Predictive Model of Lymphocyte-Specific Protein Tyrosine Kinase (LCK) Autoregulation

Cellular and Molecular Bioengineering ( 2016) DOI: 10.1007/s12195-016-0438-7 Predictive Model of Lymphocyte-Specific Protein Tyrosine Kinase (LCK) Autoregulation JENNIFER A. ROHRS,1 PIN WANG,1,2 and STACEY D. FINLEY1,2 1 Department of Biomedical Engineering, University of Southern California, 1042 Downey Way, DRB 140, Los Angeles, CA 90089, USA; and 2Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, Los Angeles, CA, USA (Received 12 February 2016; accepted 12 April 2016) Associate Editor Michael R. King oversaw the review of this article. Abstract—Lymphocyte-specific protein tyrosine kinase (LCK) is a key activator of T cells; however, little is known about the specific autoregulatory mechanisms that control its activity. We have constructed a model of LCK autophosphorylation and phosphorylation by the regulating kinase CSK. The model was fit to existing experimental data in the literature that presents an in vitro reconstituted membrane system, which provides more physiologically relevant kinetic measurements than traditional solution-based systems. The model is able to predict a robust mechanism of LCK autoregulation. It provides insights into the molecular causes of key site-specific phosphorylation differences between distinct experimental conditions. Probing the model also provides new hypotheses regarding the influence of individual binding and catalytic rates, which can be tested experimentally. This minimal model is required to elucidate the mechanistic interactions of LCK and CSK and can be further expanded to better understand T cell activation from a Address correspondence to Stacey D. Finley, Department of Biomedical Engineering, University of Southern California, 1042 Downey Way, DRB 140, Los Angeles, CA 90089, USA. Electronic mail: sfi Stacey D. Finley is the Gabilan Assistant Professor of Biomedical Engineering at the University of Southern California, where she directs the Computational Systems Biology Laboratory. Her research group develops mechanistic models of biological processes and utilizes the models to gain insight into the dynamics and regulation of biological systems and enable the development of novel therapeutics for pathological conditions. Dr. Finley’s research interests include applying computational modeling to investigate tumor angiogenesis, tumor metabolism, and cancer immunotherapy. Dr. Finley received her B.S. in Chemical Engineering from Florida A & M University and obtained her Ph.D. in Chemical Engineering from Northwestern University. She completed postdoctoral training at Johns Hopkins University in the Department of Biomedical Engineering, where she was awarded postdoctoral fellowships from the NIH National Research Service Award and the UNCF/Merck Science Initiative. Dr. Finley was named a 2015 Emerging Scholar and is currently a Keystone Symposia Fellow. Most recently, she received an NSF CAREER award. Dr. Finley has a joint appointment in the Department of Chemical Engineering and Materials Science and is an associate member of the USC Norris Comprehensive Cancer Center. This article is part of the 2016 Young Innovators Issue. systems perspective. Our computational model enables the evaluation of LCK protein interactions that mediate T cell activation on a more quantitative level, providing new insights and testable hypotheses. Keywords—Systems biology, Computational modeling, T cell signaling, Parameter estimation. INTRODUCTION Lymphocyte-specific protein tyrosine kinase (LCK) is a key regulator of T cell activation and differentiation.6,38 LCK helps to activate healthy T cells against diseased cells in the body by phosphorylating immunotyrosine activating motifs (ITAMS) on the CD3f chain of the T cell receptor (TCR).25 Mutations in the LCK gene can lead to autoimmune disease14 and contribute to cancer.7 Recently, LCK has been shown to play an important and complex role in the activation of chimeric antigen receptor (CAR) engineered T cells.22 CARs are engineered proteins that contain a variety of T cell signaling domains linked to an extracellular antibody single chain variable fragment  2016 The Author(s). This article is published with open access at Springerlink.com ROHRS et al. (scFv). These proteins can activate T cells against a tumor-associated antigen to eradicate cancer cells.22,37 As CARs are adapted and modified to more specifically target different types of cancer cells, understanding the detailed mechanisms that govern their activation has become more important. Despite its strong role in regulating T cell signaling, little is known about the specific mechanisms that control LCK catalytic activity. LCK is a multi-domain protein that can catalyze the phosphorylation of many substrates in T cells, including itself. LCK has two main phosphorylation sites, the tyrosine residues Y394 and Y505. Y394 is located close to the kinase domain, and, therefore, has been shown to play a significant role in substrate specificity.23 Y505 is located near the C-terminal tail of the protein. When phosphorylated, this tail is thought to fold up and bind in cis, locking the molecule in a ‘‘closed’’ conformation.9 Therefore, it is commonly accepted that phosphorylation at Y394 (denoted as LCK species P394U505) increases the catalytic activity of LCK and phosphorylation at Y505 (species U394P505) decreases catalytic activity.44 It has been shown that the unphosphorylated and doubly phosphorylated forms of LCK (species U394U505 and P394P505, respectively) retain an intermediate catalytic activity when acting on some substrates,15 although they may have more complex kinetics on others. These four forms of LCK distribute and aggregate differently within cells,34 and, while all four forms exist in resting T cells, efforts to calculate the exact ratios of the species have been inconclusive.2,30 Several proteins have been shown to control LCK phosphorylation. For example, C-terminal Src kinase (CSK) is a regulatory kinase that phosphorylates LCK specifically at Y505.41 In addition, several phosphatases act on LCK and CSK, most notably CD45 and PTPN22.45 It is commonly accepted that LCK can autophosphorylate at Y394,44 but it has only recently been appreciated that LCK can also autophosphorylate at Y505.15 The kinetics of these LCK phosphorylation and dephosphorylation reactions determine the pool of catalytically active LCK available to control T cell activation in vivo. Traditionally, the kinetics of these reactions are studied experimentally with recombinant proteins in solution3,33; however, inside the cell, LCK is largely bound to the plasma membrane, in a twodimensional density distribution.18,46 This binding to the plasma membrane can profoundly influence a protein’s kinetics in several ways: (i) by altering the conformation of the protein, opening or closing available binding pockets, (ii) by changing the diffusion kinetics, which can alter the rate at which the enzyme encounters i (...truncated)