Assessing structure-function effects of mutations in domains of Inducible Tyrosine Kinase Roman Levytskyy.

Assessing structure-function effects of mutations in domains of Inducible Tyrosine Kinase
Roman Levytskyy

Practical significance of Itk
Pathogenesis of asthma from W.S.F. Wong, K.P. Leong (2004)

Itk signaling Itk signaling pathway from P.L. Schwartzberg, L.D. Finkelstein, J.A. Readinger (2005)

Itk domain structure TH PH BH SH3 SH2 SH1 Btk/Tec PH BH SH3 SH1 SH2
PR1 PR2 SH3 SH2 SH1 Btk/Tec PH BH SH3 SH1 SH2 PR Itk/Emt/Tsk CCCCCCC PR SH3 SH2 SH1 Rlk PH BH SH3 SH2 SH1 Bmx/Etk SH3 SH2 SH1 Src (Lck, Fyn) SH2 SH2 SH1 Syk (Syk, ZAP-70) Tsoukas Lab, unpublished image

Conformations of Itk according to NMR data.
Itk conformations Conformations of Itk according to NMR data. Y180 phosphorylation is shown as red star Conformation B Y180 phosphorylation SH2 P287 trans SH3 Opening BH PH PPR KD Cyclophillin A Spontaneous isomerisation Conformation A SH2 P287 Cis Trans SLP-76 Conformation C

Hypotheses wtItk is distributed randomly in resting T-cells but changes localization pattern and conformation pattern upon TCR-induced activation. The above events and subsequent effects on cellular function are regulated by various domains of Itk

Phosphorylation site mutants
Y180F/E Y511F PH BH SH3 SH1 SH2 PR Y180F Y180E Y511F Y180 phosphorylation SH2 P287 trans SH3 Opening BH PH PPR KD

Poly Proline Region (PPR) and SH3 Domain Mutants
W208K PH BH SH3 SH1 SH2 PR Btk-SH3 PPR1 PPR2 Btk-SH3 W208K Y180 phosphorylation SH2 P287 trans SH3 Opening BH PH PPR KD

SH2 Domain and SH1 (Kinase) Domain Mutants
R265K P287G K390R PH BH SH3 SH1 SH2 PR R265K P287G K390R Cyclophillin A Spontaneous isomerisation SH3 BH PH PPR SH2 P287 Cis KD Trans

Btk-SH3-PPR2 Y180E-P287G Combination Mutants Y180E-P287G PH BH SH3 SH1
Btk-SH3-PRR2 Btk-SH3-PPR2 Y180E-P287G BH PH PPR KD SLP-76 SH2 P287 Trans SH3 Double mutants were created to have synergistic effect

F26S R29C Y87S Y90F F92S PH Domain Mutants Mutations PH BH SH3 SH1 SH2
PR F26S R29C Y87S Y90F F92S PH Domain mutants branched into a separate project, which is pursued by another graduate student in our lab

Aim 1. Design a battery of mutant constructs for conformation and function assessment
pME Vector constructs CMV Vector constructs CMV promoter can be used for expression in primary T cells, and can be used to transfect Itk-/- mouse cells for flow cytometry assays SR α (Large T Antigen dependent) promoter gives few high expressing cells for microscopy and biochemical assays in JTag and 293T cell lines

Aim 1. List of mutants Mutant Function 1 F26S Part of the FYF motiff in IP4 binding pocket 2 R29C Blocks PH domain interactions 3 Y87S ? 4 Y90F 5 F92S 6 Y180E False positive phosphorylation on molecular SH3 switch conf. A->conf. C 7 Y180F Nonphosphorylatable SH3 switch analog on conf. A->conf. C 8 Y511F Nonphosphorylatable analog of activation switch 9 PPR1 One critical prolin inactivated in PPR region 10 PPR2 Two critical prolins inactivated in PPR region, should disable SH3-PRR binding 11 Btk-SH3 Contains SH3 domain from Btk, should disable SH3-PRR binding 12 W208K Actin polymerization deficient 13 R265K Inactive 14 P287G Locks SH2 domain in trans 15 K390R Kinase inactive 16 Btk-SH3-PPR2 Contains mutants 10 and 11 combined. Mutations should have synergistic effect 17 Y180E-P287G Contains mutants 6 and 14 combined. Mutations should have synergistic effect

Aim 2. Assess conformation and localization changes
How does conformation and localization pattern change with introduction of different mutations?

