共查询到20条相似文献,搜索用时 31 毫秒
1.
2.
Mikiko C. Siomi Paul S. Eder Naoyuki Kataoka Lili Wan Qing Liu Gideon Dreyfuss 《The Journal of cell biology》1997,138(6):1181-1192
3.
4.
5.
6.
SPA2 encodes a yeast protein that is one of the first proteins to localize to sites of polarized growth, such as the shmoo tip and the incipient bud. The dynamics and requirements for Spa2p localization in living cells are examined using Spa2p green fluorescent protein fusions. Spa2p localizes to one edge of unbudded cells and subsequently is observable in the bud tip. Finally, during cytokinesis Spa2p is present as a ring at the mother–daughter bud neck. The bud emergence mutants bem1 and bem2 and mutants defective in the septins do not affect Spa2p localization to the bud tip. Strikingly, a small domain of Spa2p comprised of 150 amino acids is necessary and sufficient for localization to sites of polarized growth. This localization domain and the amino terminus of Spa2p are essential for its function in mating. Searching the yeast genome database revealed a previously uncharacterized protein which we name, Sph1p (Spa2p homolog), with significant homology to the localization domain and amino terminus of Spa2p. This protein also localizes to sites of polarized growth in budding and mating cells. SPH1, which is similar to SPA2, is required for bipolar budding and plays a role in shmoo formation. Overexpression of either Spa2p or Sph1p can block the localization of either protein fused to green fluorescent protein, suggesting that both Spa2p and Sph1p bind to and are localized by the same component. The identification of a 150–amino acid domain necessary and sufficient for localization of Spa2p to sites of polarized growth and the existence of this domain in another yeast protein Sph1p suggest that the early localization of these proteins may be mediated by a receptor that recognizes this small domain.Polarized cell growth and division are essential cellular processes that play a crucial role in the development of eukaryotic organisms. Cell fate can be determined by cell asymmetry during cell division (Horvitz and Herskowitz, 1992; Cohen and Hyman, 1994; Rhyu and Knoblich, 1995). Consequently, the molecules involved in the generation and maintenance of cell asymmetry are important in the process of cell fate determination. Polarized growth can occur in response to external signals such as growth towards a nutrient (Rodriguez-Boulan and Nelson, 1989; Eaton and Simons, 1995) or hormone (Jackson and Hartwell, 1990a
,
b
; Segall, 1993; Keynes and Cook, 1995) and in response to internal signals as in Caenorhabditis elegans (Goldstein et al., 1993; Kimble, 1994; Priess, 1994) and Drosophila melanogaster (St Johnston and Nusslein-Volhard, 1992; Anderson, 1995) early development.
Saccharomyces cerevisiae undergo polarized growth towards an external cue during mating and to an internal cue during budding. Polarization towards a mating partner (shmoo formation) and towards a new bud site requires a number of proteins (Chenevert, 1994; Chant, 1996; Drubin and Nelson, 1996). Many of these proteins are necessary for both processes and are localized to sites of polarized growth, identified by the insertion of new cell wall material (Tkacz and Lampen, 1972; Farkas et al., 1974; Lew and Reed, 1993) to the shmoo tip, bud tip, and mother–daughter bud neck. In yeast, proteins localized to growth sites include cytoskeletal proteins (Adams and Pringle, 1984; Kilmartin and Adams, 1984; Ford, S.K., and J.R. Pringle. 1986. Yeast. 2:S114; Drubin et al., 1988; Snyder, 1989; Snyder et al., 1991; Amatruda and Cooper, 1992; Lew and Reed, 1993; Waddle et al., 1996), neck filament components (septins) (Byers and Goetsch, 1976; Kim et al., 1991; Ford and Pringle, 1991; Haarer and Pringle, 1987; Longtine et al., 1996), motor proteins (Lillie and Brown, 1994), G-proteins (Ziman, 1993; Yamochi et al., 1994; Qadota et al., 1996), and two membrane proteins (Halme et al., 1996; Roemer et al., 1996; Qadota et al., 1996). Septins, actin, and actin-associated proteins localize early in the cell cycle, before a bud or shmoo tip is recognizable. How this group of proteins is localized to and maintained at sites of cell growth remains unclear.Spa2p is one of the first proteins involved in bud formation to localize to the incipient bud site before a bud is recognizable (Snyder, 1989; Snyder et al., 1991; Chant, 1996). Spa2p has been localized to where a new bud will form at approximately the same time as actin patches concentrate at this region (Snyder et al., 1991). An understanding of how Spa2p localizes to incipient bud sites will shed light on the very early stages of cell polarization. Later in the cell cycle, Spa2p is also found at the mother–daughter bud neck in cells undergoing cytokinesis. Spa2p, a nonessential protein, has been shown to be involved in bud site selection (Snyder, 1989; Zahner et al., 1996), shmoo formation (Gehrung and Snyder, 1990), and mating (Gehrung and Snyder, 1990; Chenevert et al., 1994; Yorihuzi and Ohsumi, 1994; Dorer et al., 1995). Genetic studies also suggest that Spa2p has a role in cytokinesis (Flescher et al., 1993), yet little is known about how this protein is localized to sites of polarized growth.We have used Spa2p green fluorescent protein (GFP)1 fusions to investigate the early localization of Spa2p to sites of polarized growth in living cells. Our results demonstrate that a small domain of ∼150 amino acids of this large 1,466-residue protein is sufficient for targeting to sites of polarized growth and is necessary for Spa2p function. Furthermore, we have identified and characterized a novel yeast protein, Sph1p, which has homology to both the Spa2p amino terminus and the Spa2p localization domain. Sph1p localizes to similar regions of polarized growth and sph1 mutants have similar phenotypes as spa2 mutants. 相似文献
7.
