Genomic Plasticity of the Human Fungal Pathogen Candida albicans

The genomic plasticity of Candida albicans, a commensal and common opportunistic fungal pathogen, continues to reveal unexpected surprises. Once thought to be asexual, we now know that the organism can generate genetic diversity through several mechanisms, including mating between cells of the opposite or of the same mating type and by a parasexual reduction in chromosome number that can be accompanied by recombination events (2, 12, 14, 53, 77, 115). In addition, dramatic genome changes can appear quite rapidly in mitotic cells propagated in vitro as well as in vivo. The detection of aneuploidy in other fungal pathogens isolated directly from patients (145) and from environmental samples (71) suggests that variations in chromosome organization and copy number are a common mechanism used by pathogenic fungi to rapidly generate diversity in response to stressful growth conditions, including, but not limited to, antifungal drug exposure. Since cancer cells often become polyploid and/or aneuploid, some of the lessons learned from studies of genome plasticity in C. albicans may provide important insights into how these processes occur in higher-eukaryotic cells exposed to stresses such as anticancer drugs.The purpose of this review is to describe the tools used to detect genome changes, to highlight recent advances in our understanding of large-scale chromosome changes that arise in Candida albicans, and to discuss the role of specific stresses in eliciting these genome changes. The types of genomic diversity that have been characterized suggest that C. albicans can undergo extreme genomic changes in order to survive stresses in the human host. We propose that C. albicans and other pathogens may have evolved mechanisms not only to tolerate but also to generate large-scale genetic variation as a means of adaptation.C. albicans is a polymorphic yeast with a 16-Mb (haploid) genome organized in 8 diploid chromosomes (140, 154, 203). The C. albicans genome displays a very high degree of plasticity. This plasticity includes the types of genomic changes frequently observed with cancer cells, including gross chromosomal rearrangements, aneuploidy, and loss of heterozygosity (reviewed in references 100, 117, and 157). Similar to somatic cancer cells, C. albicans reproduces primarily through asexual clonal division (65, 84). Nonetheless, it has retained much of the machinery needed for mating and meiosis (189), yet meiosis has never been observed (13, 120).C. albicans has two mating-type-like (MTL) alleles, MTLa and MTLα (76). The MTL locus is on the left arm of chromosome 5 (Chr5), approximately 80 kbp from the centromere. Most C. albicans isolates are heterozygous for the MTL locus, but approximately 3 to 10% of clinical isolates are naturally homozygous at MTL (104, 108). Mating can occur between strains carrying the opposite MTL locus, and most strains that were found to be naturally MTL homozygous are mating competent (104, 108). MTL-homozygous strains were also constructed from MTL-heterozygous strains by deletion of either the MTLa or MTLα locus (77) or by selection for Chr5 loss on sorbose (87, 115).Mating between these diploid strains of opposite mating type can occur both in vitro (115) and in vivo (77, 97). The products are tetraploid and do not undergo a conventional meiotic reduction in ploidy (12, 120). Rather, they undergo random loss of multiple chromosomes, a process termed “concerted chromosome loss,” until they reach a near-diploid genome content (2, 12, 53, 85). A subset of these cells also undergoes multiple gene conversion events reminiscent of meiotic recombination, and most remain trisomic for one to several chromosomes (53). While mating and concerted chromosome loss have been induced in the laboratory, the role of the parasexual cycle during the host-pathogen interaction and in the response to stresses, such as exposure to antifungal drugs, remains unclear. The prevailing model is that adaptive mutations (such as those that occur with the acquisition of drug resistance) evolve through somatic events, including point mutations, recombination, gene conversion, loss of heterozygosity, and/or aneuploidy (13).     

