scholarly journals Biophysical basis underlying dynamic Lck activation visualized by ZapLck FRET biosensor

2019 ◽  
Vol 5 (6) ◽  
pp. eaau2001 ◽  
Author(s):  
Rongxue Wan ◽  
Jenny Wu ◽  
Mingxing Ouyang ◽  
Lei Lei ◽  
Jiaming Wei ◽  
...  
Keyword(s):  
T Cells ◽  
Kinase Activity ◽  
Diffusion Rate ◽  
Live Cells ◽  
Wild Type ◽  
Tcr Signaling ◽  
Lck Kinase ◽  
Full Activation

Lck plays crucial roles in TCR signaling. We developed a new and sensitive FRET biosensor (ZapLck) to visualize Lck kinase activity with high spatiotemporal resolutions in live cells. ZapLck revealed that 62% of Lck signal was preactivated in T-cells. In Lck-deficient JCam T-cells, Lck preactivation was abolished, which can be restored to 51% by reconstitution with wild-type Lck (LckWT) but not a putatively inactive mutant LckY394F. LckWT also showed a stronger basal Lck-Lck interaction and a slower diffusion rate than LckY394F. Interestingly, aggregation of TCR receptors by antibodies in JCam cells led to a strong activation of reconstituted LckY394F similar to LckWT. Both activated LckY394F and LckWT diffused more slowly and displayed increased Lck-Lck interaction at a similar level. Therefore, these results suggest that a phosphorylatable Y394 is necessary for the basal-level interaction and preactivation of LckWT, while antibody-induced TCR aggregation can trigger the full activation of LckY394F.

scholarly journals CD8 beta chain influences CD8 alpha chain-associated Lck kinase activity.

1995 ◽  
Vol 181 (4) ◽  
pp. 1267-1273 ◽  
Author(s):  
H Y Irie ◽  
K S Ravichandran ◽  
S J Burakoff
Keyword(s):  
T Cells ◽  
Tyrosine Phosphorylation ◽  
Cd8 T Cells ◽  
Kinase Activity ◽  
Cross Linking ◽  
Beta Chain ◽  
Tyrosine Kinase Activity ◽  
Alpha Beta ◽  
Lck Kinase ◽  
Beta Gene

The CD8 molecule plays an important role in the differentiation of CD8+ T cells in the thymus and in their normal function in the periphery. CD8 exists on the cell surface in two forms, the alpha alpha homodimer and the alpha beta heterodimer. Recent studies indicate an important role for the CD8 beta chain in thymic development of CD8+ T cells and suggest that signaling via CD8 alpha beta may be distinct from CD8 alpha alpha. To better understand these differences, we introduced the CD8 beta gene into a T cell hybridoma which only expressed the CD8 alpha alpha homodimer. In the parent hybridoma, cross-linking of the CD8 alpha chain led to minimal enhancement of CD8-associated Lck tyrosine kinase activity. In the CD8 beta+ transfectants, several observations suggested that CD8 beta modifies CD8 alpha-associated Lck tyrosine kinase activity: (a) in in vitro kinase assays, antibody-mediated crosslinking of CD8 alone, or CD8 cross-linking with the TCR, resulted in 10-fold greater activation of Lck kinase activity, compared to cells expressing CD8 alpha alpha alone; (b) in vivo, markedly enhanced tyrosine phosphorylation of several intracellular proteins was observed upon CD8 cross-linking with the TCR in CD8 alpha beta-expressing cells, compared to cells expressing CD8 alpha alpha alone; and (c) Lck association with CD8 alpha was stabilized by the coexpression of CD8 beta. Thus, the differential Lck kinase activation and tyrosine phosphorylation seen with CD8 alpha alpha vs. CD8 alpha beta may reflect the unique signaling capabilities of the CD8 beta molecule. These differences in signaling may, in part, account for the diminished ability to generate CD8 single positive thymocytes in mice bearing a homozygous disruption of the CD8 beta gene.

scholarly journals Src kinase activity and SH2 domain regulate the dynamics of Src association with lipid and protein targets

2007 ◽  
Vol 178 (4) ◽  
pp. 675-686 ◽  
Author(s):  
Dmitry E. Shvartsman ◽  
John C. Donaldson ◽  
Begoña Diaz ◽  
Orit Gutman ◽  
G. Steven Martin ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Src Kinase ◽  
Sh2 Domain ◽  
Live Cells ◽  
Size Analysis ◽  
Membrane Association ◽  
Wild Type ◽  
Protein Targets ◽  
Associated Proteins ◽  
Specific Membrane

