T Cell Activation Results in Conformational Changes in the Src Family Kinase Lck to Induce Its Activation

Abstract

The lymphocyte-specific Src family protein tyrosine kinase p56(Lck) (Lck) is essential for T cell development and activation and, hence, for adaptive immune responses. The mechanism by which Lck activity is directed toward specific substrates in response to T cell receptor (TCR) activation remains elusive. We used fluorescence lifetime imaging microscopy to assess the activation-dependent spatiotemporal changes in the conformation of Lck in live human T cells. Kinetic analysis of the fluorescence lifetime of Lck biosensors enabled the direct visualization of the dynamic local opening of 20% of the total amount of Lck proteins after activation of T cells with antibody against CD3 or by superantigen-loaded antigen-presenting cells. Parallel biochemical analysis of TCR complexes revealed that the conformational changes in Lck correlated with the induction of Lck enzymatic activity. These data show the dynamic, local activation through conformational change of Lck at sites of TCR engagement.

The coreceptor CD4 is expressed in distinct nanoclusters and does not colocalize with T-cell receptor and active protein tyrosine kinase p56lck

“…Furthermore, it has only recently become clear that all four different conformational states of Lck are determined by the combined phosphorylation status of two regulating tyrosine residues (Y394 and Y505) and that these constitutively coexist in a dynamic equilibrium, regardless of the activation status of a T cell (43,49). However, it is not as clear whether or not the activation of a T cell induces changes in the relative proportions of the activated forms of Lck (43,50). Therefore, how nonspecific phosphorylation of the ITAM motif by constitutively existing active forms of Lck is regulated in nonactivated T cells, and how the constitutively existing active forms of Lck are involved in a very efficient and fast signaling propagation upon ligation of TCR, are still open questions.…”

The Late Endosomal Transporter CD222 Directs the Spatial Distribution and Activity of Lck

“…Consequently, the homeostasis of the four proposed conformational forms of Lck (3) is shifted toward the inhibitory closed conformation of Lck. Recently, it has been proposed that the intracellular distribution of Lck is regulated via its phosphorylation state and the corresponding conformations (4,6): the open active form (pY394) is thought to induce clustering, whereas the closed inactive form (pY505) prevents the formation of clusters, which has been shown to be lipid independent (4). Putting these data together one might suggest that the CD222 knockdown, which results in a 2-fold increase of inhibitory Lck pY505, does prevent Lck from clustering and T cells from getting activated.…”

“…Lck rather exists in several conformational and activity states: 1) an unphosphorylated "primed" form; 2) an active form phosphorylated at the tyrosine 394 (pY394); 3) an inactive "closed" form phosphorylated at the COOH-terminal tyrosine 505 (pY505); and 4) a double phosphorylated form (pY394 + pY505) that is thought to possess an equal kinase activity as single phosphorylated Lck at Y394 (3). Hence, active Lck is constitutively present in resting T cells, and it is its coordinated intracellular distribution that is crucial to balance T cell activity and nonreactivity (5)(6)(7). The antagonistic activities of the phosphatase CD45 and the kinase Csk control Lck's mode of action (2,8,9).…”

Ionic CD3−Lck interaction regulates the initiation of T-cell receptor signaling

“…The SH2 domain binds to phosphotyrosine, and its interaction with an inhibitory pTyr site at the Lck C terminus leads to an autoinhibitory folding (21,22). Earlier experiments show that ∼40% of Lck molecules in resting T cells are constitutively active (23) and antigen stimulation leads to local activation of Lck molecules (24,25). However, how Lck recognizes CD3 substrates and initiates ITAM phosphorylation remains elusive.…”