Identification of Nd1, a novel murine kelch family protein,involved in stabilization of actin filaments

We isolated Nd1, a novel kelch family gene that encodes two forms of proteins, Nd1-L and Nd1-S. Nd1-L contains a BTB/POZ domain in its N terminus and six kelch repeats in the C terminus. Nd1-S has the BTB/POZ domain but lacks the six kelch repeats. Nd1-L but not Nd1-S mRNA is detected ubiquitously in normal mouse tissues. Nd1-L and Nd1-S proteins can form a dimer through the BTB/POZ domain. Nd1-L colocalizes with actin filaments detected using a confocal microscope, and its kelch repeats bind to them in vitro. Overexpression of Nd1-L in NIH3T3 cells delayed cell growth by affecting the transition of cytokinesis. Furthermore, the overexpression prevented NIH3T3 cells from cell death induced by actin destabilization but not by microtubule dysfunction. These data suggest that Nd1-L functions as a stabilizer of actin filaments as an actin-binding protein and may play a role in the dynamic organization of the actin cytoskeleton.     

Actinfilin,a brain-specific actin-binding protein in postsynaptic density

The dynamic assembly and disassembly of actin-based cytoskeleton is closely linked to the changes in the postsynaptic density in both number and shape, which is thought to be important in forming long-term memory. Thus, regulation of actin filaments may play a critical role in contributing to the formation of long-term memory. Here, we report the cloning of actinfilin, a brain-specific Kelch protein, which interacts with F-actin. Actinfilin contains an amino-terminal POZ/BTB domain and carboxyl positioned six tandem Kelch repeats that presumably form six blades of beta-propeller structure of the Kelch domain. Co-immunoprecipitation analyses showed that the amino-terminal POZ domain mediated actinfilin-actinfilin interaction. The recombinant Kelch domain alone was sufficient to mediate binding to F-actin. Immunohistochemistry studies of rat brain sections suggested that actinfilin is broadly expressed in neurons of most regions of the brain. The subcellular localization of actinfilin was studied by biochemical fractionation and immunogold labeling. The results showed the postsynaptic density distribution of actinfilin. Together, these results indicate that actinfilin may be a key player in the actin-based neuronal function.     

Novel kelch-like protein, KLEIP, is involved in actin assembly at cell-cell contact sites of Madin-Darby canine kidney cells

Dynamic rearrangements of cell-cell adhesion underlie a diverse range of physiological processes, but their precise molecular mechanisms are still obscure. Thus, identification of novel players that are involved in cell-cell adhesion would be important. We isolated a human kelch-related protein, Kelch-like ECT2 interacting protein (KLEIP), which contains the broad-complex, tramtrack, bric-a-brac (BTB)/poxvirus, zinc finger (POZ) motif and six-tandem kelch repeats. KLEIP interacted with F-actin and was concentrated at cell-cell contact sites of Madin-Darby canine kidney cells, where it colocalized with F-actin. Interestingly, this localization took place transiently during the induction of cell-cell contact and was not seen at mature junctions. KLEIP recruitment and actin assembly were induced around E-cadherin-coated beads placed on cell surfaces. The actin depolymerizing agent cytochalasin B inhibited this KLEIP recruitment around E-cadherin-coated beads. Moreover, constitutively active Rac1 enhanced the recruitment of KLEIP as well as F-actin to the adhesion sites. These observations strongly suggest that KLEIP is localized on actin filaments at the contact sites. We also found that N-terminal half of KLEIP, which lacks the actin-binding site and contains the sufficient sequence for the localization at the cell-cell contact sites, inhibited constitutively active Rac1-induced actin assembly at the contact sites. We propose that KLEIP is involved in Rac1-induced actin organization during cell-cell contact in Madin-Darby canine kidney cells.     

hDKIR, a human homologue of the Drosophila kelch protein, involved in a ring-like structure

