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Undifferentiated human promyelocytic leukemia HL-60 cells show little or no superoxide production, but generate a very low O2 – concentration upon incubation with all-trans-retinoic acid (ATRA). Its production reaches a maximum within 20 h, and thereafter is maintained at an almost con-stant level. The differentiated cells show phorbol 12-myri-state 13-acetate (PMA)-stimulatedNADPHoxidase activity consistent with the amount of gp91phox(phagocytic oxid-ase) expressed in the plasma membrane. | Eur. J. Biochem. 269 2622-2629 2002 FEBS 2002 doi 10.1046 j.1432-1033.2002.02939.x Interaction between p21-activated protein kinase and Rac during differentiation of HL-60 human promyelocytic leukemia cell induced by all-fra s-retinoic acid Yukio Nisimoto1 and Hisamitsu Ogawa2 1Department of Biochemistry Aichi Medical University School of Medicine Nagakute Aichi Japan department of Biology Fujita Health University School of Medicine Toyoake Aichi Japan Undifferentiated human promyelocytic leukemia HL-60 cells show little or no superoxide production but generate a very low O2- concentration upon incubation with all-transretinoic acid ATRA . Its production reaches a maximum within 20 h and thereafter is maintained at an almost constant level. The differentiated cells show phorbol 12-myri-state 13-acetate PMA -stimulated NADPH oxidase activity consistent with the amount of gp91phox phagocytic oxidase expressed in the plasma membrane. Three isoforms of p21-activated serine threonine kinases PAK68 PAK65 and PAK62 were found in both cytosolic and membrane fractions and their contents were significantly increased during induced differentiation. The amount of Rac identified in the two fractions was also markedly enhanced by ATRA-induced differentiation. In contrast neither PAK nor Rac was seen in the plasma membrane of undifferentiated HL-60 or human neutrophil but they were abundant in the cytoplasmic fraction. Binding of Rac with PAK isoforms was shown in the membrane upon induced differentiation of HL-60 cells. Direct binding of purified Rac1 to PAK68 was quantified using a fluorescent analog of GTP methylanthraniloyl guanosine-5 - P y-imido triphosphate bound to Rac as a reporter group. Rac1 bound to PAK68 with a 1 1 stoichiometry and with a Kd value of 6.7 nM. Keywords Rac PAK HL-60 GTPase MAP kinase. A phagocytic superoxide-generating system is expressed upon induced differentiation of HL-60 human promyelo-cytic leukemia cells with dimethylsulfoxide or retinoic acid
