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

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

3. Tyrosine Phosphorylation Motifs PatternMatchesComment 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. MAJOR CONCLUSIONS 1.pS, pT and pY proportions similar to humans: ~ 85, 10 and 5% (respectively). Compare with frequency of residue in proteins: ~ 8, 6 and 3%. pY more prevalent than previously thought (no Tyr kinases in plants!) AND, protein phosphorylation as important in plants as in animals. 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) Isolate PM fraction Treat 2 h ± BL 2-DE with amazing resolution!

7. Confirmation of BR-induced Phosphorylation of BSK1

8. working γ 1.Used quantitative 2-DE to identify signal transduction component. 2.Prefractionation was essential. 3.BSKs are soluble proteins but found associated with the PM. Myristoylation likely involved. 4.Protein:protein interactions are involved in signaling: BSK:BRI1 and 14-3-3:BZR1 The New Model for BR Signaling

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

11. Protein Correlation Profiling for Label-Free Quantitation

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

13. 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)

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

15. 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.

16. 850 860 870 880 890 900 IRYILSGKAW ASLFDNRTAF TTKKDYGIGE REAQWAQAQR T LHGLQPKED VNIFPEKG S Y 910 920 930 940 RELSEIAEQA KRRAEIARLR ELHTLKGHVE SVAKLKGLDI DTAGHHY T V Novel Regulation of the H + -ATPase by C-terminal Phosphorylation Phospho Thr-881 activates in the absence of a 14-3-3 protein Phospho Thr-948 activates but only in the presence of a 14-3-3 protein