Several lines of CHO cells stably overexpressing protein kinase C (PKC) subspecies to various extents were established by the DNA-mediated transfer. Upon treatment with phorbol 12-myristate 13-acetate, the growth of the cells expressing the PKC-6 subspecies was markedl inhibited, whereas cell lines expressing PKC-a, PKC-IH, and PKC-C subspecies were not sign tly affected. Flow cytometric analysis indicated that all cell les overexpressing PKC-8 subspecies accumulated in G2/M phase in response to phorbol 12-myristate 13-acetate. In these arrete cells, dikaryons were predominant, implying that phorbol ester-induced inhibition of cell division is specific to telophase. These results suggest PKC-6 subspecies may play a role in the normal cell cycle progression.Protein kinase C (PKC) appears to play crucial roles in signal transduction leading to cell growth and differentiation. This enzyme was first shown to be activated by diacylglycerol generated by the receptor-mediated hydrolysis of inositol phospholipids and has been identified as a major receptor of tumor-promoting phorbol esters (1). Molecular cloning and biochemical studies have revealed that the PKC family is a large family, consisting ofat least eight subspecies, a, 3PI, /11, y, 8, E, A, and q (PKC-L) in mammalian'tissues (2-5). These subspecies show subtly different enzymological properties and distinct tissue distribution. Molecular genetic approaches have also been used to explore the role of this PKC family in cell proliferation by expressing defined enzyme subspecies, such as PKC-a, PKC-P, or PKC-y, in mammalian cells (6-11). In some cell lines stably overproducing PKC subspecies, growth promotion such as enhanced growth rates, increased saturation densities, and anchorageindependent growth has been observed (6,7,9,10 MATERIALS AND METHODS Construction of Expression Plasmids. cDNAs for rat PKC-a, -/311, -8, and -C subspecies were excised from plasmids pTB755, pTB708, pTB808, and pTB949, respectively (14-17), by complete or partial digestion with EcoP. These cDNAs were then separately inserted downstream of the Abelson murine leukemia virus long terminal repeat in plasmid pTB399 (18) by replacing the interleukin 2 sequence. The long terminal repeat-PKC cDNA segments between Sal I and Cla I sites ofthese plasmids were transferred to the same sites of plasmid pTB348 (18), which carried the hamster dihydrofolate reductase (DHFR) cDNA under the control of the simian virus 40 early promoter. The resulting expression plasmids for the a, . 3II,8, and ; subspecies are referred to as pTB789, pTB705, pTB1322, and pTB1324, respectively. All the procedures were done as described (19).Transfection, Selection, and Gene Amplification. DHFR-CHO cells (20) were transfected with each of the expression constructs by the calcium phosphate-DNA coprecipitation method (21). DHFR+ transformants were selected by colony formation in Dulbecco's modified Eagle's medium (DMEM) containing 10%6 (vol/vol) dialyzed fetal bovine serum and proline (35 ,Ag/ml). Gene amplificatio...
A promoter with the consensus sequence(TTGACA and TATAAT) at -35 and -10 regions was constructed, and the distance between the two consensus sequences was independently altered at two restriction sites. In an in vitro transcription system, a maximum activity was observed at the spacer length of 17 base-pairs regardless of the sites of space adjustment. Evidence was also provided that the start point of transcription was fixed by the consensus sequence at the -10 region.
A Ca2+ binding site like an EF-hand motif was designed and created in human lysozyme by replacing both Gln-86 and Ala-92 with aspartic adds by site-directed mutagenesis. The mutant human lysozyme (D86/92-lysozyme) was expressed and secreted by yeast. One Ca2+ was found to bind one molecule of the purified protein with the binding constant 5.0 x 10' M-1. The enzymatic activity of holo-D86/92-lysozyme against glycol chitin at 40'C was 2-fold higher than that of the native lysozyme. Maximal activity of the holo-D86/92-lysozyme was observed at 80"C, where its relative activity normalized to the value at 40°C was 6-fold and 17-fold higher than those of the native and apoenzymes, respectively. (2) and hydrophobic (3) and electrostatic interactions (4,5).In some proteins, metal ions are known to act as stabilizers through their electrostatic interactions. One example is alactalbumin, the tertiary structure of which is stabilized by
The unfolding and refolding kinetics of six proline mutants of the human lysozyme (h-lysozyme) were carried out and compared to that of the wild-type protein. Our results show that the slow refolding phase observed in the h-lysozyme refolding kinetics cannot be ascribed to proline isomerization reactions. The h-lysozyme contains two proline residues at positions 71 and 103, both in the trans conformation in the native state. The refolding kinetics of the P71G/P103G mutant, in which both prolines have been replaced by a glycine, were found to be similar to those of the wild-type protein. The same slow phase amplitude of about 10% was found for both proteins, and the slow phase rate constants were also identical within experimental error. Other mutants such as P103G or P71G, in which only one of the two prolines has been replaced by a glycine, and A47P with its three prolines, gave identical slow refolding phases. The X-ray structure analysis and scanning microcalorimetric study of each protein (Herning et al., unpublished experiments) have confirmed that none of the considered mutations affects significantly protein structure and that no major changes in protein stability were brought about by these mutations. Therefore, comparison of the properties of the mutant and wild-type proteins is legitimate. Interestingly, the refolding kinetics of the V110P mutant, in which a proline residue has been introduced at position 110 (N-terminus of an alpha-helix), were clearly triphasic. For this mutant an additional very slow phase with properties similar to those expected from the proline hypothesis was detected. Equilibrium denaturation studies were conducted for each protein, and the refolding pathway of h-lysozyme is partly presented. We also discuss the effect of proline mutations on the energetics of the folding pathway of the h-lysozyme in water.
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