Evolution of the vertebrate paralemmin gene family: ancient origin of gene duplicates suggests distinct functions

Paralemmin-1 is a protein implicated in plasma membrane dynamics, the development of filopodia, neurites and dendritic spines, as well as the invasiveness and metastatic potential of cancer cells. However, little is known about its mode of action, or about the biological functions of the other paralemmin isoforms: paralemmin-2, paralemmin-3 and palmdelphin. We describe here evolutionary analyses of the paralemmin gene family in a broad range of vertebrate species. Our results suggest that the four paralemmin isoform genes (PALM1, PALM2, PALM3 and PALMD) arose by quadruplication of an ancestral gene in the two early vertebrate genome duplications. Paralemmin-1 and palmdelphin were further duplicated in the teleost fish specific genome duplication. We identified a unique sequence motif common to all paralemmins, consisting of 11 highly conserved residues of which four are invariant. A single full-length paralemmin homolog with this motif was identified in the genome of the sea lamprey Petromyzon marinus and an isolated putative paralemmin motif could be detected in the genome of the lancelet Branchiostoma floridae. This allows us to conclude that the paralemmin gene family arose early and has been maintained throughout vertebrate evolution, suggesting functional diversification and specific biological roles of the paralemmin isoforms. The paralemmin genes have also maintained specific features of gene organisation and sequence. This includes the occurrence of closely linked downstream genes, initially identified as a readthrough fusion protein with mammalian paralemmin-2 (Palm2-AKAP2). We have found evidence for such an arrangement for paralemmin-1 and -2 in several vertebrate genomes, as well as for palmdelphin and paralemmin-3 in teleost fish genomes, and suggest the name paralemmin downstream genes (PDG) for this new gene family. Thus, our findings point to ancient roles for paralemmins and distinct biological functions of the gene duplicates.     

PALML, a novel paralemmin-related gene mapping on human chromosome 1p21.

We describe PALML, a novel gene encoding a 551 amino acid protein with similarity to paralemmin and the paralemmin-like amino terminal domain of AKAP2, a protein kinase A anchor protein. PALML mRNA is expressed in many tissues and is most abundant in cardiac and skeletal muscle, while absent from brain and blood. Exogenously expressed PALML fusion protein has a widespread cytoplasmic localization, and it is excluded from the nucleus. Human PALML maps on human chromosome 1p21 (between D1S2767 and D1S223). SSCP-HD analysis of exonic sequences in patients with VUR (familial non-syndromic vesicoureteral reflux syndrome) excluded mutations in the PALML gene from causing this disease. PALML, paralemmin and AKAP2 share the presence of a conserved coiled coil region that may mediate protein interactions with shared partners. Based on its resemblance to paralemmin and AKAP2, PALML is hypothesized to be involved in regulating intracellular signaling and membrane-cytoskeletal interactions.     

Determination of structural regions important for Ca(2+) uptake activity in Arabidopsis MCA1 and MCA2 expressed in yeast

MCA1 is a plasma membrane protein that correlates Ca(2+) influx and mechanosensing in Arabidopsis. MCA2 is a paralog of MCA1, and both share 72.7% amino acid sequence identity and several common structural features, including putative transmembrane (TM) segments, an EF hand-like region in the N-terminal half, a coiled-coil motif in the middle and a PLAC8 motif in the C-terminal half. To determine structural regions important for Ca(2+) uptake activity, the activity of truncated forms of MCA1 and MCA2 was assessed using yeast expression assays. The N-terminal half of MCA1 with a coiled-coil motif (MCA1(1-237)) did not have Ca(2+) uptake activity, while MCA2(1-237) did. The N-terminal half of MCA1 without the coiled-coil motif (MCA1 (1-185)) showed Ca(2+) uptake activity, as did MCA2(1-186). Both MCA1(1-173) and MCA2(1-173) having the EF hand-like region had Ca(2+) uptake activity. Deletion of a putative TM segment (Ile11-Ala33) and the Asp21 to asparagine mutation in MCA1 and MCA2 abolished Ca(2+) uptake activity. Finally, MCA1(173-421) and MCA2(173-416) lacking the N-terminal half had no Ca(2+) uptake activity. These results suggest that the N-terminal half of both proteins with the EF hand-like region is necessary and sufficient for Ca(2+) uptake and that the coiled-coil motif regulates MCA1 negatively and MCA2 positively.     

