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REMAINING CLASSES

REMAINING CLASSES. Lec 11 Wed Sept 29 Lec 12 Mon Oct 4 also EVENING SESSION! (7-9pm) Lec 13 Wed Oct 6 (2 talks) Thu Oct 7 second EVENING SESSION (7-9PM) Lec 14 Mon Oct 11 (2 talks) Lec 15 Wed Oct 13 FINAL EXAM —in class or take home? . Lecture 11—Sept 29, 2010.

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REMAINING CLASSES

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  1. REMAINING CLASSES Lec 11 Wed Sept 29 Lec 12 Mon Oct 4 also EVENING SESSION! (7-9pm) Lec 13 Wed Oct 6 (2 talks) Thu Oct 7 second EVENING SESSION (7-9PM) Lec 14 Mon Oct 11 (2 talks) Lec 15 Wed Oct 13 FINAL EXAM—in class or take home?

  2. Lecture 11—Sept 29, 2010 • Finish Sugiyama et al. discussion • Phosphorylation and BR Signaling—BSK1 • Quantitation in Mass Spec • Quantitative analysis of sucrose-induced phosphorylation: AHA1/2

  3. Tyrosine Phosphorylation Motifs Pattern Matches Comment xxxxxxYRxxxxx 14 (14.6%) Novel xxExxxYxxxxxx 12 (12.5%) Novel xxxxDxYxxxxxx 11 (11.5%) SHP1 phosphatase: [DE]xY xxxxxxYxx[ST]xx[FY] 11 (11.5%) Novel xxxxxxYx[AG]xxxx 16 (16.7%) Novel

  4. pY more prevalent than previously thought (no Tyr kinases in plants!) AND, protein phosphorylation as important in plants as in animals. MAJOR CONCLUSIONS • pS, pT and pY proportions similar to humans: ~ 85, 10 and 5% (respectively). Compare with frequency of residue in proteins: ~ 8, 6 and 3%. 2. Tyr phosphoproteome shows some distinct features from pSer/pThr. 3. Larger % of nuclear proteins are phosphorylated compared to other subcellular compartments. 4. Majority (75%) of phosphorylation sites occur outside of conserved domains. [Except for pY, with ~50% within conserved domains.] 5. Twenty ‘pY motifs’ identified. Foundation for Future Studies

  5. BSK = BR-signaling kinase Two stage model for BR signaling (2006)

  6. det2-1 plants (7 d on agar) Treat 2 h ± BL Isolate PM fraction 2-DE with amazing resolution!

  7. Confirmation of BR-induced Phosphorylation of BSK1

  8. working γ The New Model for BR Signaling • Used quantitative 2-DE to identify signal transduction component. • Prefractionation was essential. • BSKs are soluble proteins but found associated with the PM. Myristoylation likely involved. • Protein:protein interactions are involved in signaling: BSK:BRI1 and 14-3-3:BZR1

  9. Over Expression of BSKs Can Partially Rescue the bri1-5 weak allele

  10. Protein Correlation Profiling for Label-Free Quantitation

  11. Proteome Coverage for Quantitation Only a subset of the proteome is identified, and of that, only the higher abundance proteins can be quantitated!

  12. Arabidopsis Seedlings: LL + Sucrose , 7 days DD - Sucrose, 1 day Resupply 30 mM Suc in the dark: 0 to 30 min Plasma Membranes isolated Phosphpeptides enriched (IMAC) LC/MS/MS and MS3 (label free quantitation)

  13. Sucrose Supply Increases Phosphorylation of Thr-947 Activating Proton Pumping

  14. New Phosphorylation Site Identified in the C-terminus of AHA1 and AHA2: Thr-881 Expression of AHA2 in a yeast pma1- mutant strain rescues growth on glucose. The T881D mutation strongly enhances growth suggesting that Phospho Thr-881 directly activates the pump.

  15. Novel Regulation of the H+-ATPase by C-terminal Phosphorylation Phospho Thr-881 activates in the absence of a 14-3-3 protein 850 860 870 880 890 900 IRYILSGKAW ASLFDNRTAF TTKKDYGIGE REAQWAQAQR TLHGLQPKED VNIFPEKGSY 910 920 930 940 RELSEIAEQA KRRAEIARLR ELHTLKGHVE SVAKLKGLDI DTAGHHYTV Phospho Thr-948 activates but only in the presence of a 14-3-3 protein

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