14‐3‐3 Proteins regulate K2P5.1 surface expression on T lymphocytes

K_2P5.1 channels (also called TASK‐2 or Kcnk5) have already been shown to be relevant in the pathophysiology of autoimmune disease because they are known to be upregulated on peripheral and central T lymphocytes of multiple sclerosis (MS) patients. Moreover, overexpression of K_2P5.1 channels in vitro provokes enhanced T‐cell effector functions. However, the molecular mechanisms regulating intracellular K_2P5.1 channel trafficking are unknown so far. Thus, the aim of the study is to elucidate the trafficking of K_2P5.1 channels on T lymphocytes. Using mass spectrometry analysis, we have identified 14‐3‐3 proteins as novel binding partners of K_2P5.1 channels. We show that a non‐classical 14‐3‐3 consensus motif (R‐X‐X‐pT/S‐x) at the channel's C‐terminus allows the binding between K_2P5.1 and 14‐3‐3. The mutant K_2P5.1/S266A diminishes the protein‐protein interaction and reduces the amplitude of membrane currents. Application of a non‐peptidic 14‐3‐3 inhibitor (BV02) significantly reduces the number of wild‐type channels in the plasma membrane, whereas the drug has no effect on the trafficking of the mutated channel. Furthermore, blocker application reduces T‐cell effector functions. Taken together, we demonstrate that 14‐3‐3 interacts with K_2P5.1 and plays an important role in channel trafficking.      

Deoxycytidine kinase-deficient mutants of Chinese hamster ovary cells are hypersensitive to DNA alkylating agents

Chinese hamster ovary cell strains deficient in deoxycytidine kinase activity were selected by isolating mutants resistant to high concentrations of the analogue arabinosyl cytosine. Mutants isolated were deficient in the pool of dCTP, supporting earlier a suggestion that the deoxycytidine kinase may play a role in the turnover and maintenance of the dCTP pool. Consistent with earlier observations that increased intracellular levels of dTTP relative to dCTP lead to increased sensitivy to monofunctional DNA alkylating agents, deoxycytidine kinase-deficient mutants showed a 2–5-fold increase in sensitivity to the cytotoxic and mutagenic effects of one agent, ethyl methanesulfonate (EMS). The survival of the two kinase-deficient strains after mutagen treatment was clearly related to dCTP level as the strain with lowest dCTP was most sensitive to EMS. Thus hypersensitivity to this class of DNA damaging agents can result from cellular mutations decreasing the intracellular level of dCTP.     

Suppression of T lymphocyte functions by human C3 fragments. I. Inhibition of human T cell proliferative responses by a kallikrein cleavage fragment of human iC3b

Cleavage of human iC3b by kallikrein isolated from human plasma generates a fragment, C3d-K, which is capable of inhibiting mitogen-, antigen-, and alloantigen-induced T lymphocyte proliferation. Native C3, C3a, C3b, and C3c-K had no effect on lymphocyte proliferative responses. In addition to being a potent suppressor of mitogen- and antigen-induced proliferation, C3d-K is capable of inducing leukocytosis in both mice and rabbits. Intravenous injection of C3d-K, but not C3, C3a, C3b, or C3c-K, results in a twofold to threefold increase in the number of circulating leukocytes. Thus, C3d-K exhibits two apparently independent functions, namely suppression of T cell proliferation and leukocytosis. Cleavage of iC3b by kallikrein results in the production of only two fragments. The larger fragment, C3c-K, is 144,000 m.w. and has a chemical structure analogous to that of C3c obtained from the cleavage of C3 by trypsin or elastase. The smaller fragment, C3d-K, is 41,000 m.w. and contains the metastable binding site of C3. It is through this site located in the C3d region of the molecule that C3 attaches covalently to target cells. Analysis of the amino terminal region of C3d-K provided a sequence that fails to overlap with any sequence yet reported for other characterized C3 fragments, including C3d originally obtained from elastase digestion. A revised model of the C3 molecule is proposed, with locations of the C3e and C3d fragments assigned on the basis of chemical analyses.     

Structural alterations of the aprt locus induced by deoxyribonucleoside triphosphate pool imbalances in Chinese hamster ovary cells.

Mutants induced at the adenine phosphoribosyl transferase (aprt) locus by dTTP or dCTP pool imbalances were examined for alterations in genomic DNA sequences. No observable changes were detected by Southern blot analysis of most mutant DNAs, suggesting induction of base pair alterations or other events below our level of detection (approximately 30 base pairs). However, in a few strains (11 from a total collection of 125 mutant cell strains), we were able to localize these events to restriction endonuclease recognition sequences when the mutations resulted in the loss or gain of a particular site. The distribution of lost or gained sites in aprt-deficient mutants induced by the two types of pool imbalances clearly varied, with those occurring in a mutator strain with increased dCTP clustering at one end of the aprt gene. Mutants induced by dTTP also revealed novel events: multiple restriction site modifications in a small region of the aprt gene in one mutant and a small (approximately 50 base pairs) insertion or duplication of DNA sequences. As in previous studies, very few deletion or insertion mutants were detected at the aprt locus. The significance of these findings in terms of the known biochemical and genetic consequences of these pool imbalances is discussed.     

