The migration inhibitory factor-related proteins (MRPs) MRP-8 and MRP-14 were detected in differentiated human leukemia cell lines (THP-1 and HL-60) by immunocytochemical analysis. They were induced and colocalized in the cytoplasm and in lesser amounts in the nucleus when THP-1 and HL-60 cells were induced to differentiate by 1alpha,25-dihydroxyvitamin D3 or retinoic acid. In a search for a protein capable of binding MRPs, both MRPs were individually produced in insect cells (Sf21) infected with recombinant baculovirus. The purified recombinant MRPs were electrophoretically and antigenically indistinguishable from the native proteins, and their ability to form the MRP8/14 complex was retained. The presence of MRP binding sites was investigated by a binding assay using recombinant MRPs and specific monoclonal antibodies. MRP binding sites were detected on the cell membrane of the human leukemia cell lines THP-1, Raji, and MOLT-4. HL-60 cells treated with 1alpha, 25-dihydroxyvitamin D3 did not express MRP binding sites on the cell membrane, but a high level of MRPs accumulated in the cells. The occurrence of MRP binding sites on the cell surface of leukemia cell lines of monocyte and lymphocyte origin suggests that MRPs, released from neutrophils under certain conditions, may contribute to the activation and recruitment of effector cells to inflammatory lesions.
A nonhistone chromatin protein (NHCP) has been purified to homogeneity from a 0.5 M NaCl extract of Ehrlich ascites tumor cell (EAT cell) nuclei as a phosphate acceptor for casein kinase II using ion-exchange column chromatographies and Sephacryl S300 gel filtration. The purified NHCP (approximate Mr = 400,000) was found to be a tetramer of an Mr = 98,000 polypeptide (pI = 6.9) and to have high contents of glycine (15%) and serine (11.6%). This protein (designated as 400-kDa NHCP) was highly phosphorylated by casein kinase II (Mr = 130,000), but not by histone kinase. Casein kinase II phosphorylated only seryl residues of the purified 400-kDa NHCP. The NHCP bound with DNA, but not with RNAs, and the DNA binding ability of the protein was reduced when it was phosphorylated by casein kinase II. Moreover, we found that (a) the 400-kDa NHCP is present in large quantities in malignant mouse cells, such as EAT, EL-4, and Meth-A cells, but only slightly in normal tissues and cells; (b) the protein level is rapidly increased when mouse lymphocytes are treated with recombinant interleukin 2 (T cell growth factor) or concanavalin A; and (c) the kinase responsible for the 400-kDa NHCP phosphorylation in the chromatin of various mouse cells is a casein kinase II. These experimental results suggest that the 400-kDa NHCP acts as an effective phosphate acceptor for casein kinase II at the chromatin level and that an increased phosphorylation of the protein by the kinase may be implicated in the progress of cell differentiation and proliferation.
Two calcium binding proteins, MRP-8 and MRP-14, are specifically synthesized in human myeloid cells. This paper shows that Me2SO, all-trans-retinoic acid (RA) and 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3), but not 12-O-tetradecanoyl phorbol-13-acetate (PMA) are potent inducers of MRP-8/14 protein complex in human leukemic cells. Transforming growth factor-beta 1 (TGF-beta 1) is shown to enhance the inductive effect of RA and 1 alpha,25(OH)2D3. We have examined the possibility that MRP expression is regulated through the protein kinase pathway. Both cytosolic and membrane-bound protein kinase C (PKC) activities increased during differentiation by RA and 1 alpha,25(OH)2D3. PMA-treatment led to a decrease of cytosolic PKC activity and an increase of membrane-bound PKC activity in the presence of these differentiation inducers, while PMA alone resulted in low cytosolic and high membrane-bound PKC activities. PKC inhibitor H7 inhibited MRP synthesis in HL-60 cells treated with RA and 1 alpha,25(OH)2D3. These results suggest that cytosolic PKC activity may be involved in a stimulatory pathway of MRP synthesis and that protein phosphorylation reactions may play important roles in MRP expression during myelocytic differentiation.
A phosphate-incorporating protein has been highly purified from the cytosol of Ehrlich ascites tumor cells (EAT cells). The nitrocellulose membrane method was used to follow the progress of the purification by quantitation of the [32P]phosphorylated form of the protein. The purified protein was identified as an NDP-kinase since it exhibited NDP-kinase activity and had enzyme characteristics in common with other NDP-kinases from various mammalian cells. The purified NDP-kinase was found to have a molecular weight of approximately 76,000 daltons. Moreover, the enzyme appears to consist of two distinct polypeptides (18,000 and 20,000 daltons). This enzyme contained 19 amino acids, with high levels of glycine (9.8%) and lysine (9.0%). The enzyme rapidly formed a [32P]phosphoenzyme when incubated with [gamma-32P]ATP in the presence of Mg2+ (1 mM) at the optimum pH of 7.5 even at low temperature (below 4 degrees C). This phosphoenzyme is an enzyme-bound, high-energy-phosphate intermediate, because ATP was formed from it on incubation with ADP in the presence of Mg2+ (1 mM). This finding suggests that the phosphoenzyme functions as an intermediate in NDP-kinase action.
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