It has been suggested that cell-type determination in Dictyostelium discoideum is dependent on the position of a cell in the cell cycle at the time of starvation. In order to understand the molecular basis of this phenomenon, studies on the cell cycle were initiated and the isolation of a Dictyostelium gene encoding a homolog of the Cdc2 kinase has been described. Additional cdc2 genes could not be isolated from Dictyostelium using polymerase chain reaction technology, but a gene has been isolated that is highly related to cdc2. The encoded product is a protein of 33 kDa that shares over 60% identity to the cell-cycle-dependent Cdc2 kinases. However, despite this high level of identity, the gene is not capable of complementing the temperature-sensitive cdc28 mutant of Saccharomyces cerevisiae. Furthermore, the gene product shares some characteristics with the recently described PCTAIRE proteins; it contains a PCTAIRE motif instead of the Cdc2 kinase conserved PSTAIRE sequence; it does not possess the conserved GDSEID sequence that is involved in the activation of the enzyme and it has a Ser in the position equivalent to Thr-161. However, the Dictyostelium protein exhibits a slightly higher level of identity to the Cdc2 kinases than to the PCTAIRE proteins and is smaller than any of the PCTAIRE proteins thus far identified. Since the gene product has characteristics of both Cdc2 kinases and PCTAIRE proteins the gene product has been designated Crp (Cdc2-Related PCTAIRE) kinase. The gene is expressed as two transcripts of 1.5 and 1.8 kb and the expression is developmentally regulated with low levels of mRNA in vegetative cells and significantly higher levels throughout the remainder of the differentiation process. These results suggest the possibility that the gene product is involved in Dictyostelium differentiation rather than growth. This report is the first evidence for a highly-related cdc2 gene in unicellular eukaryotes. It also demonstrates for the first time that a unicellular eukaryote expresses a protein containing the PCTAIRE sequence (Michaelis, 1993).
Recent studies on the molecular mechanisms controlling the mammalian cell cycle have disclosed a large family of cdc2-related serine/threonine kinases. Among this gene family, the PCTAIRE protein kinases comprise a distinct subfamily of unknown cellular function. To analyze the genomic structure and chromosomal location of the PCTAIRE-1 and -3 genes, human cosmid clones for each gene were isolated by screening a human genomic library with murine PCTAIRE cDNA probes. Overlapping clones encompassing approximately 60 kb of genomic DNA were obtained for both PCTAIRE-1 and -3. These clones have abeen confirmed to encode authentic PCTAIRE genes by the detection of exon-intron structures and the coincidence of the nucleotide sequence of exons to that of the published human cDNAs. Using these cosmid clones as probes for FISH analyses, the chromosomal loci for PCTAIRE-1 and PCTAIRE-3 were assigned to bands Xp11 and 1q31-q32, respectively (Okuda, 1994).
The roles of the cyclin dependent kinase (Cdk) family in murine germ cell development have been examined by studying the expression of five Cdk family genes (Cdc2, Cdk2, Cdk4, Pctaire-1, and Pctaire-3) in mouse reproductive organs. Northern blot and in situ hybridization analyses revealed distinctive expression patterns for these genes, with striking cellular, lineage, and developmental stage specificity. Cdk expression is observed in cell types with proliferative activity: Cdc2 and Cdk2 expression in premeiotic spermatocytes in the testis, and Cdc2, Cdk2, and Cdk4 expression in granulosa cells of ovarian follicles. Cdc2 transcripts are most abundant in late pachytene to diplotene spermatocytes, which are soon to undergo meiosis. Surprisingly, expression of Cdk family genes is also observed in non-proliferating cell types. All five Cdk family genes examined are expressed in Sertoli cells of the adult testis; these cells are no longer mitotically active. With regard to Pctaire-1 and Pctaire-3, the highest levels of expression are observed in postmeiotic spermatids. Immunoblot analysis also reveals the presence of high levels of Pctaire-1 in postmeiotic germ cells. These results suggest that Cdk family kinases may exhibit various functions in germinal and somatic cells during gametogenesis, not only in the cell cycle but also in other regulatory processes, including differentiation (Rhee, 1995).
