Computational Model of Chimeric Antigen Receptors Explains Site-Specific Phosphorylation Kinetics

Biophys J. 2018 Sep 18;115(6):1116-1129. doi: 10.1016/j.bpj.2018.08.018. Epub 2018 Aug 22.


Chimeric antigen receptors (CARs) have recently been approved for the treatment of hematological malignancies, but our lack of understanding of the basic mechanisms that activate these proteins has made it difficult to optimize and control CAR-based therapies. In this study, we use phosphoproteomic mass spectrometry and mechanistic computational modeling to quantify the in vitro kinetics of individual tyrosine phosphorylation on a variety of CARs. We show that each of the 10 tyrosine sites on the CD28-CD3ζ CAR is phosphorylated by lymphocyte-specific protein-tyrosine kinase (LCK) with distinct kinetics. The addition of CD28 at the N-terminal of CD3ζ increases the overall rate of CD3ζ phosphorylation. Our computational model identifies that LCK phosphorylates CD3ζ through a mechanism of competitive inhibition. This model agrees with previously published data in the literature and predicts that phosphatases in this system interact with CD3ζ through a similar mechanism of competitive inhibition. This quantitative modeling framework can be used to better understand CAR signaling and T cell activation.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • CD28 Antigens / chemistry
  • CD28 Antigens / metabolism
  • Computer Simulation*
  • Kinetics
  • Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / metabolism
  • Mutation
  • Phosphorylation
  • Proteomics
  • Receptors, Antigen / chemistry
  • Receptors, Antigen / genetics
  • Receptors, Antigen / metabolism*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*
  • Substrate Specificity
  • Tyrosine / metabolism


  • CD28 Antigens
  • Receptors, Antigen
  • Recombinant Fusion Proteins
  • Tyrosine
  • Lymphocyte Specific Protein Tyrosine Kinase p56(lck)