Roman Levytskyy

Publications

• In Vivo Consequences of Disrupting SH3-mediated Interactions of the Inducible T Cell Kinase (ITK)

  Roman M. Levytskyy, Nupura Hirve, David M. Guimond, Lie Min, Amy H. Andreotti, Constantine D. Tsoukas

  Journal of Signal Transduction. 02/2012;

  ITK-SH3 mediated interactions, both with exogenous ligands and via intermolecular selfassociation with ITK-SH2, have been shown to be important for regulation of ITK activity. The biological significance of these competing SH3 interactions is not completely understood. A mutant of ITK where substitu... [more] ITK-SH3 mediated interactions, both with exogenous ligands and via intermolecular selfassociation with ITK-SH2, have been shown to be important for regulation of ITK activity. The biological significance of these competing SH3 interactions is not completely understood. A mutant of ITK where substitution of the SH3 domain with that of the related kinase BTK (ITKBTK(SH3)) was used to disrupt intermolecular self-association of ITK while maintaining canonical binding to exogenous ligands such as SLP-76. ITK-BTK(SH3) displays reduced association with SLP-76 leading to inefficient transphosphorylation, reduced phosphorylation of PLCγ1, and diminished Th2 cytokine production. In contrast, ITK-BTK(SH3) displays no defect in its localization to the T cell-APC contact site. Another mutation, Y511F, in the activation loop of ITK, impairs ITK activation. T cells expressing ITK-Y511F display defective phosphorylation of ITK and its downstream target PLCγ1, as well as significant inhibition of Th2 cytokines. In contrast, the inducible localization of ITK-Y511F to the T cell-APC contact site and its association with SLP-76 are not affected. The presented data lend further support to the hypothesis that precise interactions between ITK and its signaling partners are required to support ITK signaling downstream of the TCR.

• Structure-function effects of mutations in domains of Inducible Tyrosine Kinase

  Roman M. Levytskyy

  01/2012

  Degree: PhD

  Supervisor: Constantine D. Tsoukas

  The dissertation is studying structural mutants of various domains of the Inducible Tyrosine Kinase (Itk), and their relation to functionality of this protein in immune cells. Using cell lines as well as primary cells from Itk-/- mice, and employing systematic functional study approach this work is ... [more] The dissertation is studying structural mutants of various domains of the Inducible Tyrosine Kinase (Itk), and their relation to functionality of this protein in immune cells. Using cell lines as well as primary cells from Itk-/- mice, and employing systematic functional study approach this work is exploring effects of several mutations (Y511F, BtkSH3, FYF) on immediate, and far downstream signaling of Itk under conditions of TCR induced stimulation. Structural (FRET), as well as biochemical and molecular biology techniques were used (western blotting, flow cytometry, ELISA). It was shown that Itk does not exhibit noticeable differences neither in structure, nor in localization in resting cells, but changes conformation and localization patterns under stimulation in T-cell-APC system. This event is pronounced in statistically significant manner in wild type Itk, but is disrupted to different extent in mutants. PH domain mutant FYF is the only one from those explored that has disrupted localization pattern, and surprisingly, nonfunctional activation residue mutant Y511F preserves intact localization. However, all mutants show disruption in structural pattern when compared to wild type Itk. Another novel finding is related to Itk-SLP-76 interaction, which proved to be disrupted in BtkSH3 mutant that lacks noncanonical SH3 domain interactions, pointing on important role of noncanonical SH3 interactions in signaling of Itk in complex with SLP-76, and as a result in activation of Itk. All mutants show some extent of disruption in Y511 phosphorylation, which has a direct effect on downstream events as assessed by PLC[Gamma] phosphorylation, and reflects defects in structure and localization patterns, with mutants being significantly deficient comparing to wild type. This results in even more pronounced effect in Th2 cytokine production, and this dissertation provides the first evidence of the influence of these mutants on Th2 cytokines, which are the signature cytokines of Itk. The dissertation proposes explanations of the mentioned phenomena, underlines an importance of different parts of Itk molecule in its functionality, and makes possible to apply the data acquired here to further understand the role of Itk in deeper detail with possible clinical implications in the future.

• Functional effects of the structural mutants of Itk domains

  Levytskyy R.M., Hirve N., Tsoukas C.D.

  SDSU Graduate Research Symposium, SDSU Bioscience Center, San Diego, CA; 01/2011

  The Inducible T cell kinase (Itk) is important for T cell development and activation. In our lab we use mutational analysis in an attempt to understand ITK structure-function relationships. Here we have used three mutants, one with a mutation in the transphosphorylation target site (Y511F), anoother... [more] The Inducible T cell kinase (Itk) is important for T cell development and activation. In our lab we use mutational analysis in an attempt to understand ITK structure-function relationships. Here we have used three mutants, one with a mutation in the transphosphorylation target site (Y511F), anoother with a substitution of the ITK-SH3 domain with that of the related kinase Btk (Itk/Btk-SH3), and third one with mutated residues in PH domain binding pocket (FYF). Latter mutants are predicted to disrupt the ability of ITK to multimerize. Upon TCR engagement, wild type Itk forms a specific conformational pattern at the T cell-APC contact site. Occurrence of this pattern is disrupted in the Y511F and FYF mutants, but remains intact in the Itk/Btk-SH3 mutant. Localization of Y511F and Btk-SH3 mutants to the contact site remains similar to wild type Itk, but it is disrupted in FYF mutant. Even though the transphosphorylation and enzymatic activation of Y511F is disrupted, its association with signaling partner SLP-76, remains intact. In contrast, the interaction of Itk/Btk-SH3 shows normal phosphorylation behavior, but decreased interaction with SLP-76. All mutants showed no significant defects in their ability to associate with another signaling partner LAT. Mouse thymocytes from Itk-deficient mice, transfected with Y511F mutant display defective ITK phosphorylation and significant decrease in the phosphorylation of the ITK downstream target, PLCγ1. Btk-SH3 mutant also showed lower PLCγ1 and pY511 phosphorylation. Ongoing experiments are addressing the effects of FYF mutant on these signaling events, as well as the effects of all three mutants on cytokine production.

