IN-VITRO AUTOPHOSPHORYLATION AND CYCLIC NUCLEOTIDE-DEPENDENT DEPHOSPHORYLATION OF SEA-URCHIN SPERM HISTONE KINASE

We identified two phosphorylatable proteins (33 kDa and 48 kDa) in cru de extractes of spermatozoa from the sea urchin, Hemicentrotus pulcher rimus. While the 33 kDa protein was phosphorylated in 100 mM Mg2+-cont aining medium with cAMP or cGMP, phosphorylation of the 48 kDa protein occurred irrespective of the Mg2+-concentration if it was more than 1 0 mM. Physiological concentration if it was more than 10 mM. Physiolog ical concentrations of cAMP or cGMP triggered dephosphorylation of the [P-32]-phosphorylated 48 kDa protein in a concentration dependent man ner. The dephosphorylation of 48 kDa protein was inhibited with calycu lin A or okadaic acid. The 48 kDa protein was associated with a 39 kDa protein to form a larger oligomer with about 400 kDa. The purified 40 0 kDa protein showed cyclic nucleotide-dependent histone kinase activi ty. The histone kinase activity shifted from 400 kDa to 39 kDa on a Su perose 6 HR in the presence of 5 x 10(-6) M cAMP. Phosphorylation of t he 48 kDa protein in the purified 400 kDa protein required only Mg[P-3 2]ATP. A photoaffinity cAMP analogue, 8-N3-[P-32]cAMP, was incorporate d into the 48 kDa protein. These results suggest that the 48 kDa prote in which is a reguratory subunit of the histone kinase is autophosphor ylated, and binding of cAMP to the phosphorylated 48 kDa subunit of th e kinase results in dissociation of the 48 kDa subunit(s) from the 39 kDa subunit(s) to allow the 48 kDa subunit accessible to a protein pho sphatase.