Molecular cloning of a GTPase activating protein specific for the Krev-1 protein p21rap1

The rap1/Krev-1 gene encodes a ras-related protein that suppresses transformation by ras oncogenes. We have purified an 88 kd GTPase activating protein (GAP), specific for the rap1/Krev-1 gene product, from bovine brain. Based on partial amino acid sequences obtained from this protein, a 3.3 kb cDNA was isolated from a human brain library. Expression of the cDNA in insect Sf9 cells resulted in high level production of an 85-95 kd rap1GAP that specifically stimulated the GTPase activity of p21rap1. The complete deduced amino acid sequence is not homologous to any known protein sequences, including GAPs specific for p21ras. Northern and Western blotting analysis indicate that rap1GAP is not ubiquitously expressed and appears most abundant in fetal tissues and certain tumor cell lines, particularly the Wilms' kidney tumor, SK-NEP-1, and the melanoma, SK-MEL-3, cell lines.     

Identification of amino acid residues required for Ras p21 target activation.

The Krev-1 gene has been shown to suppress ras-mediated transformation in vitro. Both ras and Krev-1 proteins have identical effector domains (ras residues 32 to 40), which are required for biological activity and for the interaction of Ras p21 with Ras GTPase-activating protein (GAP). In this study, five amino acid residues flanking the ras effector domain, which are not conserved with the Krev-1 protein, were shown to be required for normal protein-protein interactions and biological activity. The substitution of Krev-1 p21 residues 26, 27, 30, 31, and 45 with the corresponding amino acid residues from Ras p21 resulted in a Krev-1 protein which had ras function in both mammalian and yeast biological assays. Replacement of these residues in Ras p21 with the corresponding Krev-1 p21 amino acids resulted in ras proteins which were impaired biologically or reduced in their affinity for in vitro GAP binding. Evaluation of these mutant ras proteins have implications for Ras p21-GAP interactions in vivo.     

Inhibition of ras-induced germinal vesicle breakdown in Xenopus oocytes by rap-1B

A cDNA clone (Krev-1) has recently been identified that possesses the ability to reverse the transformed phenotype when introduced into a K-ras-transformed NIH/3T3 cell line. The Krev-1 protein, also known as rap-1A, was found to share 50% homology with the ras proteins. The rap-1A protein has also been shown to block the interaction of ras with its GTPase activating protein in vitro, leading to speculation regarding its role in vivo. A closely related protein, rap-1B, has also been identified in platelets, human erythroleukemia cells, neutrophils, and aortic smooth muscle cells. Unlike rap-1A, rap-1B has been shown to be phosphorylated in platelets. Given the high degree of similarity between the amino acid sequences of rap-1A and rap-1B, we sought to investigate the effect of microinjected rap-1B on H-ras(Val12)-induced germinal vesicle breakdown in Xenopus laevis oocytes. In this assay system, equimolar concentrations of rap-1B were found to block germinal vesicle breakdown triggered by the oncogenic ras protein. However, in the presence of IGF-1, this inhibition was not observed. Moreover, rap-1B is readily phosphorylated in the oocytes.     

Molecular cloning of smg p21B and identification of smg p21 purified from bovine brain and human platelets as smg p21B

We have previously purified smg p21 from bovine brain membranes and isolated its cDNA from a bovine brain cDNA library. In the present studies, we have performed extensive screening of the bovine brain cDNA library with the cloned smg p21 cDNA as a probe and isolated another cDNA encoding a protein highly homologous to smg p21. The proteins encoded by the previously and newly isolated cDNAs are designated as smg p21A and -B, respectively. Since the partial amino acid sequences determined previously from the smg p21 purified from bovine brain were identical with the common amino acid sequences between smg p21A and -B, we have further sequenced smg p21 and identified it as smg p21B. We have also further sequenced the smg p21 purified from human platelet membranes and identified it as smg p21B. Amino acid sequence analysis indicates that smg p21A is identical with the rap1A and Krev-1 proteins and smg p21B is identical with the rap1B protein.     

Partial purification of a GTPase-activating protein for rap2b from bovine brain membranes.

Rap2b is a ras-related GTP-binding protein isolated from a human platelet cDNA library. It shares 90% similarity to the previously described rap2a and is closely related to rap1a (Krev-1, smgp21), which has been shown to possess reversion of transformation activity in Kirsten ras transformed 3T3 cells. In this study we have partially purified a protein from bovine brain membranes which stimulates the GTPase activity of rap2b. This rap2b GTPase-activating protein (GAP) activity is not immunoreactive with antibodies specific for rap1 GAP or ras GAP, yet displays limited GTPase stimulatory activity toward rap1. This result differs from the previously described rap1 GAP which is highly specific for rap1. When the rap2 GAP activity is analyzed by coomassie staining, an enrichment of a approximately 55 kDa protein is observed providing further evidence of a distinct rap2 GAP.     

