Selective activation of Akt1 by mammalian target of rapamycin complex 2 regulates cancer cell migration, invasion, and metastasis.

Mammalian target of rapamycin complex (mTORC) regulates a variety of cellular responses including proliferation, growth, differentiation and cell migration. In this study, we show that mammalian target of rapamycin complex 2 (mTORC2) regulates invasive cancer cell migration through selective activation of Akt1. Insulin-like growth factor-1 (IGF-1)-induced SKOV-3 cell migration was completely abolished by phosphatidylinositol 3-kinase (PI3K) (LY294002, 10 µM) or Akt inhibitors (SH-5, 50 µM), whereas inhibition of extracellular-regulated kinase by an ERK inhibitor (PD98059, 10 µM) or inhibition of mammalian target of rapamycin complex 1 (mTORC1) by an mTORC1 inhibitor (Rapamycin, 100 nM) did not affect IGF-1-induced SKOV-3 cell migration. Inactivation of mTORC2 by silencing Rapamycin-insensitive companion of mTOR (Rictor), abolished IGF-1-induced SKOV-3 cell migration as well as activation of Akt. However, inactivation of mTORC1 by silencing of Raptor had no effect. Silencing of Akt1 but not Akt2 attenuated IGF-1-induced SKOV-3 cell migration. Rictor was preferentially associated with Akt1 rather than Akt2, and over-expression of Rictor facilitated IGF-1-induced Akt1 activation. Expression of PIP3-dependent Rac exchanger1 (P-Rex1), a Rac guanosine exchange factor and a component of the mTOR complex, strongly stimulated activation of Akt1. Furthermore, knockdown of P-Rex1 attenuated Akt activation as well as IGF-1-induced SKOV-3 cell migration. Silencing of Akt1 or P-Rex1 abolished IGF-1-induced SKOV-3 cell invasion. Finally, silencing of Akt1 blocked in vivo metastasis, whereas silencing of Akt2 did not. Given these results, we suggest that selective activation of Akt1 through mTORC2 and P-Rex1 regulates cancer cell migration, invasion and metastasis.

Isolation and characterization of the bovine microsatellite loci.

Microsatellite loci were isolated using five repetitive probes for Korean native cattle. Eleven microsatellite loci were developed based on a biotin hybrid capture method, and enrichment of the genomic libraries (AAAT, TG, AG, T, and TGC repeats) was performed using Sau3AI adapters. The isolated markers were tested in two half-sib Korean cattle families and four imported breeds (Angus, Limousine, Holstein, and Shorthorn). Nine informative microsatellite loci were observed, and two microsatellite loci were revealed as monomorphic in Korean cattle. In the imported breeds, however, all of the markers were informative. In total, 213 alleles were obtained at the 11 loci across five breeds, and the average number of alleles found per locus, considering all populations, was 4.26. Heterozygosity was 0.71 (expected) and 0.57 (observed). The range of the polymorphic information content for the markers in all cattle populations was 0.43-0.69. Eleven percent of genetic variation was attributed to differentiation between populations as determined by the mean F (ST) values. The remaining 89% corresponded to differences among individuals. The isolated markers may be used to identify and classify the local breeds on a molecular basis.

Spin-labeling proton NMR study on aromatic amino acid residues in the guanine nucleotide binding site of human c-Ha-ras(1-171) protein.

A truncated human c-Ha-ras gene product, ras(1-171) protein, was prepared and chemically modified with maleimide spin-label (MSL). By trypsin digestion of the MSL-labeled ras(1-171) protein, MSL-labeled peptide fragments were isolated and sequenced. The cysteine residue in position 118 of the protein, but not the other cysteine residues, Cys-51 or Cys-80, was found to be specifically labeled by MSL. The ESR spectrum of the MSL-labeled ras(1-171) protein indicates that the MSL group attached to Cys-118 is strongly immobilized. Proton NMR spectra at 400-MHz were measured for this MSL-labeled ras(1-171) protein and also for a control sample of a labeled ras(1-171) protein whose MSL was reduced by sodium ascorbate. In the difference spectra for these two proteins, resonances of protons in the vicinity of the MSL group attached to Cys-118 of the ras(1-171) protein were observed. Thus, the MSL group was found to be in the vicinity of the protein-bound GDP. A phenylalanine residue and two histidine residues, which were characterized by 2D HOHAHA and DQF-COSY spectra, were also found to be in the vicinity of MSL. NOE and pH titration analyses indicate that this phenylalanine residue is close to the bound GDP and one of the two histidine residues. By carboxypeptidase digestion, the two histidine residues near MSL were identified as His-27 and His-94.(ABSTRACT TRUNCATED AT 250 WORDS)

Conformation of guanosine 5'-diphosphate as bound to a human c-Ha-ras mutant protein: a nuclear Overhauser effect study.

