- Top of page
- MATERIALS AND METHODS
The golli products of the myelin basic protein (MBP) gene are expressed in neurons and oligodendrocytes (OLs). In certain neuronal populations, golli proteins undergo translocation between the nucleus and cytoplasm/processes during development. The proteins consist of two domains, a golli domain of 133 amino acids and an MBP domain of variable length. One objective of this study was to identify the sequences responsible for nuclear targeting. Site-directed mutagenesis and deletion analyses were used to generate a series of golli-green fluorescent protein (GFP) DNA constructs that were transfected into OL and neuronal cell lines to follow localization by confocal microscopy. The results indicated that a 36-residue stretch in the MBP domain is essential for nuclear targeting, and the sequence appears to be a nontraditional localization signal motif. The studies also revealed that overexpression of golli proteins could induce dramatic changes in cell morphology. In OL lines, overexpression of intact golli proteins, or golli peptide alone, caused an increase in the length and number of processes, and the elaboration of membrane sheets. In the neuronal lines, there was a dramatic increase in number and length of extensions. The results, consistent with the timing of golli expression in cells during neural development, suggest that golli proteins may be involved in process formation/extension in OLs and neurons during development. These studies have defined two functional domains in the golli protein. Sequences in the MBP domain target the protein into the nucleus and sequences within the golli domain induce process sheet extension in OLs and neurons. © 2002 Wiley-Liss, Inc.
The myelin basic protein (MBP) gene encodes two families of proteins: the “classic” MBPs and the golli proteins (Campagnoni et al., 1993; Pribyl et al., 1993). In mouse, three golli products have been identified: BG21, J37, and TP8 (Campagnoni et al., 1993; also see Fig. 1A). Unlike the classic MBPs, which are expressed exclusively in myelin-forming cells in the nervous system, the golli proteins are expressed in myelin-forming cells as well as in neurons in the central nervous system (CNS) and peripheral nervous system (PNS; Landry et al., 1996, 1997, 1998; Pribyl et al., 1996) and in macrophages and T-cells in the immune system (Feng et al., 2000). Northern blot analyses, in situ hybridization, and immunohistochemical studies indicate that golli mRNAs and proteins are expressed in the embryonic mouse brain much earlier than the MBP mRNAs and long before myelination. Within the oligodendrocyte (OL), both in vivo and in vitro, golli proteins are detected in the nucleus, cell bodies, and proximal processes, but are not localized within the myelin membrane (Landry et al., 1996; Pribyl et al., 1996; Givogri et al., 2000; Campagnoni and Skoff, 2001). Similarly, golli proteins first appear in many neurons when they are extending processes for migration, establishment of connections and, in the case of OLs, just prior to myelination (Landry et al., 1996, 1997, 1998; Pribyl et al., 1996).
Figure 1. A: Schematic representation of the organization of the mouse mbp gene and the golli and classic MBP (shaded) products derived from the gene. The gene contains two major transcription start sites (tss) that give rise to either the golli products (tss1) or the “classic” MBPs (tss3). Tss2 is a minor start site that gives rise to the M41 MBP transcript, which encodes the 14-kDa MBP. Note that all of the “classic” MBP proteins contain residues 1–36, which are derived from part of exon 5B. B: Diagrammatic scheme of the golli-mbp:green fluorescent protein (GFP) constructs designed to study subcellular targeting of the protein in the glial cell lines. The golli protein was divided into the MBP and golli domain in order to determine whether either regions were responsible for nuclear targeting. Expression of the insert is under the cytomegalovirus (CMV) promoter. C: Mutagenic primers were designed to delete the dibasic lysine-arginine (KR) residues in the MBP domain. Two mutant constructs were generated one in which K5R6 was deleted (MBPΔ1) and the second in which K52R53 was removed (MBPΔ2). D: Two additional MBP:GFP constructs were generated to localize the nuclear targeting sequence within the MBP domain.
Download figure to PowerPoint
Subcellular targeting of the golli protein is rather unusual. In OLs and neurons, golli can be detected in the cell body, nucleus, and processes (Landry et al., 1996; Pribyl et al., 1996). However, within certain subpopulations of neurons, golli is translocated from one cellular compartment to another, which in some cases appears to be developmentally regulated. For example, in the cerebellum, immature granule cells within the external granule cell layer (EGL) target golli into their processes. However, after these cells migrate from the EGL to the internal granule cell layer (IGL), golli is localized solely within the nuclei of the mature granule cells (Landry et al., 1996).
The function of the golli proteins is only beginning to become clarified, and several lines of evidence suggest that it is involved in signaling pathways, and a putative nuclear partner for golli has been identified (Fernandes et al., 2000; Campagnoni and Skoff, 2001). Nuclear cytoplasm shuttling of proteins is a characteristic of many signaling molecules (Bhat, 1995; Hardy and Chaudhri, 1997; Kaffman and O'Shea, 1999; Jordan et al., 2000; Wegner, 2000; Teruel and Meyer, 2000) and is consistent with a signaling role for golli proteins.
Here we describe the identification and characterization of two functional domains of the golli proteins, which may be related to their biological function. We have found that the golli domain can induce dramatic morphological changes in OLs and neurons. These changes include elaboration of membrane sheets in OLs and an increase in number and length of processes in neurons. The MBP domain appears to be essential for targeting the protein into the nucleus. These results are consistent with the notion that the golli proteins may have multiple functions within the cell.
- Top of page
- MATERIALS AND METHODS
In this report, we have shown that golli proteins contain two functional domains: an MBP domain, responsible for targeting the proteins into the nucleus; and a golli domain, which can induce dramatic morphological changes in both OLs and neurons.
