Amino acid sequence of the catalytic subunit of bovine type II adenosine cyclic 3',5'-phosphate-dependent protein kinase

Biochemistry ◽  
1983 ◽  
Vol 22 (15) ◽  
pp. 3702-3709 ◽  
Author(s):  
Shozo Shoji ◽  
Lowell H. Ericsson ◽  
Kenneth A. Walsh ◽  
Edmond H. Fischer ◽  
Koiti Titani
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Amino Acid Sequence ◽  
Catalytic Subunit ◽  
Type Ii ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Bovine Type

Amino acid sequence of the regulatory subunit of bovine type II cAMP-dependent protein kinase

Biochemistry ◽  
1984 ◽  
Vol 23 (18) ◽  
pp. 4200-4206 ◽  
Author(s):  
Koji Takio ◽  
Stephen B. Smith ◽  
Edwin G. Krebs ◽  
Kenneth A. Walsh ◽  
Koiti Titani
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Amino Acid Sequence ◽  
Regulatory Subunit ◽  
Type Ii ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Bovine Type

Amino acid sequence of the regulatory subunit of bovine type I cAMP-dependent protein kinase

Biochemistry ◽  
1984 ◽  
Vol 23 (18) ◽  
pp. 4193-4199 ◽  
Author(s):  
Koiti Titani ◽  
Tatsuru Sasagawa ◽  
Lowell H. Ericsson ◽  
Santosh Kumar ◽  
Stephen B. Smith ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Amino Acid Sequence ◽  
Regulatory Subunit ◽  
Type I ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Bovine Type

scholarly journals The amino acid sequence of a hinge region in the regulatory subunit of bovine cardiac muscle cyclic AMP-dependent protein kinase II

FEBS Letters ◽  
1980 ◽  
Vol 114 (1) ◽  
pp. 83-88 ◽  
Author(s):  
Koji Takio ◽  
Kenneth A. Walsh ◽  
Hans Neurath ◽  
Stephen B. Smith ◽  
Edwin G. Krebs ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Amino Acid Sequence ◽  
Cardiac Muscle ◽  
Hinge Region ◽  
Regulatory Subunit ◽  
Dependent Protein Kinase ◽  
Protein Kinase Ii ◽  
Dependent Protein

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 Purification and characterization of the catalytic subunit of adenosine 3':5'-cyclic monophosphate-dependent protein kinase from bovine liver

1976 ◽  
Vol 159 (2) ◽  
pp. 409-422 ◽  
Author(s):  
P H Sugden ◽  
L A Holladay ◽  
E M Reimann ◽  
J D Corbin
Keyword(s):  
Protein Kinase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bovine Liver ◽  
Polyacrylamide Gel ◽  
Catalytic Subunit ◽  
Type Ii ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Gel Isoelectric Focusing

1. The catalytic subunit of bovine liver cyclic AMP-dependent protein kinase (EC2.7.1.37) was purified essentially by the method of Reimann & Corbin [(1976) Fed. Proc. Fed. Am. Soc. Exp. Biol. 35, 1384]. 2. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, sedimentation-velocity centrifugation and sedimentation-equilibrium centrifugation showed that the catalytic subunit was monodisperse. Polyacrylamide-gel isoelectric-focusing electrophoresis revealed the presence of at least three isoenzyme forms of catalytic subunit activity with slightly different pI values (6.72, 7.04 and 7.35). 3. Physical properties of the catalytic subunit were determined by several different methods. It had mol.wt. 39000-42000, Stokes radium 2.73-3.08 nm, so20.w 3.14S, f/fo 1.19-1.23 and, assuming a prolate ellipsoid, axial ration 4-5. 4. Amino acid analysis was performed on the catalytic subunit. It had one cysteine residue/molecule which was essential for activity. Inhibition by thiol-specific reagents was partially prevented by the presence of ATP-Mg2+. 5. The circular-dichroic spectrum showed the catalytic subunit contained 29% α-helical form, 18% β-form and 53% aperiodic form. Near-u.v. circular dichroism showed the presence of aromatic residues whose equivalent molar ellipticity was greatly altered by the addition of ATP-Mg2+. 6. Kinetic experiments showed that the catalytic subunit had an apparent Km for ATP of 7 muM. 5'-Adenylyl imidodiphosphate inhibitied competitively with ATP with a Ki of 60 muM. The kinetic plot for histone (Sigma, type II-A) was biphasic showing ‘high’-and ‘low’-Km segments. Under assay conditions the specific activity of the catalytic subunit was 3 × 10(6) units/mg of protein. Of various metal ions tested, the catalytic subunit was most active with Mg2+.7. When assayed with histone (Sigma, type II-A) as substrate, the activity of the catalytic subunit was increased by non-ionic detergents or urea. No such activation was observed with casein as substrate.

scholarly journals The DNA-Dependent Protein Kinase Catalytic Subunit Is Phosphorylated In Vivo on Threonine 3950, a Highly Conserved Amino Acid in the Protein Kinase Domain

2006 ◽  
Vol 27 (5) ◽  
pp. 1581-1591 ◽  
Author(s):  
Pauline Douglas ◽  
Xiaoping Cui ◽  
Wesley D. Block ◽  
Yaping Yu ◽  
Shikha Gupta ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Kinase Domain ◽  
Catalytic Subunit ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Protein Kinase Domain ◽  
Dependent Protein

ABSTRACT The protein kinase activity of the DNA-dependent protein kinase (DNA-PK) is required for the repair of DNA double-strand breaks (DSBs) via the process of nonhomologous end joining (NHEJ). However, to date, the only target shown to be functionally relevant for the enzymatic role of DNA-PK in NHEJ is the large catalytic subunit DNA-PKcs itself. In vitro, autophosphorylation of DNA-PKcs induces kinase inactivation and dissociation of DNA-PKcs from the DNA end-binding component Ku70/Ku80. Phosphorylation within the two previously identified clusters of phosphorylation sites does not mediate inactivation of the assembled complex and only partially regulates kinase disassembly, suggesting that additional autophosphorylation sites may be important for DNA-PK function. Here, we show that DNA-PKcs contains a highly conserved amino acid (threonine 3950) in a region similar to the activation loop or t-loop found in the protein kinase domain of members of the typical eukaryotic protein kinase family. We demonstrate that threonine 3950 is an in vitro autophosphorylation site and that this residue, as well as other previously identified sites in the ABCDE cluster, is phosphorylated in vivo in irradiated cells. Moreover, we show that mutation of threonine 3950 to the phosphomimic aspartic acid abrogates V(D)J recombination and leads to radiation sensitivity. Together, these data suggest that threonine 3950 is a functionally important, DNA damage-inducible phosphorylation site and that phosphorylation of this site regulates the activity of DNA-PKcs.

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