1H NMR spectra of a GDP/GTP-binding domain of human c-Ha-ras gene product (residues 1-171) in which glutamine-61 was replaced by leucine [ras(L61/1-171) protein] were analyzed. By one-dimensional and two-dimensional homonuclear Hartmann-Hahn spectroscopy and nuclear Overhauser effect (NOE) spectroscopy of the complex of the ras(L61/1-171) protein and GDP, the ribose H1', H2', H3', and H4' proton resonances of the bound GDP were identified. The guanine H8 proton resonance of the bound GDP was identified by substituting [8-2H]GDP for GDP. The dependences of the H1' and H8 proton resonance intensities on the duration of irradiation of the H1', H2', H3', and H8 protons were measured. By numerical simulation of these time-dependent NOE profiles, the conformation of the protein-bound GDP was elucidated; the guanosine moiety takes the anti form about the N-glycosidic bond with a dihedral angle of chi = -124 +/- 2 degrees and the ribose ring takes the C2'-endo form. Such an analysis of the conformation of a guanine nucleotide as bound to a GTP-binding protein will be useful for further studies on the molecular mechanism of the conformational activation of ras proteins on ligand substitution of GDP with GTP.

Biosynthesis of a protein containing a nonprotein amino acid by Escherichia coli: L-2-aminohexanoic acid at position 21 in human epidermal growth factor.

Endeavoring to develop a method to biosynthesize proteins substituted with nonprotein amino acids, we attempted the incorporation of L-2-aminohexanoic acid (Ahx) into human epidermal growth factor (hEGF). Escherichia coli YK537 strain harboring plasmid pTA1522, which has the phoA promoter-phoA signal peptide-hEGF gene, was used. Cells were cultured first in high-phosphate medium and then, for induction of the hEGF-encoding gene, transferred to low-phosphate medium containing Ahx (0.25 mg/ml). hEGF and Ahx-substituted hEGF, [Ahx21]hEGF, secreted into the periplasm were recovered. After treatment with H2O2, [Ahx21]-hEGF was clearly separated from methionine-oxidized hEGF by one-step reverse-phase HPLC. Substitution of the methionine residue of hEGF with Ahx was confirmed by the amino acid analysis of [Ahx21]hEGF. The three biological activities of [Ahx21]hEGF were the same as those of hEGF. From the successful production of [Ahx21]hEGF, a basic strategy was established for preparing proteins substituted with nonprotein amino acid (alloprotein). Induction of the phoA promoter of pho regulon and secretion of the product to the periplasm may depress heat shock-like responses and subsequent hydrolysis of the product by cytoplasmic protease.

Degradation of epidermal growth factor receptors by cathepsin L-like protease: inhibition of the degradation by c-Ha-ras gene products.

Extract of NIH3T3 mouse fibroblasts contains a protease which can cleave epidermal growth factor receptor (EGF receptor). This protease was tentatively named cathepsin X and purified to near homogeneity. The characteristics of cathepsin X were similar to those of cathepsin L and the proteolytic activity of cathepsin X was inhibited by c-Ha-ras gene products.

Inhibition of cathepsin L-induced degradation of epidermal growth factor receptors by c-Ha-ras gene products.

The inhibitory activities of c-Ha-ras gene products (p21s) toward several cysteine proteinases have been investigated. The activity of cathepsin L was inhibited by p21s most effectively while those of cathepsin B and papain were slightly inhibited by p21s. p21s did not show any inhibitory activity toward cathepsin H. In order to connect the protease-inhibitor activity of p21s with cell growth, the degradation of epidermal growth factor receptors (EGF-receptors) was investigated. EGF-receptors were preferentially cleaved by cathepsin L but not by cathepsin B or H. The cleavage of EGF-receptors by cathepsin L was inhibited by p21s dose-dependently. These results raise the possibility that p21s can suppress the degradation of growth-related proteins such as EGF-receptors and thereby affect cell growth.

Degradation of a cAMP-binding protein is inhibited by human c-Ha-ras gene products.

Incubation of the particulate fraction of cell extract prepared from NIH3T3 mouse fibroblasts resulted in preferential proteolytic degradation of a cAMP-binding protein. The proteolysis was inhibited by human c-Ha-ras gene products produced by Escherichia coli. The proteolysis was observed at pH 6 to 7, and inhibited by antipain and leupeptin. These results suggest that cAMP-binding proteins might be cleaved by thiol proteinases. In fact, c-Ha-ras gene products were proved to inhibit the cathepsin B-like activity present in the particulate fraction.

c-Ha-ras gene products are potent inhibitors of cathepsins B and L.

c-Ha-ras proteins produced by Escherichia coli inhibited the activities of cathepsins B and L which had been partially purified from rat kidney. Furthermore, amino acid sequence homology between c-Ha-ras proteins and thiol proteinase inhibitors has been found.