Purification of N-terminally truncated histone H2A-monoubiquitin conjugates from leukemic cell nuclei: probable proteolytic products of ubiquitinated H2A.
To gain insight into the significance of nuclear ubiquitinated proteins, two serial extracts prepared from various leukemic cells were analysed by western blotting with anti-ubiquitin antibody. Two previously unidentified ubiquitinated proteins with molecular masses of 10 and 17 kDa were found in 8 M urea-soluble extracts, obtained from Tris-buffer-insoluble materials, of acute myeloid leukemia OCI/AML 1a cells and the cells from the leukemia patients. Both proteins were successfully purified from the OCI/AML 1a cells and identified as monoubiquitin-truncated H2A conjugates, the 10 kDa ubiquitinated H2A(115-129) and the 17 kDa ubiquitinated H2A(54-129), suggesting that both proteins were produced by limited proteolysis of an intact form (23 kDa) of ubiquitinated H2A(1-129). The 17 kDa protein as well as the 23 kDa ubiquitinated histone H2A were localised in chromatin fractions of the OCI/AML cells and released by high concentrations of salt in a micrococcal nuclease-sensitive manner, suggesting their association with chromatin. In contrast, the 10 kDa protein remained insoluble even when the nuclei were treated with nuclease under high salt concentrations, presumably due to binding to the nuclear matrix. An antibody recognising H2A(70-81) also detected the 17 kDa protein in anti-ubiquitin immunoprecipitates obtained from the OCI/AML cell nuclei. In addition, the 17 kDa protein levels in THP-1 cells were transiently increased, concomitant with a decrease in the 23 kDa ubiquitinated H2A, by treatment with phorbol 12-myristate 13-acetate or all-trans-retinoic acid, both of which induce differentiation. This is the first report of probable proteolytic products of ubiquitinated H2A, which might have a role in nuclear functions.
Okawa
Yutaka
Y
Department of Biochemistry I, Jikei University School of Medicine, 3-25-8, Nishishinbashi, Minato-ku, Tokyo 105-8461, Japan.
Takada
Koji
K
Minami
Jiro
J
Aoki
Katsuhiko
K
Shibayama
Hirohiko
H
Ohkawa
Kiyoshi
K
D000595:Amino Acid Sequence; D002457:Cell Extracts; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry*; D002843:Chromatin / Q000378:metabolism; D006657:Histones / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D015151:Immunoblotting; D007938:Leukemia / Q000473:pathology*; D008297:Male; D008969:Molecular Sequence Data; D013347:Subcellular Fractions; D014452:Ubiquitins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*
D002457:Cell Extracts; D002843:Chromatin; D006657:Histones; D007106:Immune Sera; D014452:Ubiquitins; C021645:chromatin conjugate protein A24