Identification of structural elements involved in the interaction of simian virus 40 small tumor antigen with protein phosphatase 2A

SV40 small tumor antigen (small-t) was used as a model to identify structural elements involved in the interactions between regulatory proteins and protein phosphatase 2A (PP2A). Using mutant proteins and synthetic peptides, we identified a small domain within small-t that is a major site for interaction with the dimeric form of PP2A. A series of small-t truncation mutants identified a region surrounding the first of two conserved cysteine clusters that was critical for interaction with PP2A. These mutants also identified additional regions of small-t that contribute to high affinity interaction. Deletion of residues 110-119, which encompass the first cysteine cluster, resulted in a protein that failed to bind to PP2A. Synthetic peptides that contained residues 105-122 of small-t blocked binding of small-t to PP2A. These peptides also inhibited the phosphatase activity of PP2A in a manner analogous to full-length small-t. The active small-t peptides adopt a beta-strand structure that was essential for high affinity interaction with the PP2A dimer. Based on circular dichroism measurements, the same cysteine cluster-containing peptides that bind to PP2A also interact with zinc. Interaction with zinc required the conserved cysteines but was not required for interaction with PP2A.     

Replication protein A is the major single-stranded DNA binding protein detected in mammalian cell extracts by gel retardation assays and UV cross-linking of long and short single-stranded DNA molecules

Band shift and UV cross-linking assays were used to analyze the major single-stranded DNA (ssDNA) binding activity in lysates of primate and rodent cells. The ssDNA binding activity behaved chromatographically similar to that of replication protein A (RP-A), a multisubunit protein containing three polypeptides of molecular mass 70, 34, and 14 kDa. A 70-kDa protein was found to harbor the ssDNA binding activity when UV cross-linked to long ssDNA or to oligonucleotide probes. Monoclonal antibodies against the 70- and the 34-kDa subunits produced super-gel-shift patterns, demonstrating that the reactive protein is indeed RP-A and that the retarded native binding complex included both subunits. RP-A displayed oligonucleotide-specific binding dependent on oligomer length. Increasing oligonucleotide length led to the formation of slow migrating complexes harboring multiple RP-A molecules, suggesting that an interval of about 20-30 bases is required for the binding of RP-A molecules. While similar binding activity was detected in cell extracts derived from proliferating and quiescent cells, a sharp decline in ssDNA binding activity was observed in the SV40-transformed Chinese hamster cell line 631 following UV irradiation. The nature of this decrease in activity and its possible effect on DNA replication is discussed.     

Covalent catalysis in nucleotidyl transfer. A KTDG motif essential for enzyme-GMP complex formation by mRNA capping enzyme is conserved at the active sites of RNA and DNA ligases

Vaccinia virus RNA capping enzyme, a heterodimer of 95- and 31-kDa subunits, catalyzes transfer of GMP from GTP to the 5'-diphosphate terminus of RNA via a covalent enzyme-guanylate intermediate. The GMP residue is attached to the 95-kDa subunit through a phosphoamide bond to the epsilon-amino group of a lysine residue. The amino acid sequence of the large subunit includes a lysine-containing motif, Tyr-X-X-X-Lys260-Thr-Asp-Gly, that is conserved in the RNA guanylyltransferases encoded by Shope fibroma virus and Saccharomyces cerevisiae. The KXDG motif is also encountered at the sites of covalent adenylylation of bacteriophage T4 RNA ligase and mammalian DNA ligase I (Thogerson, H. C., Morris, H. R., Rand, K. N., and Gait, M. J. (1985) Eur. J. Biochem. 147, 325-329; Tomkinson, A. E., Totty, N. F., Ginsburg, M., and Lindahl, T. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 400-404). We find that conservative amino acid substitutions at three out of four positions within the KTDG sequence of vaccinia capping enzyme either prevent or strongly inhibit enzyme-guanylate formation. The conserved motif is therefore an essential component of the guanylyltransferase domain. Lys260 is implicated as the active site. Comparison of the sequences of capping enzymes and polynucleotide ligases from diverse sources suggests that KX(D/N)G may be a signature element for covalent catalysis in nucleotidyl transfer.     