Melanin Externalization in Candida albicans Depends on Cell Wall Chitin Structures

The fungal pathogen Candida albicans produces dark-pigmented melanin after 3 to 4 days of incubation in medium containing l-3,4-dihydroxyphenylalanine (l-DOPA) as a substrate. Expression profiling of C. albicans revealed very few genes significantly up- or downregulated by growth in l-DOPA. We were unable to determine a possible role for melanin in the virulence of C. albicans. However, we showed that melanin was externalized from the fungal cells in the form of electron-dense melanosomes that were free or often loosely bound to the cell wall exterior. Melanin production was boosted by the addition of N-acetylglucosamine to the medium, indicating a possible association between melanin production and chitin synthesis. Melanin externalization was blocked in a mutant specifically disrupted in the chitin synthase-encoding gene CHS2. Melanosomes remained within the outermost cell wall layers in chs3Δ and chs2Δ chs3Δ mutants but were fully externalized in chs8Δ and chs2Δ chs8Δ mutants. All the CHS mutants synthesized dark pigment at equivalent rates from mixed membrane fractions in vitro, suggesting it was the form of chitin structure produced by the enzymes, not the enzymes themselves, that was involved in the melanin externalization process. Mutants with single and double disruptions of the chitinase genes CHT2 and CHT3 and the chitin pathway regulator ECM33 also showed impaired melanin externalization. We hypothesize that the chitin product of Chs3 forms a scaffold essential for normal externalization of melanosomes, while the Chs8 chitin product, probably produced in cell walls in greater quantity in the absence of CHS2, impedes externalization.Candida albicans is a major opportunistic fungal human pathogen that causes a wide variety of infections (9, 68). In healthy individuals C. albicans resides as a commensal within the oral cavity and gastrointestinal and urogenital tracts. However, in immunocompromised hosts, C. albicans causes infections ranging in severity from mucocutaneous infections to life-threatening disseminated diseases (9, 68). Research into the pathogenicity of C. albicans has revealed a complex mix of putative virulence factors (7, 60), perhaps reflecting the fine balance this species strikes between commensal colonization and opportunistic invasion of the human host.Melanins are biological pigments, typically dark brown or black, formed by the oxidative polymerization of phenolic compounds. They are negatively charged hydrophobic molecules with high molecular weights and are insoluble in both aqueous and organic solvents. Their insolubility makes melanins difficult to study, and no definitive structure has yet been found for them; they probably represent an amorphous mixture of polymers (35). There are various types of melanin in nature, including eumelanin and phaeomelanin (76). Two principal types of melanin are found in the fungal kingdom. The majority are 1.8-dihydroxynapthalene (DNH) melanins synthesized from acetyl-coenzyme A (CoA) via the polyketide pathway (5). DNH melanins have been found in a wide range of opportunistic fungal pathogens of humans, including dark (dematiaceous) molds, such as Cladosporium, Fonsecaea, Phialophora, and Wangiella species, and as conidial pigments in Aspergillus fumigatus and Aspergillus niger (41, 80, 87, 88). However, several other fungal pathogens, including Blastomyces dermatitidis, Coccidioides posadasii, Cryptococcus neoformans, Histoplasma capsulatum, Paracoccidioides brasiliensis, and Sporothrix schenckii, produce eumelanin (3,4-dihydroxyphenylalanine [DOPA]-melanin) through the activity of a polyphenol oxidase (laccase) and require an exogenous o-diphenolic or p-diphenolic substrate, such as l-DOPA (16, 30, 63,–65, 67, 79).The production of melanin in humans and other mammals is a function of specialized cells called melanocytes. Particles of melanin polymers, sometimes, including more than one melanin type, are built up within membrane-bound organelles called melanosomes (76), and these are actively transported along microtubules to the tips of dendritic outgrowths of melanocytes, from where they are transferred to neighboring cells (32, 81). The mechanism of intercellular transfer of