Isolation of multiple classes of mutants of CHO cells resistant to cyclic AMP

Gottesman, Michael M.; LeCam, Alphonse; Bukowski, Maria A.; Pastan, Ira

doi:10.1007/bf01538695

Cited by 91 publications

(51 citation statements)

References 34 publications

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“…All ofthe effects ofcAMP seem to be mediated through its activation of cAMP-dependent protein kinase (30,31 32). Although these studies provide some guidance as to the possible physiological effects of EF, the responses of particular tissues and animals remain to be determined.…”

Section: Resultsmentioning

confidence: 99%

Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic AMP concentrations of eukaryotic cells.

Leppla¹

1982

Proc. Natl. Acad. Sci. U.S.A.

872

658

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Anthrax toxin is composed ofthree proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF). These proteins individually cause no known physiological effects in animals but in pairs produce two toxic actions. Injection of PA with LF causes death ofrats in 60 min, whereas PA with EF causes edema in the skin of rabbits and guinea pigs. The mechanisms of action of-these proteins have not beendetermined. It is shown here that EF is an adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] produced by Bacllus anthracis in an inactive form. Activation occurs upon contact with a heat-stable eukaryotic cell material. The specific activity of the resulting adenylate cyclase nearly, equals that of the most active known cyclase. In Chinese hamster ovary cells exposed to PA and EF, cAMP concentrations increase without a lag to values about 200-fold above normal, remain high in the continued presence of toxin, and decrease rapidly after its removal. The increase in cAMP is completely blocked by excess LF. It is. suggested that PA interacts with cells to form a receptor system by which EF and perhaps LF gain access to the cytoplasm.

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Section: Resultsmentioning

confidence: 99%

Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic AMP concentrations of eukaryotic cells.

Leppla¹

1982

Proc. Natl. Acad. Sci. U.S.A.

872

658

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“…CHO cells as well as Lec2 and Lec8 mutant CHO cells were cultured as described previously (21). Cell extracts from neuroblastoma cell lines (SKNAS and SKNSH) were gifts from Dr. Maria Tsokos, NCI, National Institutes of Health.…”

Section: Methodsmentioning

confidence: 99%

Sialylated Core 1 O-Glycans Influence the Sorting of Pmel17/gp100 and Determine Its Capacity to Form Fibrils

Valencia

Rouzaud

Julien

et al. 2007

Journal of Biological Chemistry

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Pmel17 is a melanocyte/melanoma-specific protein that is essential for the maturation of melanosomes to form mature, fibrillar, and pigmented organelles. Recently, we reported that the less glycosylated form of Pmel17 (termed iPmel17) is sorted via the plasma membrane in a manner distinct from mature Pmel17 (termed mPmel17), which is sorted directly to melanosomes. To clarify the mechanism(s) underlying the distinct processing and sorting of Pmel17, we generated a highly specific antibody (termed ␣PEP25h) against an epitope within the repeat domain of Pmel17 that is sensitive to changes in O-glycosylation. ␣PEP25h recognizes only iPmel17 and allows analysis of the processing and sorting of iPmel17 when compared with ␣PEP13h, an antibody that recognizes both iPmel17 and mPmel17. Our novel findings using ␣PEP25h demonstrate that iPmel17 differs from mPmel17 not only in its sensitivity to endoglycosidase H, but also in the content of core 1 O-glycans modified with sialic acid. This evidence reveals that iPmel17 is glycosylated differently in the Golgi and that it is sorted through the secretory pathway. Analysis of Pmel17 processing in glycosylation-deficient mutant cells reveals that Pmel17 lacking the correct addition of sialic acid and galactose loses the ability to form fibrils. Furthermore, we show that addition of sialic acid affects the stability and sorting of Pmel17 and reduces pigmentation. Alterations in sialyltransferase activity and substrates differ between normal and transformed melanocytes and may represent a critical change during malignant transformation.The human PMEL17 gene encodes two type I integral membrane proteins that are translated from alternatively spliced mRNAs. Those two proteins, known as Pmel17 and gp100, differ as to whether they include a 7-amino acid motif in the membrane proximal region of the luminal domain (1-3). Pmel17 is critical to the formation of an internal fibrillar matrix within stage II melanosomes that is essential to the maturation of that organelle and the subsequent synthesis and deposition of melanin within it. Pmel17 is synthesized as a 68.6-kDa protein, as deduced from the cloned cDNA (2). It is rapidly and quantitatively glycosylated to an "immature" ϳ95-kDa form (termed iPmel17), but only a limited amount is further processed to a "mature" ϳ115-kDa form (termed mPmel17), due at least in part to N-glycosylation at five possible sites (4). mPmel17 can then be cleaved by a furin-like proprotein convertase that generates two fragments, one small fragment (cPmel17, ϳ26 kDa) containing the transmembrane (TM) 3 and the C-terminal domains, and the other larger fragment contains the N-terminal region (5). Those fragments can remain bound to each other by S-S bonds (5, 6). However, the iPmel17 formed remains fully endoglycosidase H (EndoH)-sensitive (4) and is significantly more stable over time than either of the other forms of Pmel17 (mPmel17 and cPmel17) (5).N-Linked core glycosylation begins in the ER and is essential for the function, stability, folding, intrace...