Src functions depend on its association with the plasma membrane and with specific membrane-associated assemblies. Many aspects of these interactions are unclear. We investigated the functions of kinase, SH2, and SH3 domains in Src membrane interactions. We used FRAP beam-size analysis in live cells expressing a series of c-Src–GFP proteins with targeted mutations in specific domains together with biochemical experiments to determine whether the mutants can generate and bind to phosphotyrosyl proteins. Wild-type Src displays lipid-like membrane association, whereas constitutively active Src-Y527F interacts transiently with slower-diffusing membrane-associated proteins. These interactions require Src kinase activity and SH2 binding, but not SH3 binding. Furthermore, overexpression of paxillin, an Src substrate with a high cytoplasmic population, competes with membrane phosphotyrosyl protein targets for binding to activated Src. Our observations indicate that the interactions of Src with lipid and protein targets are dynamic and that the kinase and SH2 domain cooperate in the membrane targeting of Src.

scholarly journals Protein tyrosine kinase p56-Lck regulates lymphocyte function-associated 1 adhesion molecule expression, granule exocytosis, and cytolytic effector function in a cloned T cell.

1994 ◽  
Vol 180 (3) ◽  
pp. 1115-1127 ◽  
Author(s):  
T Torigoe ◽  
J A Millan ◽  
K W Chan ◽  
R Taichman ◽  
A A Brian ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Adhesion Molecule ◽  
Kinase Activity ◽  
Cytolytic Activity ◽  
Effector Function ◽  
Lymphocyte Function ◽  
Lck Kinase ◽  
Cell Effector

Elevated levels of p56-Lck kinase activity were achieved in an interleukin 2 (IL-2)-dependent cloned cytolytic T cell CTLL-2 through gene transfer approaches. CTLL-2-Lck cells remained dependent on IL-2 for growth and survival in culture but exhibited markedly elevated, IL-2-independent cytolytic activity against a variety of tumor targets. This immune cell effector function was similar to the non-major histocompatibility complex-restricted cytolytic activity previously described for lymphokine activated killer (LAK) cells, and involved a cytolytic mechanism that was independent of protein synthesis in either the T cells or the tumor targets. Characterization of CTLL-2-Lck cells revealed markedly elevated levels of both the alpha (CD11a) and beta (CD18) chains of the cell adhesion molecule lymphocyte function-associated 1 (LFA-1) and increased binding of these T cells to a recombinant protein representing the extracellular domain of the LFA-ligand, intercellular adhesion molecule 1 (ICAM-1). Antibodies to CD11a partially abrogated cytolytic killing of tumor target cells by CTLL-2-Lck cells, suggesting that the upregulation in LFA protein levels potentially accounts at least in part for the phenotype of these T cells. Gene transfer-mediated elevations in p56-Lck kinase activity in an IL-3-dependent myeloid cell clone 32D.3 also resulted in increased LFA-1 expression, demonstrating that the findings are not unique to CTLL-2 cells. In addition to upregulation of LFA-1 expression, CTLL-Lck cells also exhibited more efficient exocytosis of cytotoxic granules upon activation with Ca(2+)-ionophore and phorbol ester, relative to control transfected and untransfected CTLL-2 cells. The findings functionally link the Lck kinase to a T cell effector pathway involved in cell-mediated cytotoxicity.

scholarly journals Topological Requirements and Signaling Properties of T Cell–activating, Anti-CD28 Antibody Superagonists

2003 ◽  
Vol 197 (8) ◽  
pp. 955-966 ◽  
Author(s):  
Fred Lühder ◽  
Yun Huang ◽  
Kevin M. Dennehy ◽  
Christine Guntermann ◽  
Ingrid Müller ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Nuclear Factor Κb ◽  
Tcr Signaling ◽  
D Loop ◽  
Full Activation

Full activation of naive T cells requires both engagement of the T cell antigen receptor (TCR; signal 1) and costimulatory signaling by CD28 (signal 2). We previously identified two types of rat CD28-specific monoclonal antibodies (mAbs): “conventional,” TCR signaling–dependent costimulatory mAbs and “superagonistic” mAbs capable of inducing the full activation of primary resting T cells in the absence of TCR ligation both in vitro and in vivo. Using chimeric rat/mouse CD28 molecules, we show that the superagonists bind exclusively to the laterally exposed C′′D loop of the immunoglobulin-like domain of CD28 whereas conventional, costimulatory mAbs recognize an epitope close to the binding site for the natural CD80/CD86 ligands. Unexpectedly, the C′′D loop reactivity of a panel of new antibodies raised against human CD28 could be predicted solely on the basis of their superagonistic properties. Moreover, mouse CD28 molecules engineered to express the rat or human C′′D loop sequences activated T cell hybridomas without TCR ligation when cross-linked by superagonistic mAbs. Finally, biochemical analysis revealed that superagonistic CD28 signaling activates the nuclear factor κB pathway without inducing phosphorylation of either TCRζ or ZAP70. Our findings indicate that the topologically constrained interactions of anti-CD28 superagonists bypass the requirement for signal 1 in T cell activation. Antibodies with this property may prove useful for the development of T cell stimulatory drugs.

scholarly journals Mutational analysis of Lck in CD45-negative T cells: dominant role of tyrosine 394 phosphorylation in kinase activity.