Dirk G?rlich Marylena Dabrowski F. Ralf Bischoff Ulrike Kutay Peer Bork Enno Hartmann Siegfried Prehn Elisa Izaurralde 《The Journal of cell biology》1997,138(1):65-80
The importin-α/β complex and the GTPase Ran mediate nuclear import of proteins with a classical nuclear localization signal. Although Ran has been implicated also in a variety of other processes, such as cell cycle progression, a direct function of Ran has so far only been demonstrated for importin-mediated nuclear import. We have now identified an entire class of ∼20 potential Ran targets that share a sequence motif related to the Ran-binding site of importin-β. We have confirmed specific RanGTP binding for some of them, namely for two novel factors, RanBP7 and RanBP8, for CAS, Pse1p, and Msn5p, and for the cell cycle regulator Cse1p from Saccharomyces cerevisiae. We have studied RanBP7 in more detail. Similar to importin-β, it prevents the activation of Ran''s GTPase by RanGAP1 and inhibits nucleotide exchange on RanGTP. RanBP7 binds directly to nuclear pore complexes where it competes for binding sites with importin-β, transportin, and apparently also with the mediators of mRNA and U snRNA export. Furthermore, we provide evidence for a Ran-dependent transport cycle of RanBP7 and demonstrate that RanBP7 can cross the nuclear envelope rapidly and in both directions. On the basis of these results, we propose that RanBP7 might represent a nuclear transport factor that carries an as yet unknown cargo, which could apply as well for this entire class of related RanGTP-binding proteins.The nuclear pore complexes (NPC)1 are the sites where the exchange of macromolecules between nucleus and cytoplasm occurs (Feldherr et al., 1984). Transport through the NPCs is bidirectional and comprises a multitude of substrates. Small molecules can passively diffuse through the NPC. The transport of proteins and RNAs >40–60 kD is, however, generally an active process, i.e., it is energy dependent (Newmeyer et al., 1986) and mediated by saturable transport receptors (Goldfarb et al., 1986; Michaud and Goldfarb, 1991; Jarmolowski et al., 1994). To accomplish multiple rounds of transport, these transport receptors are thought to shuttle between nucleus and cytoplasm (Goldfarb et al., 1986). An import receptor, for example, has to bind its import substrate initially in the cytoplasm, release it after NPC passage into the nucleus, and return to the cytoplasm without the cargo. Conversely, an export factor has to bind the export substrate only in the nucleus and on the way out. This model predicts asymmetry in these transport cycles and implies that the binding of the transport receptor to its cargo is regulated by the different environments of nucleus and cytoplasm.The nuclear import of proteins with a classical nuclear localization signal (NLS) is to date the best characterized nucleocytoplasmic transport pathway (for reviews see Görlich and Mattaj, 1996; Koepp and Silver, 1996; Schlenstedt, 1996). The signal contains one or more clusters of basic amino acids (for review see Dingwall and Laskey, 1991) and is recognized by the importin-α/β complex (for references see Sweet and Gerace, 1995; Panté and Aebi, 1996). The α subunit provides the NLS binding site (Imamoto et al., 1995; Weis et al., 1995) and is therefore also called the NLS receptor (Adam and Gerace, 1991). The β subunit accounts for the interaction with the NPC (Görlich et al., 1995; Moroianu et al., 1995) and carries importin-α with the NLS substrate into the nucleus. The translocation into the nucleus is terminated by the disassembly of the importin complex, and both subunits are returned probably separately to the cytoplasm. Importin-α interacts with -β via its importin-β binding domain (IBB domain; Görlich et al., 1996a
; Weis et al., 1996a
). Binding to importin-β with an IBB domain is sufficient for nuclear entry, and the IBB domain can therefore be regarded as the nuclear targeting signal of importin-α. The export domain of importin-α has not yet been identified, but it is distinct from the IBB domain.The small GTPase Ran (Drivas et al., 1990; Bischoff and Ponstingl, 1991b
; Belhumeur et al., 1993) plays a key role in NLS-dependent protein import (Melchior et al., 1993; Moore and Blobel, 1993). GTP hydrolysis by Ran is absolutely essential for import (Melchior et al., 1993; Moore and Blobel, 1993; Schlenstedt et al., 1995a
