Binding of Regulatory Subunits of Cyclic AMP-Dependent Protein Kinase to Cyclic CMP Agarose

We have cloned a gene (BCY1) from the yeast Saccharomyces cerevisiae that encodes a regulatory subunit of the cyclic AMP-dependent protein kinase. The encoded protein has a structural organization similar to that of the RI and RII regulatory subunits of the mammalian cyclic AMP-dependent protein kinase. Strains of S. cerevisiae with disrupted BCY1 genes do not display a cyclic AMP-dependent protein kinase in vitro, fail to grow on many carbon sources, and are exquisitely sensitive to heat shock and starvation.

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Exocytosis in Rat Parotid Acini After in vitro Treatment with Forskolin is Accompanied by Cellular Redistribution of Regulatory Subunits of Cyclic AMP-dependent Protein Kinase

Journal of Dental Research ◽

10.1177/00220345860650080401 ◽

1986 ◽

Vol 65 (8) ◽

pp. 1057-1063

Author(s):

M.I. Mednieks ◽

L.F. Cheng ◽

A.R. Hand

Keyword(s):

Protein Kinase ◽

Cyclic Amp ◽

Dependent Protein Kinase ◽

Regulatory Subunits ◽

Dependent Protein ◽

Rat Parotid ◽

In Vitro Treatment

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Regulatory subunits of cyclic AMP-dependent protein kinase: presence in granules and secretion by exocrine and endocrine cells

Journal of Cell Science ◽

10.1242/jcs.93.4.675 ◽

1989 ◽

Vol 93 (4) ◽

pp. 675-681

Author(s):

A.R. Hand ◽

M.I. Mednieks

Keyword(s):

Protein Kinase ◽

Cyclic Amp ◽

Endocrine Cells ◽

Polyclonal Antibodies ◽

Secretory Granules ◽

Regulatory Subunit ◽

Secretory Cells ◽

Dependent Protein Kinase ◽

Regulatory Subunits ◽

Dependent Protein

Cyclic AMP-dependent protein kinase (cAPK) is the intracellular mediator of signal transduction events involving the adenylate cyclase-cyclic AMP system. A monoclonal antibody (MAb BB1) to the type II regulatory subunit (RII) of cAPK was used in a post-embedding immunogold-labeling procedure to determine the ultrastructural localization of RII in several different secretory cells of the rat. Label was present in nuclei, especially over the heterochromatin, and in the cytoplasm, particularly in areas containing rough endoplasmic reticulum. Immunolabeled RII was also present in secretory granules of the parotid gland, exocrine and endocrine pancreas, seminal vesicle, anterior and intermediate pituitary, and intestinal endocrine cells. Photoaffinity labeling of parotid saliva, pancreatic and seminal fluids with the cyclic AMP analogue, 32P-labeled-8-azido-cyclic AMP, revealed the presence of cyclic AMP-binding proteins with electrophoretic mobilities similar to those of authentic cAPK regulatory subunits. These results confirm our previous observations on the localization of cAPK regulatory subunits in the rat parotid using polyclonal antibodies, and extend them to a number of other exocrine and endocrine cells. The apparent widespread occurrence of cAPK subunits in secretory granules and secretory fluids suggests that cAPK may be involved in specific intragranular regulatory and/or phosphorylation events, or that it has an unidentified extracellular function.