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“…For some experiments, large quantities of cells were grown in suspension by using 100-ml glass bottles containing 50 ml of complete medium and incubated in a gyratory shaker (New Brunswick Scientific Co., Inc.) at 37°C and 160 rpm. The growth rates of cells in culture (11) and the drug resistance of various cell lines (5) were measured as previously described. 4 ,ug of taxol per ml, 5% dimethyl sulfoxide, and 0.1% aprotinin.…”

Section: Methodsmentioning

confidence: 99%

Mutations affecting assembly and stability of tubulin: evidence for a nonessential beta-tubulin in CHO cells.

Boggs¹,

Cabral²

1987

Mol. Cell. Biol.

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Eight strains of Chinese hamster ovary (CHO) cells having an assembly-defective ,-tubulin were found among revertants of strain Cmd 4, a mutant with a conditional lethal mutation in a 0-tubulin gene (F. Cabral, M. E. Sobel, and M. M. Gottesman, Cell 20:29-36, 1980). The altered j3-tubulins in these strains have electrophoretically silent alterations or, in some cases, an increase or a decrease in apparent molecular weight based on their migration in two-dimensional gels. The identity of these variant proteins as P-tubulin was confirmed by peptide mapping, which also revealed the loss of distinct methionine-containing peptides in the assembly-defective I8-tubulins of lower apparent molecular weight. The altered mobility of these I-tubulin polypeptides was not the result of a posttranslational modification, since the altered species could be labeled in very short incubations with [35SImethionine and were found among in vitro-translated polypeptides by using purified mRNA. In at least one strain, an altered DNA restriction fragment could be demonstrated, suggesting that an alteration occurred in one of the structural genes for 0-tubulin. Assembly-defective ,-tubulin was unstable and turned over with a half-life of only 1 to 2 h in exponentially growing cells. This rapid degradation of a tubulin gene product resulted in approximately 30% lower steady-state levels of both a-and 0-tubulin yet did not affect the growth rate of the cells or the distribution of the microtubules as judged by immunofluorescence microscopy. These results argue that CHO cells possess a ,-tubulin gene product that is not essential for survival.Microtubule assembly is a complex process in which oaand 3-tubulin heterodimers interact end to end and laterally to form hollow tubes 25 nm in diameter. Much of our understanding of microtubule assembly has come from in vitro experiments pioneered by Weisenberg (29). These and subsequent studies have demonstrated a requirement for GTP, a pH optimum of 6.6 to 6.9, and a minimum concentration of 0.2 to 1 mg of tubulin per ml. In addition, enhanced polymerization occurs in the presence of glycerol, dimethyl sulfoxide, polyethylene glycol, D20, polycations, and microtubule-associated proteins; but polymerization is inhibited by Ca2+ (10). More recent studies using proteolytic cleavage of tubulin by subtilisin have shown that a highly acidic C-terminal 4-kilodalton (kDa) fragment can be released from both oa-and ,B-tubulin, resulting in a tubulin species which assembles at a lower critical concentration, fails to bind microtubule-associated proteins, and is insensitive to Ca2+ These in vitro studies have greatly expanded our knowledge of factors that affect tubulin assembly and have provided valuable information on the function of particular subunit domains on tubulin. Comparable in vivo techniques for approaching similar questions have not been well developed. In particular, a genetic approach toward studying assembly has not yet been used, even though such an approach has proved to be invaluable for under...

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Isolation of multiple classes of mutants of CHO cells resistant to cyclic AMP

Cited by 91 publications

References 34 publications

Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic AMP concentrations of eukaryotic cells.

Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic AMP concentrations of eukaryotic cells.

Sialylated Core 1 O-Glycans Influence the Sorting of Pmel17/gp100 and Determine Its Capacity to Form Fibrils

Mutations affecting assembly and stability of tubulin: evidence for a nonessential beta-tubulin in CHO cells.

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