Facilitated geranylgeranylation of shrimp ras-encoded p25 fusion protein by the binding with guanosine diphosphate

A cDNA was isolated from the shrimp Penaeus japonicus by homology cloning. Similar to the mammalian Ras proteins, this shrimp hepatopancreas cDNA encodes a 187-residue polypeptide whose predicted amino acid sequence shares 85% homology with mammalian KB-Ras proteins and demonstrates identity in the guanine nucleotide binding domains. Expression of the cDNA of shrimp in Escherichia coli yielded a 25-kDa polypeptide with positive reactivity toward the monoclonal antibodies against Ras of mammals. As judged by nitrocellulose filtration assay, the specific GTP binding activity of ras-encoded p25 fusion protein was approximately 30,000 units/mg of protein, whereas that of GDP was 5,000 units/mg of protein. In other words, the GTP bound form of ras-encoded p25 fusion protein prevails. Fluorography analysis demonstrated that the prenylation of both shrimp Ras-GDP and shrimp Ras-GTP by protein geranylgeranyltransferase I of shrimp Penaeus japonicus exceeded that of nucleotide-free form of Ras by 10-fold and four-fold, respectively. That is, the protein geranylgeranyl transferase I prefers to react with ras-encoded p25 fusion protein in the GDP bound form.     

Disrupting the geranylgeranylation at the C-termini of the shrimp Ras by depriving guanine nucleotide binding at the N-terminal

In order to assess the effects of guanine nucleotide binding on the geranylgeranylation at the CAAX box of the shrimp Ras, we experimented with the shrimp Penaeus japonicus Ras (S-Ras) which is geranylgeranylated at the C-termini, shares 85% homology with mammalian K(B)-Ras protein and demonstrates identity in the guanine nucleotide binding domains (Huang C-F, Chuang N-N. 1999. J Exp Zool 283:510-521). Several point mutations in the S-ras gene were generated at codons 12 (G12V), 61 (Q61K), and 116 (N116I). The bacterially expressed mutant S-Ras proteins, G12V and Q61K, were bound with GTP without hydrolysis. In contrast, the mutant S-Ras N116I was defective in its ability to bind any guanine nucleotides. Autoradiography studies showed that the purified shrimp protein geranylgeranyltransferase I (Lin R-S, Chuang N-N. 1998. J Exp Zool 281:565-573) was unable to catalyze the transfer of [(3)H]-geranylgeranylpyrophosphate to this mutant N116I but very competently caused the geranylgeranylation of GTP-locked mutants, G12V and Q61K. These results demonstrate that the geranylgeranylation at the CAAX box of the shrimp Ras protein requires the proper binding of guanine nucleotide at its N-terminal region. J. Exp. Zool. 286:441-449, 2000.     

GTPase-activating protein SH2-SH3 domains induce gene expression in a Ras-dependent fashion.

The p21ras GTPase-activating protein (GAP) is thought to function as both a negative regulator and a downstream target of p21ras. Here, we have investigated the role of GAP by using a transient expression assay with a fos luciferase reporter plasmid. We used GAP deletion mutants that lack the domain involved in interaction with p21ras and encode essentially only the SH2-SH3 domains. When these GAP deletion mutants were expressed, we observed a marked induction of fos promoter activity similar to induction by activated p21ras. Expression of a full-length GAP construct had no effect on the activity of the fos promoter. Activation of the fos promoter by these GAP SH2-SH3 regions was inhibited by cotransfection of a dominant inhibitory mutant of p21ras, Ras(Asn-17). Thus, the induction of gene expression by GAP SH2-SH3 domains is dependent on p21ras activity. Moreover, induction of fos promoter activity by GAP SH2-SH3 domains is increased severalfold after cotransfection of an activated mutant of p21ras, Ras(Leu-61), or insulin stimulation of A14 cells, both leading to an increase in the levels of GTP-bound p21ras. The combined effect of Ras(Leu-61) and the GAP deletion mutants was not inhibited by Ras(Asn-17), indicating that GAP SH2-SH3 domains do not function to activate endogenous p21ras but cooperate with another signal coming from active p21ras. These data suggest that GAP SH2-SH3 domains serve to induce gene expression by p21ras but that additional signals coming from p21ras are required for them to function.     