• Spatiotemporal behavior, intracellular signaling and interactions of Itk in T cells.

  Levytskyy R.M., Tsoukas C.D.

  36th La Jolla Immunology Conference, Salk Institute, La Jolla, CA; 01/2010

  We have used two mutants, one with a mutation in the transphosphorylation target site (Y511F), the other with a substitution of the ITK-SH3 domain with that of the related kinase Btk (Itk/Btk-SH3). This latter mutant is known to disrupt the ability of ITK to multimerize. Upon TCR engagement, wild ty... [more] We have used two mutants, one with a mutation in the transphosphorylation target site (Y511F), the other with a substitution of the ITK-SH3 domain with that of the related kinase Btk (Itk/Btk-SH3). This latter mutant is known to disrupt the ability of ITK to multimerize. Upon TCR engagement, wild type Itk forms a specific conformational pattern at the T cell-APC contact site. Occurrence of this pattern is disrupted in the Y511F mutant, but remains intact in the Itk/Btk-SH3 mutant. Localization of both mutants to the contact site remains similar to wild type Itk. Even though the transphosphorylation and enzymatic activation of Y511F is disrupted, its association with signaling partners, SLP-76 and LAT, remains intact. In contrast, the interaction of Itk/Btk-SH3 shows normal phosphorylation behavior, but decreased interaction with SLP-76. In transfected Itk-KO mouse thymocytes, Y511F shows decreased PLCγ1 and ERK1/2 phosphorylation. Ongoing experiments are addressing the effects of ITK/Btk-SH3 on these signaling events, as well as the effects of both mutants on cytokine production.

• Method of detection of nitrite ions in solutions

  Levytskyy R.M., Hryvul T.M., Stoika R.S.

  Ref. No: UA76576

  Year: 01/2005

• Correlation between generation of nitric oxide and cell viability in human peripheral blood mononuclear cells and leukemic Jurkat T-cell line.

  Roman M Levytskyy, Yevgeniy Z Filyak, Rostislav S Stoika

  Experimental oncology. 10/2004; 26(3):217-20.

  AIM: To measure nitric oxide (NO) production in the form of nitrite derivative in relation to cell viability and apoptosis development in human peripheral blood mononuclear cells compared to that processes in human leukemic Jurkat T-cell line. METHODS: Apoptosis was induced by dexamethasone (1 micro... [more] AIM: To measure nitric oxide (NO) production in the form of nitrite derivative in relation to cell viability and apoptosis development in human peripheral blood mononuclear cells compared to that processes in human leukemic Jurkat T-cell line. METHODS: Apoptosis was induced by dexamethasone (1 microg/ml) or NaNO(2) (7 microg/ml) added in the presence or absence of NO-synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) (27 microg/ml) during cell culturing. Cell viability was determined by trypan blue assay. Apoptosis was measured using DNA "ladder" assay. RESULTS: Dexamethazone and NaNO(2) were shown to cause DNA "laddering" in both cell types. L-NAME prevented the appearance of apoptosis in both normal mononuclear cells of peripheral blood and leukemic Jurkat T-cell in the case of dexamethasone action, but it could not prevent it in the case of NaNO(2) action. The results of cell viability showed that both the dexamethasone and NaNO(2) significantly increased the percentage of dead cells. Their effect was better expressed in Jurkat T-cell line. The levels of nitrite production were higher in the leukemic T-cells comparing to such levels in the normal mononuclear cells. CONCLUSION: Strong positive correlation was demonstrated between NO production and apoptosis development in both studied cell types, however leukemic Jurkat T-cell line responses were better expressed than such responses in normal mononuclear cells of peripheral blood. Potential significance of that correlation as well as possible mechanisms of appearing differences are discussed.

• Impact of dexametazone on nitrite production by T-leukemic cells of Jurkat T-cell line

  Levytskyy R.M., Panchyshyn O.J., Hryvul О.Т., Makukh J.М., Stoika R.S.

  All-Ukrainian students’ conference in Lviv State Academy of Veterinary Medicine, Lviv State Academy of Veterinary Medicine; 01/2003

• Dualism of nitric oxide action in apoptosis

  Levytskyy R.M., Lozinsky R.P., Panchyshyn O.J., Negrych T.I., Stoika R.S.

  The Animal Biology. 01/2003; 5:39-51.

• Blood indices of bloodstock bulls

  Hryvul О.Т., Makukh J.М., Tsarik R.V., Levytskyy R.M., Korobov V.M.

  International students’ conference in Lviv State Academy of Veterinary Medicine, Lviv State Academy of Veterinary Medicine; 01/2001