; Palacios et al., 1996) and is possibly even its sole source of energy (Weis et al., 1996b
). Although the molecular mechanism of import is far from being understood, it appears that Ran fulfils at least two distinct functions in this process: first, Ran''s GTP cycle probably drives translocation into the nucleus (Melchior et al., 1993; Moore and Blobel, 1993; Weis et al., 1996b
), which appears to involve the binding of (cytoplasmic) RanGDP to the NPC, followed by nucleotide exchange and GTP hydrolysis, but it does not involve binding of RanGTP to importin-β (Görlich et al., 1996b
). Unfortunately, nothing is known of how Ran''s GTP cycle would translate into a directed movement through the NPC. Secondly, Ran regulates the interaction between importin-α and -β (Rexach and Blobel, 1995; Chi et al., 1996; Görlich et al., 1996b
). Binding of RanGTP to importin-β disassembles the importin-α/β complex at the nuclear side of the NPC, thereby terminating translocation (Görlich et al., 1996b
). The asymmetric distribution of Ran''s principal GDP/GTP exchange factor (RCC1; Bischoff and Ponstingl, 1991a
) and GTPase activating protein (RanGAP1, or RNA1 in yeast; Bischoff et al., 1995a
; Becker et al., 1995) crucially determines where the importin heterodimer can form and where it is forced to dissociate. RCC1 is a nuclear, chromatin-bound protein (Ohtsubo et al., 1987, 1989) that generates RanGTP in the nucleus, whereas free RanGTP is depleted from the cytoplasm by RanGAP1, which is excluded from the nucleoplasm (Hopper et al., 1990; Matunis et al., 1996; Mahajan et. al, 1997). Thus, low RanGTP levels in the cytoplasm allow importin-α to bind -β, and the high RanGTP concentration in the nuclear compartment dissociates the importin complex. The concentration of free RanGTP can, in this model, be regarded as a marker for cytoplasmic identity (low RanGTP) and nuclear identity (high RanGTP), which is “sensed” by the Ran-binding site in importin-β.It is likely that at least some properties of importin-β are shared by the mediators of the other nucleocytoplasmic transport pathways. This is emphasized by the recent identification of the importin-β–related transportin (Pollard et al., 1996) as an import receptor recognizing the M9 domain, the nuclear targeting signal in hnRNP A1 (Michael et al., 1995), and of yeast transportin (Kap 104p) as an import receptor for mRNA binding proteins (Aitchison et al., 1996). Furthermore, importin-β or its NPC-binding domain cross-compete with other pathways, such as M9-dependent import, NES-mediated nuclear export, and the export of mRNA and U snRNA (Kutay et al., 1997). This would suggest that these other transport receptors share at least some binding sites at the NPC and take a similar path through the nuclear pore complex as importin-β.In addition to importin-β, a number of other Ran-binding proteins are detectable in eukaryotic cells, e.g., in overlay blots using Ran γ-[32P]GTP as a probe. These can be grouped into two classes (Lounsbury et al., 1994, 1996): first, those with a RanBP1 homology domain including the Ran binding protein 1 (RanBP1) itself (Coutavas et al., 1993; Bischoff et al., 1995b
) and the nuclear pore protein RanBP2, which has four RanBP1 homology domains (Wu et al., 1995; Yokoyama et al., 1995). Their binding to Ran can be competed by RanBP1. Second, importin-β and so far unidentified protein(s) of ∼120 kD whose Ran-binding is competed by importin-β but not by excess of RanBP1 (Lounsbury et al., 1994, 1996). Both RanBP1 and importin-β inhibit the nucleotide exchange on RanGTP (Coutavas et al., 1993; Lounsbury et al., 1994, 1996; Bischoff et al., 1995b
; Görlich et al., 1996b
). However, they do not cross-compete with each other for Ran binding but instead bind to different, nonoverlapping sites on Ran (Chi et al., 1996; Kutay et al., 1997; Lounsbury and Macara, 1997). Another striking difference is that RanBP1 facilitates the activation of Ran''s GTPase by RanGAP1 (Beddow et al., 1995; Bischoff et al., 1995b
), whereas the importin-β/RanGTP complex is entirely GAP resistant (Floer and Blobel, 1996; Görlich et al., 1996b