Cloning and sequencing of cDNA for the rat plasminogen activator inhibitor-1

A cDNA encoding rat plasminogen activator-inhibitor (PAI-1) has been isolated from an HTC rat hepatoma cell cDNA library constructed in phage lambda gt10. The cDNA contains 118 bp of 5'-untranslated sequence, 1206 bp encoding a 402-amino acid (aa) protein and 1747 bp of 3'-untranslated sequence. The protein-coding sequence and the derived amino acid sequence share 82% and 81% identity, respectively, with human PAI-1 cDNA and protein. The rat cDNA encodes a preprotein with a 23-aa leader peptide and a predicted N-terminal serine for the mature protein. Three of four potential N-glycosylation acceptor sites as well as the active site of rat PAI-1 are identical to the human protein. The 3'-untranslated region contains a number of unusual regions, including 80 bp of tandemly repeated GpA dinucleotides, a 115-bp stretch which shares greater than 90% sequence identity with a region within the 3'-untranslated cDNA of human PAI-1, and two 70-bp stretches of highly T-rich sequence located close to the 3'-terminus of the cDNA.     

A ras-related gene from the lower eukaryote Dictyostelium that is highly conserved relative to the human rap genes.

The cellular slime mold Dictyostelium discoideum contains two ras genes, DdrasG and Ddras that are differentially expressed during development. We have characterized a gene that hybridized to both Ddras and DdrasG under low, but not under high stringency conditions. The deduced amino acid sequence is highly conserved with respect to the human rap (Krev-1, smg21) proteins and the corresponding gene has been designated Ddrap1. The Ddrap1 gene is expressed at all stages during development but is expressed maximally during the aggregation and culmination periods when the expression of Ddras and DdrasG is declining. During vegetative growth and early development Ddrap1 cDNA hybridizes to a single mRNA of 1.1 kb. As development progresses the level of this mRNA declines and messages of 1.0 and 1.3 kb appear.     

Molecular Cloning of Two Additional Members of the Neural Visinin-Like Ca2+-Binding Protein Gene Family

Abstract: We have isolated a rat cDNA clone encoding a neural visinin-like Ca²⁺-binding protein (NVP), which we designate NVP-1. To identify additional molecular forms of NVP, a rat brain cDNA library was screened for their presence using an NVP-1 cDNA probe under low-stringency hybridization conditions. Two types of cDNA clones encoding structurally related proteins, designated NVP-2 and NVP-3, have been isolated. The deduced amino acid sequences of NVP-2 and NVP-3 are 89.0% and 68.6% identical to that of NVP-1, respectively, and contain consensus sequences for EF-hand Ca²⁺-binding sites. Northern blot analysis shows that NVP-1, NVP-2, and NVP-3 mRNAs are most highly expressed in brain and are differentially expressed in various regions of rat brain. These results suggest that NVP-2 and NVP-3 are additional members of the NVP gene family.     

RSR1, a ras-like gene homologous to Krev-1 (smg21A/rap1A): role in the development of cell polarity and interactions with the Ras pathway in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae ras-like gene RSR1 is particularly closely related to the mammalian gene Krev-1 (also known as smg21A and rap1A). RSR1 was originally isolated as a multicopy suppressor of a cdc24 mutation, which causes an inability to bud or establish cell polarity. Deletion of RSR1 itself does not affect growth but causes a randomization of bud position. We have now constructed mutant alleles of RSR1 encoding proteins with substitutions of Val for Gly at position 12 (analogous to constitutively activated Ras proteins) or Asn for Lys at position 16 (analogous to a dominant-negative Ras protein). rsr1Val-12 could not restore a normal budding pattern to an rsr1 deletion strain but could suppress a cdc24 mutation when overexpressed. rsr1Asn-16 could randomize the budding pattern of a wild-type strain even in low copy number but was not lethal even in high copy number. These and other results suggest that Rsr1p functions only in bud site selection and not in subsequent events of polarity establishment and bud formation, that this function involves a cycling between GTP-bound and GDP-bound forms of the protein, and that the suppression of cdc24 involves direct interaction between Rsr1p[GTP] and Cdc24p. Functional homology between Rsr1p and Krev-1 p21 was suggested by the observations that expression of the latter protein in yeast cells could both suppress a cdc24 mutation and randomize the budding pattern of wild-type cells. As Krev-1 overexpression can suppress ras-induced transformation of mammalian cells, we looked for effects of RSR1 on the S. cerevisiae Ras pathway. Although no suppression of the activated RAS2Val-19 allele was observed, overexpression of rsr1Val-12 suppressed the lethality of strains lacking RAS gene function, apparently through a direct activation of adenyl cyclase. This interaction of Rsr1p with the effector of Ras in S. cerevisiae suggests that Krev-1 may revert ras-induced transformation of mammalian cells by affecting the interaction of ras p21 with its effector.     