We have previously purified and cloned an apoptosis-inducing protein (AIP) derived from fish infected with the anisakis simplex. Recently, we identified a series of AIP-responsive genes in the HL-60 cell line using a subtractive hybridization method. Here we report the molecular cloning and characterization of one of these genes, which encodes a novel human kelch protein containing 568 amino acid residues, termed hDKIR. The Drosophila Kelch protein localizes to a ring canal structure, which is required for oocyte development. When hDKIR was expressed in cultured-mammalian cells, hDKIR localized to a ring-like structure. Furthermore, when coexpressed with Mayven or Keap1, hDKIR bound to Mayven and recruited Mayven into ring-like structures perfectly. This indicates that kelch homologues can interact with each other in a specific manner and such interaction can affect the subcellular localization of kelch proteins. Finally, domain analysis revealed that both the N-terminal POZ (poxviruses and zinc fingers) and intervening region (IVR) domains of hDKIR are essential for ring-like structure activity, suggesting that the development of the ring-like structure is independent of the ability to bind actin.     

Reversible, activity-dependent targeting of profilin to neuronal nuclei

The actin cytoskeleton in pyramidal neurons plays a major role in activity-dependent processes underlying neuronal plasticity. The small actin-binding protein profilin shows NMDA receptor-dependent accumulation in dendritic spines, which is correlated with suppression of actin dynamics and long-term stabilization of synaptic morphology. Here we show that following NMDA receptor activation profilin also accumulates in the nucleus of hippocampal neurons via a process involving rearrangement of the actin cytoskeleton. This simultaneous targeting to dendritic spines and the cell nucleus suggests a novel mechanism of neuronal plasticity in which profilin both tags activated synapses and influences nuclear events.     

The BACK domain in BTB-kelch proteins

A novel conserved motif--the BACK (for BTB and C-terminal Kelch) domain--is found in the majority of proteins that contain both the BTB domain and kelch repeats. Many kelch-repeat proteins are involved in organization of the cytoskeleton via interaction with actin and intermediate filaments, whereas BTB domains have multiple cellular roles, including recruitment to E3 ubiquitin ligase complexes. The identification of the BACK domain in BTB and kelch proteins, and its high conservation across metazoan genomes, suggest an important function for this domain with a possible role in substrate orientation in Cullin3-based E3 ligase complexes.     

Process elongation of oligodendrocytes is promoted by the Kelch-related actin-binding protein Mayven

Rearrangement of the cytoskeleton leading to the extension of cellular processes is essential for the myelination of axons by oligodendrocytes. We observed that the actin-binding protein, Mayven, is expressed during all stages of the oligodendrocyte lineage, and that its expression is up-regulated during oligodendrocyte differentiation. Mayven is localized in the cytoplasm and along the cell processes. Mayven also binds actin, and is involved in the cytoskeletal reorganization in oligodendrocyte precursor cells (O-2A cells) that leads to process elongation. Mayven overexpression resulted in an increase in the process outgrowth of O-2A cells and in the lengths of the processes, while microinjection of Mayven-specific antibodies inhibited process extension in these cells. Furthermore, O-2A cells transduced with recombinant retroviral sense Mayven (pMIG-W-Mayven) showed an increase in the number of oligodendrocyte processes with outgrowth, while recombinant retroviral antisense Mayven (pMIG-W-Mayven-AS) blocked O-2A process extension. Interestingly, co-localization and association of Mayven with Fyn kinase were found in O-2A cells, and these interactions were increased during the outgrowth of oligodendrocyte processes. This association was mediated via the SH3 domain ligand (a.a. 1-45) of Mayven and the SH3 domain of Fyn, suggesting that Mayven may act as a linker to bind Fyn, via its N-terminus. Thus, Mayven plays a role in the dynamics of cytoskeletal rearrangement leading to the process extension of oligodendrocytes.     

Drosophila Kelch Is an Oligomeric Ring Canal Actin Organizer

Drosophila kelch has four protein domains, two of which are found in kelch-family proteins and in numerous nonkelch proteins. In Drosophila, kelch is required to maintain ring canal organization during oogenesis. We have performed a structure–function analysis to study the function of Drosophila kelch. The amino-terminal region (NTR) regulates the timing of kelch localization to the ring canals. Without the NTR, the protein localizes precociously and destabilizes the ring canals and the germ cell membranes, leading to dominant sterility. The amino half of the protein including the BTB domain mediates dimerization. Oligomerization through the amino half of kelch might allow cross-linking of ring canal actin filaments, organizing the inner rim cytoskeleton. The kelch repeat domain is necessary and sufficient for ring canal localization and likely mediates an additional interaction, possibly with actin.     

