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Biochemical Characterization of Tyrosine Kinase and Phosphotyrosine Phosphatase Activities of HL-60 Leukemia Cells¹

Department of Pharmacology and Developmental Therapeutics Program, Comprehensive Cancer Center, Yale University School of Medicine, New Haven, Connecticut 06510

The cellular phosphotyrosine content of the HL-60 promyelocytic leukemia markedly decreased during the induced granulocytic and monocytic maturation of these cells. This occurs in the face of major increases in tyrosine kinase and protein phosphotyrosine phosphatase activities (D. A. Frank and A. C. Sartorelli, Biochem. Biophys. Res. Commun., 140: 440–447, 1986). In the present work, these two activities were characterized in the particulate fraction of HL-60 cells, since both enzymes are membrane bound. The tyrosine kinase activity utilized ATP as a phosphate donor, although GTP and other nucleotides were competitive with ATP. The enzyme was temperature sensitive, had a pH optimum of 6.5, and required Mg²⁺ or Mn²⁺ for activity, with additional stimulation of activity being produced by Zn²⁺. Agents such as epidermal growth factor and insulin, which stimulate other tyrosine kinase enzymes, were without effect on the tyrosine kinase activity of HL-60 cells. Enzyme activity was stimulated, however, by non-ionic detergents and was inhibited by quercetin. The protein phosphotyrosine phosphatase activity was paralleled by that of p-nitrophenyl phosphatase, was inhibited by VO, Zn²⁺ and F^-, and was maximally active at a pH of 7 to 8. The characteristics of the tyrosine kinase and the protein phosphotyrosine phosphatase activities were distinct from those of other known proteins of these classes.

Tyrosine kinase activity was predominantly located on the plasma membrane, while the protein phosphotyrosine phosphatase activity was concentrated on internal membranes. The activities of both enzymes present on the plasma membrane appeared to exist on the cytoplasmic face of this membrane.

Further characterization of the activities of these enzyme systems and their contribution to the regulation of tyrosine phosphorylation would appear to be important to an understanding of the control of cellular proliferation and differentiation.

¹ This investigation was supported in part by USPHS Grant CA-02817 from the National Cancer Institute.

² To whom requests for reprints should be addressed, at the Yale Comprehensive Cancer Center, Yale University School of Medicine, P. O. Box 3333, New Haven, CT 06510.

Received 12/29/87. Revised 4/11/88. Accepted 4/27/87.

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