Specific tyrosine (Y) residues on LAT function as binding sites for SH2 domains of different effector proteins when phosphorylated. Analysis of mutant LAT binding properties show that Y171, Y191, Y226 and Y132 are binding sites while a separate tyrosine residue Y110 must be phosphorylated for full activation of Ras by Grb2 [1]. It is important to appreciate that SH2 domains of different proteins have different binding specificities for the amino acid residues around the phosphorylated tyrosine [2]. Thus, LAT has different binding sites for the SH2 domains of Grb2, GADS and PLC-γ1.
The sequences around Y171 (YVNV), 191 (YVNV), and 226 (YENL) are classical Grb2 SH2 domain binding motifs (YXN) [1,3]. Y110 (SYENE) might also be a recognition site for SH2 domain of Grb2 [3]. Mutating individual tyrosine residues to phenylalanine does not prevent Grb2 binding and this is consistent with the model that there are multiple binding sites for Grb2 on LAT [1].
GADS has a SH2 domain that is quite similar to that of Grb2 [4]. Y171 and Y191 are binding sites for Gads SH2 domain but Y226 is not a binding site for Gads [4]. The experimental evidence consistent with this model was that double mutation of Y171 and Y191 was sufficient to completely abolish Gads binding [4].
Y132 (YLVV) is a PLC-γ1 SH2 domain binding motif [1,4]. Evidence suggests that it is the N-terminal SH2 domain of that PLC-γ1 binds to Y132 [4]. Mutation of Y132 to phenylalanine was sufficient to abolish PLC-γ1 binding [4].
As seen in figure 1, fluorescence microscope images of LAT, GADS and Grb2 in vivo suggest that downstream effector proteins form a multi-protein activated complex with LAT after TCR activation [5].
Figure 1: Fluorescence microscopy of Grb2, GADS and LAT showing that the proteins are associated in clusters during signal transduction [5]. Image taken from Balagopalan,L et al. (2010). “The LAT story: a tale of cooperativity, coordination, and choreography.” Cold Spring Harbor perspectives in biology 2(8): a005512. Original figure 3: LAT signaling complexes and microclusters.
Additionally, it appears that the binding of PLC-γ1 is stabilized by binding of Grb2. Mutations in LAT at tyrosines 171, 191, and 226 result in a reduction of PLC-γ1 binding to LAT, even though these residues are not binding sites for the SH2 domain of PLC-γ1 [4,6]. This suggests that different effector proteins show cooperative binding to LAT. One hypothesis for how cooperative binding occurs is that the binding of an effector e.g. Grb2 to its appropriate binding site creates conformational changes in LAT which stabilizes lower affinity binding of SH2 domains of other effectors e.g. PLC-γ1 to various sites [4]. The formation of activated signaling clusters by cooperative binding might afford a greater sensitivity of control of TcR pathways [5]. A weakly activating signal might be less effective in recruiting multiple proteins to LAT, possibly resulting in a weaker response [5].
Interaction with Gads-SH2
Figure 2. A-weighted 2Fo–Fc electron density isosurfaces around phosphotyrosyl residues in Gads-SH2/pLAT complexes. Taken from Cho, S. Structural basis for differential recognition of tyrosine-phosphorylated sites in the linker for activation of T cells (LAT) by the adaptor Gads. EMBO J. 23, 1441-51 (2004).
A: Gads-SH2 in complex with pLAT171 (1.8 Å resolution, 1.5 contour level).
B: Gads-SH2 in complex with pLAT191 (1.8 Å resolution, 1.5 contour
level)
C: Gads-SH2 in complex with pLAT226 (1.9 Å resolution, 1.5 contour
level)
Overall description: The crystal structures of Gads-SH2 in complex with three LAT phosphopeptides to high resolution show that the phosphate group of the LAT peptides is located in a deep hydrophilic cavity, where it is stabilized by an intricate network of hydrogen bonds and ionic interactions. The SH2 domain of Gads (Gads-SH2) displays a higher affinity for phosphopeptides representing LAT sites 171 and 191 [7].
Interaction with PLC-γ
The LAT molecule is also responsible for binding to and activating phospholipase C-γ1 (PLC-γ1) on the cell membrane. The activation of PLC-γ1 is necessary for the formation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) from phosphatidylinositol 4,5-bisphosphate. IP3 and DAG are important 2nd messengers in many signalling pathways downstream of receptor-mediated tyrosine kinase activators [8].
