scholarly journals Genetic and biochemical analysis of the adenylyl cyclase-associated protein, cap, in Schizosaccharomyces pombe.

1992 ◽  
Vol 3 (2) ◽  
pp. 167-180 ◽  
Author(s):  
M Kawamukai ◽  
J Gerst ◽  
J Field ◽  
M Riggs ◽  
L Rodgers ◽  
...  
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Adenylyl Cyclase ◽  
Biochemical Analysis ◽  
Slow Growth ◽  
Proper Function ◽  
Adenylyl Cyclase Activity ◽  
Yeast Saccharomyces Cerevisiae ◽  
Terminal Domain ◽  
Cellular Responsiveness ◽  
Entire Gene

We have identified, cloned, and studied a gene, cap, encoding a protein that is associated with adenylyl cyclase in the fission yeast Schizosaccharomyces pombe. This protein shares significant sequence homology with the adenylyl cyclase-associated CAP protein in the yeast Saccharomyces cerevisiae. CAP is a bifunctional protein; the N-terminal domain appears to be involved in cellular responsiveness to RAS, whereas loss of the C-terminal portion is associated with morphological and nutritional defects. S. pombe cap can suppress phenotypes associated with deletion of the C-terminal CAP domain in S. cerevisiae but does not suppress phenotypes associated with deletion of the N-terminal domain. Analysis of cap disruptants also mapped the function of cap to two domains. The functional loss of the C-terminal region of S. pombe cap results in abnormal cellular morphology, slow growth, and failure to grow at 37 degrees C. Increases in mating and sporulation were observed when the entire gene was disrupted. Overproduction of both cap and adenylyl cyclase results in highly elongated large cells that are sterile and have measurably higher levels of adenylyl cyclase activity. Our results indicate that cap is required for the proper function of S. pombe adenylyl cyclase but that the C-terminal domain of cap has other functions that are shared with the C-terminal domain of S. cerevisiae CAP.

Comparison of human CAP and CAP2, homologs of the yeast adenylyl cyclase-associated proteins

1994 ◽  
Vol 107 (6) ◽  
pp. 1671-1678 ◽  
Author(s):  
G. Yu ◽  
J. Swiston ◽  
D. Young
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Schizosaccharomyces Pombe ◽  
Adenylyl Cyclase ◽  
Normal Function ◽  
Temperature Sensitive ◽  
Related Protein ◽  
Adenylyl Cyclase Activity ◽  
Terminal Domain ◽  
Associated Proteins ◽  
Terminal Domains

We previously reported the identification of human CAP, a protein that is related to the Saccharomyces cerevisiae and Schizosaccharomyces pombe adenylyl cyclase-associated CAP proteins. The two yeast CAP proteins have similar functions: the N-terminal domains are required for the normal function of adenylyl cyclase, while loss of the C-terminal domains result in morphological and nutritional defects that are unrelated to the cAMP pathways. We have amplified and cloned cDNAs from a human glioblastoma library that encode a second CAP-related protein, CAP2. The human CAP and CAP2 proteins are 64% identical. Expression of either human CAP or CAP2 in S. cerevisiae cap- strains suppresses phenotypes associated with deletion of the C-terminal domain of CAP, but does not restore hyper-activation of adenylyl cyclase by RAS2val19. Similarly, expression of either human CAP or CAP2 in S. pombe cap- strains suppresses the morphological and temperature-sensitive phenotypes associated with deletion of the C-terminal domain of CAP in this yeast. In addition, expression of human CAP, but not CAP2, suppresses the propensity to sporulate due to deletion of the N-terminal domain of CAP in S. pombe. This latter observation suggests that human CAP restores normal adenylyl cyclase activity in S. pombe cap- cells. Thus, functional properties of both N-terminal and C-terminal domains are conserved between the human and S. pombe CAP proteins.