).Although a direct involvement of Ran has so far only been demonstrated in the importin-dependent transport pathway, perturbations in the Ran system have severe effects on a great variety of cellular functions, such as RNA processing, RNA export, regulation of chromosome structure, cell cycle progression, initiation of replication, microtubule structure, etc. (for review see Dasso, 1993; Sazer, 1996). One could argue that these effects are all secondary consequences from an impaired NLS-dependent protein import. However, it is also possible that these defects are more direct and that eukaryotic cells contain many immediate targets of Ran function.Here we describe a novel superfamily of Ran-binding proteins, which includes about a dozen factors in yeast and probably even more in higher eukaryotes. The members of this superfamily share with importin-β an NH2-terminal sequence motif that appears to account for RanGTP binding. Indeed we could confirm the interaction with Ran for the following factors: RanBP7 and RanBP8, two novel, related proteins described here, Cse1p, a cell cycle regulator in yeast, CAS, which is required for apoptosis in cultured human cells, and for Msn5p and Pse1p from yeast. Of these we have characterized RanBP7 and RanBP8 in more detail. Both resemble closely importin-β in their interaction with Ran, and both bind directly to nuclear pore complexes. RanBP7 can cross the nuclear membrane rapidly and in both directions. We provide evidence for a transport cycle in which RanBP7 first enters the nucleus, binds RanGTP inside the nucleus as a prerequisite for rapid re-export to the cytoplasm, after which the RanBP7/RanGTP complex becomes finally disassembled by the concerted action of RanBP1 and RanGAP1 in the cytoplasm. We propose that during these transport cycles, RanBP7 would normally carry an as yet unidentified cargo. This means, RanBP7 and possibly also the other members of the RanBP7/Cse1p/ importin-β superfamily could function as transport receptors that shuttle between nucleus and cytoplasm. RanBP7 and importin-β form an abundant, heterodimeric complex in the cytoplasm that appears to have a function different from nuclear import of proteins with a classical NLS. It might be a way to regulate either RanBP7 or importin-β function. Alternatively, the RanBP7/importin-β complex might constitute an import receptor with a substrate specificity different from that of the importin-α/β complex. 相似文献
8.
Andrew P. Kowalczyk Elayne A. Bornslaeger Jeffrey E. Borgwardt Helena L. Palka Avninder S. Dhaliwal Connie M. Corcoran Mitchell F. Denning Kathleen J. Green 《The Journal of cell biology》1997,139(3):773-784
The desmosome is a highly organized plasma membrane domain that couples intermediate filaments to the plasma membrane at regions of cell–cell adhesion. Desmosomes contain two classes of cadherins, desmogleins, and desmocollins, that bind to the cytoplasmic protein plakoglobin. Desmoplakin is a desmosomal component that plays a critical role in linking intermediate filament networks to the desmosomal plaque, and the amino-terminal domain of desmoplakin targets desmoplakin to the desmosome. However, the desmosomal protein(s) that bind the amino-terminal domain of desmoplakin have not been identified. To determine if the desmosomal cadherins and plakoglobin interact with the amino-terminal domain of desmoplakin, these proteins were co-expressed in L-cell fibroblasts, cells that do not normally express desmosomal components. When expressed in L-cells, the desmosomal cadherins and plakoglobin exhibited a diffuse distribution. However, in the presence of an amino-terminal desmoplakin polypeptide (DP-NTP), the desmosomal cadherins and plakoglobin were observed in punctate clusters that also contained DP-NTP. In addition, plakoglobin and DP-NTP were recruited to cell–cell interfaces in L-cells co-expressing a chimeric cadherin with the E-cadherin extracellular domain and the desmoglein-1 cytoplasmic domain, and these cells formed structures that were ultrastructurally similar to the outer plaque of the desmosome. In transient expression experiments in COS cells, the recruitment of DP-NTP to cell borders by the chimera required co-expression of plakoglobin. Plakoglobin and DP-NTP co-immunoprecipitated when extracted