N., Stroud, M. R., Hakomori, S., and Fenderson, Stimulation by various factors as above produces a mar B. A. (1995) Monoclonal antibody directed to Ley oligosaccharide ginal increase (10-20%) of total Cer. It is difficult to inhibits implantation in the mouse. Biol. Rep rod. 52, 903-912 distinguish this de novo appearing Cer from the pre-exist 11. Yamakawa, T. (1966) Glycolipids of mammalian red blood cells ing large quantity of Cer within cells in terms of intracel in Lipoide: 16 Colloquium der Gesellschaft Physiologische

Vol. 118, No. 6, 1995 1100 S . Hakomori and Y. Igarashi

Chemie (Schute, E., ed.) pp. 87-111, Springer-Verlag, Berlin Nakao, T., Murase, K., Kiso, M., Hasegawa, A., and Tadano 12. Ito, K., Handa, K., and Hakomori, S. (1994) Species-specific Aritomi, K. (1993) Sulfated glycolipids are ligands for a lym

expression of sialosyl-Lex on polymorphonuclear leukocytes phocyte homing receptor, L-selectin (LECAM-1): Binding (PMN), in relation to selectin-dependent PMN responses. epitope in sulfated sugar chain. Biochem. Biophys. Res. Commun. Glycoconjugate J. 11, 232-237 190,426-434 13. Muroi, K., Suda, T., Nojiri, H., Ema, H., Amemiya, Y., Miura, 34. Needham, L. K. and Schnaar, R. L. (1993) The HNK-1 reactive Y., Nakauchi, H., Singhal, A. K., and Hakomori, S. (1992) sulfoglucuronyl glycolipids are ligands for L-selectin and P-selec Reactivity profiles of leukemic myeloblasts with monoclonal tin but not E-selectin. Proc. Natl. Acad. Sci. USA 90, 1359-1363 antibodies directed to sialosyl-Lex and other lacto-series type 2 35. Kanda, T., Yamawaki, M., Ariga, T., and Yu, R. K. (1995) chain antigens: Absence of reactivity with normal hematopoietic Interleukin 1ƒÀ up-regulates the expression of sulfoglucuronosyl progenitor cells. Blood 79, 713-719 paragloboside, a ligand for L-selectin, in brain microvascular 14. Varki, A. (1994) Selectin ligands. Proc. Natl. Acad. Sci. USA 91, endothelial cells. Proc. Natl. Acad. Sci. USA 92, 7897-7901 7390-7397 36. Kleinman, H. K., Martin, G. R., and Fishman, P. H. (1979) 15. Fishman, P. H. (1980) Mechanism of action of cholera toxin: Ganglioside inhibition of fibronectin-mediated cell adhesion to Studies on the lag period. J. Membr. Biol. 54, 61-72 collagen. Proc. Natl. Acad. Sci. USA 76, 3367-3371 16. Fishman, P. H. and Brady, R. O. (1976) Biosynthesis and function 37. Rauvala, H., Carter, W. G., and Hakomori, S. (1981) Studies on of gangliosides. Science 194, 906-915 cell adhesion and recognition: I. Extent and specificity of cell 17. Karlsson, K.-A. (1989) Animal glycosphingolipids as membrane adhesion triggered by carbohydrate-reactive proteins (glyco attachment sites for bacteria. Annu. Rev. Biochem. 58, 309-350 sidases and lectins) and by fibronectin. J. Cell Biol. 88, 127-137 18. Tillack, T. W., Allietta, M., Moran, R. E., and Young, W. W., Jr. 38. Cheresh, D. A., Pytela, R., Pierschbacher, M. D., Klier, F. G., (1983) Localization of globoside and Forssman glycolipids on Ruoslahti, E., and Reisfeld, R.A. (1987) An Arg Gly-Asp direct erythrocyte membranes. Biochim. Biophys. Acta 733, 15-24 ed receptor on the surface of human melanoma cells exists in a 19. Rock, P., Allietta, M., Young, W. W., Jr., Thompson, T. E., and divalent cation-dependent functional complex with the disialo