melanosomes has not yet been established, but the export process probably involves the fusion of cell and vesicular membranes rather than secretion of naked melanin (82). In pathogenic fungi, melanins are often reported to be associated with or “in” the cell wall (35, 36, 50, 72, 79). However, there is variation between species: the melanin may be located external to the wall, e.g., in P. brasiliensis (79); within the wall itself (reviewed in reference 42); or as a layer internal to the wall and external to the cell membrane, e.g., in C. neoformans (22, 45, 85). However, mutants of C. neoformans bearing disruptions of three CDA genes involved in the biosynthesis of cell wall chitosan, or of CHS3, encoding a chitin synthase, or of CSR2, which probably regulates Chs3, all released melanin into the culture supernatant, suggesting a role for chitin or chitosan in retaining the pigment polymer in its normal intracellular location (3, 4). However, vesicles externalized from C. neoformans cells also show laccase activity (21), so the effect of chitin may be on vesicle externalization rather than on melanin itself. Internal structures compatible with mammalian melanosomes have been observed in Cladosporium carrionii (73) and in Fonsecaea pedrosoi (2, 26). Remarkably, F. pedrosoi also secretes melanin and locates the polymer within the cell wall (1, 2, 25, 27, 74).Melanization has been found to play an important role in the virulence of several human fungal pathogens, such as C. neoformans, A. fumigatus, P. brasiliensis, S. schenckii, H. capsulatum, B. dermatitidis, and C. posadasii (among recent reviews are references 29, 42, 62, 74, and 79). From these and earlier reviews of the extensive literature, melanin has been postulated to be involved in a range of virulence-associated properties, including interactions with host cells; protection against oxidative stresses, UV light, and hydrolytic enzymes; resistance to antifungal agents; iron-binding activities; and even the harnessing of ionizing radiation in contaminated soils (15). The most extensively studied fungal pathogen for the role of melanization is C. neoformans, which possesses two genes, LAC1 and LAC2, encoding melanin-synthesizing laccases (52, 69, 90). It has been known since early studies with naturally occurring albino variants of C. neoformans (39) that melanin-deficient strains are attenuated in mouse models of cryptococcosis. Deletion of both the LAC1 and LAC2 genes reduced survival of C. neoformans in macrophages (52), and a study based on otherwise isogenic LAC1⁺ and LAC1⁻ strains confirmed the importance of LAC1 in experimental virulence (66). Other genes in the regulatory pathway for LAC1 are similarly known to be essential to virulence (12, 84).C. albicans has been shown to produce melanin with DOPA as a substrate for production of the polymer (53). The cells could be treated with hot acids to produce typical melanin “ghosts,” and antibodies specific for melanin reacted with the fungal cells by immunohistochemistry with tissues from experimentally infected mice, demonstrating that C. albicans produces melanin in vivo (53). However, no candidate genes encoding laccases have yet been identified in the C. albicans genome (http://www.candidagenome.org/). In this study, we investigated the production of melanin by C. albicans and showed that its normal externalization from wild-type cells, including formation of melanosomes, can be altered to an intracellular and intrawall location by mutation of genes involved in chitin synthesis. C. albicans has four genes encoding chitin synthase enzymes. CHS1 is an essential gene under normal conditions (59), and its product is the main enzyme involved in septum formation (83). Chs3 forms the bulk of the chitin in the cell wall and the chitinous ring at sites of bud emergence (8, 51, 57), while Chs2 contributes to differential chitin levels found between yeast and hyphal forms of the fungus, and Chs8 influences the architecture of chitin microfibrils (43, 51, 55, 57, 58). We found that melanin externalization was unaffected in a chs8Δ mutant but was reduced or abrogated in chs2Δ and chs3Δ mutants. Expression profiles of melanin-producing cells grown in the presence of l-DOPA did not identify any potential laccase-synthesizing genes.     

