Cyclic-AMP-dependent activation of an inter-phylum hybrid histone-kinase complex reconstituted from sea urchin sperm-regulatory subunits and bovine heart catalytic subunits.
A cAMP-dependent histone kinase was purified and characterized from spermatozoa of the sea urchin Hemicentrotus pulcherrimus. The molecular mass of the kinase was estimated to be 178 kDa by native PAGE and 400 kDa by gel chromatography on a Superose 6 HR 10/30 column. The enzyme, composed of two 39-kDa catalytic subunits and two 48-kDa regulatory subunits, phosphorylates the lysine-rich histone subspecies (H1 and H2B) isolated from H. pulcherrimus spermatozoa. We isolated cDNA clones encoding a 39-kDa catalytic subunit and a 48-kDa regulatory subunit of the enzyme. The cDNA clone for the 39-kDa subunit was 3881 bp, and the 352-residue deduced amino acid sequence showed 78% similarity with the catalytic subunit of/mammalian cAMP-dependent protein kinase (PKA). The cDNA for the 48-kDa subunit was 4589 bp and the 368-residue deduced amino acid sequence showed 57% similarity with the regulatory subunit of mammalian PKA, although the N-terminal 77 residues showed poor similarity. The mRNAs encoding both the catalytic subunit (7.5 kb) and the regulatory subunit (4.6 kb) were expressed in testis, ovary and egg. An inter-phylum hybrid enzyme, reconstituted from the regulatory subunit of cAMP-dependent histone kinase of sea urchin sperm and the catalytic subunit of bovine heart PKA, has a cAMP-dependent histone kinase activity. Thus, we suggest that the N-terminal 77-amino-acid residues of the regulatory subunit are not essential for inhibition by the regulatory subunit of the catalytic subunit, and that cAMP-dependent inhibitory activity of the regulatory subunit resides in the sequence between the inhibitory site and the C-terminus.
Hoshino
K
K
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Japan.
Nomura
K
K
Suzuki
N
N
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002384:Catalysis; D002417:Cattle; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000502:physiology*; D018076:DNA, Complementary / Q000302:isolation & purification; D004789:Enzyme Activation; D008297:Male; D008969:Molecular Sequence Data; D009206:Myocardium / Q000737:chemistry / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D006656:Protamine Kinase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D012617:Sea Urchins; D013094:Spermatozoa / Q000737:chemistry / Q000201:enzymology; D013347:Subcellular Fractions / Q000201:enzymology / Q000378:metabolism
D018076:DNA, Complementary; D010446:Peptide Fragments; D000242:Cyclic AMP; D006656:Protamine Kinase