Tillack, T. W. (1990) Organization of glycosphingolipids in ganglioside GD2. J. Cell Biol. 105, 1163-1173 phosphatidylcholine bilayers: Use of antibody molecules and Fab 39. Okada, Y., Mugnai, G., Bremer, E. G., and Hakomori, S. (1984) fragments as morphologic markers. Biochemistry 29, 8484-8490 Glycosphingolipids in detergent-insoluble substrate attachment 20. Rock, P., Allietta, M., Young, W. W., Jr., Thompson, T. E., and matrix (DISAM) prepared from substrate attachment material

Tillack, T. W. (1991) Ganglioside GM and asialo-GM1 at low (SAM): Their possible role in regulating cell adhesion. Exp. Cell concentration are preferentially incorporated into the gel phase in Res. 155, 448-456 two-component, two-phase phosphatidylcholine bilayers. Bio 40. Zheng, M., Fang, H., Tsuruoka, T., Tsuji, T., Sasaki, T., and chemistry 30, 19-25 Hakomori, S. (1993) Regulatory role of GM3 ganglioside in ƒ¿5ƒÀ1 21. Boldt, D. H., Speckart, S. F., Richards, R. L., and Alving, C.R. integrin receptor for fibronectin-mediated adhesion of FUA169 (1977) Interaction of plant lectins with glycolipid in liposomes. cells. J. Biol. Chem. 268, 2217-2222 Biochem. Biophys. Res. Commun. 74, 208-214 41. Dea, I. C. M., McKinnon, A. A., and Rees, D. A. (1972) Tertiary 22. Barondes, S. H., Cooper, D. N. W., Gitt, M.A., and Leffler, H. and quaternary structure in aqueous polysaccharide systems (1994) Galectins: Structure and function of a large family of which model cell wall cohesion: Reversible changes in conforma animal lectins. J. Biol. Chem. 269, 20807-20810 tion and association of agarose, carrageenan and galacto-man 23. Lasky, L. A. (1995) Selectin-carbohydrate interactions and the nans. J. Mol. Biol. 68, 153-172 initiation of the inflammatory response. Annu. Rev. Biochem. 64, 42. Valent, B. S. and Albersheim, P. (1974) The structure of plant 113-139 cell walls: V. On the binding of xyloglucan to cellulose fibers. 24. Tiemeyer, M., Yasuda, Y., and Schnaar, R. L. (1989) Ganglio Plant Physiol. 54, 105-108 side-specific binding protein on rat brain membranes. J Biol. 43. Bryce, T. A., McKinnon, A. A., Morris, E. R., Rees, D. A., and Chem. 264, 1671-1681 Thom, D. (1974) Chain conformations in the sol-gel transitions 25. Tiemeyer, M. and Schnaar, R. L. (1990) Receptors for gang for polysaccharide systems, and their characterisation by spectro liosides on rat brain membranes: Specificity, regional and subcel scopic methods. Faraday Disc. Chem. Soc. 57, 221-229 lular distribution in Trophic Factors and the Nervous System 44. Arnott, S., Fulmer, A., Scott, W. E., Dea, I. C. M., Moorhouse, R., (Horrocks, L. A., Neff, N. H., Yates, A. J., and Hadjiconstantinou, and Rees, D. A. (1974) The agarose double helix and its function M., eds.) pp. 119-133, Raven Press, New York in agarose