Anti-Cdc25 antibodies inhibit guanyl nucleotide-dependent adenylyl cyclase of Saccharomyces cerevisiae and cross-react with a 150-kilodalton mammalian protein

1992 ◽  
Vol 12 (6) ◽  
pp. 2653-2661
Author(s):  
E Gross ◽  
I Marbach ◽  
D Engelberg ◽  
M Segal ◽  
G Simchen ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Protein ◽  
Adenylyl Cyclase ◽  
Gene Product ◽  
Cyclase Activity ◽  
Yeast Saccharomyces Cerevisiae ◽  
Terminal Domain ◽  
Yeast Homolog ◽  
Cdc25 Protein ◽  
Beta Galactosidase

The CDC25 gene product of the yeast Saccharomyces cerevisiae has been shown to be a positive regulator of the Ras protein. The high degree of homology between yeast RAS and the mammalian proto-oncogene ras suggests a possible resemblance between the mammalian regulator of Ras and the regulator of the yeast Ras (Cdc25). On the basis of this assumption, we have raised antibodies against the conserved C-terminal domain of the Cdc25 protein in order to identify its mammalian homologs. Anti-Cdc25 antibodies raised against a beta-galactosidase-Cdc25 fusion protein were purified by immunoaffinity chromatography and were shown by immunoblotting to specifically recognize the Cdc25 portion of the antigen and a truncated Cdc25 protein, also expressed in bacteria. These antibodies were shown both by immunoblotting and by immunoprecipitation to recognize the CDC25 gene product in wild-type strains and in strains overexpressing Cdc25. The anti-Cdc25 antibodies potently inhibited the guanyl nucleotide-dependent and, approximately 3-fold less potently, the Mn(2+)-dependent adenylyl cyclase activity in S. cerevisiae. The anti-Cdc25 antibodies do not inhibit cyclase activity in a strain harboring RAS2Val-19 and lacking the CDC25 gene product. These results support the view that Cdc25, Ras2, and Cdc35/Cyr1 proteins are associated in a complex. Using these antibodies, we were able to define the conditions to completely solubilize the Cdc25 protein. The results suggest that the Cdc25 protein is tightly associated with the membrane but is not an intrinsic membrane protein, since only EDTA at pH 12 can solubilize the protein. The anti-Cdc25 antibodies strongly cross-reacted with the C-terminal domain of the Cdc25 yeast homolog, Sdc25. Most interestingly, these antibodies also cross-reacted with mammalian proteins of approximately 150 kDa from various tissues of several species of animals. These interactions were specifically blocked by the beta-galactosidase-Cdc25 fusion protein.

Identification of a human cDNA encoding a protein that is structurally and functionally related to the yeast adenylyl cyclase-associated CAP proteins

1992 ◽  
Vol 12 (11) ◽  
pp. 5033-5040
Author(s):  
H Matviw ◽  
G Yu ◽  
D Young
Keyword(s):  
Polymerase Chain Reaction ◽  
Adenylyl Cyclase ◽  
Polymerase Chain Reaction Method ◽  
Proper Function ◽  
Chain Reaction ◽  
Terminal Domain ◽  
Human Cdna ◽  
Acid Protein ◽  
Polymerase Chain ◽  
Terminal Domains

The adenylyl cyclases of both Saccharomyces cerevisiae and Schizosaccharomyces pombe are associated with related proteins named CAP. In S. cerevisiae, CAP is required for cellular responses mediated by the RAS/cyclic AMP pathway. Both yeast CAPs appear to be bifunctional proteins: the N-terminal domains are required for the proper function of adenylyl cyclase, while loss of the C-terminal domains results in morphological and nutritional defects that appear to be unrelated to the cAMP pathways. Expression of either yeast CAP in the heterologous yeast suppresses phenotypes associated with loss of the C-terminal domain of the endogenous CAP but does not suppress loss of the N-terminal domain. On the basis of the homology between the two yeast CAP proteins, we have designed degenerate oligonucleotides that we used to detect, by the polymerase chain reaction method, a human cDNA fragment encoding a CAP-related peptide. Using the polymerase chain reaction fragment as a probe, we isolated a human cDNA clone encoding a 475-amino-acid protein that is homologous to the yeast CAP proteins. Expression of the human CAP protein in S. cerevisiae suppresses the phenotypes associated with loss of the C-terminal domain of CAP but does not suppress phenotypes associated with loss of the N-terminal domain. Thus, CAP proteins have been structurally and, to some extent, functionally conserved in evolution between yeasts and mammals.

scholarly journals Genetic and biochemical analysis of the adenylyl cyclase of Schizosaccharomyces pombe.