Arachidonic Acid Stimulates Cell Adhesion through a Novel p38 MAPK-RhoA Signaling Pathway That Involves Heat Shock Protein 27

Rho GTPases are critical components of cellular signal transduction pathways. Both hyperactivity and overexpression of these proteins have been observed in human cancers and have been implicated as important factors in metastasis. We previously showed that dietary n-6 fatty acids increase cancer cell adhesion to extracellular matrix proteins, such as type IV collagen. Here we report that in MDA-MB-435 human melanoma cells, arachidonic acid activates RhoA, and inhibition of RhoA signaling with either C3 exoenzyme or dominant negative Rho blocked arachidonic acid-induced cell adhesion. Inhibition of the Rho kinase (ROCK) with either small molecule inhibitors or ROCK II-specific small interfering RNA (siRNA) blocked the fatty acid-induced adhesion. However, unlike other systems, inhibition of ROCK did not block the activation of p38 mitogen-activated protein kinase (MAPK); instead, Rho activation depended on p38 MAPK activity and the presence of heat shock protein 27 (HSP27), which is phosphorylated downstream of p38 after arachidonic acid treatment. HSP27 associated with p115RhoGEF in fatty acid-treated cells, and this association was blocked when p38 was inhibited. Furthermore, siRNA knockdown of HSP27 blocked the fatty acid-stimulated Rho activity. Expression of dominant negative p115-RhoGEF or p115RhoGEF-specific siRNA inhibited both RhoA activation and adhesion on type IV collagen, whereas a constitutively active p115RhoGEF restored the arachidonic acid stimulation in cells in which the p38 MAPK had been inhibited. These data suggest that n-6 dietary fatty acids stimulate a set of interactions that regulates cell adhesion through RhoA and ROCK II via a p38 MAPK-dependent association of HSP27 and p115RhoGEF.The ability of tumor cells to metastasize to secondary sites is a hallmark of neoplastic disease. Unfortunately, this propensity to spread is the primary cause of morbidity and death in cancer patients (1). Metastasis is clearly a highly regulated, multistep process that occurs in a spatiotemporal manner (2–4). To escape the restrictive compartment boundaries characteristic of adult tissue, separate intravasation and extravasation steps requiring alterations in co-adhesion, adhesion, invasion, and migration must occur. Execution of these biological processes, involving multiple proteins and cellular organelles, require highly coordinated cell signaling mechanisms.The Rho family of small GTPases regulates many facets of cytoskeletal rearrangements that facilitate cell attachment and migration (5–7). Rho GTPases act as molecular switches by changing from an inactive GDP-bound conformation to an active GTP-bound conformation, thereby regulating a signaling pathway. These proteins are directly regulated by Rho guanine nucleotide exchange factors (GEFs),² Rho GTPase activating proteins, and Rho GDP-dissociation inhibitors (8–12). RhoGEFs bind to the GTPase to catalyze the dissociation of GDP, allowing the binding of GTP and thereby promoting Rho activation (8). The RGS (regulators of G protein signaling) domain-containing RhoGEFs are a recently described family of GEFs. Currently, there are three members of this family, PDZ-RhoGEF, LARG, and p115RhoGEF (13–15), in which the RGS domains function as a heterotrimeric GTPase-activating domain (13, 15, 16). The RGS family of RhoGEFs has been shown to regulate Rho during several processes including cytoskeletal rearrangements, cell adhesion, and cancer progression (17–21).There is significant interplay between the activity of small GTPases and signaling derived from fatty acid metabolism (22–28). Linoleic acid, which is metabolized to arachidonic acid, is an n-6 polyunsaturated fatty acid that is present at high levels in most western diets (29). In animal models, diets high in n-6 polyunsaturated fatty acids have been shown to enhance tumor progression and metastasis (30, 31). Additionally, arachidonic acid is stored in cell membranes and is made available by phospholipases under conditions of increased inflammatory response (32). Arachidonic acid is further metabolized by cyclooxygenases (COX), lipoxygenases (LOX), and cytochrome P450 monooxygenases to yield bioactive products that have myriad effects on cells, and altered metabolism of arachidonic acid by COX, LOX, and P450 has been implicated in cancer progression (31, 33–36).We have studied mechanisms of cell adhesion using the MDA-MB-435 cells as a model of a highly metastatic human cancer cell line (37). These cells have been extensively studied for their ability to recapitulate the metastatic cascade in vivo and in vitro, although recent work indicates that the cells currently in use are most likely a human melanoma line (38). We initially observed that arachidonic acid (AA) enhanced adhesion of MDA-MB-435 cells to type IV collagen through specific integrin-mediated pathways (37). Exogenous AA led to the activation of mitogen-activated protein kinase (MAPK)-activated protein kinase 2 and the phosphorylation of heat shock protein 27 (HSP27) via a p38 MAPK-dependent process (39). Inhibition of p38 MAPK activation blocked cell adhesion as did function-blocking antibodies specific for subunits of the collagen receptor (40). More recently, we identified the key metabolite of AA (15-(S)- hydroxyeicosatetraenoic acid) and the upstream kinases (TAK1 and MKK6) that are responsible for activation of p38 MAPK in this system (41).In this study we investigated the role of Rho activation in the MDA-MB-435 cells after exposure to arachidonic acid. Several aspects of the regulation of Rho signaling in these cells provide insights into the cross-talk between important signaling pathways.     