gel structure. J. Mol. Biol. 90, 269-284 26. Watanabe, K., Hakomori, S., Powell, M. E., and Yokota, M. 45. Fenderson, B. A., Zehavi, U., and Hakomori, S. (1984) A (1980) The amphipathic membrane proteins associated with multivalent lacto-N-fucopentaose III-lysyllysine conjugate de gangliosides: The Paul-Bunnell antigen is one of the gangliophilic compacts preimplantation mouse embryos, while the free oligo proteins. Biun. 92, 638-646 saccharide is ineffective. J. Exp. Med. 160, 1591-1596 27. Hattori, M., Horiuchi, R., Hosaka, K., Hayashi, H., and Kojima, 46. Rosenman, S. J., Fenderson, B. A., and Hakomori, S. (1988) The 1. (1995) Sialyllactose-mediated cell interaction during granulosa role of glycoconjugates in embryogenesis in Glycoconjugates in cell differentiation. J. Biol. Chem. 270, 7858-7863 Medicine (Ohyama, M. and Muramatsu, T., eds.) pp. 43-50, 28. Roberts, D. D., Wewer, U. M., Liotta, L. A., and Ginsburg, V. Professional Postgraduate Services, Tokyo (1988) Laminin-dependent and laminin-independent adhesion of 47. Eggens, I., Fenderson, B. A., Toyokuni, T., Dean, B., Stroud, human melanoma cells to sulfatides. Cancer Res. 48, 3367-3373 M. R., and Hakomori, S. (1989) Specific interaction between Lex 29. Roberts, D. D., Rao, C. N., Liotta, L. A., Gralnick, H. R., and and Lex determinants: A possible basis for cell recognition in Ginsburg, V. (1986) Comparison of the specificities of laminin, preimplantation embryos and in embryonal carcinoma cells. J. thrombospondin, and von Willebrand factor for binding to Biol. Chem. 264, 9476-9484 sulfated glycolipids. J. Biol. Chem. 261, 6872-6877 48. Kojima, N., Fenderson, B. A., Stroud, M. R., Goldberg, R. I., 30. Roberts, D. D. and Ginsburg, V. (1988) Sulfated glycolipids and Habermann, R., Toyokuni, T., and Hakomori, S. (1994) Further cell adhesion. Arch. Biochem. Biophys. 267, 405-415 studies on cell adhesion based on Lex-Lex interaction, with new 31. Aruffo, A., Kolanus, W., Walz, G., Freedman, P., and Seed, B. approaches: Embryoglycan aggregation of F9 teratocarcinoma (1991) CD62/P-selectin recognition of myeloid and tumor cell cells, and adhesion of various tumour cells based on Lex expres sulfatides. Cell 67, 35-44 sion. Glycoconjugate J. 11, 238-248 32. Kajihara, J., Guoji, Y., Kato, K., and Suzuki, Y. (1995) Sul 49. Lindenberg, S., Sundberg, K., Kimber, S. J., and Lundblad, A. fatide, a specific sugar ligand for L-selectin, blocks CC14-induced (1988) The milk oligosaccharide, lacto-N-fucopentaose I, inhibits liver in rats. Biosci. Biotechnol. Biochem. 59, 155 attachment of mouse blastocysts on endometrial monolayers. J. - 157 Rep rod. Fertil. 83, 149-158 33. Suzuki, Y., Toda, Y., Tamatani, T., Watanabe, T., Suzuki, T., 50. Kojima, N. and Hakomori, S. (1989) Specific interaction between