1991 ◽  
Vol 2 (2) ◽  
pp. 155-164 ◽  
Author(s):  
M Kawamukai ◽  
K Ferguson ◽  
M Wigler ◽  
D Young
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Adenylyl Cyclase ◽  
Regulatory Protein ◽  
Wild Type ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Amino Terminal Domain ◽  
Camp Levels ◽  
Adh1 Promoter

The adenylyl cyclase gene, cyr1, of Schizosaccharomyces pombe has been cloned. We have begun an analysis of the function and regulation of adenylyl cyclase by disrupting this gene and by over-expressing all or parts of this gene in various strains. cyr1- strains are viable and contain no measurable cyclic AMP. They conjugate and sporulate under conditions that normally inhibit wild-type strains. Strains containing the cyr1 coding sequences transcribed from the strong adh1 promoter contain greatly elevated adenylyl cyclase activity, as measured in vitro, but only modestly elevated cAMP levels. Such strains conjugate and sporulate less frequently than wild-type cells upon nutrient limitation. Strains which carry the wild-type cyr1 gene but that also express high levels of the amino terminal domain of adenylyl cyclase behave much like cyr1-strains, suggesting that the amino terminal domain can bind a positive regulator. A protein that copurifies with the adenylyl cyclase of S. pombe cross-reacts to antiserum raised against the S. cerevisiae adenylyl cyclase-associated regulatory protein, CAP.

scholarly journals Identification of a human cDNA encoding a protein that is structurally and functionally related to the yeast adenylyl cyclase-associated CAP proteins.

1992 ◽  
Vol 12 (11) ◽  
pp. 5033-5040 ◽  
Author(s):  
H Matviw ◽  
G Yu ◽  
D Young
Keyword(s):  
Polymerase Chain Reaction ◽  
Adenylyl Cyclase ◽  
Polymerase Chain Reaction Method ◽  
Proper Function ◽  
Chain Reaction ◽  
Terminal Domain ◽  
Human Cdna ◽  
Acid Protein ◽  
Polymerase Chain ◽  
Terminal Domains

The adenylyl cyclases of both Saccharomyces cerevisiae and Schizosaccharomyces pombe are associated with related proteins named CAP. In S. cerevisiae, CAP is required for cellular responses mediated by the RAS/cyclic AMP pathway. Both yeast CAPs appear to be bifunctional proteins: the N-terminal domains are required for the proper function of adenylyl cyclase, while loss of the C-terminal domains results in morphological and nutritional defects that appear to be unrelated to the cAMP pathways. Expression of either yeast CAP in the heterologous yeast suppresses phenotypes associated with loss of the C-terminal domain of the endogenous CAP but does not suppress loss of the N-terminal domain. On the basis of the homology between the two yeast CAP proteins, we have designed degenerate oligonucleotides that we used to detect, by the polymerase chain reaction method, a human cDNA fragment encoding a CAP-related peptide. Using the polymerase chain reaction fragment as a probe, we isolated a human cDNA clone encoding a 475-amino-acid protein that is homologous to the yeast CAP proteins. Expression of the human CAP protein in S. cerevisiae suppresses the phenotypes associated with loss of the C-terminal domain of CAP but does not suppress phenotypes associated with loss of the N-terminal domain. Thus, CAP proteins have been structurally and, to some extent, functionally conserved in evolution between yeasts and mammals.

scholarly journals Adenylyl cyclase activity of the fission yeast Schizosaccharomyces pombe is not regulated by guanyl nucleotides

FEBS Letters ◽  
1990 ◽  
Vol 261 (2) ◽  
pp. 413-418 ◽  
Author(s):  
David Engelberg ◽  
Enrique Poradosu ◽  
Giora Simchen ◽  
Alexander Levitzki
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Adenylyl Cyclase ◽  
Fission Yeast ◽  
Cyclase Activity ◽  
Adenylyl Cyclase Activity

scholarly journals Anti-Cdc25 antibodies inhibit guanyl nucleotide-dependent adenylyl cyclase of Saccharomyces cerevisiae and cross-react with a 150-kilodalton mammalian protein.