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.     

CDC43 and RAM2 encode the polypeptide subunits of a yeast type I protein geranylgeranyltransferase.

The question regarding the identity of the alpha and beta subunits of the yeast type I protein geranylgeranyltransferase was explored using prokaryotic expression of candidate genes. The Saccharomyces cerevisiae CDC43 and RAM2 genes were expressed in Escherichia coli and cell extracts examined for the ability to transfer [3H]geranylgeranyl diphosphate to an appropriate CaaX protein substrate. Individual expression of each gene yielded no activity; however, co-expression of the two genes resulted in high levels of [3H] geranylgeranyl incorporation into the substrate protein Ras-Cys-Val-Val-Leu. The activity was partially purified yielding approximately 12,600 units/liter. The partially purified enzyme geranylgeranylated the Ras-Cys-Val-Val-Leu, Ras-Cys-Ala-Ile-Leu, Ras-Cys-Ile-Ile-Leu, and Ras-Cys-Thr-Ile-Leu substrates but not the Ras-Cys-Val-Leu-Ser or Ras-Ser-Val-Leu-Ser substrates. The protein geranylgeranyltransferase was highly specific for geranylgeranyl diphosphate and poorly transferred farnesyl. The recombinant enzyme was indistinguishable from the native type I geranylgeranyltransferase in yeast extracts. As has been reported for the protein farnesyltransferase, the yeast type I protein geranylgeranyltransferase is also a magnesium-requiring, zinc metalloenzyme. Interestingly, the recombinant enzyme functioned with calcium as the only divalent cation, although addition of zinc increased calcium-dependent activity 2-fold.     

Activation of Ras by insulin in 3T3 L1 cells does not involve GTPase-activating protein phosphorylation.

Insulin-induced differentiation of 3T3 L1 cells to adipocytes can be mimicked by the expression of transfected ras oncogenes but not of the tyrosine-kinase oncogenes src and trk. Expression of two different transfected, dominant inhibitory ras mutants resulted in significant inhibition of insulin-induced differentiation, suggesting that endogenous Ras proteins are mediators of insulin signaling in these cells. Exposure of untransfected 3T3 L1 cells to insulin resulted in significant formation of the active Ras.GTP complex, at levels comparable with those resulting from exposure to platelet-derived growth factor. However, whereas exposure of the same cells to platelet-derived growth factor resulted in significant tyrosine phosphorylation of the p21ras GTPase-activating protein (GAP), insulin-treated cells did not show any detectable levels of de novo GAP tyrosine phosphorylation. Interestingly, insulin caused tyrosine phosphorylation of the p62 polypeptide coprecipitated with GAP by anti-GAP antibodies. Insulin-induced activation of cytosolic MAP kinase activity in untransfected 3T3 L1 cells was also mimicked by Ras expression (in the absence of insulin) in the same cells transfected with an inducible ras construct. These results confirm that Ras proteins participate in insulin signaling pathways in these mammalian cells and indicate that activation of cytosolic MAP kinases is an early event occurring downstream from Ras activation. However, tyrosine phosphorylation of GAP appears not to be a significant upstream regulatory event in the activation of Ras by insulin.     

