scholarly journals Evolution of the eukaryotic protein kinases as dynamic molecular switches

2012 ◽  
Vol 367 (1602) ◽  
pp. 2517-2528 ◽  
Author(s):  
Susan S. Taylor ◽  
Malik M. Keshwani ◽  
Jon M. Steichen ◽  
Alexandr P. Kornev
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinases ◽  
Molecular Switches ◽  
Regulatory Subunits ◽  
Eukaryotic Protein ◽  
Activation Segment ◽  
Dynamic Assembly ◽  
Dependent Protein ◽  
Eukaryotic Protein Kinases

Protein kinases have evolved in eukaryotes to be highly dynamic molecular switches that regulate a plethora of biological processes. Two motifs, a dynamic activation segment and a GHI helical subdomain, distinguish the eukaryotic protein kinases (EPKs) from the more primitive eukaryotic-like kinases. The EPKs are themselves highly regulated, typically by phosphorylation, and this allows them to be rapidly turned on and off. The EPKs have a novel hydrophobic architecture that is typically regulated by the dynamic assembly of two hydrophobic spines that is usually mediated by the phosphorylation of an activation loop phosphate. Cyclic AMP-dependent protein kinase (protein kinase A (PKA)) is used as a prototype to exemplify these features of the PKA superfamily. Specificity in PKA signalling is achieved in large part by packaging the enzyme as inactive tetrameric holoenzymes with regulatory subunits that then are localized to macromolecular complexes in close proximity to dedicated substrates by targeting scaffold proteins. In this way, the cell creates discrete foci that most likely represent the physiological environment for cyclic AMP-mediated signalling.

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

1990 ◽  
Vol 270 (1) ◽  
pp. 181-188 ◽  
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.

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

1987 ◽  
Vol 7 (4) ◽  
pp. 1371-1377 ◽  
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.

scholarly journals Selective regulation of the amount of catalytic subunit of cyclic AMP-dependent protein kinases during isoprenaline-induced growth of the rat parotid gland

1987 ◽  
Vol 248 (1) ◽  
pp. 243-250 ◽  
Author(s):  
G Schwoch
Keyword(s):  
Cyclic Amp ◽  
Dna Synthesis ◽  
Protein Kinases ◽  
Catalytic Subunit ◽  
Regulatory Subunits ◽  
Rat Parotid Gland ◽  
C Subunit ◽  
Dependent Protein ◽  
Rat Parotid ◽  
Stimulation Of

Stimulation of growth of the rat parotid gland by repeated injection of the beta-agonist isoprenaline led to a significant decrease in the activity of cyclic AMP-dependent protein kinases. Immunochemical quantification of the catalytic (C) and regulatory (RI and RII) subunits of the cyclic AMP-dependent protein kinases type I and type II revealed a loss of 65% of the immunochemically measurable amount of catalytic subunit C. The amount of the regulatory subunits, however, remained constant. The observed decrease in C-subunit was not due to a translocation of the molecule to cellular membranes or to an inhibiting effect of the heat-stable inhibitor of cyclic AMP-dependent protein kinases. A selective decrease in only the C-subunit was also observed after a brief exposure to isoprenaline leading to the stimulation of DNA synthesis. Under these conditions, the decrease was observed at the onset of DNA synthesis (17 h after injection), but not at the the time of an earlier small cyclic AMP peak (13 h after injection) or at the time of maximal DNA synthesis (24 h after injection). The results indicate that the amount of the catalytic subunit of cyclic AMP-dependent protein kinases can be regulated independently from that of the regulatory subunits. The time-limited occurrence of the specific change in the amount of the C-subunit suggests that such a regulation is of physiological significance and that it may participate in cyclic AMP-mediated events involved in the control of cellular proliferation.

scholarly journals Antiserum against the catalytic subunit of adenosine 3′:5′-cyclic monophosphate-dependent protein kinase. Reactivity towards various protein kinases

1980 ◽  
Vol 192 (1) ◽  
pp. 223-230 ◽  
Author(s):  
G Schwoch ◽  
A Hamann ◽  
H Hilz
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinases ◽  
Rat Liver ◽  
Catalytic Subunit ◽  
Type Ii ◽  
Dependent Protein Kinase ◽  
Subunit C ◽  
Bovine Heart ◽  
Dependent Protein

