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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^{1 ,2}

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

Contact between lymphokine-activated killer (LAK) cells and natural killer-resistant tumor targets SK-Mel-1 (human melanoma) or Raji (human lymphoma) stimulates phosphorylation of two M_r 65,000 LAK proteins (pp65a and pp65b) with nearly identical isoelectric points. Phosphoamino acid analysis of pp65a and pp65b detected phosphorylation exclusively on serine residues. Phosphotyrosine could not be detected on either substrate after immunoblotting with an antiphosphotyrosine antibody, and herbimycin A treatment failed to inhibit p65 phosphorylation induced by target contact. However, phorbol myristate acetate treatment alone induced LAK pp65a and pp65b phosphorylation, suggesting phosphorylation may be mediated by protein kinase C or a protein kinase C-regulated kinase. The molecular weight and isoelectric points of pp65a and pp65b are similar to that reported for the human actin-bundling protein, L-plastin (L-fimbrin). On two-dimensional SDS-PAGE gel immunoblots, a peptide specific anti-L-plastin antiserum bound to pp65a and pp65b, suggesting that the phosphoproteins are similar or identical to L-plastin. In addition, two adjacent M_r 65,000 LAK proteins were also detected by the antiserum and may correspond to unphosphorylated forms of L-plastin. On the basis of known properties of phosphorylated L-plastin, it is hypothesized that p65 phosphorylation in LAKs may regulate adhesion to tumor targets.

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 second in a series. See Frederick, M. J., Rodriguez, L.V., Johnston, D.A., and Grimm, E.A. Tumor target binding induces phosphorylation of two M_r 65,000 lymphokine-activated killer proteins. Cancer Res., 56: 127–137, 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.

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