A rat PCTAIRE-1 cDNA clone was isolated by immunoscreening of a PC12 cDNA library, followed by 5' RACE (rapid amplification of cDNA ends) to determine the 5' end. The rat PCTAIRE-1 cDNA sequence is 96% identical to mouse PCTAIRE-1 and contains an alternatively spliced exon of 131 bp near the 5' end. Although a mouse cDNA containing this exon has been reported, examination of several mouse cell lines has provided no evidence for expression of the corresponding mRNA. In contrast, reverse transcription and polymerase chain reaction (RT/PCR) across this region using RNA from proliferating, differentiated, and apoptotic PC12 cells demonstrates that alternatively spliced forms of PCTAIRE-1 mRNA, with and without this exon, are expressed. Both forms of PCTAIRE-1 mRNA are also expressed in vivo in neonatal rat brain, although other tissues examined contained only the form lacking the alternatively spliced exon. In the absence of the alternatively spliced exon PCTAIRE-1 mRNA contains an open reading frame of 1488 bp, corresponding to a 55-kDa protein that is 97% identical to mouse PCTAIRE-1 protein. When the alternatively spliced exon is present, this open reading frame is terminated by a stop codon and a second open reading frame is initiated, predicting a second PCTAIRE-1 protein of 52 kDa. The two predicted PCTAIRE-1 proteins are identical downstream of the splice site, but share no homology at their N-terminal ends (Gao. 1996).
PCTAIRE are members of a subfamily of Cdc2-related kinases that have been shown to be preferentially expressed in post-mitotic cells. To examine the neural functions of PCTAIRE, rat cDNA clones encoding PCTAIRE 1, 2, and 3 were isolated, and their expression patterns in the brain were analyzed. Among the three rat PCTAIREs, only PCTAIRE 2 was found to be specifically expressed in the brain. Furthermore, its expression is transiently increased during brain development, peaking 7-15 days after birth. Within the brain, PCTAIRE 2 is concentrated in the neuronal layers of the hippocampus and olfactory bulb, which mostly consist of post-mitotic neurons. In an immunocytochemical experiment, immunoreactivity for PCTAIRE 2 was detected in the cell bodies and extended neurites of neurons, but not in astrocytes. The PCTAIRE 2 protein was recovered in the particulate fraction and resistant to solubilization with non-ionic detergent, suggesting that PCTAIRE 2 might be present as a component of a large protein complex. An immunoprecipitation assay revealed that the PCTAIRE 2 is associated with Ser/Thr-phosphorylating activity for histone H1, and that its activity depends on association with a regulatory partner that can be released under high-salt conditions. These findings suggest that PCTAIRE 2 is a Ser/Thr kinase that might play a unique role in terminally differentiated neurons (Hirose, 1997).
A cDNA encoding a cdc2-related protein kinase, named PFTAIRE, has been cloned and characterized. It is expressed primarily in the postnatal and adult nervous system. Several populations of terminally differentiated neurons and some neuroglia express PFTAIRE mRNA and protein. In neurons, PFTAIRE protein is localized in the nucleus and cytoplasm of cell bodies. The anatomical, cellular, and ontogenic patterns of PFTAIRE expression in the nervous system differ from those of p34cdc2 and cdk5, which are expressed in brain and several other mitotic tissues. Proteins of approximately 58-60 kDa coprecipitate specifically with PFTAIRE from cytosolic protein preparations of adult mouse brain and transfected cells. These proteins appear to be the major endogenous substrates associated with this kinase activity. The temporal and spatial expression patterns of PFTAIRE in the postnatal and adult nervous system suggest that PFTAIRE kinase activity may be associated with the postmitotic and differentiated state of cells in the nervous system and that its function may be distinct from those of p34cdc2 and cdk5 (Lazzaro, 1997).
