Phorbol diester-induced phosphorylation of nuclear matrix proteins in HL60 promyelocytes. Possible role in differentiation studied by cationic detergent gel electrophoresis
Immortal HL60 promyelocytes are induced to differentiate to mortal adherent cells by a variety of agents which activate protein kinase C, including 12-O-tetradecanoylphorbol 13-acetate (TPA). In order to investigate the mechanism of this effect, we incubated HL60 cells with (/sup 32/P)orthophosphate with or without TPA and extracted their proteins with the cationic detergent benzyldimethyl-n-hexadecylammonium chloride prior to electrophoresis in a discontinuous polyacrylamide gel system in the first dimension. In this system, proteins migrate toward the cathode as a function of their molecular weight, and they are separated from other radioactive components which can obscure the pattern of protein phosphorylation on sodium dodecyl sulfate (SDS) gels. SDS gel electrophoresis was used in the second dimension, resulting in the clear resolution of a large number of proteins. TPA caused many changes in the pattern of protein phosphorylation in intact cells. Two proteins which prominently increased their incorporation of /sup 32/P were investigated in particular, and they were both found to be retained in the nuclear matrix following successive extraction of cells with Triton, digestion with DNase and RNase, and extraction with 2 M NaCl. These proteins migrated with apparent molecular weights of 80,000 and 33,000 on SDS gels, and are designated NP80 and NP33, respectively. NP80 was half-maximally phosphorylated after 7 min exposure to TPA, and half-maximally phosphorylated by 10 nM TPA. NP80 co-migrated with a faint Coomassie Blue-stained protein, and NP33 co-migrated with a more prominent protein. Several proteins incorporated less /sup 32/P when the cells were exposed to TPA, including one which was extracted from nuclei with the core histones and which co-migrated with histone H2A.
- Research Organization:
- Veterans Administration Hospital, Iowa City, IA
- OSTI ID:
- 5686945
- Journal Information:
- J. Biol. Chem.; (United States), Journal Name: J. Biol. Chem.; (United States) Vol. 15; ISSN JBCHA
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
560301* -- Chemicals Metabolism & Toxicology-- Cells-- (-1987)
59 BASIC BIOLOGICAL SCIENCES
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ADHESION
ANIMAL CELLS
AUTORADIOGRAPHY
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL EFFECTS
CARCINOGENS
CELL DIFFERENTIATION
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
ELECTROPHORESIS
ENZYMES
ESTERS
ISOTOPES
KINETICS
LIGHT NUCLEI
MOLECULAR WEIGHT
NUCLEI
NUCLEOPROTEINS
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
OXYGEN COMPOUNDS
PHORBOL ESTERS
PHOSPHATES
PHOSPHORUS 32
PHOSPHORUS COMPOUNDS
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHORYLATION
PHOSPHOTRANSFERASES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
TRANSFERASES