Structure and Function of Glycosylated Tandem Repeats from Candida albicans Als Adhesins

Tandem repeat (TR) regions are common in yeast adhesins, but their structures are unknown, and their activities are poorly understood. TR regions in Candida albicans Als proteins are conserved glycosylated 36-residue sequences with cell-cell aggregation activity (J. M. Rauceo, R. De Armond, H. Otoo, P. C. Kahn, S. A. Klotz, N. K. Gaur, and P. N. Lipke, Eukaryot. Cell 5:1664–1673, 2006). Ab initio modeling with either Rosetta or LINUS generated consistent structures of three-stranded antiparallel β-sheet domains, whereas randomly shuffled sequences with the same composition generated various structures with consistently higher energies. O- and N-glycosylation patterns showed that each TR domain had exposed hydrophobic surfaces surrounded by glycosylation sites. These structures are consistent with domain dimensions and stability measurements by atomic force microscopy (D. Alsteen, V. Dupres, S. A. Klotz, N. K. Gaur, P. N. Lipke, and Y. F. Dufrene, ACS Nano 3:1677–1682, 2009) and with circular dichroism determination of secondary structure and thermal stability. Functional assays showed that the hydrophobic surfaces of TR domains supported binding to polystyrene surfaces and other TR domains, leading to nonsaturable homophilic binding. The domain structures are like “classic” subunit interaction surfaces and can explain previously observed patterns of promiscuous interactions between TR domains in any Als proteins or between TR domains and surfaces of other proteins. Together, the modeling techniques and the supporting data lead to an approach that relates structure and function in many kinds of repeat domains in fungal adhesins.Yeast adhesins are a diverse set of cell adhesion proteins that mediate adhesion to host cells, environmental substrates, other fungi, and coinfecting bacteria (6, 8, 20, 21, 23, 29). The adhesins share common features, including compact N-terminal domains similar to Ig or lectin domains, Thr-rich midpieces, often in tandem repeats, and long highly glycosylated Ser/Thr-rich C-terminal regions that extend the functional domains out from the cell surface. No structures for the Thr-rich midpieces are known, but they can mediate aggregation of fungal cells (33, 35, 47). The prevalence and conservation of such repeats argue that they are functionally important, despite limited data on their structure and function.In Candida albicans, the Als adhesins are homologous proteins, products of 8 loci that encode numerous alleles of cell surface adhesins (16). In each mature Als protein, there are, from the N terminus, three tandem Ig-like domains, a β-sheet-rich conserved 127-residue amyloid-forming T region, a variable number of 36-residue tandem repeats (TRs), and a highly glycosylated stalk region that extends the N-terminal domains away from the cell surface (Fig. 1) (16, 33, 41). The C termini of these and other wall-associated adhesins are covalently cross-linked into the cell wall through transglycosylation of a modified glycosylphosphatidylinositol (GPI) anchor (18, 25). This modular design, including tandem repeats, is typical of fungal adhesins (8).Open in a separate windowFig. 1.Schematic diagram of the sequence of Als5p. The regions are named above, and the number of amino acid residues in each region is shown below. The modeled sequences are in the TR region.The Als protein Ig-like region, T region, and TR region all have protein-protein interaction activities (26, 33, 35). The Ig-like regions can interact with diverse mammalian proteins, presumably in a way analogous to antibody-antigen binding, as has been shown in the homologous protein α-agglutinin from Saccharomyces cerevisiae (8, 24, 26, 35). The T regions interact through formation of amyloid-like structures both in vivo and in vitro (33, 34a, 36). An insight into the function of the tandem repeats followed from observations that Als proteins initiate and maintain cell-to-cell aggregations, either spontaneously (“autoaggregation”) or following adhesion to a bead-bound defined ligand (10, 11, 36). Aggregation is more extensive for Als proteins with more tandem repeats (26, 35). This result suggested that the tandem repeats are uniquely structured to facilitate or mediate the aggregative function. Circular dichroism spectroscopy of the TR region of Als5p shows a β-sheet-rich structure in the soluble protein (35).In support of their direct involvement in