J. Biochem. Glycosphingolipid Function 1101

gangliotriaosylceramide (Gg3) and sialosyllactosylceramide plasma membranes. Biochem. Biophys. Res. Commun. 172, 77 (GM3) as a basis for specific cellular recognition between lym 84 phoma and melanoma cells. J. Biol. Chem. 264, 20159-20162 69. Weis, F. M.B. and Davis, R. J. (1990) Regulation of epidermal 51. Kojima, N. and Hakomori, S. (1991) Cell adhesion , spreading, growth factor receptor signal transduction: Role of gangliosides. and motility of GM3-expressing cells based on glycolipid-glyco J. Biol. Chem. 265, 12059-12066 lipid interaction. J. Biol. Chem. 266, 17552-17558 70. Tsuruoka, T., Tsuji, T., Nojiri, H., Holmes, E. H., and Hakomori, 52. Otsuji, E., Park, Y. S., Tashiro, K., Kojima, N., Toyokuni, T., and S. (1993) Selection of a mutant cell line based on differential Hakomori, S. (1995) Inhibition of B16 melanoma metastasis by expression of glycosphingolipid, utilizing anti- lactosylceramide administration of GM3 or Gg3-liposomes: Blocking adhesion of antibody and complement. J. Biol. Chem. 268, 2211-2216 melanoma cells to endothelial cells (anti-adhesion therapy) via 71. Nojiri, H., Stroud, M. R., and Hakomori, S. (1991) A specific type inhibition of GM3-Gg3Cer or GM3-LacCer interaction. Int. J. of ganglioside as a modulator of insulin-dependent cell growth Oncol. 6, 319-327 and insulin receptor tyrosine kinase activity: Possible association 53. Brewer, G. J. and Thomas, P. D. (1984) Role of gangliosides in of ganglioside-induced inhibition of insulin receptor function and adhesion and conductance change in large spherical model monocytic differentiation induction in HL60 cells. J. Biol. Chem. membranes. Biochim. Biophys. Acta 776, 279-287 266,4531-4537 54. Brewer, G. J. (1992) Polarity decrease at the adhesive junction 72. Bremer, E. G. and Hakomori, S. (1982) GM3 ganglioside induces between two model membranes containing gangliosides. Bio hamster fibroblast growth inhibition in chemically-defined chemistry 31, 1809-1815 medium: Ganglioside may regulate growth factor receptor func 55. Spiegel, S., Handler, J. S., and Fishman, P. H. (1986) Ganglio tion. Biochem. Biophys. Res. Commun. 106, 711-718 sides modulate sodium transport in cultured toad kidney epi 73. Bremer, E. G., Hakomori, S., Bowen-Pope, D. F., Raines, E. W., thelia. J. Biol. Chem. 261, 15755-15760 and Ross, R. (1984) Ganglioside-mediated modulation of cell 56. Goldenring, J. R., Otis, L. C., Yu, R. K., and DeLorenzo, R. J. growth, growth factor binding, and receptor phosphorylation. J. (1985) Calcium/ganglioside-dependent protein kinase activity in Biol. Chem. 259, 6818-6825 rat brain membrane. J. Neurochem. 44, 1229-1234 74. Yates, A. J., VanBrocklyn, J., Saqr, H. E., Guan, Z., Stokes, B. T., 57. Chan, K.-F. J. (1988) Ganglioside-modulated protein phospho and O'Dorisio, M. S. (1993) Mechanisms through which ganglio rylation: Partial purification and characterization of a ganglio sides inhibit PDGF-stimulated mitogenesis in intact Swiss 3T3 side-inhibited protein kinase in brain. J. Biol. Chem. 263, 568 cells: Receptor tyrosine phosphorylation, intracellular calcium, - 574 and receptor binding. Exp. Cell Res. 204, 38-45 58. Chorev, M., Feigenbaum, A., Keenan, A. K., Gilon, C., and 75. Mutoh, T., Tokuda, A., Miyada, T., Hamaguchi, M., and Fujiki, Levitzki, A. (1985) N-Bromoacetyl-amino-cyanopindolol: A N. (1995) Ganglioside GM1 binds to the Trk protein and regu highly potent ƒÀ-adrenergic affinity label blocks irreversibly a lates receptor function. Proc. Natl. Acad. Sci. USA 92, 5087 non-protein component tightly associated with the receptor. Eur. 5091 J. Biochem. 146, 9-14 76. Hanai, N., Dohi, T., Nores, G.A., and Hakomori, S. (1988) A 59. Levitzki, A. (1988) From epinephrine to cyclic AMP. Science novel ganglioside, de-N-acetyl-GM, (II3NeuNH2LacCer), acting 241,800-806 as a strong promoter for epidermal growth factor receptor kinase 60. Masters, S. B., Sullivan, K. A., Miller, R. T., Beiderman, B., and as a stimulator for cell growth. J. Biol. Chem. 263, 6296 Lopez, N. G., Ramachandran, J., and Bourne, H. R. (1988) 6301 Carboxyl terminal domain of Gs ƒ¿ specifies coupling of receptors 77. Sjoberg, E. R., Chammas, R., Ozawa, H., Kawashima, I., Khoo, to stimulation of adenylyl cyclase. Science 241, 448-451 K.-H., Morris, H. R., Dell, A., Tai, T., and Varki, A. (1995) 61. Yajima, M., Hosoda, K., Kanbayashi, Y., Nakamura, T., Taka Expression of de-N-acetyl-gangliosides in human melanoma cells hashi, I., and Ui, M. (1978) Biological properties of islets-activat is induced by genistein or nocodazole. J. Biol. Chem. 270, 2921 ing protein (IAP) purified from the culture medium of Bordetella - 2930