Aim 2. FRET technique 535 nm 435 nm YFP CFP 500 nm 470 nm

Aim 2. C-Itk-Y in resting JTag cell
CFP localization YFP localization FRET EFRET map. Gives an idea which conformation Itk has in different locations FP localization maps. Give an idea where Itk molecules are localized

Antigen Presenting Cell –
Aim 2. JTag-Raji system MHC II SEE TCR T B Superantigen Antigen Presenting Cell – B lymphocyte (Raji) T lymphocyte (JTag) T B Immunological conjugate

Aim 2. Conjugate FRET patterns
CFP YFP FRET * λ * * * V * * * * V-shape FRET pattern is a predominant pattern for wtItk

Aim 2: FRET V-shape pattern
V Shape patterns, % control

Assess conformation changes and localization in other mutant groups
Aim 2. Future directions Assess conformation changes and localization in other mutant groups

Aim 3. Assess biochemical changes
How do biochemical properties of Itk and other signaling proteins change with introduction of mutations into Itk?

Aim 2. Transphosphorylation in Jurkat cells
wt Itk 2xFP- Itk NS S CITY Y87S Btk-SH3-PRR2 IP: αItk WB: αpY WB: αItk

Aim 3. In vitro kinase assay in 293T cells
K390R Itk-GFP C-Itk-Y IP: αItk WB: αpY 2xFP Itk FP Itk noFP Itk IP: αItk WB: αItk 2xFP Itk FP Itk noFP Itk

JTag PhosphoFlow: pERK
Nonstimulated Ab Stimulated PMA/IM Stimulated 12.0 10.0 0.1

JTag PhosphoFlow: pY511 Nonstimulated Stimulated 1.0 19.7

Wt mouse PhosphoFlow: pERK
Nonstimulated Ab Stimulated PMA/IM Stimulated 13.4 16.0 1.1

Wt mouse PhosphoFlow: pPLCγ
Nonstimulated Stimulated 0.3 16.1

Biochemical changes in other molecules caused by Itk mutations require Itk-/- environment
Primary issue: Endogenous wtItk in cell lines prevents from assessing the functions of the mutant Itk unless the mutant is dominant negative Secondary issue: Cell lines are abnormal, primary cells are much more preferred

Itk KO Mouse system Thymus Spleen Stimulate Itk-/- mouse + + Mutant
GFP-tagged Itk Transfect Isolate lymphocytes Culture

Selecting the source of lymphocytes
Nontransfected GFP control GFP-wtItk Splenocytes 0.1 0.3 0.1 Thymocytes 0.1 11.5 2.0

KO Mouse Thymocyte transfections
Nontransfected GFP control GFP-wtItk Nontransfected GFP control GFP-wtItk 0.1 % 40.9 % 24.1%

KO mouse transfected with GFP-wtItk PhosphoFlow: GFP+/pERK1/2+

KO mouse transfected with GFP-wtItk PhosphoFlow: GFP+/pY511+

Aim 3. Future directions PhosphoFlow of mutant groups in Itk KO mouse thymocytes pY511 pERK1/2 PLCγ Transphosphorylation of mutant groups in JTag by WB pY180 CoIP of mutant groups in JTag SLP-76 Endogenous wtItk

Aim 4. Assess biologically relevant functions
How does introduction of mutations influence actin polymerization and cytokine production?

Aim 4. Actin polymerization with Phalloidin in JTag

Aim 4. Future Directions Address actin polymerization in Itk-/- primary mouse T cells transfected with wt or mutant forms of Itk Address cytokine expression in Itk-/- primary mouse T cells transfected with wt or mutant forms of Itk IL-4 IL-13 IFNγ TNFβ

Acknowledgements Adviser and Chair of the Committee Brett Hilton
Dr. Constantine Tsoukas (SDSU) Committee members: Dr. Ananda Goldrath (UCSD) Dr. Cornelis Murre (UCSD) Dr. Ralph Feuer (SDSU) Dr. Roland Wolkowicz (SDSU) Current and former lab members (SDSU) Jason Rudy David Guimond Dr. Juris Grasis Ritz Zhang Roma Munday Alexa Spilsbury Brett Hilton Dr. Robert Zeller

Assessing structure-function effects of mutations in domains of Inducible Tyrosine Kinase Roman Levytskyy.

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