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Characterization of the protein kinase activities of human platelet supernatant and particulate fractions

Biochemical Journal ◽

10.1042/bj2180285 ◽

1984 ◽

Vol 218 (2) ◽

pp. 285-294 ◽

Cited By ~ 10

Author(s):

S E Salama ◽

R J Haslam

Keyword(s):

Protein Kinase ◽

Cyclic Amp ◽

Soluble Fraction ◽

Deae Cellulose ◽

Type I ◽

Type Ii ◽

Dependent Protein Kinase ◽

Regulatory Subunits ◽

Dependent Protein ◽

Triton X

After human platelets were lysed by freezing and thawing in the presence of EDTA, about 35% of the total cyclic AMP-dependent protein kinase activity was specifically associated with the particulate fraction. In contrast, Ca2+-activated phospholipid-dependent protein kinase was found exclusively in the soluble fraction. Photoaffinity labelling of the regulatory subunits of cyclic AMP-dependent protein kinase with 8-azido-cyclic [32P]AMP indicated that platelet lysate contained a 4-fold excess of 49 000-Da RI subunits over 55 000-Da RII subunits. The RI and RII subunits were found almost entirely in the particulate and soluble fractions respectively. Chromatography of the soluble fraction on DEAE-cellulose demonstrated a single peak of cyclic AMP-dependent activity with the elution characteristics and regulatory subunits characteristic of the type-II enzyme. A major enzyme peak containing Ca2+-activated phospholipid-dependent protein kinase was eluted before the type-II enzyme, but no type-I cyclic AMP-dependent activity was normally observed in the soluble fraction. The particulate cyclic AMP-dependent protein kinase and associated RI subunits were solubilized by buffers containing 0.1 or 0.5% (w/v) Triton X-100, but not by extraction with 0.5 M-NaCl, indicating that this enzyme is firmly membrane-bound, either as an integral membrane protein or via an anchor protein. DEAE-cellulose chromatography of the Triton X-100 extracts demonstrated the presence of both type-I cyclic AMP-dependent holoenzyme and free RI subunits. These results show that platelets contain three main protein kinase activities detectable with histone substrates, namely a membrane-bound type-I cyclic AMP-dependent enzyme, a soluble type-II cyclic AMP-dependent enzyme and Ca2+-activated phospholipid-dependent protein kinase, which was soluble in lysates containing EDTA.

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Dissociation of rabbit muscle cyclic AMP-dependent protein kinase into catalytic and regulatory subunits by p -chloromercuribenzoate and methylmercuric chloride

FEBS Letters ◽

10.1016/0014-5793(74)80476-x ◽

1974 ◽

Vol 48 (2) ◽

pp. 238-240 ◽

Cited By ~ 5

Author(s):

A.W. Murray ◽

M. Froscio ◽

A. Rogers

Keyword(s):

Protein Kinase ◽

Cyclic Amp ◽

Rabbit Muscle ◽

Dependent Protein Kinase ◽

Regulatory Subunits ◽

Methylmercuric Chloride ◽

Dependent Protein

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Cloning and characterization of BCY1, a locus encoding a regulatory subunit of the cyclic AMP-dependent protein kinase in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.7.4.1371-1377.1987 ◽

1987 ◽

Vol 7 (4) ◽

pp. 1371-1377 ◽

Cited By ~ 2

Author(s):

T Toda ◽

S Cameron ◽

P Sass ◽

M Zoller ◽

J D Scott ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Kinase ◽

Cyclic Amp ◽

Carbon Sources ◽

Regulatory Subunit ◽

Dependent Protein Kinase ◽

Yeast Saccharomyces Cerevisiae ◽

Regulatory Subunits ◽

Dependent Protein

We have cloned a gene (BCY1) from the yeast Saccharomyces cerevisiae that encodes a regulatory subunit of the cyclic AMP-dependent protein kinase. The encoded protein has a structural organization similar to that of the RI and RII regulatory subunits of the mammalian cyclic AMP-dependent protein kinase. Strains of S. cerevisiae with disrupted BCY1 genes do not display a cyclic AMP-dependent protein kinase in vitro, fail to grow on many carbon sources, and are exquisitely sensitive to heat shock and starvation.

