Sidney Pestka, MD

Professor, Medicine, RWJUH RWJMS Research Tower 675 Hoes Lane, Room 727 Piscataway, NJ 08854 pestka@umdnj.edu

Publications

• Pestka, S. and Nirenberg, M.W. Regulatory Mechanisms and Protein Synthesis. X. Codon Recognition on 30S Ribosomes J. Mol. Biol.. 1966; 21:145.
• Langlois, R., Lee, C.C., Cantor, C.R., Vince, R., and Pestka, S. The Distance Between Two Functionally Significant Regions of the 50S Escherichia coli Ribosome: The Erythromycin Binding Site and Proteins L7/L12 J. Mol. Biol.. 1976; 106:297.
• Rubinstein, M., Rubinstein, S., Familletti, P.C., Gross, M.S., Miller, R.S., Waldman, A.A., and Pestka, S. Human Leukocyte Interferon Purified to Homogeneity Science. 1978; 202:1289.
• Miller, D.L., Kung, H.-F., and Pestka, S. Crystallization of Recombinant Human Leukocyte Interferon A Science. 1982; 215:689.
• Greiner, J.W., Guadagni, F., Noguchi, P., Pestka, S., Colcher, D., Fisher, P.B., and Schlom, J Recombinant Interferon Enhances Monoclonal Antibody-Targeting of Carcinoma Lesions in Vivo Science. 1987; 235:895.
• Cook, J.R., Emanuel, S.L., Donnelly, R.J., Soh, J., Mariano, T.M., Schwartz, B., Rhee, S., and Pestka, S. Sublocalization of the Human Interferon-gamma Receptor Accessory Factor Gene and Characterization of Accessory Factor Activity by Yeast Artificial Chromosomal Fragmentation J. Biol. Chem.. 1994; 269:7013.
• Pestka, S., Kotenko, S.V., Muthukumaran, G., Izotova, L.S., Cook, J.R., and Garotta, G. The Interferon Gamma (IFN-y) Receptor: A Paradigm for the Multichain Cytokine Receptor Cytokine and Growth Factor Reviews. 1997; 8:189.
• Kotenko, S.V., Krause, C.D., Izotova, L.S., Pollack, B.P., Wu, W., and Pestka, S. dentification and Functional Characterization of a Second Chain of the Interleukin-10 Receptor Complex, EMBO J.. 1997; 16:5894.
• Livnah, O., Johnson, D.L., Stura, E.A., Farrell, F.X., Barbone, F.P., You, Y., Liu, K.D., Goldsmith, M.A., He, W., Krause, C., Pestka, S., Jolliffe, L.K., and Wilson, I.A. An Antagonist Peptide - EPO Receptor Complex Suggests that Receptor Dimerization is not Sufficient for Activation Nat. Struct. Biol. 1998; 5:993.
• Kotenko, S.V., Izotova, L.S., Mirochnitchenko, O.V., Lee, C., and Pestka, S. The Intracellular Domain of Interferon-alpha Receptor 2c (IFN-aR2c) Chain is Responsible for Stat Activation Proc. Natl. Acad. Sci. U.S.A.. 1999; 96:5007.
• Kotenko, S.V., Saccani, S., Izotova, L.S., Mirochnitchenko, O.V., and Pestka, S. Human Cytomegalovirus Harbors its Own Unique IL-10 Homolog (cmvIL-10) Proc. Natl. Acad. Sci.U.S.A.. 2000; 97:1695.
• Krause, C. D., Mei, E., Xie, J., Jia, Y., Bopp, M. A., Hochstrasser, R. M., Pestka, S. Seeing the Light: Preassembly and Ligand-Induced Changes of the Interferon Gamma Receptor Complex in Cells Molecular & Cellular Proteomics. 2002; 1:.
• Brewer, G., Saccani, S., Sarkar, S., Lewis, A., and Pestka, S. Increased interleukin-10 mRNA stability in melanoma cells is associated with decreased levels of A+U-rich element binding factor AUF1 J. Interferon Cytokine Res.. 2003; 23:553.
