Lymphocyte Activation Receptor signal transduction David Straus dbstraus@vcu

1. Lymphocyte Activation • Receptor signal transduction • David Straus • dbstraus@vcu.edu • Reading: Janeway text; 6-1 to 6-17

2. Specificity of T lymphocyte activation relies upon TCR recognition of MHC-peptide: a relatively low affinity interaction compared to immunoglobulin. TCR MHC- peptide MHC I TCR

3. CD4/8 coreceptors CD4 and CD8 molecules act as “co-receptors”: they recognize MHC II or MHC I, and help stabilize the TCR-MHC interaction by forming a tripartite complex

4. TCR signals enhance integrin binding Affinity of integrin interaction is enhanced following successful antigen receptor engagement - “inside-out” signaling

5. Molecular rearrangements w/synapse formation T cell receptor engagement with MHC-peptide induces reorganization of cell surface molecules.

7. How does receptor engagement lead to lymphocyte activation? Y ?

8. Basic signal transduction -signals are transmitted through changes in specific activity or location of proteins -these changes in protein function are mediated by protein-protein interactions, by binding small molecules (e.g. Ca++), or by covalent modifications (e.g. phosphorylation).

9. Interaction domains found in signaling proteins can modify function through changes in activity and/or localization. PO4- Tyr Pro Pro Pro Domain Ligand Phosphotyrosine SH2 Proline-rich sequences SH3 Phosphatidylinositol Triphosphate (PIP3) PH also see Fig 6.2

10. Membrane protein localization may be determined by segregation within the plasma membrane, based on the existence of “ lipid raft” domains

11. -P-P-P- Enzymes can modify protein function by covalent modification. Protein tyrosine kinases (PTKs) play a major role in antigen receptor signaling by phosphorylating target proteins. Src kinase family (e.g. Lck, Fyn, Lyn, Blk) Syk/ZAP-70 family Tec kinase family (e.g. Itk,Btk,Rlk) PH SH3 SH2 Catalytic domain PTKs have conserved catalytic domains and protein interaction domains

12. Tyrosine kinase activity is regulated by tyrosine phosphorylation and protein interaction domains. Src-family kinases are regulated by intramolecular interactions, the CD45 tyrosine phosphatase, and the Csk kinase. SH2 domain binding to a phosphotyrosine inhibits enzyme activity. The CD45 phosphatase can dephosphorylate this tyrosine, while the Csk kinase phosphorylates it. Src kinases autophosphorylate an activating tyrosine in the catalytic domain.

13. Ligand binding changes the activity of receptors, resulting in the transduction of a signal across the cell membrane

14. Receptors use scaffolds to activate intracellular signaling pathways. Scaffolds and adaptors have no catalytic activity, but provide binding sites which bring together effector and target proteins, promoting efficient and specific signal transduction.

15. Intracellular signaling pathways include kinase cascades and second messenger flux

16. Initiation of TCR signaling remains uncertain, but involves receptor aggregation and/or conformational changes upon binding of MHC-peptide.

17. Src-family kinases initiate TCR signaling by phosphorylating receptor ITAM motifs and the ZAP-70 kinase following its recruitment to the receptor. CD4 and CD8 co-receptor binding to MHC helps direct the Lck kinase to the receptor.

19. ~P ~P ~P The LAT and SLP-76 adaptors co-localize PLCg and the Tec family kinase ITK. Activation of PLCg by ITK induces cleavage of PIP2 to DAG and IP3. DAG PLCg1 IP3 ITK LAT P~ SLP-76

20. Phosphatidylinositol signaling pathway: Phospholipase C activity generates second messengers, inositol trisphosphate (IP3) and diacylglycerol (DAG) which increase levels of intracellular calcium, and recruits Protein kinase C (PKC) and RasGRP to the membrane.

21. R R a a s s Ras Ras G G T T P P GDP GDP Raf Y-PO 4 RasGRP SOS MEK ERK The transmembrane adaptor LAT recruits SOS, a GTP exchange factor, to the membrane where it helps activate Ras and initiate the (ERK) MAP kinase pathway GRB2 LAT MEK

22. Ras activation initiates the sequential phosphorylation and activation of a series of MAP kinases leading to increased gene transcription. There are several distinct MAP kinase cascades, only one of which is shown here.

23. P~ ~P P~ ~P LAT LAT SOS SOS GRB2 GRB2 GRB2 GRB2 Each SOS exchange factor can bind two Grb2 adaptor molecules Houtman, et al. Nature Structural Biology 13: 798-805, 2006

24. Mutation of the tyrosine phosphorylation sites in the carboxy-terminus of LAT that mediate Grb2 binding prevents LAT clustering following TCR stimulation

25. Signaling complex (signalsome) formation mediated by multivalent protein interactions

26. If wildtype LAT is overexpressed, how will this effect TCR signaling?

27. Regulation of gene expression through increased transcription factor activity NFAT: nuclear localization regulated by calcium-activated phosphatase AP-1: expression and activity regulated by MAPK pathways NFkB: nuclear localization regulated by degradation

28. MAP kinase pathways increase AP-1 dependent transcription by increasing expression of the Fos subunit, and by increasing activity of the Jun subunit Two different MAP Kinase pathways are involved in receptor signaling

29. The NFAT transcription factor is normally phosphorylated and sequestered in the cytosol. The PI pathway induces calcium influx which activates Calcineurin phosphatase activity and promotes nuclear translocation of the NFAT transcription factor.

30. The NFkB transcription factor is initially sequestered in the cytosol by IkB. PKC-mediated assembly of a CARMA - Bcl10 - MALT complex leads to IK Kinase activation and phosphorylation of IkB, which is then degraded. Once released, NFkB translocates to the nucleus.

31. A combination of transcription factors respond to antigen receptor signals to regulate IL-2 gene expression and promote T cell activation.

32. Stimulated antigen receptors utilize tyrosine kinases to induce intracellular signaling pathways leading to transcription factor activation and the induction of cell growth and differentiation. Y PTKs adaptors PI pathway MAP kinase pathways Interleukin-2, and effector functions