pertussis. J. Biochem. 83, 305-312 78. Nudelman, E. D., Levery, S. B., Igarashi, Y., and Hakomori, S. 62. Hoshino, S., Kikkawa, S., Takahashi, K., Rob, H., Kaziro, Y., (1992) Plasmalopsychosine, a novel plasmal (fatty aldehyde) Kawasaki, H., Suzuki, K., Katada, T., and Ui, M. (1990) conjugate of psychosine with cyclic acetyl linkage: Isolation and Identification of sites for alkylation by N-ethylmaleimide and characterization from human brain white matter. J. Biol. Chem. pertussis toxin-catalyzed ADP-ribosylation on GTP-binding 267,11007-11016 proteins. FEBS Lett. 276, 227-231 79. Kreutter, D., Kim, J. Y. H., Goldenring, J. R., Rasmussen, H., 63. Aktories, K., Mohr, C., and Koch, G. (1992) Clostrid Ukomadu, C., DeLorenzo, R. J., and Yu, R. K. (1987) Regulation m C3 ADP-ribosyltransferase. Curr. Top. Microbiol. Im of protein kinase C activity by gangliosides. J. Biol. Chem. 262, munol. 175, 115-131 1633-1637 64. Hara-Yokoyama, M., Hirabayashi, Y., Irie, F., Syuto, B., 80. Hannun, Y. A. and Bell, R.M. (1987) Lysosphingolipids inhibit Moriishi, K., Sugiya, H., and Furuyama, S. (1995) Identification protein kinase C: Implications for the sphingolipidoses. Science of gangliosides as inhibitors of ADP-ribosyltransferases of 235,670-674 pertussis toxin and exoenzyme C3 from Clostridium botulinum. 81. Hannun, Y. A., Loomis, C.R., Merrill, A. H., Jr., and Bell, R. M. J. Biol. Chem. 270, 8115-8121 (1986) Sphingosine inhibition of protein kinase C activity and of 65. Goodemote, K. A., Mattie, M.E., Berger, A., and Spiegel, S. phorbol dibutyrate binding in vitro and in human platelets. J. (1995) Involvement of a pertussis toxin-sensitive G protein in the Biol. Chem. 261, 12604-12609 mitogenic signaling pathways of sphingosine 1-phosphate. J. 82. Merrill, A. H., Jr., Nimkar, S., Menaldino, D., Hannun, Y. A., Biol. Chem. 270, 10272-10277 Loomis, C. R., Bell, R. M., Tyagi, S. R., Lambeth, J. D., Stevens, 66. Bremer, E. G., Schlessinger, J., and Hakomori, S. (1986) Gan V. L., Hunter, R., and Liotta, D. C. (1989) Structural require glioside-mediated modulation of cell growth: Specific effects of ments for long-chain (sphingoid) base inhibition of protein kinase GM3 on tyrosine phosphorylation of the epidermal growth factor C in vitro and for the cellular effects of these compounds. receptor. J. Biol. Chem. 261, 2434-2440 Biochemistry 28, 3138-3145 67. Zhou, Q., Hakomori, S., Kitamura, K., and Igarashi, Y. (1994) 83. Igarashi, Y., Hakomori, S., Toyokuni, T., Dean, B., Fujita, S., GM3directly inhibits tyrosine phosphorylation and de-N-acetyl Sugimoto, M., Ogawa, T., El-Ghendy, K., and Racker, E. (1989) GM3 directly enhances serine phosphorylation of epidermal Effect of chemically well-defined sphingosine and its N-methyl growth factor receptor, independently of receptor-receptor inter derivatives on protein kinase C and arc kinase activities. Bio action. J. Biol. Chem. 269, 1959-1965 chemistry 28, 6796-6800 68. Igarashi, Y., Kitamura, K., Zhou, Q., and Hakomori, S. (1990) A 84. Endo, K., Igarashi, Y., Nisar, M., Zhou, Q., and Hakomori, S. role of lyso-phosphatidylcholine in GM3-dependent inhibition of (1991) signaling as target in cancer therapy: epidermal growth factor receptor autophosphorylation in A431 Inhibitory effect of N, N-dimethyl and N, N, N-trimethyl sphin