from L-cells, and yeast two hybrid analysis indicated that DP-NTP binds directly to plakoglobin but not Dsg1. These results identify a role for desmoplakin in organizing the desmosomal cadherin–plakoglobin complex and provide new insights into the hierarchy of protein interactions that occur in the desmosomal plaque.Desmosomes are highly organized adhesive intercellular junctions that couple intermediate filaments to the cell surface at sites of cell–cell adhesion (Farquhar and Palade, 1963; Staehelin, 1974; Schwarz et al., 1990; Garrod, 1993; Collins and Garrod, 1994; Cowin and Burke, 1996; Kowalczyk and Green, 1996). Desmosomes are prominent in tissues that experience mechanical stress, such as heart and epidermis, and the disruption of desmosomes or the intermediate filament system in these organs has devastating effects on tissue integrity (Steinert and Bale, 1993; Coulombe and Fuchs, 1994; Fuchs, 1994; McLean and Lane, 1995; Stanley, 1995; Bierkamp et al., 1996; Ruiz et al., 1996). Desmosomes are highly insoluble structures that can withstand harsh denaturing conditions (Skerrow and Matoltsy, 1974; Gorbsky and Steinberg, 1981; Jones et al., 1988; Schwarz et al., 1990). This property of desmosomes facilitated early identification of desmosomal components but has impaired subsequent biochemical analysis of the protein complexes that form between desmosomal components. Ultrastructurally, desmosomes contain a core region that includes the plasma membranes of adjacent cells and a cytoplasmic plaque that anchors intermediate filaments to the plasma membrane. The plaque can be further divided into an outer dense plaque subjacent to the plasma membrane and an inner dense plaque through which intermediate filaments appear to loop.Molecular genetic analysis has revealed that the desmosomal glycoproteins, the desmogleins and desmocollins, are members of the cadherin family of cell–cell adhesion molecules (for review see Buxton et al., 1993, 1994; Cowin and Mechanic, 1994; Kowalczyk et al., 1996). The classical cadherins, such as E-cadherin, mediate calcium-dependent, homophilic cell–cell adhesion (Nagafuchi et al., 1987). The mechanism by which the desmosomal cadherins mediate cell–cell adhesion remains elusive (Amagai et al., 1994; Chidgey et al., 1996; Kowalczyk et al., 1996), although heterophilic interactions have recently been detected between desmogleins and desmocollins (Chitaev and Troyanovsky, 1997). Both classes of the desmosomal cadherins associate with the cytoplasmic plaque protein plakoglobin (Kowalczyk et al., 1994; Mathur et al., 1994; Roh and Stanley, 1995b
; Troyanovsky et al., 1994), which is part of a growing family of proteins that share a repeated motif first identified in the Drosophila protein Armadillo (Peifer and Wieschaus, 1990). This multigene family also includes the desmosomal proteins band 6/plakophilin 1, plakophilin 2a and 2b, and p0071, which are now considered to comprise a subclass of the armadillo family of proteins (Hatzfeld et al., 1994; Heid et al., 1994; Schmidt et al., 1994; Hatzfeld and Nachtsheim, 1996; Mertens et al., 1996).The most abundant desmosomal plaque protein is desmoplakin, which is predicted to be a homodimer containing two globular end domains joined by a central α-helical coiled-coil rod domain (O''Keefe et al., 1989; Green et al., 1990; Virata et al., 1992). Previous studies have demonstrated that the carboxyl-terminal domain of desmoplakin interacts with intermediate filaments (Stappenbeck and Green, 1992; Stappenbeck et al., 1993; Kouklis et al., 1994; Meng et al., 1997), and the amino-terminal domain of desmoplakin is required for desmoplakin localization to the desmosomal plaque (Stappenbeck et al., 1993). Direct evidence supporting a role for desmoplakin in intermediate filament attachment to desmosomes was provided recently when expression of an amino-terminal polypeptide of desmoplakin was found to displace endogenous desmoplakin from cell borders and disrupt intermediate filament attachment to the cell surface in A431 epithelial cell lines (Bornslaeger et al., 1996).The classical cadherins, such as E-cadherin, bind directly to both β-catenin and plakoglobin (Aberle et al., 1994; Jou et al., 1995; for review see Cowin and Burke, 1996). β-Catenin is