FEZ2 has acquired additional protein interaction partners relative to FEZ1: functional and evolutionary implications

Background

The FEZ (fasciculation and elongation protein zeta) family designation was purposed by Bloom and Horvitz by genetic analysis of C. elegans unc-76. Similar human sequences were identified in the expressed sequence tag database as FEZ1 and FEZ2. The unc-76 function is necessary for normal axon fasciculation and is required for axon-axon interactions. Indeed, the loss of UNC-76 function results in defects in axonal transport. The human FEZ1 protein has been shown to rescue defects caused by unc-76 mutations in nematodes, indicating that both UNC-76 and FEZ1 are evolutionarily conserved in their function. Until today, little is known about FEZ2 protein function.

Methodology/Principal Findings

Using the yeast two-hybrid system we demonstrate here conserved evolutionary features among orthologs and non-conserved features between paralogs of the FEZ family of proteins, by comparing the interactome profiles of the C-terminals of human FEZ1, FEZ2 and UNC-76 from C. elegans. Furthermore, we correlate our data with an analysis of the molecular evolution of the FEZ protein family in the animal kingdom.

Conclusions/Significance

We found that FEZ2 interacted with 59 proteins and that of these only 40 interacted with FEZ1. Of the 40 FEZ1 interacting proteins, 36 (90%), also interacted with UNC-76 and none of the 19 FEZ2 specific proteins interacted with FEZ1 or UNC-76. This together with the duplication of unc-76 gene in the ancestral line of chordates suggests that FEZ2 is in the process of acquiring new additional functions. The results provide also an explanation for the dramatic difference between C. elegans and D. melanogaster unc-76 mutants on one hand, which cause serious defects in the nervous system, and the mouse FEZ1 -/- knockout mice on the other, which show no morphological and no strong behavioural phenotype. Likely, the ubiquitously expressed FEZ2 can completely compensate the lack of neuronal FEZ1, since it can interact with all FEZ1 interacting proteins and additional 19 proteins.     

Identification of a tissue-non-specific homologue of axonal fasciculation and elongation protein zeta-1

Fasciculation and elongation protein zeta-1 (FEZ1) is a mammalian orthologue of the Caenorhabditis elegans UNC-76 protein involved in the axonal outgrowth and fasciculation and promotes neurite extension of PC12 cells through interaction with protein kinase C zeta (PKCzeta). The gene coding for FEZ2, a homologue of FEZ1, has also been reported in rat and human. In this study, we compared mRNA expression of FEZ1 and FEZ2 in adult rat tissues and mouse embryos by Northern blot and in situ hybridization analyses. In contrast to FEZ1 whose mRNA is expressed almost exclusively in rat brain and temporarily around the neurogenesis stage of mouse embryos, the message for FEZ2 is detected weakly in most tissues and abundantly throughout the mouse embryonic stages. Similar to FEZ1, FEZ2 interacted with PKCzeta and induced neurite extension of PC12 cells when coexpressed with a constitutively active mutant of PKCzeta. These results suggest that FEZ2 plays an important role in the morphological changes of various cells by associating with PKCzeta in a tissue-non-specific manner.     

Functional regulation of FEZ1 by the U-box-type ubiquitin ligase E4B contributes to neuritogenesis

E4B (also known as UFD2a) is a mammalian homolog of Saccharomyces cerevisiae Ufd2, which was originally described as a ubiquitin chain assembly factor (E4). E4B is a U-box-type ubiquitin-protein isopeptide ligase (E3) and likely functions as either an E3 or an E4. With a yeast two-hybrid screen, we have now identified FEZ1 (fasciculation and elongation protein zeta 1) as a protein that interacts with E4B. FEZ1 is implicated in neuritogenesis when phosphorylated by protein kinase Czeta (PKCzeta). Interaction between E4B and FEZ1 in mammalian cells was enhanced by coexpression of constitutively active PKCzeta. E4B mediated the polyubiquitylation of FEZ1 but did not affect its intracellular stability, suggesting that such modification of FEZ1 is not a signal for its proteolysis. Polyubiquitylation of FEZ1 by E4B required Lys(27) of ubiquitin. Expression of a dominant-negative mutant of E4B in rat pheochromocytoma PC12 cells resulted in inhibition of neurite extension induced either by nerve growth factor or by coexpression of FEZ1 and constitutively active PKCzeta. These findings indicate that E4B serves as a ubiquitin ligase for FEZ1 and thereby regulates its function but not its degradation.     

