REMAINING CLASSESREMAINING CLASSES

Lec 11 Wed Sept 29

Lec 12 Mon Oct 4 also EVENING SESSION! (7-9pm)EVENING SESSION! (7-9pm)

Lec 13 Wed Oct 6 (2 talks)

Thu Oct 7 second EVENING SESSION (7-9PM)EVENING SESSION (7-9PM)

Lec 14 Mon Oct 11 (2 talks)

Lec 15 Wed Oct 13 FINAL EXAMFINAL EXAM—in class or take home?

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, 2010Lecture 11—Sept 29, 2010

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

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

BSK = BR-signaling kinase

Two stage model for BR signaling (2006)

det2-1 plants (7 d on agar)

Isolate PM fraction

Treat 2 h ± BL

2-DE with amazing resolution!

Confirmation of BR-induced Phosphorylation of BSK1

workingγ

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

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

Protein Correlation Profiling for Label-Free Quantitation

Proteome Coverage for Quantitation

Only a subset of the proteome is identified, and of that, only the higher abundance proteins can be quantitated!

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)

Sucrose Supply Increases Phosphorylation of Thr-947 Activating Proton Pumping

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.

850 860 870 880 890 900 IRYILSGKAW ASLFDNRTAF TTKKDYGIGE REAQWAQAQR TLHGLQPKED VNIFPEKGSY

910 920 930 940 RELSEIAEQA KRRAEIARLR ELHTLKGHVE SVAKLKGLDI DTAGHHYTV

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