Vol. 118, No. 6, 1995 1102 S. Hakomori and Y. Igarashi

gosine derivatives on in vitro and in vivo growth of human tumor 100. Mangeney, M., Lingwood, C. A., Taga, S., Caillou, B., Tursz, T., cells in nude mice. Cancer Res. 51, 1613-1618 and Wiels, J. (1993) Apoptosis induced in Burkitt's lymphoma 85. Okoshi, H., Hakomori, S., Nisar, M., Zhou, Q., Kimura, S., cells via Gb3/CD77, a glycolipid antigen. Cancer Res. 53, 5314 Tashiro, K., and Igarashi, Y. (1991) Cell membrane signaling as - 5319 target in cancer therapy II: Inhibitory effect of N, N, N-trimethyl 101. Chatterjee, S. K. (1991) Lactosylceramide stimulates aortic sphingosine on metastatic potential of murine B16 melanoma cell smooth muscle cell proliferation. Biochem. Biophys. Res. Com line through blocking of tumor cell-dependent platelet aggrega mun. 181, 554-561 tion. Cancer Res. 51, 6019-6024 102. Marsh, N. L., Elias, P. M., and Holleran, W. M. (1995) Glucosyl 86. Murohara, T., Buerke, M., Margiotta, J., Ruan, F., Igarashi, Y., ceramides stimulate murine epidermal hyperproliferation. J. Hakomori, S., and Lefer, A. M. (1995) Myocardial and endo Clin. Invest. 95, 2903-2909 thelial protection by TMS in ischemia-reperfusion injury. Am. J. 103. Dyer, C. A. and Benjamins, J. A. (1990) Glycolipids and trans Physiol. (Heart Circ. Physiol.) 269, H504-H514 membrane signaling: Antibodies to galactocerebroside cause an 87. Handa, K., Igarashi, Y., Nisar, M., and Hakomori, S. (1991) influx of calcium in oligodendrocytes. J. Cell Biol. 111, 625-633 Down-regulation of GMP-140 (CD62 or PADGEM) expression on 104. Desai, N. N., Zhang, H., Olivera, A., Mattie, M. E., and Spiegel, platelets by N, N-dimethyl and N, N, N-trimethyl derivatives of S. (1992) Sphingosine-1 -phosphate, a metabolite of sphingosine, sphingosine. Biochemistry 30, 11682-11686 increases phosphatidic acid levels by phospholipase D activation. 88. Tsuji, S., Arita, M., and Nagai, Y. (1983) GQ1b, a bioactive J. Biol. Chem. 267, 23122-23128 ganglioside that exhibits novel nerve growth factor (NGF) -like 105. Yatomi, Y., Ruan, F., Hakomori, S., and Igarashi, Y. (1995) activities in the two neuroblastoma cell lines. J. Biochem.94, Sphingosine-1-phosphate: A platelet-activating sphingolipid 303-306 released from agonist-stimulated human platelets. Blood 86, 89. Tsuji, S., Yamashita, T., and Nagai, Y. (1988) A novel, carbo 193-202 hydrate signal-mediated cell surface protein phosphorylation: 106. Sadahira, Y., Ruan, F., Hakomori, S., and Igarashi, Y. (1992) Ganglioside GQ1b stimulates ecto-protein kinase activity on the Sphingosine 1-phosphate, a specific endogenous signaling mole cell surface of a human neuroblastoma cell line, GOTO. J. cule controlling cell motility and tumor cell invasiveness. Proc. Biochem. 104, 498-503 Natl. Acad. Sci. USA 89, 9686-9690 90. Ledeen, R. W., Wu, G., Vaswani, K. K., and Cannella, M. S. 107. Bornfeldt, K. E., Graves, L. M., Raines, E. W., Igarashi, Y.,