Phosphorylation mutants of JC virus agnoprotein are unable to sustain the viral infection cycle

Many eukaryotic and viral regulatory proteins are known to undergo posttranslational modifications including phosphorylation, which plays a critical role in many aspects of cell function. Previous studies from our and other laboratories indicated that the JC virus (JCV) late regulatory protein, agnoprotein, plays an important role in the JCV life cycle. Agnoprotein contains several potential phosphorylation sites, including Ser7, Ser11, and Thr21, which are potential targets for the serine/threonine-specific protein kinase C (PKC). In this study, we investigated the functional significance of these phosphorylation sites for the activity of agnoprotein. In vitro and in vivo kinase assays demonstrated that agnoprotein is a target for phosphorylation by PKC. In addition, each of the PKC phosphorylation sites was mutated to Ala singly and in combination, and the effects of these mutations on the JCV life cycle were analyzed. Although the expression of each mutant agnoprotein was detectable during the infection cycle, virus containing each of these mutations failed to propagate. These results contrast with those obtained with an agnoprotein start codon point (Pt) mutant where agnoprotein expression was completely inhibited. The Pt mutant was viable but replicates less efficiently than the wild type (WT). Moreover, conservative substitutions at PKC phosphorylation sites (Ser7, Ser11, and Thr21 to Asp) resulted in a viable virus, which further demonstrate the importance of these sites on agnoprotein function. Further analysis of the mutants by viral release assay and electron microscopy studies revealed that viral particles were efficiently released from infected cells and morphologically indistinguishable from those of WT but were deficient in DNA content. This may account for the defective propagation of the mutants. These results imply that phosphorylated forms of agnoprotein may have essential functions in the viral life cycle and serve as potential targets for therapeutic interventions to limit JCV propagation and JCV-induced diseases.     

FEZ1/LZTS1 protein expression in ovarian cancer

The FEZ1/LZTS1 (FEZ1) gene maps to chromosome 8p22 and is frequently altered in human cancer. FEZ1 has been proposed as a candidate tumour suppressor gene and its loss may contribute to tumour progression. We have analysed the expression of FEZ1 protein in tissues from ovarian carcinomas in relation to clinico‐pathological variables, response to chemotherapy and disease‐free and overall survival. FEZ1 status was assessed by immunohistochemistry. Cytoplasmic staining for FEZ1 protein was absent or drastically reduced in 38% of tumours. FEZ1 protein expression was not related to tumour grade, histotype, disease‐free survival, or overall survival. On the contrary, it was significantly correlated with age and with FIGO stage of disease. This finding indicates that FEZ1 is involved in ovarian carcinogenesis. Moreover, loss of FEZ1 protein significantly predicted a complete treatment response in patients who received taxane‐based chemotherapy. In conclusion, the reduction or loss of FEZ1 protein could be an aid to the clinical management of patients affected by ovarian carcinoma. J. Cell. Physiol. 222: 382–386, 2010. © 2009 Wiley‐Liss, Inc.     

Fasciculation and elongation protein zeta-1 (FEZ1) participates in the polarization of hippocampal neuron by controlling the mitochondrial motility

The fasciculation and elongation protein zeta-1 (FEZ1), a mammalian orthologue of Caenorhabditis elegans UNC-76 protein, is a 45-kDa protein with four coiled-coiled domains and efficiently promotes the neurite elongation in the rat phaeochromocytoma PC12 cells. UNC-76 proteins of C. elegans and Drosophila have been genetically demonstrated to be involved in the axonal guidance. We here show that FEZ1 RNA interference (RNAi) represses the formation of axon in rat embryo hippocampal neurons. An anterograde mitochondrial movement is also retarded in neurites of the RNAi-treated hippocampal neurons. Moreover, the size of mitochondria is considerably elongated by the RNAi treatment. The transport of mitochondria from soma to axon or dendrites is essential for the neuronal differentiation. Therefore, our results strongly suggest that FEZ1 participates in the establishment of neuronal polarity by controlling the mitochondrial motility along axon.     

