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Quantitative interactomics of Lck-TurboID in living human T cells unveils T cell receptor stimulation-induced proximal Lck interactors

ABSTRACT: While Lck has been widely recognized to play a pivotal role in the initiation of the T cell receptor (TCR) signaling pathway, an understanding of the precise regulation of Lck in T cells upon TCR activation remains elusive. Investigation of protein-protein interaction (PPI) using proximity labeling techniques such as TurboID has the potential to provide valuable molecular insights into Lck regulatory networks. By expressing Lck-TurboID in Jurkat T cells, we have uncovered a dynamic, short-range Lck protein interaction network upon 30 minutes of TCR stimulation. In this novel application of TurboID, we detected 27 TCR stimulation-induced Lck-proximal interactors in living T cells, including both bona fide and uncanonical Lck interactors, validating the discovery power of this tool. Our results revealed previously unappreciated Lck PPI which may be associated with cytoskeletal rearrangement, ubiquitination of TCR signaling proteins, activation of the MAPK cascade, coalescence of the LAT signalosome, and formation of the immunological synapse. In this study, we demonstrated for the first time in immune cells and for the kinase Lck that TurboID can be utilized to unveil PPI dynamics in living cells at a time scale consistent with TCR signaling.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): T Cell

SUBMITTER: Xien Yu Chua

LAB HEAD: Arthur, Salomon

PROVIDER: PXD020759 | Pride | 2020-11-12

REPOSITORIES: Pride

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			Action	DRS
	MQ_evidence.txt	Txt
	SLP76_wt_LckTurbo_biotin_1mM_C305_rep1.raw	Raw
	SLP76_wt_LckTurbo_biotin_1mM_C305_rep2.raw	Raw
	SLP76_wt_LckTurbo_biotin_1mM_C305_rep3.raw	Raw
	SLP76_wt_LckTurbo_biotin_1mM_C305_rep4.raw	Raw

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Vav1 regulates T cell activation through a feedback mechanism and crosstalk between the T cell receptor and CD28 in CD3-stimulated T cells

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Lck phosphorylation status of Lck Tyr192 and Tyr192/Tyr505 mutantsLck phosphorylation status of Lck Tyr192 and Tyr192/Tyr505 mutants

Project description:Lymphocyte-specific protein tyrosine kinase (Lck) is crucial for signaling from the T cell receptor (TCR) and is controlled through tyrosine phosphorylations. Phosphorylated Tyr505 (pTyr505) promotes a closed, inactive conformation of Lck, while pTyr394 is critical for kinase activity. Additionally, pTyr192 has been suggested to regulate Lck activity by changing the specificity of the Lck Src-homology 2 (SH2) domain and/or by affecting Lck association with CD45 thus drastically increasing pTyr505. However, little is known about how pTyr192 affects endogenously expressed Lck. Here we used CRISPR/Cas9 genome editing to generate Jurkat cell lines expressing Lck Glu192 mimicking Lck pTyr192, or Lck Phe192 mimicking unphosphorylated Lck Tyr192. We confirmed that Lck Glu192 is hyperphosphorylated on Tyr505, possibly explaining reduced association of Lck Glu192 with prototypic Lck-SH2 ligands. To isolate the effect of Lck Tyr192 mutations from the effect on Lck pTyr505, we subsequently generated Jurkat cells doubly mutated on Lck Tyr192 and Lck Tyr505. Both Lck Phe192/Phe505 and Lck Glu192/Phe505 mutants co-precipitated similar amounts of binding partners. Moreover, both mutants displayed hyperphosphorylation of Tyr394. Our results indicate that CD45 is the main phosphatase controlling Lck pTyr394 in steady-state T cells. Additionally, our data demonstrate that the prototypic specificity of the Lck SH2 domain (owned by the Lck Phe192 mutants) promotes transphosphorylation of Tyr394. These observations pinpoint the fundamental role of Tyr192 in regulation of Lck activity and simultaneously reveal the most potent Lck mutants so far described

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Lck promotes Zap70-dependent LAT phosphorylation by bridging Zap70 to LAT

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2024-01-04 | PXD043454 | Pride

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The phosphatase DUSP22/JKAP inhibits UBR2-mediated K63-ubiquitination and activation of Lck in TCR signaling

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