An antiserum against the catalytic subunit C of cyclic AMP-dependent protein kinase, isolated from bovine heart type II protein kinase, was produced in rabbits. Reaction of the catalytic subunit with antiserum and separation of the immunoglobulin G fraction by Protein A-Sepharose quantitatively removed the enzyme from solutions. Comparative immunotitration of protein kinases showed that the amount of antiserum required to eliminate 50% of the enzymic activity was identical for pure catalytic subunit, and for holoenzymes type I and type II. The reactivity of the holoenzymes with the antiserum was identical in the absence or the presence of dissociating concentrations of cyclic AMP. Most of the holoenzyme (type II) remains intact when bound to the antibodies as shown by quantification of the regulatory subunit in the supernatant of the immunoprecipitate. Titration with the antibodies also revealed the presence of a cyclic AMP-independent histone kinase in bovine heart protein kinase I preparations obtained by DEAE-cellulose chromatography. Cyclic AMP-dependent protein kinase purified from the particulate fraction of bovine heart reacted with the antiserum to the same degree as the soluble enzyme, whereas two cyclic AMP-independent kinases separated from the particle fraction neither reacted with the antiserum nor influenced the reaction of the antibodies with the cyclic AMP-dependent protein kinase. Immunotitration of the protein kinase catalytic subunit C from rat liver revealed that the antibodies had rather similar reactivities towards the rat liver and the bovine heart enzyme. This points to a relatively high degree of homology of the catalytic subunit in mammalian tissues and species. Broad applicability of the antiserum to problems related to cyclic AMP-dependent protein kinases is thus indicated.

scholarly journals Immunofluorescent localization of cyclic nucleotide-dependent protein kinases on the mitotic apparatus of cultured cells.

1980 ◽  
Vol 87 (2) ◽  
pp. 336-345 ◽  
Author(s):  
C L Browne ◽  
A H Lockwood ◽  
J L Su ◽  
J A Beavo ◽  
A L Steiner
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinases ◽  
Cyclic Gmp ◽  
Cyclic Nucleotides ◽  
Mitotic Spindle ◽  
Cyclic Nucleotide ◽  
Dependent Protein Kinase ◽  
Mitotic Apparatus ◽  
Dependent Protein

Cyclic nucleotides and cyclic nucleotide-dependent protein kinases have been implicated in the regulation of cell motility and division, processes that depend on the cell cytoskeleton. To determine whether cyclic nucleotides or their kinases are physically associated with the cytoskeleton during cell division, fluorescently labeled antibodies directed against cyclic AMP, cyclic GMP, and the cyclic nucleotide-dpendent protein kinases were used to localize these molecules in mitotic PtK1 cells. Both the cyclic GMP-dependent protein kinase and the type II regulatory subunit of the cyclic AMP-dependent protein kinase were localized on the mitotic spindle. Throughout mitosis, their distribution closely resembled that of tubulin. Antibodies to cyclic AMP, cyclic GMP, and the type I regulatory and catalytic subunits of the cyclic AMP-dependent protein kinase did not label the mitotic apparatus. The association between specific components of the cyclic neucleotide system and the mitotic spindle suggests that cyclic nucleotide-dependent phosphorylation of spindle proteins, such as those of microtubules, may play a fundamental role in the regulation of spindle assembly and chromosome motion.

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

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.

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

1984 ◽  
Vol 218 (2) ◽  
pp. 285-294 ◽  
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.

scholarly journals Differential responses of cyclic GMP-dependent and cyclic AMP-dependent protein kinases to synthetic peptide inhibitors

1983 ◽  
Vol 213 (1) ◽  
pp. 159-164 ◽  
Author(s):  
D B Glass
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinases ◽  
Cyclic Gmp ◽  
Synthetic Peptide ◽  
Peptide Inhibitors ◽  
Catalytic Subunit ◽  
Dependent Protein Kinase ◽  
Peptide Substrates ◽  
Dependent Protein

The peptide Arg-Lys-Arg-Ala-Arg-Lys-Glu was synthesized and tested as an inhibitor of cyclic GMP-dependent protein kinase. This synthetic peptide is a non-phosphorylatable analogue of a substrate peptide corresponding to a phosphorylation site (serine-32) in histone H2B. The peptide was a competitive inhibitor of cyclic GMP-dependent protein kinase with respect to synthetic peptide substrates, with a Ki value of 86 microM. However, it did not inhibit phosphorylation of intact histones by cyclic GMP-dependent protein kinase under any conditions tested. Arg-Lys-Arg-Ala-Arg-Lys-Glu competitively inhibited the phosphorylation of either peptides or histones by the catalytic subunit of cyclic AMP-dependent protein kinase, with similar Ki values (550 microM) for both of these substrates. The peptide Leu-Arg-Arg-Ala-Ala-Leu-Gly, which was previously reported to be a selective inhibitor of both peptide and histone phosphorylation by cyclic AMP-dependent protein kinase, was a poor inhibitor of cyclic GMP-dependent protein kinase acting on peptide substrates (Ki = 800 microM), but did not inhibit phosphorylation of histones by cyclic GMP-dependent protein kinase. The selectivity of these synthetic peptide inhibitors toward either cyclic GMP-dependent or cyclic AMP-dependent protein kinases is probably based on differences in the determinants of substrate specificity recognized by these two enzymes. It is concluded that histones interact differently with cyclic GMP-dependent protein kinase from the way they do with the catalytic subunit of cyclic AMP-dependent protein kinase.

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