• Pestka, S. A Dance Between Interferon -?/? and p53 Demonstrate Collaborations in Tumor Suppression and Antiviral Activities Cancer Cell. 2003; 4:85.
• Pestka, S., Krause, C. D., Sarkar, C., Walter, M., Shi, Y., and Fisher, P. IL-10 and Related Cytokines and Receptors Annual Review of Immunology. 2004; 22:929.
• Wu, W., Kerrigan, J.E., Yadav, P., Schwartz, B., Izotova, L., Lavoie, T.B., and Pestka, S. Design and Construction of a Phosphorylatable Chimeric Monoclonal Antibody with a Highly Stable Phosphate Oncology Research / Incorporating Anti-Cancer Drug Design. 2004; 14:541.
• Lee, J-H., Cook, J.R., Yang, Z-H., Mirochnitchenko, O., Gunderson, S., Felix, A.M., Herth, N., Hoffmann, R., Pestka, S. PRMT7: A new protein arginine methyltransferase that synthesizes symmetric dimethylarginine J Biol Chem. 2004; 280:3656.
• Krause, C.D. and Pestka, S. Evolution of the class 2 cytokines and receptors, and discovery of new friends and relatives, Pharmacol. Ther.. 2005; 106:269.
• Krause, C.D., Mei, E. Mirochnitchenko, O.V., Lavnikova, N., Xie, J., Jia, Y., Hochstrasser, R.M., and Pestka, S. Interactions Among the Components of the Interleukin-10 Receptor Complex Biochem Biophys Res Commun. 2005; 340:377.
• Pestka, S. and Krause, C.D. Interferon and Related Receptors,” Chapter 5 in The Interferons: Characterization and Application Wiley-VCH. 2006; 113.
• Krause, C. D., Lavnikova. N., Xie, J., Mei, E., Mirochnitchenko, O.V., Jia, Y., Hochstrasser, R.M.and Pestka, S. Preassembly and Ligand-Induced Restructuring of the Chains of the IFN-? Receptor Complex: The Roles of Jak Kinases, Stat1 and the Receptor Chains Cell research. 2006; 16:55.
• Krause, C.D., He, W., Kotenko, S., and Pestka, S. Modulation of the Activation of Stat1 by the Interferon-gamma Receptor Complex Cell research. 2006; 16:113.
• Cook, J.R., Lee, J-H., Yang, Z-H., Krause, C.D., Herth, N., Hoffmann, R., Pestka, S. FBXO11/PRMT9, a New Protein Arginine Methyltransferase, Symmetrically Dimethylates Arginine Residues Biochem Biophys Res Commun. 2006; 342:472.
• Krause, C.D., Yang, Z-H., Kim, Y-S., Lee, J-H., Cook, J.R., and Pestka, S. Protein arginine methyltransferases: Evolution and assessment of their pharmacological and therapeutic potential Pharmacology and Theraputics. 2006;

Research

• For the past two decades, interferon research has been a major focus of the laboratory. The research has led to the isolation, purification and characterization of the alpha, beta and gamma interferons, and subsequently their cloning and expression in bacteria. Interferons developed in the laboratory are currently available as approved therapeutics in the United States and worldwide for the treatment of various malignancies and viral diseases. During the past year, members of the laboratory have continued our endeavors to understand the interferons and their receptors, and to use these potent cytokines and immunomodulators to develop new strategies for the treatment and diagnosis of various diseases. The efforts have led to a number of new paths and unexpected directions many of which are described in this summary. Differences in Receptor Interactions of Type I (IFN-a, IFN-ß and IFN-?) Human Interferons. Chinese hamster ovary (CHO) cells expressing the two chains, Hu-IFN-aR1 and Hu-IFN-aR2, of the Type 1 interferon receptor respond to all Type I human interferons including IFN-a, IFN-ß and IFN-?. A splice variant of the Hu-IFN-aR1 chain, designated Hu-IFN-aR1s, was also isolated and used to reconstitute CHO cells. With these cells, Cook, Cleary, Mariano, and Izotova identified two Type I interferons which can interact with the splice variant (Hu-IFN-aR1s) and with the Hu-IFN-aR1 chains: Hu-IFN-aA and IFN-?. Two other Type I interferons, Hu-IFN-aB2 and Hu-IFN-aF, are capable of signaling through the complete Hu-IFN-aR1 chain only, but cannot utilize the splice variant Hu-IFN-aR1s. Hu-IFN-aR1 and Hu-IFN-aR1s differ in that the latter is missing a single subdomain of the receptor extracellular domain encoded by exons 4 and 5 of the Hu-IFN-aR1 gene. Therefore, different Type I interferons require different subdomains of the Hu-IFN-aR1 