Homology between regulatory subunits of type I cyclic AMP-dependent protein kinases from bovine and murine cells

1984 ◽  
Vol 234 (2) ◽  
pp. 546-551 ◽  
Author(s):  
Robert A. Steinberg
Keyword(s):  
Cyclic Amp ◽  
Protein Kinases ◽  
Type I ◽  
Regulatory Subunits ◽  
Dependent Protein

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.

Possible Roles for Protein Phosphorylation in Platelet Responses

1979 ◽  
Author(s):  
R.J. Haslam ◽  
J.E.B. Fox ◽  
S.E. Salama ◽  
J.A. Lynham
Keyword(s):  
Cyclic Amp ◽  
Protein Phosphorylation ◽  
Protein Kinases ◽  
Platelet Function ◽  
Subcellular Fractionation ◽  
Human Platelets ◽  
Type I ◽  
Sds Page ◽  
Dependent Protein ◽  
32P Incorporation

The relationships between the phosphorylation of specific platelet polypeptides and platelet function were studied using washed human platelets labelled by preincubation with [32p] Pi. Platelet polypeptides were separated by SDS-PAGE and 32P incorporation into them determined by autoradiography. Whereas induction of platelet aggregation alone did not affect protein phosphorylation, induction of the release reaction increased 3P incorporation into several polypeptides (P75,P47,P40,P27,P20,P19), including the P-light chain of platelet myosin (P20). These changes were inhibited by drugs that blocked Ca2 movements and may be due to activation of Ca2+-dependent protein kinases. Compounds that inhibited platelet function by increasing cyclic AMP (e.g. PCE1) also suppressed these reactions but, in addition, increased phosphorylation of other polypeptides (P50,P49,P36,P24,P22). Type I and Type II cyclic AMP-dependent protein kinases were present in platelets and may mediate Che latter effects of cyclic AMP. Subcellular fractionation of 32p-labelled platelets that had been exposed to PCE1 showed that P24 was present in membranes that could take up Ca2+ by an ATP-dependent mechanism. Membranes from PCE1-treated platelets took up Ca2+ more rapidly than control membranes. Thus, the cyclic AMP-dependent phosphorylation of P24 may stimulate the removal of Ca2+ from platelet cytosol and suppress Ca2+-dependent phosphorylation reactions necessary for release of granule constituents.

Retinoylation of the cAMP-binding regulatory subunits of type I and type II cAMP-dependent protein kinases in HL60 cells

1991 ◽  
Vol 290 (2) ◽  
pp. 293-302 ◽  
Author(s):  
Noriko Takahashi ◽  
Charis Liapi ◽  
Wayne B. Anderson ◽  
Theodore R. Breitman
Keyword(s):  
Protein Kinases ◽  
Type I ◽  
Type Ii ◽  
Regulatory Subunits ◽  
Hl60 Cells ◽  
Dependent Protein

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 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.

Activation of type I cyclic AMP-dependent protein kinases is impaired by a point mutation in cyclic AMP binding sites

1989 ◽  
Vol 172 (3) ◽  
pp. 263-271 ◽  
Author(s):  
Takayoshi Kuno ◽  
Hisato Shuntoh ◽  
Takehiko Takeda ◽  
Akira Ito ◽  
Motoyoshi Sakaue ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Protein Kinases ◽  
Point Mutation ◽  
Binding Sites ◽  
Type I ◽  
Dependent Protein

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.

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