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REMAINING CLASSES

This summary highlights findings from lectures on phosphorylation dynamics and BR signaling in Arabidopsis. Key discussions include the quantitation of sucrose-induced phosphorylation in key proteins, validation of BSK1's role in BR signaling through innovative methods, and novel phosphorylation sites identified on AHA1 and AHA2. Results reveal significant similarities in phosphorylation profiles between plants and humans and emphasize the importance of protein interactions in signaling pathways. Finally, implications for understanding the phosphoproteome relevant to plant growth and development are presented.

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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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