Our data indicate that the nuclear localization signal for the golli proteins is found within the first 36 amino acids of the MBP domain, corresponding to exon 5b (or exon 1 of the MBP gene using the old numbering system). We did not identify a conventional NLS within the MBP domain of the golli proteins, so the mechanism for nuclear targeting of the golli proteins is probably regulated by sequences that do not involve traditional NLS motifs (Dytrych et al., 1998; Sherman and Brophy, 2000).
The MBP residues 1 through 36 contain numerous potential PKC phosphorylation sites. This is interesting because MBP proteins have been reported to be phosphorylated, at least partially, in vivo (Ulmer and Braun, 1986a,b) and in vitro (Miyamoto et al., 1974; Miyamoto and Kakiuchi, 1974). Pedraza et al. (1997) has shown that the addition of 12-O-tetradecanoyl phorbol 13-acetate (TPA), a potent activator of most PKC isoforms, can abolish nuclear targeting of the 21.5-kDa MBP isoform transfected into HeLa cells. Therefore it is possible that the phosphorylated state of the MBP domain in the golli products contributes to determining the subcellular location of the protein.
The minor isoforms of the classic MBPs, 21.5 kDa and 17 kDa MBP, have been observed in nuclei of OLs in the developing mouse brain (Allinquant et al., 1991; Hardy et al., 1996; Staugaitis et al., 1996). It has been shown that MBP sequences encoded by exon 6 (old exon 2) are necessary for this nuclear translocation in the classic MBP isoforms. However, exon 6 is not present in the golli products, so the golli proteins utilize a different NLS than the minor MBP isoforms. Since the golli NLS is present in all the classic MBP isoforms, then why are they not targeted to the nucleus? The context in which the NLS is found and other factors influence whether or not it functions as a NLS (Tagawa et al., 1995; Yoneda, 2000), and the structures of the golli and classic MBPs are very different. Perhaps, more importantly, targeting of the classic MBPs to the myelin membrane is achieved by polyribosome translocation, which effectively bypasses the effects of nuclear targeting signals in these molecules (for recent review see Landry and Campagnoni, 1998).
The golli domain alone was responsible for the ability of the golli proteins to induce the elaboration of processes and membranous sheets when overexpressed in OL and neuronal cell lines. Unlike the J37 and BG21 golli isoforms, which contain the entire 133 aa golli domain, TP8 contains only the first 47 aa of this sequence and is lacking any MBP domain. Thus, these data suggest that the first 47 residues of the golli domain are sufficient to induce the observed changes in cell morphology.
The findings that overexpression of the golli proteins induced elaboration of membrane sheets in the OL cell lines is significant. While the mechanism for membrane sheet elaboration in OLs is not fully understood, in vitro studies indicate that sheet formation involves the activation of PKA and PKC (Vartanian et al., 1986; Althaus et al., 1990, 1991; He and McCarthy, 1994; Yong et al., 1994; Bhat, 1995; Yong and Oh, 1997). Furthermore, the extracellular signal-regulated protein kinases (ERKs), members of the mitogen-activated protein kinase (MAPK) family, and Fyn, a member of the Src tyrosine kinase family, have been shown to have an important role in the initiation of process formation in OLs (Umemori et al., 1994; Bhat, 1995; Stariha et al., 1997; Osterhout et al., 1999; Seiwa et al., 2000). Therefore, membrane sheet formation in the N19 and N20.1 lines transfected with the golli constructs suggests that the golli proteins might be involved in some way with a signal transduction pathway involved in this process.
Similarly, overexpression of golli proteins in PC12m cells led to the induction of processes, similar to the induction seen with NGF. Like the OL lines, golli is also expressed abundantly in PC12m cells. In contrast to sheet extension in OLs, neurite extension in PC12 cells has been well characterized in vitro (Tischler and Greene, 1975, 1978, 1980). Numerous studies have shown that cells treated with NGF differentiate and extend neurites (Greene and Tischler, 1976; Szeberenyi and Erhardt, 1994; Friedman and Greene, 1999), mediated by the binding of NGF to the TrkA receptor, tyrosine kinase receptor (Barbacid, 1994; Saltiel and Decker, 1994; Greene and Kaplan, 1995; Kaplan, 1998; Friedman and Greene, 1999). Activation of the receptor leads to the initiation of several signal transduction pathways, including the Ras-MAPK pathway which is necessary for neurite extension (Pang et al., 1995; Robinson and Cobb, 1997; Korhonen et al., 1999). Interestingly, Katoh et al. (2000) have shown that overexpression of RhoG in PC12 can also induce neurite extension in the absence of NGF. RhoG is a member of the Rho GTPase family that can serve as activators of several signal transduction pathways (Hall, 1998; Bar-Sagi and Hall, 2000). These data suggest that overexpression of a modulator within the signaling pathway can bypass NGF activation to initiate neurite extension. Overexpression of golli might be acting in a similar fashion in PC12m cells to induce process extension.
Our results suggest that golli proteins may be involved in process elaboration/extension in both OLs and in neurons. In OLs, golli is expressed very early, prior to myelin formation, when the cells are beginning to extend processes (Pribyl et al., 1996). These in vitro results suggest that myelination may be a multiple stage process: extension of cell processes, the beginning of sheet elaboration, followed rapidly by delivery of myelin components to the membrane. Since golli is not a component of myelin, it may be involved in setting up process/sheet formation prior to the delivery of the myelin components to the growing membrane.
In this study, we report our findings that the golli protein contains two functional domains. Sequences located in the MBP domain targets the protein into the nucleus while the golli domain induces cell-specific morphological changes. Although the function of the golli proteins is not yet known, the present studies suggest that golli may be involved in signaling mechanisms within the cell and that this activity resides within the golli domain of the molecule.