cAMP-dependent protein kinases in the rat testis: regulatory and catalytic subunit associations

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.

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

Biochemical Journal ◽

10.1042/bj1920223 ◽

1980 ◽

Vol 192 (1) ◽

pp. 223-230 ◽

Cited By ~ 31

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.

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Microinjection of catalytic subunit of cyclic AMP-dependent protein kinases triggers acute morphological changes in thyroid epithelial cells

FEBS Letters ◽

10.1016/0014-5793(88)80781-6 ◽

1988 ◽

Vol 232 (2) ◽

pp. 409-413 ◽

Cited By ~ 18

Author(s):

Pierre P. Roger ◽

Fabienne Rickaert ◽

Georges Huez ◽

Michèle Authelet ◽

Franz Hofmann ◽

...

Keyword(s):

Epithelial Cells ◽

Cyclic Amp ◽

Protein Kinases ◽

Morphological Changes ◽

Catalytic Subunit ◽

Dependent Protein

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Hormonal Regulation and Age-Dependent Changes in mRNA Levels for Regulatory Subunits of cAMP-Dependent Protein Kinases in Rat Testis

Annals of the New York Academy of Sciences ◽

10.1111/j.1749-6632.1987.tb25076.x ◽

1987 ◽

Vol 513 (1 Cell Biology) ◽

pp. 473-476

Author(s):

O. ØYEN ◽

W. ESKILD ◽

A. FRØYSA ◽

M. SANDBERG ◽

F. O. LEVY ◽

...

Keyword(s):

Protein Kinases ◽

Hormonal Regulation ◽

Mrna Levels ◽

Regulatory Subunits ◽

Rat Testis ◽

Age Dependent ◽

Dependent Protein

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Immunoelectron microscopical localization of the catalytic subunit of cAMP-dependent protein kinases in brain microtubules and neurofilaments

FEBS Letters ◽

10.1016/0014-5793(90)80835-7 ◽

1990 ◽

Vol 277 (1-2) ◽

pp. 167-170 ◽

Cited By ~ 4

Author(s):

Bernhard Trinczek ◽

Gerhild Schwoch

Keyword(s):

Protein Kinases ◽

Catalytic Subunit ◽

Dependent Protein ◽

Microscopical Localization ◽

Brain Microtubules

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

Biochemical Journal ◽

10.1042/bj2130159 ◽

1983 ◽

Vol 213 (1) ◽

pp. 159-164 ◽

Cited By ~ 39

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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Distribution and Activation of cAMP- and cGMP-Dependent Protein Kinases in Highly Purified Human Platelet Plasma and Intracellular Membranes

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650707 ◽

1996 ◽

Vol 76 (06) ◽

pp. 1063-1071 ◽

Cited By ~ 16

Author(s):

Samer S El-Daher ◽

Martin Eigenthaler ◽

Ulrich Walter ◽

Teiichi Furuichi ◽

Atsushi Miyawaki ◽

...

Keyword(s):

Protein Kinases ◽

Human Platelet ◽

Protein Band ◽

Human Platelets ◽

Catalytic Subunit ◽

Ip3 Receptor ◽

Intracellular Membranes ◽

Exogenous Addition ◽

Dependent Protein ◽

Camp And Cgmp

SummaryPreviously cAMP- and cGMP-dependent protein kinases (cAMP-PK, cGMP-PK) have been found predominantly associated with the particulate fraction in human platelets. We now report the distribution and activation of cAMP-PK and cGMP-PK in highly purified fractions of human platelet plasma (PM) and intracellular membranes (IM) prepared using high voltage free flow electrophoresis. Two non-hydrolys-able analogues of cAMP and cGMP namely Sp-5,6-DCl-cBiMPS and 8-p-CPT-cGMP have been used to activate cAMP-PK and cGMP-PK respectively. Addition of either agonist with [γ32P]ATP stimulated the endogenous activity of cAMP-PK or cGMP-PK in PM but not in IM. With PM Sp-5,6-DCl-cBiMPS stimulated the phosphorylation of protein substrates of Mr 16,22,24,46-50,66,90,160 and 250 kDa. A specific peptide inhibitor of cAMP-PK inhibited the phosphorylation of all of the substrates by Sp-5,6-DCl-cBiMPS. 8-pCPT-cGMP also induced the phosphorylation of a number of substrates particularly 16,22, 46-50, 90 and 250 kDa proteins. Inclusion of the cAMP-PK inhibitor peptide totally blocked the phosphorylation of the 16 and 22 kDa proteins, partially inhibited phosphorylation of 46-50 and 90 kDa proteins and had no effect on the 250 kDa protein indicating the 46-50, 90 and 250 kDa proteins were also cGMP-PK substrates. Western blotting with antibodies to cGMP-PK and the catalytic subunit of cAMP-PK revealed the presence of the kinases to be exclusively associated with PM with no detection in IM.The presence of cAMP-PK substrates in IM was investigated by exogenous addition of catalytic subunit of cAMP-PK. Phosphoproteins of Mr 16, 22, 27, 30,45, 75,116 and 250 kDa were detected. A range of antibodies to cAMP-PK substrates were used to identify and localise the substrates. These antibodies revealed GPIb and VASP to be exclusively associated with PM fractions. Rap IB was also predominantly associated with PM with a small level detected in IM. Antibodies to the IP3 receptor (18A10 and 4C11) revealed the protein to be predominantly associated with IM. Additionally the antibody 4C11 recognised a 230 kDa protein band in PM that was not seen in IM. From the known specificity of these antibodies the results confirm the presence of a type IIP3 receptor in IM and a distinct (possible type III) IP3 receptor with the PM. the 16, 22, 27, 30, 75 and 116 kDa proteins in IM represent nwly detected substartes for camp-pk of presently unknown identity.

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