Lipids Regulate Lck Protein Activity through Their Interactions with the Lck Src Homology 2 Domain

Ren Sheng,Da-Jung Jung,Antonina Silkov,Hyunjin Kim,Indira Singaram,Zhi-Gang Wang,Yao Xin,Eui Kim,Mi-Jeong Park,Pallavi Thiagarajan-Rosenkranz,Sean Smrt,Barry Honig,Kwanghee Baek,Sungho Ryu,Justin Lorieau,You-Me Kim,Wonhwa Cho

doi:10.1074/jbc.m116.720284

Abstract

Lymphocyte-specific protein-tyrosine kinase (Lck) plays an essential role in T cell receptor (TCR) signaling and T cell development, but its activation mechanism is not fully understood. To explore the possibility that plasma membrane (PM) lipids control TCR signaling activities of Lck, we measured the membrane binding properties of its regulatory Src homology 2 (SH2) and Src homology 3 domains. The Lck SH2 domain binds anionic PM lipids with high affinity but with low specificity. Electrostatic potential calculation, NMR analysis, and mutational studies identified the lipid-binding site of the Lck SH2 domain that includes surface-exposed basic, aromatic, and hydrophobic residues but not the phospho-Tyr binding pocket. Mutation of lipid binding residues greatly reduced the interaction of Lck with the ζ chain in the activated TCR signaling complex and its overall TCR signaling activities. These results suggest that PM lipids, including phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, modulate interaction of Lck with its binding partners in the TCR signaling complex and its TCR signaling activities in a spatiotemporally specific manner via its SH2 domain.

Highlights

Lymphocyte-specific protein-tyrosine kinase (Lck) is a 56-kDa Src family kinase that plays an essential role in T cell receptor (TCR)4 signaling and T cell development [1, 2]
Lck-Src homology 2 (SH2) and the full-length Lck had comparable affinity for plasma membrane (PM) mimetic vesicles (Table 1), indicating that lipid binding activity of Lck lies within its SH2 domain and that the lipidbinding site of Lck SH2 domain (Lck-SH2) is fully exposed in the intact protein
When cells were stimulated with OKT3, which should trigger the release of the Lck SH2 domain from intramolecular tethering (Fig. 8) [1, 2], dynamic Lck-␨ colocalization was significantly enhanced within 5 min, which lasted for Ն10 min (Fig. 7, A and B, and a black line in D)

Summary

Lipid Regulation of Lck Activity

Lck is constitutively localized via N-terminal acylation to the plasma membrane (PM) where the TCR-CD3 complex is located [1, 2]. Structural analysis of a wide range of SH2 domains and their complexes with Tyr(P)-peptides has revealed that SH2 domains have a common architecture made of two ␣-helices (␣A and ␣B) flanking antiparallel ␤ strands (␤A to ␤G) [13, 15]. They recognize Tyr(P) and a few residues immediately C-terminal to Tyr(P) using a Tyr(P) binding pocket and a secondary binding site, respectively [13]. We explored the possibility that lipids bind the Lck SH2 domain (Lck-SH2) and control the cellular activity of Lck through this interaction

Results

Discussion

Experimental Procedures