Lck associates with and is activated by Kit in a small cell lung cancer cell line: inhibition of SCF-mediated growth by the Src family kinase inhibitor PP1

At least 70% of small cell lung cancers (SCLCs) express the Kit receptor tyrosine kinase and its ligand, stem cell factor (SCF). In an effort to define the signal transduction pathways activated by Kit in SCLC, we focused on Src family kinases and, in particular, Lck, a Src-related tyrosine kinase that is expressed in hemopoietic cells and certain tumors, including SCLC. SCF treatment of the H526 cell line induced a physical association between Kit and Lck that, in vitro, was dependent on phosphorylation of the juxtamembrane domain of Kit. Stimulation of Kit with recombinant SCF resulted in a rapid 3-6-fold increase in the specific activity of Lck, which was similar in magnitude to the activation of Lck resulting from the cross-linking of the T-cell receptor complex of Jurkat cells. Lck activity peaked by 5 min after SCF addition, and the elevated activity persisted for at least 30 min in the presence of SCF, with kinetics similar to the activation of mitogen-activated protein kinase. PP1, an inhibitor of Src family kinases with selectivity for Lck, completely inhibited SCF-mediated growth but had little effect on insulin-like growth factor-I-mediated growth. PP1 antagonized both SCF-mediated proliferation and inhibition of apoptosis. PP1 had no effect on Kit kinase activity but was shown to block total Lck activity by at least 90% by immune complex kinase assay. Low levels of Src, Hck, and Yes were also expressed in the H526 cell line; only Yes showed a consistent increase in specific activity, which was also inhibited by PP1 following SCF treatment. These data demonstrate that, in the H526 SCLC cell line, Lck and, possibly, Yes are downstream of Kit in a signal transduction pathway; the inhibition by PP1 of SCF-mediated proliferation and inhibition of apoptosis suggests that Src family kinases are intermediates in the signaling pathways that regulate these processes.     

A 28-fold increase in secretory protein synthesis is associated with DNA puff activity in the salivary gland of Bradysia hygida (Diptera, Sciaridae)

When the first group of DNA puffs is active in the salivary gland regions S1 and S3 of Bradysia hygida larvae, there is a large increase in the production and secretion of new salivary proteins demonstrable by [3H]-Leu incorporation. The present study shows that protein separation by SDS-PAGE and detection by fluorography demonstrated that these polypeptides range in molecular mass from about 23 to 100 kDa. Furthermore, these proteins were synthesized mainly in the S1 and S3 salivary gland regions where the DNA puffs C7, C5, C4 and B10 are conspicuous, while in the S2 region protein synthesis was very low. Others have shown that the extent of amplification for DNA sequences that code for mRNA in the DNA puffs C4 and B10 was about 22 and 10 times, respectively. The present data for this group of DNA puffs are consistent with the proposition that gene amplification is necessary to provide some cells with additional gene copies for the production of massive amounts of proteins within a short period of time.     

Decreased frequency of rearrangement due to the synergistic effect of nucleotide changes in the heptamer and nonamer of the recombination signal sequence of the V kappa gene A2b, which is associated with increased susceptibility of Navajos to Haemophilus influenzae type b disease

Navajos and genetically related populations have a 10-fold increased incidence of Haemophilus influenzae type b (Hib) disease compared with control populations. The Vkappa gene A2 is used to encode the majority of anti-Hib Abs, and these are the highest affinity anti-Hib Abs. Navajos carry a different allele of the A2 gene segment (A2b) that is defective in its ability to undergo V-J recombination. The A2b allele has only three nucleotide changes from the commonly occurring A2a allele, two of which could potentially affect its ability to recombine. In this study we used two independent in vitro assays to test whether the nucleotide change found in the A2b promoter and/or in the A2b recombination signal sequence (RSS) might be responsible for the decrease in recombination frequency observed in vivo. Using a luciferase reporter gene assay, we found no significant difference between A2a and A2b promoter activities. However, the competition recombination substrate assay showed a 4.5-fold reduction in the relative frequency of recombination of the A2b RSS compared with A2a. We show that this decreased frequency is due to a synergistic effect of the unique nucleotide change present in the heptamer of the A2b RSS and the shared nucleotide change present in the nonamer of both A2b and A2a. This in vitro relative frequency of rearrangement is not significantly different from that observed in vivo; therefore, the A2b RSS is probably the factor associated with the increased susceptibility to Hib disease among individuals carrying the A2b allele.