A murine cDNA encoding for a novel putative Cdk-related protein kinase, which has been named Pftaire-1, has been isolated by screening a testis cDNA library for new serine/threonine kinases. Pftaire-1 shows 50% and 49% amino acid identity with Cdk5 and Pctaire-3, respectively, and contains the eleven subdomains characteristic of the protein kinases. By Northern blot analysis two transcripts of approximately 5.5 and 4.9 kb in size have been detected. These transcripts are expressed at low levels in all murine tissues tested, except in the brain, testis and embryo, where high expression is detected. Cellular localization of the mRNAs by in situ hybridization analysis shows that Pftaire-1 is expressed in late pachytene spermatocytes in the testis and in post mitotic neuronal cells both in the brain and the embryo, suggesting a role of Pftaire-1 both in the process of meiosis as well as neuron differentiation and/or function (Besset, 1998).
PCTAIRE-1 is a member of the cyclin-dependent kinase (cdk) family whose function is unknown. The pattern of PCTAIRE-1 protein expression was examined in a number of normal and transformed cell lines of various origins and it was found that the kinase is ubiquitous. PCTAIRE-1 exhibits cytoplasmic distribution throughout the cell cycle. Confocal microscopy shows that PCTAIRE-1 does not colocalize with components of the cytoskeleton or with the endoplasmic reticulum. Endogenous PCTAIRE-1 and ectopically expressed PCTAIRE-1 display kinase activity when myelin basic protein is used as an acceptor substrate. Similar to other members of the cyclin-dependent kinase family, PCTAIRE-1 seems to require binding to a regulatory subunit to display kinase activity. PCTAIRE-1 activity is cell cycle dependent and displays a peak in the S and G2 phases. The low level of kinase activity observed until the onset of S phase correlates with elevated tyrosine phosphorylation of the molecule (Charrasse, 1999).
A selective antibody to a synthetic peptide corresponding to an N-terminal sequence of the PCTAIRE-1 protein has been developed. In rodent brain extracts it recognizes only the protein doublet characteristic of PCTAIRE-1, and this signal is completely abolished by preincubation of the antibody with the immunopeptide. Immunolabeling experiments done with this PCTAIRE-1-specific antibody reveal that the protein is widely distributed in the rodent brain as are the mRNAs visualized using an antisense riboprobe corresponding to the entire PCTAIRE-1 open reading frame. Two types of PCTAIRE-1 protein localizations were observed: (1) a diffuse labeling of almost all brain regions, particularly intense in the molecular layer of the cerebellum and the mossy fiber region of the hippocampus, and (2) a spot-like localization in the nuclei of large neurons, such as cerebellar Purkinje cells and pyramidal cells of the hippocampus. Colocalization with the B23 protein allows the identification of these compartments as nucleoli. These results suggest a nucleolar function of PCTAIRE-1 in large neurons and a role in regions containing important granule cell projections (Le Bouffant, 2000).
PCTAIRE-1 is a member of the cyclin-dependent kinase (cdk)-like class of proteins, and is localized mainly in the mammalian brain. Using the yeast two-hybrid system a mouse brain cDNA library was screened with PCTAIRE-1 as bait, and several clones were isolated coding for the mouse homologs of the following proteins: p11 (also known as calpactin I light chain) and the eta, theta (also known as tau) and zeta isoforms of 14-3-3 proteins. That these four proteins interact with PCTAIRE-1 was confirmed by demonstrating the biochemical interactions using the pure recombinant proteins. The fact that 14-3-3 proteins are known to interact with many other intracellular proteins (such as C-kinase, Raf, Bcr, P13-kinase) and p11 with annexin II (a major pp60[v-src] and C-kinase substrate) suggests that PCTAIRE-1 might be part of multiple signal transduction cascades and cellular protein networks (Sladeczek. 1997).
