View
224
Download
0
Category
Preview:
Citation preview
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!
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
Recommended