receptor chain and the splice variant chain discovered (Hu-IFN-aR1s) is functional. Because only one human recombinant interferon (IFN-aA, also called IFN-a2) is used in therapy, these results have implications for introducing additional human interferons for therapeutic use as they may have different therapeutic and side-effect profiles because they interact with the receptors differently. The Interferon Gamma Receptor (IFN-?R) Complex. Each cytokine which utilizes the Jak-Stat signal transduction pathway activates a distinct combination of members of the Jak and Stat families. Thus, either the Jaks, the Stats or both could contribute to the specificity of ligand action. With the use of chimeric receptors involving the interferon gamma receptor (IFN-?R) complex as a model system, we demonstrated that Jak2 activation is not an absolute requirement for IFN? signaling. Other members of the Jak family can functionally substitute for Jak2. IFN-? can signal through the activation of Jak family members other than Jak2 as measured by Statl homodimerization and MHC class I antigen expression. This indicates that Jaks are interchangeable in the Jak-Stat signal transduction pathway and that the Jaks do not contribute to the specificity of signal transduction in the Jak-Stat pathway. During these experiments an orphan receptor, CRFB4, was discovered to have an intracellular domain with functional activity. These studies also demonstrated that only the long form of the IFN-a receptor chain 2 (IFN-aR2C) was functional in enabling the Type I interferon complex to respond to Type I interferons. Our results with chimeric receptors demonstrate how chimeric receptors can be used to determine the function of receptors and have permitted us to discover a new functional receptor chain, CRFB4. Identification of a Second Chain of the IL-10 Receptor Complex. Studying the IFN-[alpha] and IFN-[gamma] receptor complexes, Kotenko, Krause, Izotova, Pollack and Wu focused on the orphan receptor, CRFB4, from this family of receptors as noted above. Based on our previous observation that Tyk2 tyrosine kinase associates with the CRFB4 intracellular domain, we proposed that CRFB4 could be the second chain of the IL-10 receptor complex. Only one chain, that for ligand binding (IL-10R1), was previously identified. We demonstrated that, although human IL-10 binds to the human IL-10R1 chain expressed in hamster cells, it does not induce signal transduction. However, the coexpression of CRFB4 together with the IL-10R1 chain renders hamster cells sensitive to IL-10. The IL-10:CRFB4 complex was detected by crosslinking to labeled IL-10. In addition, the IL-10R1 chain was coimmunoprecipitated with anti-CRF antibody when peripheral blood mononuclear cells were treated with IL-10. These results demonstrated that the CRFB4 chain is part of the IL-10 receptor signaling complex. Thus, the CRFB4 chain, which we designated as the IL-10R2 chain, serves as an accessory chain essential for the initiation of IL-10 induced signal transduction events. Paradigm for Cytokine Class II Receptor Complexes. Based on our studies of the IFN-[gamma] and IL-10 receptor complexes we observed specific features of this family of receptors. The binding of a ligand to the ligand binding subunit causes homodimerization of the receptor subunit, one, IFN-[gamma]R1, however, is not sufficient for initiation of a signal transduction cascade. The presence of an additional accessory subunit, IFN-[gamma]R1, in the ligand-receptor complex is required for signaling. The function of this second receptor chain, which we designated the helper receptor, is to bring an additional tyrosine kinase activity to the receptor complex. Any Jak family member can provide this function. Thus, the helper receptor does not determine the specificity of signaling and is specific only for cytokine binding. Identification of Amino Acid Residues that Recruit STAT2 to the Hu-IFN-[alpha]R2a Receptor Chain. The type I IFN family members (IFN-[alpha], IFN-[beta], IFN-[omega], and IFN-[chi]) are involved in a variety of physiological responses. They induce antiviral