1996 ◽  
Vol 16 (9) ◽  
pp. 4996-5003 ◽  
Author(s):  
U D'Oro ◽  
K Sakaguchi ◽  
E Appella ◽  
J D Ashwell
Keyword(s):  
T Cells ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Mutational Analysis ◽  
Dominant Role ◽  
Tyrosine Phosphatase ◽  
Double Mutation ◽  
Tyrosine Residues ◽  
Lck Kinase

The CD45 tyrosine phosphatase has been reported to activate the src family tyrosine kinases Lck and Fyn by dephosphorylating regulatory COOH-terminal tyrosine residues 505 and 528, respectively. However, recent studies with CD45- T-cell lines have found that despite the fact that Lck and Fyn were constitutively hyperphosphorylated, the tyrosine kinase activity of both enzymes was actually increased. In the present study, phosphoamino acid analysis revealed that the increased phosphorylation of Lck in CD45- YAC-1 T cells was restricted to tyrosine residues. To understand the relationship between tyrosine phosphorylation and Lck kinase activity, CD45- YAC-1 cells were transfected with forms of Lck in which tyrosines whose phosphorylation is thought to regulate enzyme activity (Tyr-192, Tyr-394, Tyr-505, or both Tyr-394 and Tyr-505) were replaced with phenylalanine. While the Y-to-F mutation at position 192 (192-Y-->F) had little effect, the 505-Y-->F mutation increased enzymatic activity. In contrast, the 394-Y-->F mutation decreased the kinase activity to very low levels, an effect that the double mutation, 394-Y-->F and 505Y-->F, could not reverse. Phosphopeptide analysis of tryptic digests of Lck from CD45- YAC-1 cells revealed that it is hyperphosphorylated on two tyrosine residues, Tyr-505 and, to a lesser extent, Tyr-394. The purified and enzymatically active intracellular portion of CD45 dephosphorylated Lck Tyr-394 in vitro. These results demonstrate that in addition to Tyr-505, CD45 can dephosphorylate Tyr-394, and that in the absence of CD45 the hyperphosphorylation of Tyr-394 can cause an increase in the kinase activity of Lck despite the inhibitory hyperphosphorylation of Tyr-505. Therefore, Lck kinase activity is determined by the balance of activating and inhibitory tyrosine phosphorylations that are, in turn, regulated by CD45.

scholarly journals Naive arthritogenic SKG T cells have a defect in anergy and a repertoire pruned by superantigen

2022 ◽  
Author(s):  
Judith F Ashouri ◽  
Elizabeth McCarthy ◽  
Steven Yu ◽  
Noah Perlmutter ◽  
Charles Lin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
Antigen Receptor ◽  
Cell Subpopulation ◽  
Cytokine Signaling ◽  
Wild Type ◽  
Tcr Signaling

How autoreactive CD4 T cells develop to cause rheumatoid arthritis remains unknown. We used a reporter for antigen-receptor signaling in the SKG autoimmune arthritis model to profile a T cell subpopulation enriched for arthritogenic naive CD4 T cells before arthritis onset by bulk and single cell RNA and T cell antigen-receptor (TCR) sequencing. Our analyses reveal that despite their impaired proximal TCR signaling, a subset of SKG naive CD4 T cells that have recently encountered endogenous antigen upregulate gene programs associated with positive regulation of T cell activation and cytokine signaling at higher levels than wild type cells in the pre-disease state. These arthritogenic cells also induce genes associated with negative regulation of T cell activation but do so less efficiently than wild type cells. Furthermore, their TCR sequences exhibit a previously unrecognized biased peripheral TCR Vβ repertoire likely driven by endogenous viral superantigens. These particular Vβs, known to recognize endogenous mouse mammary tumor virus (MMTV) superantigen, are further expanded in arthritic joints. Our results demonstrate that autoreactive naive CD4 T cells which recognize endogenous viral superantigens are poised to cause disease by their altered transcriptome.