Cloning and sequencing of a cDNA encoding the precursor to the 24 kDa iron-sulfur protein of human mitochondrial NADH dehydrogenase

1. We have isolated a cDNA encoding the 24 kDa subunit, an iron-sulfur protein, of mitochondrial NADH dehydrogenase from a human fibroblast cDNA library by colony hybridization using a rat 24 kDa subunit cDNA as a probe. 2. The presequence predicted from the human cDNA sequence is typical of precursors to mitochondrial proteins in a high content of basic residues and in the absence of acidic ones. 3. The mature form of the human 24 kDa subunit shows 95% homology with its rat counterpart. Five cysteine residues are conserved among human, rat and bovine; four of these are expected to be involved in the binding of a binuclear iron-sulfur cluster.     

Isolation and characterization of a cDNA encoding a chicken beta thyroid hormone receptor.

We have isolated and characterized a cDNA encoding a chicken beta homolog of c-erbA, or thyroid hormone receptor (TR). Chicken liver cDNA libraries were screened with a rat TR beta-1 cDNA probe, and several cDNA inserts were isolated and characterized. The sequence of one cDNA predicts a 369-amino-acid open reading frame (ORF), with a protein sequence that possesses 96% identity with that of rat TR beta-1, but only 88% identity with chicken TR alpha. These data indicate that the cDNA likely encodes a beta form of TR that has the expected putative DNA and T3 binding domains. The chicken TR beta (chTR beta) in vitro translated protein binds T3 with high affinity, and binds both the thyroid hormone response element (TRE) from the rat growth hormone gene and the Xenopus vitellogenin A2 gene estrogen response element (ERE), similarly to that of the rat TR beta-1. Northern blot analysis revealed the expression of a 7.0-kb RNA in several tissues including cerebellum, pituitary, kidney, and liver. This chicken liver TR beta cDNA sequence varies in both the 5' and 3' untranslated regions from the chicken kidney TR beta cDNA sequence recently reported (Forrest et al., 1990). The 5' untranslated cDNA sequence divergence occurs near a potential splice site junction of the human TR beta gene, suggesting that this chicken liver cDNA may represent an alternatively spliced RNA product of the chicken TR beta gene.     

Cloning and expression of a novel angiotensin II receptor subtype.

Angiotensin II (AII) is a major regulator of cardiovascular function and fluid homeostasis. Recently, the cDNA for an AII receptor (AT1) was cloned from rat smooth muscle and bovine adrenal. To search for AII receptor subtypes, we amplified rat adrenal cortex cDNA by PCR using primers based on the AT1 receptor. The product was distinct from the AT1 receptor as indicated by restriction enzyme analysis and DNA sequencing. A full-length cDNA clone (2.2 kilobase pairs) encoding a novel AII receptor (AT3) was obtained by screening an adrenal cortex library. The AT3 cDNA encodes a Mr 40,959 protein with 95% amino acid identity to the rat smooth muscle receptor, but the overall nucleotide similarity is 71% due to low homology in the 5'- (58%) and 3'- (62%) untranslated regions. Expressed AT3 receptors in Xenopus oocytes and COS-7 cells mediate agonist-induced Ca2+ mobilization but are pharmacologically distinct from the AT1 receptors. AT3 mRNA is most abundant in the adrenal cortex and pituitary and differs from AT1 mRNA in its tissue distribution. The structural features of the AT3 receptor, including two additional potential phosphorylation sites for protein kinase C, could be related to the distinctive binding properties of the adrenal and vascular receptors and to their differential regulation during altered sodium intake.     

Molecular cloning of cDNA coding for rat proliferating cell nuclear antigen (PCNA)/cyclin.

The 'proliferating cell nuclear antigen' (PCNA), also known as cyclin, appears at the G1/S boundary in the cell cycle. Because of its possible relationship with cell proliferation, PCNA/cyclin has been receiving attention. PCNA/cyclin is a non-histone acidic nuclear protein with an apparent mol. wt of 33000-36000. The amino acid composition and the sequence of the first 25 amino acids of rabbit PCNA/cyclin are known. Using an oligonucleotide probe corresponding to the sequence of the first five amino acids, a cDNA clone for PCNA/cyclin was isolated from rat thymocyte cDNA library. The cDNA (1195 bases) contains an open reading frame of 813 nucleotides coding for 261 amino acids. The 3'-non-coding region is 312 nucleotides long and contains three putative polyadenylation signals. The mol. wt of rat PCNA/cyclin was calculated to be 28 748. The deduced amino acid sequence and composition of rat PCNA/cyclin are in excellent agreement with the published data. Using the cDNA probe, two species of mRNA (1.1 and 0.98 kb) were detected in rat thymocyte RNA. Southern blot analysis of total human genomic DNA suggests that there is a single gene coding for PCNA/cyclin. The deduced amino acid sequence of rat PCNA/cyclin has a similarity with that of herpes simplex virus type-1 DNA binding protein.