1992 ◽  
Vol 12 (6) ◽  
pp. 2653-2661 ◽  
Author(s):  
E Gross ◽  
I Marbach ◽  
D Engelberg ◽  
M Segal ◽  
G Simchen ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Protein ◽  
Adenylyl Cyclase ◽  
Gene Product ◽  
Cyclase Activity ◽  
Yeast Saccharomyces Cerevisiae ◽  
Terminal Domain ◽  
Yeast Homolog ◽  
Cdc25 Protein ◽  
Beta Galactosidase

The CDC25 gene product of the yeast Saccharomyces cerevisiae has been shown to be a positive regulator of the Ras protein. The high degree of homology between yeast RAS and the mammalian proto-oncogene ras suggests a possible resemblance between the mammalian regulator of Ras and the regulator of the yeast Ras (Cdc25). On the basis of this assumption, we have raised antibodies against the conserved C-terminal domain of the Cdc25 protein in order to identify its mammalian homologs. Anti-Cdc25 antibodies raised against a beta-galactosidase-Cdc25 fusion protein were purified by immunoaffinity chromatography and were shown by immunoblotting to specifically recognize the Cdc25 portion of the antigen and a truncated Cdc25 protein, also expressed in bacteria. These antibodies were shown both by immunoblotting and by immunoprecipitation to recognize the CDC25 gene product in wild-type strains and in strains overexpressing Cdc25. The anti-Cdc25 antibodies potently inhibited the guanyl nucleotide-dependent and, approximately 3-fold less potently, the Mn(2+)-dependent adenylyl cyclase activity in S. cerevisiae. The anti-Cdc25 antibodies do not inhibit cyclase activity in a strain harboring RAS2Val-19 and lacking the CDC25 gene product. These results support the view that Cdc25, Ras2, and Cdc35/Cyr1 proteins are associated in a complex. Using these antibodies, we were able to define the conditions to completely solubilize the Cdc25 protein. The results suggest that the Cdc25 protein is tightly associated with the membrane but is not an intrinsic membrane protein, since only EDTA at pH 12 can solubilize the protein. The anti-Cdc25 antibodies strongly cross-reacted with the C-terminal domain of the Cdc25 yeast homolog, Sdc25. Most interestingly, these antibodies also cross-reacted with mammalian proteins of approximately 150 kDa from various tissues of several species of animals. These interactions were specifically blocked by the beta-galactosidase-Cdc25 fusion protein.

Calcium Entry Attenuates Adenylyl Cyclase Activity

CHEST Journal ◽  
1995 ◽  
Vol 107 (5) ◽  
pp. 1420-1425 ◽  
Author(s):  
William B. Abernethy ◽  
John F. Butterworth ◽  
Richard C. Prielipp ◽  
Jian P. Leith ◽  
Gary P. Zaloga
Keyword(s):  
Adenylyl Cyclase ◽  
Calcium Entry ◽  
Cyclase Activity ◽  
Adenylyl Cyclase Activity

scholarly journals The Capacity to Secrete Insulin Is Dose-Dependent to Extremely High Glucose Concentrations: A Key Role for Adenylyl Cyclase

Metabolites ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 401
Author(s):  
Katherine M. Gerber ◽  
Nicholas B. Whitticar ◽  
Daniel R. Rochester ◽  
Kathryn L. Corbin ◽  
William J. Koch ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Adenylyl Cyclase ◽  
Human Islets ◽  
Adenylyl Cyclase Activity ◽  
World Record ◽  
Glucokinase Activator ◽  
Amplifying Pathway ◽  
Dose Dependent ◽  
Adenylyl Cyclase Inhibitor ◽  
Cyclase Activator

Insulin secretion is widely thought to be maximally stimulated in glucose concentrations of 16.7-to-30 mM (300-to-540 mg/dL). However, insulin secretion is seldom tested in hyperglycemia exceeding these levels despite the Guinness World Record being 147.6 mM (2656 mg/dL). We investigated how islets respond to 1-h exposure to glucose approaching this record. Insulin secretion from human islets at 12 mM glucose intervals dose-dependently increased until at least 72 mM glucose. Murine islets in 84 mM glucose secreted nearly double the insulin as in 24 mM (p < 0.001). Intracellular calcium was maximally stimulated in 24 mM glucose despite a further doubling of insulin secretion in higher glucose, implying that insulin secretion above 24 mM occurs through amplifying pathway(s). Increased osmolarity of 425-mOsm had no effect on insulin secretion (1-h exposure) or viability (48-h exposure) in murine islets. Murine islets in 24 mM glucose treated with a glucokinase activator secreted as much insulin as islets in 84 mM glucose, indicating that glycolytic capacity exists above 24 mM. Using an incretin mimetic and an adenylyl cyclase activator in 24 mM glucose enhanced insulin secretion above that observed in 84 mM glucose while adenylyl cyclase inhibitor reduced stimulatory effects. These results highlight the underestimated ability of islets to secrete insulin proportionally to extreme hyperglycemia through adenylyl cyclase activity.

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