KLHL1/MRP2 mediates neurite outgrowth in a glycogen synthase kinase 3beta-dependent manner

The actin-based cytoskeleton is essential for the generation and maintenance of cell polarity, cellular motility, and the formation of neural cell processes. MRP2 is an actin-binding protein of the kelch-related protein family. While MRP2 has been shown to be expressed specifically in brain, its function is still unknown. Here, we report that in neuronal growth factor (NGF)-induced PC12 cells, MRP2 was expressed along the neurite processes and colocalized with Talin at the growth cones. MRP2 mRNA and protein levels were up-regulated in PC12 cells following NGF stimulation. Moreover, treatment of PC12 cells with interfering RNAs for MRP2 and glycogen synthase kinase 3beta (GSK3beta) resulted in the inhibition of neurite outgrowth. A significant decrease in MRP2 expression levels was observed following GSK3beta inhibition, which was correlated with the inhibited neurite outgrowth, while GSK3beta overexpression was found to increase MRP2 expression levels. MRP2 interacted with GSK3beta through its NH2 terminus containing the BTB domain, and these molecules colocalized along neurite processes and growth cones in differentiated PC12 cells and rat primary hippocampal neurons. Additionally, increased associations of MRP2 with GSK3beta and MRP2 with actin were observed in the NGF-treated PC12 cells. Thus, this study provides, for the first time, insights into the involvement of MRP2 in neurite outgrowth, which occurs in a GSK3beta-dependent manner.     

Overexpression of Nd1-s, a variant form of new kelch family protein, perturbs the cell cycle progression of fibroblasts

The murine Nd1 gene encodes two forms of protein, Nd1-L and Nd1-S, both of which share the BTB/POZ domain, but Nd1-S lacks the kelch repeats. Although Nd1-L ubiquitously expresses, localizes in the cytoplasm and functions as a stabilizer of actin filaments, expression and function of Nd1-S were unknown. Here we show that Nd1-S were expressed in all tissues examined and localized in the nucleus as a speckled-like pattern. Furthermore, overexpression of Nd1-S perturbed cell growth of NIH3T3 cells at the G1/S phase of the cell cycle. These results suggest that Nd1-S may play a role in cell cycle progression in the nucleus.     

A Highlights from MBoC Selection: LIM and SH3 protein 1 localizes to the leading edge of protruding lamellipodia and regulates axon development

The actin cytoskeleton drives cell motility and is essential for neuronal development and function. LIM and SH3 protein 1 (LASP1) is a unique actin-binding protein that is expressed in a wide range of cells including neurons, but its roles in cellular motility and neuronal development are not well understood. We report that LASP1 is expressed in rat hippocampus early in development, and this expression is maintained through adulthood. High-resolution imaging reveals that LASP1 is selectively concentrated at the leading edge of lamellipodia in migrating cells and axonal growth cones. This local enrichment of LASP1 is dynamically associated with the protrusive activity of lamellipodia, depends on the barbed ends of actin filaments, and requires both the LIM domain and the nebulin repeats of LASP1. Knockdown of LASP1 in cultured rat hippocampal neurons results in a substantial reduction in axonal outgrowth and arborization. Finally, loss of the Drosophila homologue Lasp from a subset of commissural neurons in the developing ventral nerve cord produces defasciculated axon bundles that do not reach their targets. Together, our data support a novel role for LASP1 in actin-based lamellipodial protrusion and establish LASP1 as a positive regulator of both in vitro and in vivo axon development.     