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Association of catalytic and regulatory subunits of cyclic AMP-dependent protein kinase requires a negatively charged side group at a conserved threonine

Molecular and Cellular Biology ◽

10.1128/mcb.10.3.1066-1075.1990 ◽

1990 ◽

Vol 10 (3) ◽

pp. 1066-1075

Author(s):

L R Levin ◽

M J Zoller

Keyword(s):

Protein Kinase ◽

Cyclic Amp ◽

Yeast Cells ◽

Wild Type ◽

Dependent Protein Kinase ◽

Regulatory Subunits ◽

C Subunit ◽

Dependent Protein ◽

Tight Association

In Saccharomyces cerevisiae, as in higher eucaryotes, cyclic AMP (cAMP)-dependent protein kinase is a tetramer composed of two catalytic (C) subunits and two regulatory (R) subunits. In the absence of cAMP, the phosphotransferase activity of the C subunit is inhibited by the tight association with R. Mutation of Thr-241 to Ala in the C1 subunit of S. cerevisiae reduces the affinity of this subunit for the R subunit approximately 30-fold and results in a monomeric cAMP-independent C subunit. The analogous residue in the mammalian C subunit is known to be phosphorylated. Peptide maps of in vivo 32P-labeled wild-type C1 and mutant C1(Ala241) suggest that Thr-241 is phosphorylated in yeast cells. Substituting Thr-241 with either aspartate or glutamate partially restored affinity for the R subunit. Uncharged and positively charged residues substituted at this site resulted in C subunits that failed to associate with the R subunit. Replacement with the phosphorylatable residue serine resulted in a C subunit with wild-type affinity for the R subunit. Analysis of this protein revealed that it appears to be phosphorylated on Ser-241 in vivo. These data suggest that the interaction between R and C involves a negatively charged phosphothreonine at position 241 of yeast C1, which can be mimicked by either aspartate, glutamate, or phosphoserine.

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Localization of catalytic and regulatory subunits of cyclic AMP-dependent protein kinases in mitochondria from various rat tissues

Biochemical Journal ◽

10.1042/bj2700181 ◽

1990 ◽

Vol 270 (1) ◽

pp. 181-188 ◽

Cited By ~ 56

Author(s):

G Schwoch ◽

B Trinczek ◽

C Bode

Keyword(s):

Protein Kinase ◽

Cyclic Amp ◽

Protein Kinases ◽

Type I ◽

Matrix Space ◽

Dependent Protein Kinase ◽

Inner Membrane ◽

Regulatory Subunits ◽

C Subunit ◽

Dependent Protein

Observation and quantification of the catalytic subunit C of cyclic AMP-dependent protein kinases by immuno-gold electron microscopy suggested a high concentration of cyclic AMP-dependent protein kinases in mitochondria from liver, kidney, heart and skeletal muscle, pancreas, parotid gland and brain cells. The position of gold particles pointed to a localization in the inner membrane/matrix space. A similar distribution was obtained by immunolocalization of the cyclic AMP-dependent protein kinase regulatory subunits RI and RII in liver, pancreas and heart cells. The results indicated the presence of both the type I and the type II cyclic AMP-dependent protein kinases in mitochondria of hepatocytes, and the preferential occurrence of the type I protein kinase in mitochondria from exocrine pancreas and heart muscle. The immunocytochemical results were confirmed by immunochemical determination of cyclic AMP-dependent protein kinase subunits in fractionated tissues. Determinations by e.l.i.s.a. of the C-subunit in parotid gland cell fractions indicated about a 4-fold higher concentration of C-subunit in the mitochondria than in a crude 1200 g supernatant. Immunoblot analysis of subfractions from liver mitochondria supported the localization in situ of cyclic AMP-dependent protein kinases in the inner membrane/matrix space and suggested that the type I enzyme is anchored by its regulatory subunit to the inner membrane. In accordance with the immunoblot data, the specific activity of cyclic AMP-dependent protein kinase measured in the matrix fraction was about twice that measured in whole mitochondria. These findings indicate the importance of cyclic AMP-dependent protein kinases in the regulation of mitochondrial functions.

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