An antibody directed against the C-terminal part of PCTAIRE-1 recognizes three proteins in rodent brain. The high-molecular-mass band is most abundant in the cerebellum, hippocampus and cortex. It migrates at the same apparent molecular mass as recombinant PCTAIRE-1 and interacts, like recombinant PCTAIRE-1, with p11 (also known as calpactin I light chain) and 14-3-3 proteins. Combination of p11 or 14-3-3 affinity resins with immunoprecipitation and peptide elution allow for a purification of a full-length PCTAIRE-1 preparation having significant kinase activity. These results suggest that PCTAIRE-1 is an active kinase in brain. The catalytic core region of PCTAIRE-1, which is common for all cyclin-dependent kinases, does not interact with p11 and 14-3-3 proteins in the two-hybrid assay. Full interaction with p11 and 14-3-3 proteins requires both, the N-terminal and C-terminal ends of PCTAIRE-1, suggesting that complex three-dimensional arrangements are responsible for these interactions. A low-molecular-mass protein (migrating at about 30 kDa) that was also recognized by the antibody directed against the carboxy-terminal part of PCTAIRE-1, is abundant and almost homogeneously distributed in all brain areas investigated. Database searches starting with the amino acid sequences of two peptides obtained by tryptic digestion of this protein have yielded cDNA and genomic sequences, allowing for the composition of a DNA sequence coding for a putative 26 kDa protein containing both peptides. This protein has no important sequence similarity with any other known protein. But many DNA sequences are found in databases with an almost 100% identity with parts of the 26 kDa protein coding sequence. These results allow these widely distributed cDNA sequences to be attributed to an existing 26-kDa protein and the gene has been localized within two recently published genomic sequences (Le Bouffant, 1998).
PCTAIRE 2 is a Cdc2-related kinase that is predominantly expressed in the terminally differentiated neuron. To elucidate the function of PCTAIRE 2, proteins that associate with PCTAIRE 2 were screened by the yeast two-hybrid system. A positive clone was found to encode a novel protein that could bind to PCTAIRE 2 in vitro as well as in vivo, and was designated as Trap (tudor repeat associator with PCTAIRE 2). The overall structure of Trap shows no significant homology to any proteins, but contains five repeated domains (the tudor-like domain), conserved in Drosophila Tudor protein. Trap associates with the N-terminal domain of PCTAIRE 2 through its C-terminal domain, which contains two tudor-like domains. PCTAIRE 1, but not PCTAIRE 3, can also associate with Trap. Trap is predominantly expressed in brain and testis, and gradually increases during brain development throughout life, consistent with the expression pattern of PCTAIRE 2. Immunoreactivities for PCTAIRE 2 and Trap were colocalized to the mitochondria in COS 7 cells. Immunohistochemical analyses shows that PCTAIRE 2 and Trap are distributed in the same cell layer of the cerebral cortex and cerebellum. These findings suggest that Trap is a physiological partner of PCTAIRE 2 in terminally differentiated neurons (Hirose, 2000).
Previously reported data on the X inactivation status of the ubiquitin activating enzyme E1 (UBE1) gene have been contradictory, and the issue has remained unsettled. Three lines of evidence are presented that UBE1 is expressed from the inactive X chromosome and therefore escapes X inactivation. (1) By RNA in situ hybridization, UBE1 RNA is detected from both the active and inactive X chromosomes in human female fibroblasts. (2) UBE1 is expressed in a large panel of somatic cell hybrids retaining inactive human X chromosomes, including two independent hybrids that do not require UBE1 expression for survival. (3) Sites at the 5' end of UBE1 are unmethylated on both active and inactive X chromosomes, consistent with the gene escaping inactivation. In order to address whether other genes that escape inactivation map to the same region of the X chromosome, the expression of genes mapping adjacent to UBE1 was also examined. The gene for PCTAIRE-1 (PCTK1) maps within 5 kb of UBE1 and similarly escapes X inactivation by the somatic cell hybrid assay, whereas six other genes that are within 1 Mb of UBE1 in Xp11.23 are silenced on the inactive X chromosome. Comparative mapping studies of the homologous loci in mouse establish that Ube1-x and Pctk1 are also within close physical proximity on the murine X chromosome, and expression studies of the Pctk1 gene determine that, similar to Ube1-x, it is subject to X inactivation in mouse. Methylation of CpG residues at restriction sites at the 5' end of both genes on the murine inactive X chromosome are consistent with both genes being subject to X inactivation in mouse, in contrast to their expression status in humans (Carrel, 1996).
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