pp60c-src variants containing lesions that affect phosphorylation at tyrosines 416 and 527

1989 ◽  
Vol 9 (9) ◽  
pp. 3647-3656
Author(s):  
R Harvey ◽  
K M Hehir ◽  
A E Smith ◽  
S H Cheng
Keyword(s):  
Kinase Activity ◽  
Biochemical Properties ◽  
T Antigen ◽  
Wild Type ◽  
Biochemical Basis ◽  
Tyrosine Residues ◽  
Transforming Activity ◽  
Middle T Antigen ◽  
Full Activation

The biological and biochemical properties of pp60c-src are regulated, in part, by phosphorylation at Tyr-416 and Tyr-527. The tyrosine kinase and transforming activities of pp60c-src are suppressed by phosphorylation at Tyr-527, whereas full activation of pp60c-src requires phosphorylation at Tyr-416. To test specifically the significance of the negatively charged phosphate moieties on these tyrosine residues, we have substituted the codons for both residues with codons for either Glu or Gln. A negatively charged Glu at position 527 was unable to mimic a phosphorylated Tyr at this position, and, in consequence, the mutated pp60c-src was activated and transforming. Similarly, substitution of Tyr-416 with Glu was unable to stimulate the activities of the enzyme. However, mutagenesis of Tyr-416 to Gln (to form the mutant 416Q) activated the kinase activity approximately twofold over that observed for wild-type pp60c-src. When introduced into the mutant 527F (containing Phe-527 instead of Tyr), the double mutant 416Q-527F exhibited weak transforming activity. This is in contrast to the other double mutants 416E-527F and 416F-527F, which were nontransforming. The biochemical basis by which 416Q activates pp60c-src is not understood but probably involves some local conformational perturbation. Deletion of residues 519 to 524 (RH5), a region previously shown to be necessary for association with middle-T antigen, led to loss of phosphorylation at Tyr-527 and activation of the enzymatic and focus-forming activities of pp60c-src. Hence, the sequences necessary for complex formation with middle-T antigen may also be required by the kinase(s) which phosphorylates Tyr-527 in vivo. This suggests that normal cells contain cellular proteins which are analogous to middle-T antigen and whose action regulates the activity of pp60c-src by controlling phosphorylation or dephosphorylation at residue 527.

scholarly journals pp60c-src variants containing lesions that affect phosphorylation at tyrosines 416 and 527.

1989 ◽  
Vol 9 (9) ◽  
pp. 3647-3656 ◽  
Author(s):  
R Harvey ◽  
K M Hehir ◽  
A E Smith ◽  
S H Cheng
Keyword(s):  
Kinase Activity ◽  
Biochemical Properties ◽  
T Antigen ◽  
Wild Type ◽  
Biochemical Basis ◽  
Tyrosine Residues ◽  
Transforming Activity ◽  
Middle T Antigen ◽  
Full Activation

The biological and biochemical properties of pp60c-src are regulated, in part, by phosphorylation at Tyr-416 and Tyr-527. The tyrosine kinase and transforming activities of pp60c-src are suppressed by phosphorylation at Tyr-527, whereas full activation of pp60c-src requires phosphorylation at Tyr-416. To test specifically the significance of the negatively charged phosphate moieties on these tyrosine residues, we have substituted the codons for both residues with codons for either Glu or Gln. A negatively charged Glu at position 527 was unable to mimic a phosphorylated Tyr at this position, and, in consequence, the mutated pp60c-src was activated and transforming. Similarly, substitution of Tyr-416 with Glu was unable to stimulate the activities of the enzyme. However, mutagenesis of Tyr-416 to Gln (to form the mutant 416Q) activated the kinase activity approximately twofold over that observed for wild-type pp60c-src. When introduced into the mutant 527F (containing Phe-527 instead of Tyr), the double mutant 416Q-527F exhibited weak transforming activity. This is in contrast to the other double mutants 416E-527F and 416F-527F, which were nontransforming. The biochemical basis by which 416Q activates pp60c-src is not understood but probably involves some local conformational perturbation. Deletion of residues 519 to 524 (RH5), a region previously shown to be necessary for association with middle-T antigen, led to loss of phosphorylation at Tyr-527 and activation of the enzymatic and focus-forming activities of pp60c-src. Hence, the sequences necessary for complex formation with middle-T antigen may also be required by the kinase(s) which phosphorylates Tyr-527 in vivo. This suggests that normal cells contain cellular proteins which are analogous to middle-T antigen and whose action regulates the activity of pp60c-src by controlling phosphorylation or dephosphorylation at residue 527.

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