aggregation, the repeat region of the C. albicans adhesin Als5p mediates cell-cell aggregation in the absence of the Ig-like and T domains (35). Moreover, the repeats can also potentiate binding of Als5p to fibronectin (35). Thus, the TR domains mediate cellular aggregation and increased binding to fibronectin. In addition, TR domains and their amino acid sequences are highly conserved across several Candida species (3). These properties need to be explained by their three-dimensional structure.Because there are no homologous structures known, we modeled by two independent ab initio methods. Rosetta assembles structures by combining short peptide structures extracted from the protein structural database PDB (38), then combines structures in a Monte Carlo approach, and assesses energetics of assembled structures. Rosetta has recently been shown to generate accurate models for protein-sized domains (40). We also predicted structures with LINUS, which generates randomized structures and rapidly estimates energetics to choose low-energy models (45). The models were supported by structural analyses with atomic force microscopy and circular dichroism spectroscopy. Functional assays showed that the TR domains can mediate binding activities predicted from the calculated structures.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Tyrosine Phosphorylation at a Site Highly Conserved in the L1 Family of Cell Adhesion Molecules Abolishes Ankyrin Binding and Increases Lateral Mobility of Neurofascin

This paper presents evidence that a member of the L1 family of ankyrin-binding cell adhesion molecules is a substrate for protein tyrosine kinase(s) and phosphatase(s), identifies the highly conserved FIGQY tyrosine in the cytoplasmic domain as the principal site of phosphorylation, and demonstrates that phosphorylation of the FIGQY tyrosine abolishes ankyrin-binding activity. Neurofascin expressed in neuroblastoma cells is subject to tyrosine phosphorylation after activation of tyrosine kinases by NGF or bFGF or inactivation of tyrosine phosphatases with vanadate or dephostatin. Furthermore, both neurofascin and the related molecule Nr-CAM are tyrosine phosphorylated in a developmentally regulated pattern in rat brain. The FIGQY sequence is present in the cytoplasmic domains of all members of the L1 family of neural cell adhesion molecules. Phosphorylation of the FIGQY tyrosine abolishes ankyrin binding, as determined by coimmunoprecipitation of endogenous ankyrin and in vitro ankyrin-binding assays. Measurements of fluorescence recovery after photobleaching demonstrate that phosphorylation of the FIGQY tyrosine also increases lateral mobility of neurofascin expressed in neuroblastoma cells to the same extent as removal of the cytoplasmic domain. Ankyrin binding, therefore, appears to regulate the dynamic behavior of neurofascin and is the target for regulation by tyrosine phosphorylation in response to external signals. These findings suggest that tyrosine phosphorylation at the FIGQY site represents a highly conserved mechanism, used by the entire class of L1-related cell adhesion molecules, for regulation of ankyrin-dependent connections to the spectrin skeleton.Vertebrate L1, neurofascin, neuroglial cell adhesion molecule (Ng-CAM),¹ Ng-CAM–related cell adhesion molecule (Nr-CAM), and Drosophila neuroglian are members of a family of nervous system cell adhesion molecules that possess variable extracellular domains comprised of Ig and fibronectin type III domains and a relatively conserved cytoplasmic domain (Grumet, 1991; Hortsch and Goodman, 1991; Rathgen and Jessel, 1991; Sonderegger and Rathgen, 1992; Hortsch, 1996). Members of this family, including a number of alternatively spliced forms, are abundant in the nervous system during early development as well as in adults. Neurofascin and Nr-CAM, for example, constitute ∼0.5% of the total membrane protein in adult brain (Davis et al., 1993; Davis and Bennett, 1994). Cellular functions attributed to the L1 family include axon fasciculation (Stallcup and Beasley, 1985; Landmesser et al., 1988; Brummendorf and Rathjen, 1993; Bastmeyer et al., 1995; Itoh et al., 1995; Magyar-Lehmann et al., 1995), axonal guidance (van den Pol and Kim, 1993; Liljelund et al., 1994; Brittis and Silver, 1995; Brittis et al., 1995; Lochter et al., 1995; Wong et al., 1996), neurite extension (Chang et al., 1987; Felsenfeld et al., 1994; Hankin and Lagenaur, 1994; Ignelzi et al., 1994; Williams et al., 1994a ,b,c,d; Doherty