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Predictive Model of Lymphocyte-Specific Protein Tyrosine Kinase (LCK) Autoregulation.

ABSTRACT: Lymphocyte-specific protein tyrosine kinase (LCK) is a key activator of T cells; however, little is known about the specific autoregulatory mechanisms that control its activity. We have constructed a model of LCK autophosphorylation and phosphorylation by the regulating kinase CSK. The model was fit to existing experimental data in the literature that presents an in vitro reconstituted membrane system, which provides more physiologically relevant kinetic measurements than traditional solution-based systems. The model is able to predict a robust mechanism of LCK autoregulation. It provides insights into the molecular causes of key site-specific phosphorylation differences between distinct experimental conditions. Probing the model also provides new hypotheses regarding the influence of individual binding and catalytic rates, which can be tested experimentally. This minimal model is required to elucidate the mechanistic interactions of LCK and CSK and can be further expanded to better understand T cell activation from a systems perspective. Our computational model enables the evaluation of LCK protein interactions that mediate T cell activation on a more quantitative level, providing new insights and testable hypotheses.

SUBMITTER: Rohrs JA

PROVIDER: S-EPMC4978775 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Predictive Model of Lymphocyte-Specific Protein Tyrosine Kinase (LCK) Autoregulation.

Rohrs Jennifer A JA Wang Pin P Finley Stacey D SD

Cellular and molecular bioengineering 20160426

Lymphocyte-specific protein tyrosine kinase (LCK) is a key activator of T cells; however, little is known about the specific autoregulatory mechanisms that control its activity. We have constructed a model of LCK autophosphorylation and phosphorylation by the regulating kinase CSK. The model was fit to existing experimental data in the literature that presents an <i>in vitro</i> reconstituted membrane system, which provides more physiologically relevant kinetic measurements than traditional solu ...[more]

PMID: 27547268

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Project description:BackgroundUpon engagement of the T-cell receptor (TCR), the Src-family protein tyrosine kinase p56Lck phosphorylates components of the TCR (e.g. the TCRζ chains), thereby initiating T-cell activation. The enzymatic activity of Lck is primarily regulated via reversible and dynamic phosphorylation of two tyrosine residues, Y394 and Y505. Lck possesses an additional highly conserved tyrosine Y192, located within the SH2 domain, whose role in T-cell activation is not fully understood.MethodsKnock-in mice expressing a phospho-mimetic (Y192E) form of Lck were generated. Cellular and biochemical characterization was performed to elucidate the function of Y192 in primary T cells. HEK 293T and Jurkat T cells were used for in vitro studies.ResultsCo-immunoprecipitation studies and biochemical analyses using T cells from LckY192E knock-in mice revealed a diminished binding of LckY192E to CD45 and a concomitant hyperphosphorylation of Y505, thus corroborating previous data obtained in Jurkat T cells. Surprisingly however, in vitro kinase assays showed that LckY192E possesses a normal enzymatic activity in human and murine T cells. FLIM/FRET measurements employing an LckY192E biosensor further indicated that the steady state conformation of the LckY192E mutant is similar to Lckwt. These data suggest that Y192 might regulate Lck functions also independently from the Lck/CD45-association. Indeed, when LckY192E was expressed in CD45-/-/Csk-/- non-T cells (HEK 293T cells), phosphorylation of Y505 was similar to Lckwt, but LckY192E still failed to optimally phosphorylate and activate the Lck downstream substrate ZAP70. Furthermore, LckY19E was recruited less to CD3 after TCR stimulation.ConclusionsTaken together, phosphorylation of Y192 regulates Lck functions in T cells at least twofold, by preventing Lck association to CD45 and by modulating ligand-induced recruitment of Lck to the TCR.Major findingsOur data change the current view on the function of Y192 and suggest that Y192 also regulates Lck activity in a manner independent of Y505 phosphorylation. Video Abstract.

Project description:Soluble extracts prepared from bovine thymus contain an angiotensin-I-phosphorylating activity that is activated several-fold by high concentrations of NaCl. Fractionation of this protein-tyrosine kinase activity by chromatography on DEAE-cellulose yields a major diffuse peak of activity. The enzymes responsible for this activity are found at much higher levels in extracts from bovine thymus than from bovine spleen. The peak of activity from the DEAE-cellulose column can be further separated into two major peaks by chromatography on heparin-agarose. The second peak to elute from the heparin-agarose column was previously purified through several chromatographic steps to yield a 40 kDa protein-tyrosine kinase (p40). We have now partially purified the early-eluting peak of kinase activity by chromatography on columns of butyl-agarose, protamine-agarose and Sephacryl S200. The enzyme was identified following covalent modification with 5'-fluorosulphonylbenzoyladenosine (FSBA) by reactivity with anti-FSBA antibodies. This procedure labelled a series of 52-56 kDa proteins. These proteins were also recognized by polyclonal anti-peptide antibodies raised against the C-terminal 33 amino acids of p56lck, a major T lymphocyte protein-tyrosine kinase. Peptide maps of the partially purified enzyme were identical to maps generated from p56lck obtained from LSTRA cells. These data suggest that bovine thymus p56lck is responsible for the activity found in the early-eluting peak from heparin-agarose. Antibodies raised against a peptide corresponding to amino acids 39-64 of p56lck, a sequence found near the N-terminus, recognized the slower-migrating, but not the faster-migrating, form of the enzyme, indicating that a fraction of the protein had been proteolysed near the N-terminus during purification. The partially purified bovine enzyme exhibited a restricted substrate specificity in vitro and did not readily phosphorylate human erythrocyte band 3, casein or histone, but was able to phosphorylate acid-treated enolase. The dilute enzyme present in fractions eluting from chromatography columns was unable to catalyse an autophosphorylation reaction. Autophosphorylation could be detected in more concentrated enzyme samples and was readily observed in immune-complex assays. The phosphorylation of angiotensin I by bovine thymus p56lck was weakly activated by polyionic compounds such as heparin and polylysine, and was strongly activated by high concentrations of NaCl.

Dataset Information

Predictive Model of Lymphocyte-Specific Protein Tyrosine Kinase (LCK) Autoregulation.

Publications

Predictive Model of Lymphocyte-Specific Protein Tyrosine Kinase (LCK) Autoregulation.

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