Primary central nervous system lymphoma expressing the human neurotropic polyomavirus, JC virus, genome

B lymphocytes are known as a potential site for latency and reactivation of the human neurotropic polyomavirus, JC virus (JCV). In light of recent studies on the oncogenicity of JCV and the transforming ability of the JCV early protein, T antigen, we investigated the association of JCV with B-cell lymphomas of the central nervous system. Examination of 27 well-characterized clinical specimens by gene amplification and immunohistochemistry revealed the presence of DNA sequences corresponding to the JCV early genome and the late Agnoprotein in 22 samples and the JCV late genome encoding the viral capsid proteins in 8 samples. Expression of T antigen and that of Agnoprotein by immunohistochemistry were each detected in six specimens. No evidence of the production of viral capsid proteins was observed, ruling out productive infection of JCV in the tumor cells. The results from laser capture microdissection verified the presence of JCV T-antigen sequences in tumor cells with positive immunoreactivity to antibodies against the viral proteins T antigen and Agnoprotein. Due to previous reports demonstrating an association of the Epstein-Barr virus (EBV) with transformation of B lymphocytes, EBV DNA sequences and the EBV transforming protein, latent membrane protein 1 (LMP1), were analyzed in parallel. EBV LMP1 DNA sequences were detected in 16 of 23 samples, and LMP1 expression was detected in 16 samples, 5 of which exhibited positive immunoreactivity to JCV proteins. Double labeling demonstrated coexpression of JCV T antigen and EBV LMP1 in the same cells. The detection of the JCV genome in large numbers of B-cell lymphomas and its coexistence with EBV suggest a potential role for JCV in the pathogenesis of primary CNS lymphoma.     

JC virus agnoprotein inhibits in vitro differentiation of oligodendrocytes and promotes apoptosis

Productive infection of oligodendrocytes, which are responsible for the formation of myelin sheath in the central nervous system, with the human neurotropic virus JC virus (JCV) causes the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). In addition to encoding T antigen and the capsid proteins, which are produced at the early and late phases of the infection cycle, respectively, JCV encodes a small regulatory protein named agnoprotein that is important for successful completion of the virus life cycle. Here we used bipotential CG-4 cells to examine the impact of agnoprotein on oligodendrocyte differentiation and survival in the absence of JCV lytic infection. We demonstrate that the expression of agnoprotein delayed the formation of complex outgrowth networks of the cells during oligodendrocyte differentiation. These alterations were accompanied by high levels of DNA damage, induction of proapoptotic proteins, and suppression of prosurvival signaling. Accordingly, apoptosis was significantly increased upon the induction of CG-4 cells toward differentiation in cells expressing agnoprotein. These observations provide the first evidence for the possible involvement of agnoprotein, independent from its role in viral replication, in a series of biological events that may contribute to the pathological features seen in PML lesions.     

Role of JC virus agnoprotein in virion formation

JC virus (JCV) belongs to the polyomavirus family of double-stranded DNA viruses and causes progressive multifocal leukoencephalopathy in humans. JCV encodes early proteins (large T antigen, small T antigen, and T' antigen) and four late proteins (agnoprotein, and three viral capsid proteins, VP1, VP2, and VP3). In the current study, a novel function for JCV agnoprotein in the morphogenesis of JC virion particles was identified. It was found that mature virions of agnoprotein-negative JCV are irregularly shaped. Sucrose gradient sedimentation and cesium chloride gradient ultracentrifugation analyses revealed that the particles of virus lacking agnoprotein assemble into irregularly sized virions, and that agnoprotein alters the efficiency of formation of VP1 virus-like particles. An in vitro binding assay and immunocytochemistry revealed that agnoprotein binds to glutathione S-transferase fusion proteins of VP1 and that some fractions of agnoprotein colocalize with VP1 in the nucleus. In addition, gel filtration analysis of formation of VP1-pentamers revealed that agnoprotein enhances formation of these pentamers by interacting with VP1. The present findings suggest that JCV agnoprotein plays a role, similar to that of SV40 agnoprotein, in facilitating virion assembly.     