also an armadillo family member (McCrea et al., 1991; Peifer et al., 1992), and both plakoglobin and β-catenin bind directly to α-catenin (Aberle et al., 1994, 1996; Jou et al., 1995; Sacco et al., 1995; Obama and Ozawa, 1997). α-Catenin is a vinculin homologue (Nagafuchi et al., 1991) and associates with both α-actinin and actin (Knudson et al., 1995; Rimm et al., 1995; Nieset et al., 1997). Through interactions with β- and α-catenin, E-cadherin is coupled indirectly to the actin cytoskeleton, and this linkage is required for the adhesive activity of E-cadherin (Ozawa et al., 1990; Shimoyama et al., 1992). In addition, E-cadherin association with plakoglobin appears to be required for assembly of desmosomes (Lewis et al., 1997), underscoring the importance of E-cadherin in the overall program of intercellular junction assembly. However, the hierarchy of molecular interactions that couple the desmosomal cadherins to the intermediate filament cytoskeleton is largely unknown, although the desmocollin cytoplasmic domain appears to play an important role in recruiting components of the desmosomal plaque (Troyanovsky et al., 1993, 1994). Since desmosomal cadherins form complexes with plakoglobin and because the amino-terminal domain of desmoplakin is required for desmoplakin localization at desmosomes, we hypothesized that the amino-terminal domain of desmoplakin interacts with the desmosomal cadherin– plakoglobin complex.In previous studies, we used L-cell fibroblasts to characterize plakoglobin interactions with the cytoplasmic domains of the desmosomal cadherins and found that the desmosomal cadherins regulate plakoglobin metabolic stability (Kowalczyk et al., 1994) but do not mediate homophilic adhesion (Kowalczyk et al., 1996). To test the ability of the desmoplakin amino-terminal domain to interact with the desmosomal cadherin–plakoglobin complex, we established a series of L-cell lines expressing the desmosomal cadherins in the presence or absence of a desmoplakin amino-terminal polypeptide (DP-NTP).1 The results indicate that one important function of the desmoplakin amino-terminal domain is to cluster desmosomal cadherin–plakoglobin complexes. In addition, DP-NTP and plakoglobin were found to form complexes that could be co-immunoprecipitated from L-cell lysates. Using the yeast two hybrid system, DP-NTP was found to bind directly to plakoglobin but not Dsg1. These data suggest that plakoglobin couples the amino-terminal domain of desmoplakin to the desmosomal cadherins and that desmoplakin plays an important role in organizing the desmosomal cadherin–plakoglobin complex into discrete plasma membrane domains. 相似文献
9.
A Requirement for Cyclin D3–Cyclin-dependent Kinase (cdk)-4 Assembly in the Cyclic Adenosine Monophosphate–dependent Proliferation of Thyrocytes
下载免费PDF全文
![点击此处可从《The Journal of cell biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Fabienne Depoortere Alexandra Van Keymeulen Jiri Lukas Sabine Costagliola Jirina Bartkova Jacques E. Dumont Jiri Bartek Pierre P. Roger Sarah Dremier 《The Journal of cell biology》1998,140(6):1427-1439
10.
11.
Timothy D. Garver Qun Ren Shmuel Tuvia Vann Bennett 《The Journal of cell biology》1997,137(3):703-714
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. 相似文献
12.
13.
KSA Antigen Ep-CAM Mediates Cell–Cell Adhesion of Pancreatic Epithelial Cells: Morphoregulatory Roles in Pancreatic Islet Development
下载免费PDF全文
![点击此处可从《The Journal of cell biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
V. Cirulli L. Crisa G.M. Beattie M.I. Mally A.D. Lopez A. Fannon A. Ptasznik L. Inverardi C. Ricordi T. Deerinck M. Ellisman R.A. Reisfeld A. Hayek 《The Journal of cell biology》1998,140(6):1519-1534
14.
A Natural Hepatocyte Growth Factor/Scatter Factor Autocrine Loop in Myoblast Cells and the Effect of the Constitutive Met Kinase Activation on Myogenic Differentiation 总被引:7,自引:0,他引:7
下载免费PDF全文
![点击此处可从《The Journal of cell biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Sergio Anastasi Silvia Giordano Olga Sthandier Giovanna Gambarotta Rossella Maione Paolo Comoglio Paolo Amati 《The Journal of cell biology》1997,137(5):1057-1068
15.
16.
17.
18.
19.
Mathilde Waser Nasrin Mesaeli Charlotte Spencer Marek Michalak 《The Journal of cell biology》1997,138(3):547-557