The PACT domain, a conserved centrosomal targeting motif in the coiled-coil proteins AKAP450 and pericentrin

AKAP450 (also known as AKAP350, CG-NAP or Hyperion) and pericentrin are large coiled-coil proteins found in mammalian centrosomes that serve to recruit structural and regulatory components including dynein and protein kinase A. We find that these proteins share a well conserved 90 amino acid domain near their C-termini that is also found in coiled-coil proteins of unknown function from Drosophila and fission yeast. Fusion of the C-terminal region from either protein to a reporter protein confers a centrosomal localization, and overexpression of the domain from AKAP450 displaces endogenous pericentrin, suggesting recruitment to a shared site. When isolated from transfected cells the C-terminal domain of AKAP450 was associated with calmodulin, suggesting that this protein could contribute to centrosome assembly.     

Isolation and chromosomal assignment of a novel human gene, CORO1C, homologous to coronin-like actin-binding proteins

We have isolated a gene, termed CORO1C (human coronin-like actin-binding protein 1C), that encodes a new member of the coronin-like family of proteins. The cDNA consists of 3,857 nucleotides, with an open reading frame of 1,422 bp encoding a 474 amino acid protein. The deduced amino acid sequence shared 65% identity with p57 (human coronin-like actin-binding protein), as well as 46% identity with coronin, a protein first isolated from the slime mold Dictyostelium discoideum. Computer analysis predicted that the product of the CORO1C gene would contain five WD repeats in its N-terminal region and a coiled-coil motif in its C-terminal region, both of which are conserved among coronin-like proteins. CORO1C was ubiquitously expressed in all human tissues examined, in contrast to other known coronin-like molecules, each of which is expressed in a tissue-specific manner. Immunocytochemical staining demonstrated that CORO1C was co-localized with F-actin; therefore, the gene product is likely to be important in cytokinesis, motility, and signal transduction, as are the other members of this molecular family. We assigned this novel gene to chromosome 12q24.1 by fluorescence in situ hybridization.     

Identification of a PDZ domain containing Golgi protein, GOPC, as an interaction partner of frizzled

The frizzled gene is evolutionally conserved in a wide variety of organisms including mammals, and in Drosophila, frizzled is implicated in the development of planar polarity. We describe here the isolation and characterization of a Golgi protein, GOPC, as a frizzled interacting protein. GOPC comprises one PDZ domain, two coiled-coil motifs and two evolutionally conserved regions. Immunofluorescence studies indicated that a significant fraction of GOPC protein was localized in the Golgi apparatus. Using a series of deletion mutants, we show that both coiled-coil motifs and a C-terminal conserved region were required for its Golgi localization. Interestingly, deletion mutants that lack a N-terminal conserved region or coiled-coil motifs formed aggresome-like perinuclear structure. Interaction of GOPC and frizzled was observed both in vivo and in vitro, and the PDZ domain of GOPC and the C-terminal Ser/Thr-X-Val motif of frizzled were required for their interaction. Immunofluorescence studies indicated that, although frizzled was a membrane protein, it was localized at the Golgi apparatus as well, and colocalization of GOPC and frizzled at the Golgi apparatus was observed. Furthermore, when GOPC was coexpressed with frizzled, translocation of GOPC to the plasma membrane was observed. Importantly, brefeldin A interrupted not only the localization of GOPC to the Golgi apparatus but also the translocation of frizzled to the plasma membrane, indicating that the Golgi structure was required for the proper subcellular localization of frizzled. Taken together, these results indicate that GOPC may play a role in the vesicle transport of frizzled from the Golgi apparatus to the plasma membrane.     

Domain analysis of cortexillin I: actin-bundling, PIP(2)-binding and the rescue of cytokinesis

Cortexillins are actin-bundling proteins that form a parallel two-stranded coiled-coil rod. Actin-binding domains of the alpha-actinin/spectrin type are located N-terminal to the rod and unique sequence elements are found in the C-terminal region. Domain analysis in vitro revealed that the N-terminal domains are not responsible for the strong actin-filament bundling activity of cortexillin I. The strongest activity resides in the C-terminal region. Phosphatidylinositol 4,5-bisphosphate (PIP(2)) suppresses this bundling activity by binding to a C-terminal nonapeptide sequence. These data define a new PIP(2)-regulated actin-bundling site. In vivo the PIP(2)-binding motif enhances localization of a C-terminal cortexillin I fragment to the cell cortex and improves the rescue of cytokinesis. This motif is not required, however, for translocation to the cleavage furrow. A model is presented proposing that cortexillin translocation is based on a mitotic cycle of polar actin polymerization and midzone depolymerization.     