The agnoprotein of polyomaviruses: a multifunctional auxiliary protein

The late region of the three primate polyomaviruses (JCV, BKV, and SV40) encodes a small, highly basic protein known as agnoprotein. While much attention during the last two decades has focused on the transforming proteins encoded by the early region (small and large T-antigens), it has become increasingly evident that agnoprotein has a critical role in the regulation of viral gene expression and replication, and in the modulation of certain important host cell functions including cell cycle progression and DNA repair. The importance of agnoprotein is underscored by its expression during lytic infection of glial cells by JCV that occurs in progressive multifocal leukoencephalopathy (PML), and also in some JCV-associated human neural tumors particularly medulloblastoma. In this review, we will discuss the structure and function of agnoprotein in the viral life cycle during the course of lytic infection and the consequences of agnoprotein expression for the host cell.     

Over-expression of GFP-FEZ1 causes generation of multi-lobulated nuclei mediated by microtubules in HEK293 cells

FEZ1 (Fasciculation and elongation protein zeta 1) is an ortholog of the Caenorhabditis elegans protein UNC-76, involved in neuronal development and axon outgrowth, in that worm. Mammalian FEZ1 has already been reported to cooperate with PKC-zeta in the differentiation and polarization of PC12 neuronal cells. Furthermore, FEZ1 is associated with kinesin 1 and JIP1 to form a cargo-complex responsible for microtubule based transport of mitochondria along axons. FEZ1 can also be classified as a hub protein, since it was reported to interact with over 40 different proteins in yeast two-hybrid screens, including at least nine nuclear proteins. Here, we transiently over-expressed GFP-FEZ1full in human HEK293 and HeLa cells in order to study the sub-cellular localization of GFP-FEZ1. We observed that over 40% of transiently transfected cells at 3 days post-transfection develop multi-lobulated nuclei, which are also called flower-like nuclei. We further demonstrated that GFP-FEZ1 localizes either to the cytoplasm or the nuclear fraction, and that the appearance of the flower-like nuclei depends on intact microtubule function. Finally, we show that FEZ1 co-localizes with both, α- and especially with γ-tubulin, which localizes as a centrosome like structure at the center of the multiple lobules. In summary, our data suggest that FEZ1 has an important centrosomal function and supply new mechanistic insights to the formation of flower-like nuclei, which are a phenotypical hallmark of human leukemia cells.     

Structural Analysis of Intermolecular Interactions in the Kinesin Adaptor Complex Fasciculation and Elongation Protein Zeta 1/ Short Coiled-Coil Protein (FEZ1/SCOCO)

Cytoskeleton and protein trafficking processes, including vesicle transport to synapses, are key processes in neuronal differentiation and axon outgrowth. The human protein FEZ1 (fasciculation and elongation protein zeta 1 / UNC-76, in C. elegans), SCOCO (short coiled-coil protein / UNC-69) and kinesins (e.g. kinesin heavy chain / UNC116) are involved in these processes. Exploiting the feature of FEZ1 protein as a bivalent adapter of transport mediated by kinesins and FEZ1 protein interaction with SCOCO (proteins involved in the same path of axonal growth), we investigated the structural aspects of intermolecular interactions involved in this complex formation by NMR (Nuclear Magnetic Resonance), cross-linking coupled with mass spectrometry (MS), SAXS (Small Angle X-ray Scattering) and molecular modelling. The topology of homodimerization was accessed through NMR (Nuclear Magnetic Resonance) studies of the region involved in this process, corresponding to FEZ1 (92-194). Through studies involving the protein in its monomeric configuration (reduced) and dimeric state, we propose that homodimerization occurs with FEZ1 chains oriented in an anti-parallel topology. We demonstrate that the interaction interface of FEZ1 and SCOCO defined by MS and computational modelling is in accordance with that previously demonstrated for UNC-76 and UNC-69. SAXS and literature data support a heterotetrameric complex model. These data provide details about the interaction interfaces probably involved in the transport machinery assembly and open perspectives to understand and interfere in this assembly and its involvement in neuronal differentiation and axon outgrowth.