et al., 1995; Zhao and Siu, 1995), a role in long term potentiation (Luthl et al., 1994), synaptogenesis (Itoh et al., 1995), and myelination (Wood et al., 1990). The potential clinical importance of this group of proteins has been emphasized by the findings that mutations in the L1 gene on the X chromosome are responsible for developmental anomalies including hydrocephalus and mental retardation (Rosenthal et al., 1992; Jouet et al., 1994; Wong et al., 1995).The conserved cytoplasmic domains of L1 family members include a binding site for the membrane skeletal protein ankyrin. This interaction was first described for neurofascin (Davis et. al., 1993) and subsequently has been observed for L1, Nr-CAM (Davis and Bennett, 1994), and Drosophila neuroglian (Dubreuil et al., 1996). The membrane-binding domain of ankyrin contains two distinct sites for neurofascin and has the potential to promote lateral association of neurofascin and presumably other L1 family members (Michaely and Bennett, 1995). Nodes of Ranvier are physiologically relevant axonal sites where ankyrin and L1 family members collaborate, based on findings of colocalization of a specialized isoform of ankyrin with alternatively spliced forms of neurofascin and NrCAM in adults (Davis et al., 1996) as well as in early axonal developmental intermediates (Lambert, S., J. Davis, P. Michael, and V. Bennett. 1995. Mol. Biol. Cell. 6:98a).L1, after homophilic and/or heterophilic binding, participates in signal transduction pathways that ultimately are associated with neurite extension and outgrowth (Ignelzi et al., 1994; Williams et al., 1994a ,b,c,d; Doherty et al., 1995). L1 copurifies with a serine–threonine protein kinase (Sadoul et al., 1989) and is phosphorylated on a serine residue that is not conserved among other family members (Wong et al., 1996). L1 pathway(s) may also involve G proteins, calcium channels, and tyrosine phosphorylation (Williams et al., 1994a ,b,c,d; Doherty et al., 1995). After homophilic interactions, L1 directly activates a tyrosine signaling cascade after a lateral association of its ectodomain with the fibroblast growth factor receptor (Doherty et al., 1995). Antibodies against L1 have also been shown to activate protein tyrosine phosphatase activity in growth cones (Klinz et al., 1995). However, details of the downstream substrates of L1-promoted phosphorylation and dephosphorylation and possible roles of the cytoplasmic domain are not known.Tyrosine phosphorylation is well established to modulate cell–cell and cell–extracellular matrix interactions involving integrins and their associated proteins (Akiyama et al., 1994; Arroyo et al., 1994; Schlaepfer et al., 1994; Law et al., 1996) as well as the cadherins (Balsamo et al., 1996; Krypta et al., 1996; Brady-Kalnay et al., 1995; Shibamoto et al., 1995; Hoschuetzky et al., 1994; Matsuyoshi et al., 1992). For example, the adhesive functions of the calciumdependent cadherin cell adhesion molecule are mediated by a dynamic balance between tyrosine phosphorylation of β-catenin by TrkA and dephosphorylation via the LARtype protein tyrosine phosphatase (Krypta et al., 1996). In this example the regulation of binding among the structural proteins is the result of a coordination between classes of protein kinases and protein phosphatases.This study presents evidence that neurofascin, expressed in a rat neuroblastoma cell line, is a substrate for both tyrosine kinases and protein tyrosine phosphatases at a tyrosine residue conserved among all members of the L1 family. Site-specific tyrosine phosphorylation promoted by both tyrosine kinase activators (NGF and bFGF) and protein tyrosine phosphatase inhibitors (dephostatin and vanadate) is a strong negative regulator of the neurofascin– ankyrin binding interaction and modulates the membrane dynamic behavior of neurofascin. Furthermore, neurofascin and, to a lesser extent Nr-CAM, are also shown here to be tyrosine phosphorylated in developing rat brain, implying a physiological relevance to this phenomenon. These results indicate that neurofascin may be a target for the coordinate control over phosphorylation that is elicited by protein kinases and phosphatases during in vivo tyrosine phosphorylation cascades. The consequent decrease in ankyrin-binding capacity due to phosphorylation of neurofascin could represent a general mechanism among the L1 family members for regulation of membrane–cytoskeletal interactions in both developing and adult nervous systems.