DISTINCTIVE LOCALIZATION OF GROUP 3 LATE EMBRYOGENESIS ABUNDANT SYNTHESIZING CELLS DURING BRINE SHRIMP DEVELOPMENT

Despite numerous studies on late embryogenesis abundant (LEA) proteins, their functions, roles, and localizations during developmental stages in arthropods remain unknown. LEA proteins protect crucial proteins against osmotic stress during the development and growth of various organisms. Thus, in this study, fluorescence in situ hybridization was used to determine the crucial regions protected against osmotic stress as well as the distinctive localization of group 3 (G3) LEA⁺ cells during brine shrimp development. Several cell types were found to synthesize G3 LEA RNA, including neurons, muscular cells, APH‐1⁺ cells, and renal cells. The G3 LEA⁺ neuronal cell bodies outside of the mushroom body projected their axonal bundles to the central body, but those inside the mushroom body projected their axonal bundles toward the deutocerebrum without innervating the central body. The cell bodies inside the mushroom body received axons of the G3 LEA⁺ sensory cells at the medial ventral cup of the nauplius eye. Several glands were found to synthesize G3 LEA RNA during the nauplius stages of brine shrimp, including the sinus, antennal I and II, salt, and three ectodermal glands. This study provides the first demonstration of the formation of G3 LEA⁺ sinus glands at the emergence stages of brine shrimp. These results suggest that G3 LEA protein is synthesized in several cell types. In particular, specific glands play crucial roles during the emergence and nauplius stages of brine shrimp.     

Increased p21(ras) activity in human fibroblasts transduced with survivin enhances cell proliferation

Survivin is critically involved in mitosis and when overexpressed enhances the activity of the Aurora B kinase, a serine-threonine kinase belonging to the family of oncogenic Aurora/IpI1p-related kinases. Both proteins interact with Ras GTPase-activating protein suggesting an impact on the Ras pathway. This study aimed at defining the role of survivin in proliferation and potential transformation of cells. When survivin was overexpressed in normal human lung fibroblasts, the characteristic track lanes of fibroblasts were disturbed and the rate of cell proliferation was increased. An enhanced level of p21(ras) mRNA and protein expression and concomitant rise in levels of activated p21(ras) were observed. Despite increased proliferation cell survival remained dependent on serum and cells were not able to form colonies in soft agar assays. These data suggest that overexpression of survivin increases cell growth but, despite the increase in active p21(ras), is not sufficient to transform primary cells. Yet, in addition to its anti-apoptotic function it might contribute to the accelerated growth of tumour cells by increasing p21(ras) activity.     

癌基因Ha-ras在转化和未转化细胞中表达和调控的差异

以前的工作曾用人胃癌基因Ha-ras转化了大鼠全胚细胞系Ratl细胞,得到转化细胞Rat3-3。克隆了Ha-ras癌基因6.6kb及其上游区2.5kb DNA片段,并发现2.5kb有Alu重复顺序,说明这个片段是来源于人胃癌细胞,虽族观察到p21蛋白编码12位点突变,我们又发现转化的Rat3-3细胞的Ha-ras mRNA水平比未转化的Rat1高大约五倍;通过DNase I超敏感实验证明只有转化细胞核中的Ha-ras基因对DNaseI敏感,1μg/mL的DNaseI就有明显的降解,而未转化细胞Rat1细胞核的Ha-ras基因在15μg/mL的DNaseI中也未发现有任何降解;另外还发现转化细胞核有一种能为Ha-ras基因上游区2.5kb特异结合的核蛋白,分子量大约35kD,此核蛋白不能与6.6kb Ha-ras基因本身结合,在未转化细胞中未发现此蛋白。从这些结果推测,癌基因Ha-ras的活化,除了点突变外,还可能存在另一条活化途径,即它的上游区可能有类似增强子的调控区。     

DNA-dependent RNA-polymerases from Artemia embryos. characterization of polymerases I and II from nauplius larvae.

Partial purification and characterization of DNA-dependent RNA-polymerases from nauplius larvae of the brine shrimp, Artemia salina, are described. Fractionation of solubilized RNA-polymerases on columns of DEAE-cellulose yielded partially purified preparations of RNA polymerases I and II. The properties of these enzymes were found to be similar to properties of corresponding enzymes from other animal sources. A significant change in the relative amounts of polymerases I and II occurs between 36 and 72 hr of development. Polymerase activity obtained from 36-hr nauplii consisted of approximately equal amounts of polymerases I and II, whereas polymerase II accounted for more than 80% of the activity recovered from 72-hr nauplii. Total polymerase activity was lower at 72 than at 36 hr. The significance of these changes in relation to the decrease in RNA synthesis in vivo that occurs after 36 hr is discussed.