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Tumor Target Binding Induces Phosphorylation of Two M_r 65,000 Lymphokine-activated Killer Proteins^{1 ,2}

Departments of Tumor Biology [M. J. F., E. A. G.], Molecular Pathology [L. V. R.], and Biomathematics [D. A. J.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

Effector-target cell conjugate formation is an essential step during lymphokine-activated killer (LAK) cell-mediated cytotoxicity. Protein phosphorylation changes in human LAKs after contact with NK-resistant (LAK-sensitive) tumors were examined by two-dimensional SDS-PAGE. Exposure to either SK-Mel-1 (melanoma) or Raji (lymphoma) targets led to increased phosphorylation of two M_r 65,000 LAK proteins, pp65a and pp65b, with isoelectric points of 5.1 and 5.2, respectively. Phosphorylation of both substrates was initiated between 1 and 5 min after coincubation with tumor targets. Contact between LAKs and targets was required for p65 phosphorylation because soluble tumor factors failed to induce phosphorylation. Normal peripheral blood lymphocyte targets, which are bound very poorly by LAKs and are resistant to killing, failed to induce LAK p65 phosphorylation. The broad protein kinase inhibitor staurosporine inhibited phosphorylation of pp65a and pp65b, supporting the hypothesis that activation of a LAK protein kinase leads to p65 phosphorylation. Cross-linking of CD16 (FcRIIIA), which mediates antibody-dependent cellular cytotoxicity in LAKs, also led to increased pp65a and pp65b phosphorylation. Collectively, these data provide correlative evidence that p65 phosphorylation may be involved in the cytolytic function of LAKs.

¹ Supported by National Cancer Institute Award R01-CA45225 (E. A. G.) and NIH Grant CA 33305 (L. V. R.).

² This manuscript is the first in a series. See M. J. Frederick, L. V. Rodriguez, D. A. Johnson, B. G. Darnay, and E. A. Grimm, Characterization of the M_r 65,000 lymphokine-activated killer proteins phosphorylated after tumor target binding: evidence that pp65a and pp65b are phosphorylated forms of L-plastin. Cancer Res., 56: 138–144, 1996.

³ To whom requests for reprints should be addressed, at Department of Tumor Biology, Box 79, M. D. Anderson Cancer Center, 1515 Holcombe Avenue, Houston, TX 77030.

Received 8/10/95. Accepted 10/31/95.