(1990) Comparison of exogenous and endogenous gangliosides as Wayman, G., Yamamura, S., Yatomi, Y., Sidhu, J. S., Krebs, modulators of neuronal differentiation in Trophic Factors and the E. G., Hakomori, S., and Ross, R. (1995) Sphingosine-l-phos

Nervous System (Horrocks, L. A., Neff, N. H., Yates, A. J., and phate inhibits PDGF-induced chemotaxis of human arterial Hadjiconstantinou, M., eds.) pp. 17-34, Raven Press, New York smooth muscle cells: Spatial and temporal modulation of PDGF 91. Sasaki, K., Kurata, K., Kojima, N., Kurosawa, N., Ohta, S., chemotactic signal transduction. J. Cell Biol. 130, 193-206 Hanai, N., Tsuji, S., and Nishi, T. (1994) Expression cloning of 108. Higashi, H., Omori, A., and Yamagata, T. (1992) Calmodulin, a a GM3-specific ƒ¿-2, 8-sialyltransferase (GD3 synthase). J. Biol. ganglioside-binding protein: Binding of gangliosides to calmodu Chem. 269, 15950-15956 lin in the presence of calcium. J. Biol. Chem. 267, 9831-9838 92. Nara, K., Watanabe, Y., Maruyama, K., Kasahara, K., Nagai, 109. Higashi, H. and Yamagata, T. (1992) Mechanism for ganglio Y., and Sanai, Y. (1994) Expression cloning of a CMP-NeuAc: side-mediated modulation of a calmodulin-dependent enzyme: NeuAca2-3Ga1ƒÀl-4G1cIil-1•LCer ƒ¿2, 8-sialyltransferase (GD3 Modulation of calmodulin-dependent cyclic nucleotide phospho synthase) from human melanoma cells. Proc. Natl. Acad. Sci. diesterase activity through binding of gangliosides to calmodulin USA 91, 7952-7956 and the enzyme. J. Biol. Chem. 267, 9839-9843 93. Kojima, N., Kurosawa, N., Nishi, T., Hanai, N., and Tsuji, S. 110. Abdel-Ghany, M., Osusky, M., Igarashi, Y., Hakomori, S., (1994) Induction of cholinergic differentiation with neurite Shalloway, D., and Racker, E. (1992) Substrate-specific modula sprouting by de novo biosynthesis and expression of GD3 and tion of Src-mediated phosphorylation of Ras and caseins by b-series gangliosides in Neuro2a cells. J. Biol. Chem. 269, sphingosines and other substrate modulators. Biochim. Biophys. 30451-30456 Acta 1137, 349-355 94. Nojiri, H., Takaku, F., Terui, Y., Miura, Y., and Saito, Y. (1986) 111. Ohta, H., Yatomi, Y., Sweeney, E. A., Hakomori, S., and Ganglioside GM,: An acidic membrane component that increases Igarashi, Y. (1994) A possible role of sphingosine in induction of during -like cell differentiation can induce monocytic apoptosis by tumor necrosis factor-ƒ¿ in human neutrophils. differentiation of human myeloid and monocytoid leukemic cell FEBS Lett. 355, 267-270 lines HL-60 and U937. Proc. Natl. Acad. Sci. USA 83,95. 112. Ohta, H., Sweeney, E. A., Masamune, A., Yatomi, Y., Hako Nojiri, H., Kitagawa, S., Nakamura, M., Kirito, K., Enomoto, Y., mori, S., and Igarashi, Y. (1995) Induction of apoptosis by and Saito, M. (1988) Neolacto-series gangliosides induce granulo sphingosine in human leukemic HL-60 cells: A possible endo

