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LETTERS TO THE EDITOR |
Institut für Biochemie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany
In a recent paper, Tavano et al. (1) reported the essential role of a C-terminal 208PXXPP212 motif in CD28 for the recruitment of Lck to the immunological synapse, and we would like to comment on their results in the light of the affinity of different CD28-Lck interactions that we have studied in vitro by biophysical methods.
Peptides comprising residues 206–220 of human CD28 and 205–215 of murine CD28, respectively, bind to the SH3 domain of Lck only with very low affinity (Kd > 1 mM). Although we agree that this interaction can nevertheless be of physiological relevance in a situation in which both proteins are membrane associated and located in lipid rafts, we want to draw attention to an alternative CD28 interaction that can be formed after phosphorylation of Tyr209 of human CD28 with the Lck SH2 domain (Fig. 1). This interaction (Kd = 2.13 µM) is in vitro at least three orders of magnitude stronger than the SH3 interaction and is in the range of those observed for other SH2-ligand interactions (2).
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Phe mutation at this position severely impaired the ability of mCD28 to deliver a costimulus for the expression of CD69 and the production of IL-2 (3). Although tyrosine phosphorylation at this position could not be associated with any specific signaling event up to now, our data suggest that this residue is likely involved in Lck SH2 binding. We feel that this finding has important implications for the design of new experiments that should also take into account the existence of a second mode of CD28-Lck interaction involving the SH2 domain in addition to the SH3 interaction reported by Tavano et al. (1).
References
Venetian Institute of Molecular Medicine, University of Padua, Padua, Italy
In our recent paper titled "CD28 and Lipid Rafts Coordinate Recruitment of Lck to the Immunological Synapse of Human T Lymphocytes," we demonstrated the essential role of the C-terminal 208PXXPP212 motif in CD28 for the recruitment of lipid rafts, and therefore of the raft-associated protein Lck, to the immunological synapse (IS) (R1 ). We do not believe that the lower recruitment of Lck to the IS of T cells expressing the CD28-3A mutant is the consequence of a lower affinity of the CD28-Lck interaction. First, we demonstrated that not only Lck but also a myristoylated and palmitoylated fluorescent protein, used as raft marker, is less recruited to the IS of CD28-3A-expressing T cells (Fig. 2 of our paper). Second, in agreement with the model proposed by Hofinger and Sticht, we have data indicating that the direct interaction between CD28 and Lck does not require the CD28 C-terminal 208PXXPP212 motif (our manuscript in preparation).
Our paper indicates that a large fraction of Lck is recruited to the IS of T cells engaging a B7+ APC as a result of lipid rafts mobilization, an event requiring CD28 signaling through its C-terminal 208PXXPP212 motif.
References
This article has been cited by other articles:
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  M. Libra, A. Gloghini, P. M. Navolanic, A. L. Zignego, V. De Re, A. Carbone, and E. H. Sasso JH6 Gene Usage among HCV-Associated MALT Lymphomas Harboring t(14;18) Translocation J. Immunol., April 1, 2005; 174(7): 3839 - 3839. [Full Text] [PDF]
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) for the Lck SH2 domain: Changes of the relative fluorescence were measured in a competitive binding study that was performed as described in Ref. 
) (Kd = 8.99 µM). Titration curves for a high-affinity SH2 ligand (•) containing a YEEI binding motif (Kd = 0.10 µM) and for the Lck C terminus were reprinted with permission from Ref.