The mechanism by which PLC-γ1 is recruited on LAT for activation is incompletely understood, but the use of real-time fluorescent imaging has shown that the N-terminal SH2 domain of PLC-γ1 is necessary but not sufficient for its recruitment [9]. Either the SH3 or C-terminal SH2 domain of PLC-γ1 are also required for stable recruitment of PLC-γ1 to LAT. All three PLC-γ1 SH domains are required for phosphorylation of PLC-γ1 Y783, which in turn is critical for enzyme activation and the healthy development of T-cells [9].
The importance of LAT for the recruitment and eventual activation of PLC-γ1 complex can be studied by analysing LAT-deficient T-cells expressing transgenic LAT2/LAB (linker for the activation of B cells). Although LAT and LAB share similar structural features, none of the tyrosine residues in LAB resemble known PLC-γ1 binding motifs equivalent to Y136 in LAT [10]. LAT deficient naïve T-cells cannot differentiate fully and expression of the LAB transgene in these T-cells could only restore development partially [11]. The presence of functional LAB in these cells even allowed for a blunted calcium response to TCR cross-linking [12]. The partial phenotypic rescue of LAT-deficient T-cells by the LAB transgene is possibly due to efficient recruitment of Grb2 but inefficient recruitment of PLC-γ1, resulting in suboptimal signalling [11]. Ultimately, mice with LAT-deficient T-cells still develop lymphoproliferative disease regardless of the presence of LAB [11].
Figure 3. A. Crystal structure of Activated FGFR1 in Complex with a PLCγ FragmentRibbon diagram of the complex structure FGFR1-3P in green, N-SH2 and C-SH2 domains are in cyan and blue, respectively, the linker connecting the two SH2 domains is in pale green and the C-terminal extension is in orange. B. Schematic Representation of PLCγ. Tyr residue 783 is located between the C-SH2 and SH3 domains. Adapted from Bae, J.H, Lew, E.D., Yuzawa, S., Tomé , F., Lax, .I, and Schlessinger J. (2009). The selectivity of receptor tyrosine kinase signaling is controlled by a secondary SH2 domain binding site. Cell. 138, 514-24.
References
[1]. Lin, J. and Weiss, A. (2001). “Identification of the minimal tyrosine residues required for linker for activation of T cell function.” The Journal of biological chemistry 276(31): 29588-29595.
[2]. Songyang, Z. (1999). “Recognition and regulation of primary-sequence motifs by signaling modular domains.” Progress in biophysics and molecular biology 71(3-4): 359-372.
[3]. Sánchez, I.E. et al. (2008). “Genome-wide prediction of SH2 domain targets using structural information and the FoldX algorithm.” PLoS computational biology 4(4): e1000052.
[4]. Zhang, W et al. (2000). “Association of Grb2, Gads, and phospholipase C-gamma 1 with phosphorylated LAT tyrosine residues.” The Journal of biological chemistry 275(30): 23355-23361.
[5]. Balagopalan, L et al. (2010). “The LAT story: a tale of cooperativity, coordination, and choreography.” Cold Spring Harbor perspectives in biology 2(8): a005512.
[6]. Houtman, Jon C.D. et al. (2004). “Binding specificity of multiprotein signaling complexes is determined by both cooperative interactions and affinity preferences.” Biochemistry 43(14): 4170-8.
[7]. Cho, S. Structural basis for differential recognition of tyrosine-phosphorylated sites in the linker for activation of T cells (LAT) by the adaptor Gads. EMBO J. 23, 1441-51 (2004).
[8] Phillips-Mason PJ, Kaur H, Burden-Gulley SM, Craig SE, Brady-Kalnay SM (2011)."Identification of phospholipase C gamma1 as a protein tyrosine phosphatise substrate that regulates cell migration.". J Cell Biochem 112 (1): 39–48.
[9] Braiman A, Barda-Saad M, Sommers CL, Samelson LE. (2006) Recruitment and activation of PLCgamma1 in T cells: a new insight into old domains. Epub, 25(4):774-84
[10] Songyang, Z., S.Shoelson, M.Chaudhuri, G.Gish, T.Pawson, W. Haser, F. King, T. Roberts, S. Ratnofsky, R.Lechleider, et al 1993. SH2 domains recognize specific phosphopeptide sequences. Cell72:767.
[11] Orr S and McVicar D, (2011) LAB/NTAL/Lat2: a force to be reckoned with in all leukocytes? Journal of Leukocyte Biologyvol. 89 no. 1 11-19
[12] Janssen E, Zhu M, Craven B, and Zhang W. (2004) Linker for Activation of B Cells: A Functional Equivalent of a Mutant Linker for Activation of T Cells Deficient in Phospholipase C-γ1 Binding. The Journal of Immunology, 172: 6810-6819.