cytic differentiation of human promyelocytic leukemia cell line genous modulator of apoptotic DNA fragmentation occurring HL-60. J. Biol. Chem. 263, 7443-7446 during phorbol ester-induced differentiation. Cancer Res. 55, 96. Felding-Habermann, B., Igarashi, Y., Fenderson, B. A., Park, 691-697 L. S., Radin, N. S., Inokuchi, J., Strassmann, G., Hands, K., and 113. Megidish, T., White, T., Takio, K., Titani, K., Igarashi, Y., and Hakomori, S. (1990) A ceramide analogue inhibits T cell prolifer Hakomori, S. The signal modulator protein 14-3-3 is a target of ative response through inhibition of glycosphingolipid synthesis sphingosine- or N, N-dimethylsphingosine-dependent kinase in and enhancement of N, N-dimethylsphingosine synthesis. Bio 3T3(A31) cells. Biochem. Biophys. Res. Commun., in press chemistry 29, 6314-6322 114. Olivera, A. and Spiegel, S. (1993) Sphingosine-l-phosphate as 97. Nakamura, K., Suzuki, H., Hirabayashi, Y., and Suzuki, A. second messenger in cell proliferation induced by PDGF and FCS

(1995) IV3 (NeuGcƒ¿2-8NeuGc)-Gg4Cer is restricted to CD4+ T mitogens. Nature 365, 557-560 cells producing interleukin-2 and a small population of mature 115. Okazaki, T., Bell, R. M., and Hannun, Y. A. (1989) Sphingo thymocytes in mice. J. Biol. Chem. 270, 3876-3881 myelin turnover induced by vitamin D3 in HL-60 cells: Role in 98. Saggioro, D., Sorio, C., Calderazzo, F., Callegaro, L., Panozzo, cell differentiation. J. Biol. Chem. 264, 19076-19080 M., Berton, G., and Chieco-Bianchi, L. (1993) Mechanism of 116. Hannun, Y. A. (1994) The sphingomyelin cycle and the second action of the monosialoganglioside GM1 as a modulator of CD4 messenger function of ceramide. J. Biol. Chem. 269, 3125-3128 expression: Evidence that GM1 -CD4 interaction triggers dissocia 117. Kolesnick, R. N. and Golde, D. W. (1994) The sphingomyelin tion of p56lck from CD4, and CD4 internalization and degrada pathway in tumor necrosis factor and interleukin-1 signaling. tion. J. Biol. Chem. 268, 1368-1375 Cell 77, 325-328 99. Mangeney, M., Richard, Y., Coulaud, D., Tursz, T., and Wiels, J. 118. Ueki, K., Matsuda, S., Tobe, K., Gotoh, Y., Tamemoto, H., (1991) CD77: an antigen of germinal center B cells entering Yachi, M., Akanuma, Y., Yazaki, Y., Nishida, E., and Kadowaki, apoptosis. Eur. J. Immunol. 21, 1131-1140 T. (1994) Feedback regulation of mitogen-activated protein

J. Biochem. Glycosphingolipid Function 1103

kinase kinase kinase activity of c-Raf-1 by insulin and phorbol MacDonald, S. G., Ross, R., and Krebs, E. G. (1993) Protein ester stimulation. J. Biol. Chem. 269, 15756-15761 kinase A antagonizes platelet-derived growth factor-induced 119. Kochh, W., Heidecker, G., Kochs, G. , Hummel, R., Vahidi, H., Mi signaling by mitogen-activated protein kinase in human arterial schak, H., Finkenzeller, G., Marine , D., and Rapp, U.R. (1993) smooth muscle cells. Proc. Natl. Acad. Sci. USA 90, 10300 Protein kinase Cƒ¿ activates RAF-1 by direct phosphorylation. - 10304 Nature 364, 249-252 122. Sakakura, C., Igarashi, Y., Anand, J. K., Sadozai, K. K., and 120. Wu, J., Dent, P., Jelinek, T., Wolfman , A., Weber, M. J., and S Hakomori, S. (1996 in press) Plasmalopsychosine of human brain turgill, T. W. (1993) Inhibition of the EGF-activated MAP mimics the effect of nerve growth factor by activating its receptor kinase signaling pathway by adenosine 3•L, 5•L-monophosphate. kinase and mitogen-activated protein kinase in PC12 cells: Science 262, 1065-1068 Induction of neurite outgrowth and prevention of apoptosis. J. 121. Graves, L. M., Bornfeldt, K. E., Raines, E. W., Potts, B. C., Biol. Chem.

Vol. 118, No. 6, 1995