DNA sequence determination of gamma-radiation-induced mutations of the hamster aprt locus

From a collection of 85 independent gamma-radiation hamster aprt- mutants, 27 having no major structural alterations were analysed at the nucleotide level by using the polymerase chain reaction to amplify mutant exons and then directly sequencing the double-stranded products. The majority of these mutations were simple base substitutions of all types, particularly transversions (11/27). Frameshifts and small deletions were also induced. The 'spectrum' of mutations produced by gamma-radiation was not significantly different from that occurring spontaneously at this locus. Differences with respect to the target and structure of frameshifts and small deletions occurring in the two collections were apparent.     

Reassignment of residue 122 in the myoglobin from the killer whale,Orcinus orca

Summary The complete amino acid sequence of the major component myoglobin from killer whale,Orcinus orca, was determined by automated Edman degradation. In this study residue 122 was found to be glutamic acid instead of glutamine as was originally reported (Castillo et al. 1977). This reassignment affects the phylogenetic relationship of killer whale myoglobin with the myoglobins from other closely related cetacean species and also affects studies concerned with the physical parameters of the protein.This is the 114th paper in a series dealing with coordination complexes and catalytic properties of proteins and related substances. For the preceding paper see Neireiter et al. 1979. This work was supported by Public Health Service Research Grant HL-05556     

Covalent linkage between ribonucleic Acid primer and deoxyribonucleic Acid product of the avian myeloblastosis virus deoxyribonucleic Acid polymerase

Initiation of deoxyribonucleic acid (DNA) synthesis by the avian myeloblastosis virus DNA polymerase was previously suggested to involve a ribonucleic acid (RNA) primer, the initial product being a DNA molecule joined by a phosphodiester bond to the RNA primer. The existence and nature of such an RNA-DNA joint was investigated by assaying for transfer of a ³²P atom from an α-³²P-deoxyribonucleotide to a 2′(3′)-ribonucleotide after alkaline hydrolysis of the polymerase product. Such a transfer was observed, but only from α-³²P-deoxyadenosine triphosphate and only to 2′(3′)-adenosine monophosphate. This same transfer was observed in both the endogenous DNA polymerase reaction of purified virions and the reconstructed reaction of purified DNA polymerase plus purified 60 to 70S viral RNA. These results indicate a high level of specificity for the initiation process and support the idea of a low-molecular-weight initiator RNA as part of the 60 to 70S RNA complex.     

Loss of heterozygosity and base substitution at the APRT locus in mismatch-repair-proficient and -deficient colorectal carcinoma cell lines.

We determined the nature of mutations occurring at the autosomal APRT locus in mismatch-repair-proficient and -deficient colorectal carcinoma cell lines. The analysis of mutations that result in APRT deficiency in a mismatch-repair-deficient strain of DLD-1 heterozygous for this locus enabled us to measure the rate of loss of the wild-type gene through deletion, recombination, or gene conversion as well as the rate of point mutation. The overall rate of mutation at the APRT locus in DLD-1 was elevated 100-fold compared with the mismatch-repair-proficient colorectal carcinoma cell line SW620. Loss of heterozygosity (LOH) at APRT accounted for only 4 to 9% of mutant strains derived from DLD-1, indicating a rate for these types of events of 4 x 10(-7) to 9 x 10(-7). In SW620 the rate of LOH at APRT was about 10-fold higher. LOH was not found at polymorphic markers within the same chromosome subband as APRT, indicating that only a limited portion of the chromosome was affected by these alterations. Chromosome painting of SWS620 mutants revealed that the loss of APRT occurred together with a substantial portion of the long arm of chromosome 16. Differences in the nature of base substitutions at APRT (e.g., the proportion of mutations resulting from transitions or transversions) in these tumor cell lines were also detected. There was also an important similarity---the presence of a mutant APRT gene with multiple base substitutions that may be the result of some sort of error-prone DNA synthesis.     

Increased rate of base substitution in a hamster mutator strain obtained during serial selection for gene amplification.

The pattern of mutations produced by a mutator gene (obtained during serial selection for amplification of the dihydrofolate reductase [dhfr] locus) shows a pronounced shift from that found in wild-type cells. The rate of certain types of base substitutions (particularly transitions) is dramatically increased, while gene rearrangements constitute a lower proportion of mutations. These data suggest a lower fidelity of the replication process in the mutator strain.