and antiproliferative activities; stimulation of cytotoxic activity of lymphocytes, natural killer cells, and macrophages; modulation of cellular differentiation; stimulation of MHC class I antigens and other surface markers. Type I IFNs activate the Jak-Stat signal transduction pathway. After the ligand binding, Tyk2 and Jak1 kinases are activated, leading to activation of Stat1 and Stat2, latent transcriptional factors. After phosphorylation they form the active transcriptional complex ISGF-3 (IFN-stimulated gene factor-3) through the association of the Stat1/Stat2 heterodimer with the p48 protein. So far, the detailed activation mechanism of Stat2 by the type I IFN receptor complex is still unclear. Unlike other Stats that have been shown to interact with corresponding receptors through the highly specific interaction between Stat SH2 domains and the phosphorylated tyrosine motifs within the intracellular domain of the receptors, Stat2 was shown to associate with the IFN-[alpha]R2c chain through its N-terminal domain. Ge and Kotenko are in the process of identifying the amino acid residues, of the IFN-[alpha]R2 chain that are responsible for interacting with and activating Stat2. In order to avoid the cross species activity of human type I IFNs on hamster cells, the Hu-IFN-[gamma]R1/[alpha]R2c hybrid derivatives were expressed in CHO derived Q21 cells that carry Hu-IFN-[gamma]R2. Thus, Hu-IFN-[gamma], which is not active on hamster cells, was used to activate signal transduction through the chimeric receptor complex. The obtained cell lines were used to study IFN--induced MHC class I expression and Stat1 activation. The results so far indicate that the recruitment site for Stat2 on the IFN-[alpha]2Rc intracellular domain is localized within the C-terminal quarter of the IFN-[alpha]2Rc intracellular domain. Construction of Phosphorylatable Monoclonal Antibodies to Tumor Associated Antigens. The mouse monoclonal antibody B72.3 recognizes the tumor associated antigen TAG-72 that is expressed on the surface of human adenocarcinomas. This monoclonal antibody has begun to be used in clinical research protocols for the diagnosis and therapy of adenocarcinomas. To develop more effective methods for labeling this monoclonal antibody we introduced a phosphorylation site into chimeric monoclonal antibody B72.3 (MAb-chB72.3) by site-specific mutation of the coding sequence. The chimeric molecule contains the mouse variable region fused to the human IgG1 constant region. The phosphorylation site for the cAMP-dependent protein kinase was positioned at the carboxyl terminus of the heavy chain constant region of the MAb chB72.3. The resultant modified MAb chB72.3-P was expressed in 293 cells and purified. The MAb-chB72.3-P protein was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase with [gamma-32P]ATP to high radiospecific activity. The P-labeled MAb-chB72.3-P protein bound to cells expressing the TAG-72 antigen and was stable in serum. To improve the monoclonal antibody for use in humans, a second generation monoclonal antibody with greater affinity, CC49, was used and a chimeric version, chCC49, with the human IgG1 constant region was used. Phosphorylation sites were introduced into this chimeric monoclonal antibody CC49 (MAb-chCC49) by inserting three synthetic fragments encoding two phosphorylation sites into an expression vector, pdHL7. The resultant modified antibody, MAb-chCC49-6P, containing six phosphorylation sites per heavy chain, was expressed in NS0 cells and purified. The MAb-chCC49-6P protein can be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase with [gamma-32P]ATP to exceptionally high specific activity. The 32P-labeled MAb-chCC49-6P protein binds to cells expressing TAG-72 antigens. The introduction of phosphorylation sites into monoclonal antibodies (MAb) provides a procedure for labeling MAb for the diagnosis and treatment of cancers. Additional work in this area will focus on evaluating the introduction of new protein kinase recognition sites and their characterization to find the most effective ones for use in diagnosis and therapy of cancer.