Characterization of Rab45/RASEF containing EF-hand domain and a coiled-coil motif as a self-associating GTPase

Rab-family GTPases function as key regulators for membrane traffic. Among them, Rab45/RASEF is an atypical GTPase in that it contains a coiled-coil motif at the mid region and a distinct N-terminal EF-hand domain with C-terminal Rab-homology domain. Here, we provide the initial biochemical characterization and intracellular localization of human Rab45. Rab45 bound guanine nucleotide tri- and di-phosphates through the C-terminal Rab domain. Rab45 was capable of self-interacting, and the self-interaction required the mid region containing the coiled-coil motif. Rab45 expressed in HeLa cells was localized in a small patch in the perinuclear area of the cell, and the localization was regulated by the guanine nucleotide-bound states of Rab45. Interestingly, the mid region, together with Rab domain, appeared to be essential for the characteristic perinuclear localization of Rab45, indicating that the self-interaction may be involved in the intracellular localization of Rab45.     

Functional importance of the coiled-coil of the Ebola virus glycoprotein

Ebola virus contains a single glycoprotein (GP) that is responsible for receptor binding and membrane fusion and is proteolytically cleaved into disulfide-linked GP1 and GP2 subunits. The GP2 subunit possesses a coiled-coil motif, which plays an important role in the oligomerization and fusion activity of other viral GPs. To determine the functional significance of the coiled-coil motif of GP2, we examined the effects of peptides corresponding to the coiled-coil motif of GP2 on the infectivity of a mutant vesicular stomatitis virus (lacking the receptor-binding/fusion protein) pseudotyped with the Ebola virus GP. A peptide corresponding to the C-terminal helix reduced the infectivity of the pseudotyped virus. We next introduced alanine substitutions into hydrophobic residues in the coiled-coil motif to identify residues important for GP function. None of the substitutions affected GP oligomerization, but some mutations, two in the N-terminal helix and all in the C-terminal helix, reduced the ability of GP to confer infectivity to the mutant vesicular stomatitis virus without affecting the transport of GP to the cell surface, its incorporation into virions, and the production of virus particles. These results indicate that the coiled-coil motif of GP2 plays an important role in facilitating the entry of Ebola virus into host cells and that peptides corresponding to this region could act as efficient antiviral agents.     

Maize ROP7 GTPase contains a unique, CaaX box-independent plasma membrane targeting signal

Signals in the carboxy-terminal hypervariable region (HVR) of Rho and Ras GTPases target these proteins to specific membrane compartments, where they function in signal transduction. ROP6 and ROP7 are closely related maize Rops (a plant-specific Rho subgroup) that share HVR sequences divergent from other Rho HVRs. Both ROPs terminate in CAA, instead of the consensus C-terminal CaaX motif required for membrane association of all characterized Ras and Rho GTPases. The ROP6/7 HVR contains two additional cysteines, potential sites for post-translational modification that leads to membrane association; one is in an internal CaaX motif, which would be at the C-terminus if the final intron in both genes were not removed. Transient expression of a GFP-ROP7 fusion revealed its near-total association with the plasma membrane (PM). Furthermore, the ROP7 HVR is sufficient to target GFP to the PM. Surprisingly, the cysteine in the terminal CAA is not required for PM targeting of GFP-ROP7. In contrast, an internal HVR cysteine is essential for proper targeting of the fusion, and the cysteine in the internal CaaX is required for complete membrane association. Interestingly, this CaaX motif can also direct PM association when placed at the fusion C-terminus by addition of an internal stop codon. Fractionation experiments confirm that maize ROPs associate with membranes in maize seedlings. Our analysis suggests that the ROP7 HVR directs PM localization by a mechanism independent of a C-terminal CaaX motif; this mechanism may have evolved through addition of 3' intron/exon sequences to a rop progenitor.     

