The pool of preactivated Lck in the initiation of T‐cell signaling: a critical re‐evaluation of the Lck standby model

The initiation of T‐cell receptor (TCR) signaling, based on the cobinding of TCR and CD4‐Lck heterodimer to a peptide–major histocompatibility complex II on antigen presenting cells, represents a classical model of T‐cell signaling. What is less clear however, is the mechanism which translates TCR engagement to the phosphorylation of immunoreceptor tyrosine‐based activation motifs on CD3 chains and how this event is coupled to the delivery of Lck function. Recently proposed ‘standby model of Lck’ posits that resting T‐cells contain an abundant pool of constitutively active Lck (pY394 Lck ) required for TCR triggering, and this amount, upon TCR engagement, remains constant. Here, we show that although maintenance of the limited pool of pY394 Lck is necessary for the generation of TCR proximal signals in a time‐restricted fashion, the total amount of this pool, ~2%, is much smaller than previously reported (~40%). We provide evidence that this dramatic discrepancy in the content of pY394 Lck is likely the consequence of spontaneous phosphorylation of Lck that occurred after cell solubilization. Additional discrepancies can be accounted for by the sensitivity of different pY394 Lck ‐specific antibodies and the type of detergents used. These data suggest that reagents and conditions used for the quantification of signaling parameters must be carefully validated and interpreted. Thus, the limited size of pY394 Lck pool in primary T‐cells invites a discussion regarding the adjustment of the quantitative parameters of the standby model of Lck and reevaluation of the mechanism by which this pool contributes to the generation of proximal TCR signaling.