NRP/B, a Novel Nuclear Matrix Protein, Associates With p110RB and Is Involved in Neuronal Differentiation

The nuclear matrix is defined as the insoluble framework of the nucleus and has been implicated in the regulation of gene expression, the cell cycle, and nuclear structural integrity via linkage to intermediate filaments of the cytoskeleton. We have discovered a novel nuclear matrix protein, NRP/B (nuclear restricted protein/brain), which contains two major structural elements: a BTB domain–like structure in the predicted NH₂ terminus, and a “kelch motif” in the predicted COOH-terminal domain. NRP/B mRNA (5.5 kb) is predominantly expressed in human fetal and adult brain with minor expression in kidney and pancreas. During mouse embryogenesis, NRP/B mRNA expression is upregulated in the nervous system. The NRP/B protein is expressed in rat primary hippocampal neurons, but not in primary astrocytes. NRP/B expression was upregulated during the differentiation of murine Neuro 2A and human SH-SY5Y neuroblastoma cells. Overexpression of NRP/B in these cells augmented neuronal process formation. Treatment with antisense NRP/B oligodeoxynucleotides inhibited the neurite development of rat primary hippocampal neurons as well as the neuronal process formation during neuronal differentiation of PC-12 cells. Since the hypophosphorylated form of retinoblastoma protein (p110^RB) is found to be associated with the nuclear matrix and overexpression of p110^RB induces neuronal differentiation, we investigated whether NRP/B is associated with p110^RB. Both in vivo and in vitro experiments demonstrate that NRP/B can be phosphorylated and can bind to the functionally active hypophosphorylated form of the p110^RB during neuronal differentiation of SH-SY5Y neuroblastoma cells induced by retinoic acid. Our studies indicate that NRP/B is a novel nuclear matrix protein, specifically expressed in primary neurons, that interacts with p110^RB and participates in the regulation of neuronal process formation.     

Actin-binding properties and colocalization with actin during spermiogenesis of mammalian sperm calicin

The nucleus of mammalian spermatozoa is surrounded by a rigid layer, the perinuclear theca, which is divided into a subacrosomal layer and a postacrosomal calyx. Among the proteins characterized in the perinuclear theca, calicin is one of the main components of the calyx. Its sequence contains three kelch repeats and a BTB/POZ domain. We have studied the association of boar calicin with F-actin and the distribution of boar and human calicin during spermiogenesis compared with the distribution of actin. Calicin was purified from boar sperm heads under nondenaturating conditions. The molecule bound actin with high affinity (K(d) = approximately 5 nM), and a stoichiometry of approximately one calicin per 12 actin monomers was observed. Gel filtration studies showed that calicin forms homomultimers (tetramers and higher polymers). According to immunocytochemical results, calicin is present (together with actin) in the acrosomal region of round spermatids and is mainly localized in the postacrosomal region of late spermatids and spermatozoa. Taken together, the results suggest that the affinity of calicin to F-actin allows targeting of calicin at the subacrosomal space of round spermatids, and that its ability to form homomultimers contributes to the formation of a rigid calyx.     

Molecular characterization of KLHL3, a human homologue of the Drosophila kelch gene

The Drosophila kelch protein is a structural component of ring canals and is required for oocyte maturation. Here, we report the cloning and genomic structure of a new human homologue of kelch, KLHL3. At the amino acid level, KLHL3 shares 77% similarity with Drosophila kelch and 89% similarity with Mayven (KLHL2), another human kelch homolog. The approximately 6.5-kb mRNA has a single open reading frame encoding a protein of 587 amino acids with a predicted molecular mass of 650 kDa. Like kelch and KLHL2, the KLHL3 protein contains a poxvirus and zinc finger domain at the N-terminus and six tandem repeats (kelch repeats) at the C-terminus. At least three isoforms, which differ in the length of the N-terminus, are produced and may be the result of alternative promoter usage. We also identified alternative polyadenylation sites and alternative splicing; thus, as many as 12 mRNA variants and six putative protein isoforms could be produced. The KLHL3 gene is mapped to human chromosome 5, band q31, contains 17 exons, and spans approximately 120 kb of genomic DNA. KLHL3 maps within the smallest commonly deleted segment in myeloid leukemias characterized by a deletion of 5q; however, we detected no inactivating mutations of KLHL3 in malignant myeloid disorders with loss of 5q.