Identification, cloning and characterization of a novel nuclear protein, HA95, homologous to A-kinase anchoring protein 95

Previously, we have identified and characterized nuclear AKAP95 from man which targets cyclic AMP (cAMP)-dependent protein kinase (PKA)-type II to the condensed chromatin/spindle region at mitosis. Here we report the cloning of a novel nuclear protein with an apparent molecular mass of 95 kDa that is similar to AKAP95 and is designated HA95 (homologous to AKAP95). HA95 cDNA sequence encodes a protein of 646 amino acids that shows 61% homology to the deduced amino acid sequence of AKAP95. The HA95 gene is located on chromosome 19p13.1 immediately upstream of the AKAP95 gene. Both HA95 and AKAP95 genes contain 14 exons encoding similar regions of the respective proteins, indicating a previous gene duplication event as the origin of the two tandem genes. Despite their apparent similarity, HA95 does not bind RII in vitro. HA95 contains a putative nuclear localization signal in its N-terminal domain. It is localized exclusively into the nucleus as demonstrated in cells transfected with HA95 fused to either green fluorescence protein or the c-myc epitope. In the nucleus, the HA95 protein is found as complexes directly associated with each other or indirectly associated via other nuclear proteins. In interphase, HA95 is co-localized with AKAP95, but the two proteins are not biochemically associated. At metaphase, both proteins co-localize with condensed chromosomes. The similarity in sequence and localization of HA95 and AKAP95 suggests that the two molecules constitute a novel family of nuclear proteins that may exhibit related functions.     

Transmembrane and Coiled-Coil Domain Family 1 Is a Novel Protein of the Endoplasmic Reticulum

The endoplasmic reticulum (ER) is a continuous membrane network in eukaryotic cells comprising the nuclear envelope, the rough ER, and the smooth ER. The ER has multiple critical functions and a characteristic structure. In this study, we identified a new protein of the ER, TMCC1 (transmembrane and coiled-coil domain family 1). The TMCC family consists of at least 3 putative proteins (TMCC1–3) that are conserved from nematode to human. We show that TMCC1 is an ER protein that is expressed in diverse human cell lines. TMCC1 contains 2 adjacent transmembrane domains near the C-terminus, in addition to coiled-coil domains. TMCC1 was targeted to the rough ER through the transmembrane domains, whereas the N-terminal region and C-terminal tail of TMCC1 were found to reside in the cytoplasm. Moreover, the cytosolic region of TMCC1 formed homo- or hetero-dimers or oligomers with other TMCC proteins and interacted with ribosomal proteins. Notably, overexpression of TMCC1 or its transmembrane domains caused defects in ER morphology. Our results suggest roles of TMCC1 in ER organization.     

Polarized targeting of peripheral membrane proteins in neurons

Differential targeting of neuronal proteins to axons and dendrites is essential for directional information flow within the brain, however, little is known about this protein-sorting process. Here, we investigate polarized targeting of lipid-anchored peripheral membrane proteins, postsynaptic density-95 (PSD-95) and growth-associated protein-43 (GAP-43). Whereas the N-terminal palmitoylated motif of PSD-95 is necessary but not sufficient for sorting to dendrites, the palmitoylation motif of GAP-43 is sufficient for axonal targeting and can redirect a PSD-95 chimera to axons. Systematic mutagenesis of the GAP-43 and PSD-95 palmitoylation motifs indicates that the spacing of the palmitoylated cysteines and the presence of nearby basic amino acids determine polarized targeting by these two motifs. Similarly, the axonal protein paralemmin contains a C-terminal palmitoylated domain, which resembles that of GAP-43 and also mediates axonal targeting. These axonally targeted palmitoylation motifs also mediate targeting to detergent-insoluble glycolipid-enriched complexes in heterologous cells, suggesting a possible role for specialized lipid domains in axonal sorting of peripheral membrane proteins.     

一种新的蛋白酪氨酸磷酸酶基因的克隆、定位和组织表达谱研究

从人胎肝cDNA库分离出一长度为5248bp的cDNA克隆,该基因包含26个外显子和25个内含子,染色体定位于在某些肿瘤细胞中易缺失的3p21.1-21.33。其可读框编码1636个氨基酸,该蛋白属于蛋白酪氨酸磷酸酶（PTP）家族,其C端有一个典型的PTP结构域,N端含有约800氨基酸残基的BRO1样结构域及随后2个可能的SH3结构域结合位点,在这两个结构域之间及C末端还各有一个脯氨酸富集区。Northern杂交和点杂交分析显示,该基因以大约5.4kb的单一转录物广泛表达于人体各种组织,而且在人部分肿瘤细胞中高表达。结果提示,人源PTP－TD14是一个新的蛋白酪氨酸磷酸酶。